InteractiveFly: GeneBrief

Insulin-like peptide 2: Biological Overview | References

BIOLOGICAL OVERVIEW

The insulin/IGF-like signalling (IIS) pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs) in Drosophila are expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs) in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, RNA interference was used to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. It was found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. A compensatory increase was observed in dilp3 and dilp5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, it was showm that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. This study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps (Broughton, 2008).

The insulin/IGF-like signalling (IIS) pathway, present throughout multicellular animals, has functions including the regulation of growth, development and metabolic homeostasis, as well as determination of adult lifespan, resistance to stress and fecundity, in C. elegans, Drosophila and mouse. Mutations that reduce the activity of IIS can increase lifespan in all three organisms, often in conjunction with associated alterations in growth, stress resistance, metabolic phenotypes and fecundity. Identifying the precise modulations of IIS that are required for lifespan-extension is thus important for determining which, if any, of the associated phenotypes are either causal in extension of lifespan or unavoidably associated with it (Broughton, 2008).

Intracellular components of IIS are encoded by single genes in the invertebrates C. elegans and Drosophila, with the exception of the triplication of the protein kinase B, Akt, SGK-1 in C. elegans, resulting in a relatively simple intracellular signalling pathway. In contrast, mammals have several versions of the intracellular components of IIS. In contrast, there are multiple genes for the insulin-like ligands in the two invertebrates, with seven detected in the Drosophila genome and 38 in C. elegans. The seven Drosophila insulin-like peptides (DILPs) are predicted to resemble preproinsulin at the structural level, and are therefore considered orthologous to mammalian insulin. The genes encoding the Drosophila insulin-like peptides are independently transcriptionally regulated in response to nutrition, as well as in a tissue- and stage-specific manner during development (Brogiolo, 2001; Ikeya, 2002). Thus, in these invertebrates, the multiple functions of IIS may be mediated in part by functional diversification of the ligands (Broughton, 2008).

Reducing the levels of a subset of the DILPs, by ablation of DILP2, 3 and 5-producing mNSCs in the pars intercerebralis of the brain late in the final larval instar, leads to an array of phenotypes including increased fasting glucose levels in the adult hemolymph, increased storage of lipid and carbohydrate, reduced fecundity, extension of median and maximal lifespan and increased resistance to oxidative stress and starvation (Broughton, 2005). Ablation of these cells earlier in larval development resulted in developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph (Rulifson, 2002). These findings imply that reduction in the level of one or more of the three DILPs produced in these cells causes this diverse array of phenotypes, but direct proof of this, together with information on possible functional diversification and redundancy of the DILPs, requires direct manipulation of the levels of individual DILPs (Broughton, 2008).

Of the DILPs produced by the mNSCs, DILP2 is currently thought to be the most important. It is the most highly expressed, the most potent growth stimulator and over-expression of it alone can rescue the diabetic phenotypes of mNSC-ablated larvae (Broughton, 2005: Rulifson, 2002: Ikeya, 2002). Furthermore, DILP2 has been suggested to play a prominent role in lifespan-extension by reduced IIS (Hwangbo, 2004; Min, 2008). Hwangbo (2004), and Min (2008), reported a reduction in dilp2, but not in dilp3 and 5 mRNA, in response to activated FOXO in head fat body, and proposed that this reduction mediated lifespan-extension. dilp2 expression also responds to JNK activation in the mNSCs, and the resulting lifespan-extension was suggested to be mediated by Foxo-dependent repression of dilp2 (Wang, 2005). Bauer (2007) showed that expression of a dominant negative form of p53 in adult neurons extended lifespan and reduced dilp2 transcript levels, and again suggested that the reduction of dilp2 expression was responsible for the increase in lifespan. However, none of these studies experimentally manipulated dilp2 expression and nor did they establish whether DILP protein levels were affected by the changes in transcript level (Broughton, 2008).

To test experimentally the hypothesis that a reduction in dilp2 alone produces lifespan extension, and to ascertain if other phenotypes regulated by DILP2 could be determinants of lifespan, dilp2 expression was reduced using RNAi specifically against dilp2 in the mNSCs, and the consequences for lifespan-extension and other phenotypes associated with mNSC ablation were examined. It was found that reducing dilp2 alone in the adult fly to a level similar to that due to mNSC ablation and observed in the above-mentioned studies correlating dilp2 levels and lifespan had no effect on lifespan, fecundity, stress resistance, hemolymph carbohydrate levels or glycogen levels. A possible explanation of this finding is compensation by increased expression of one or more of the other dilps, if there can be functional redundancy between them. Indeed, increases in dilp3 and 5 mRNA were observed following dilp2 knock down, suggesting compensatory regulation of dilp3 and 5. In the case of dilp3, this up-regulation may be due to reduced IIS in the mNSCs upon dilp2 knock down, since it was showm that dilp3 is up-regulated upon down regulation of IIS via manipulation of the insulin receptor activity and that it requires FOXO for its basal expression. Knock down of dilp2 did, however, lead to an increase in total trehalose content of the same magnitude as that resulting from mNSC ablation. dilp2 expression is therefore not limiting for lifespan, and it plays an individual role only in the increase in trehalose storage among the phenotypes affected by mNSC ablation. Furthermore, the data show that increased trehalose storage alone is not sufficient to extend lifespan (Broughton, 2008).

This study successfully and specifically reduced both the levels of RNA and protein; DILP2 levels were limiting for only one of the phenotypes resulting from mNSC ablation, increased whole body trehalose content. It remains quite possible that the lifespan extensions due to the FOXO, JNK or p53 manipulations are mediated by reductions in one or more of DILPs 2, 3 and 5, because the context in which the level of dilp2 was lowered was different in each case. This could, for instance, have resulted in different levels of transcript for other dilps or different states of the intracellular IIS pathway. These findings do show clearly, however, that neither reduction in dilp2 RNA and DILP2 protein expression alone nor increased trehalose storage alone is sufficient to extend lifespan (Broughton, 2008).

Of the phenotypes observed on mNSC-ablation, it was found that reduction in DILP2 had an effect only on whole body trehalose levels. This result could indicate that reduction in DILP2 per se is required for increased stored tehalose upon mNSC-ablation. Alternatively, if there is functional redundancy between the 3 DILPs produced in the mNSCs, it is possible that trehalose storage is the most sensitive of the phenotypes to a reduction in the overall expression of DILPs in the mNSCs. The compensation observed in transcript levels of dilps 3 and 5 upon reduction in dilp2 transcript may not be sufficient to bring overall dilp transcript levels in the mNSCs back to normal, because dilp2 is much more highly expressed than the other two (Broughton, 2005; Hwangbo, 2004). Similar arguments apply to the lack of effect of reduction in DILP2 levels on the other phenotypes associated with mNSC-ablation. Because of the compensation in dilps 3 and 5, the result could indicate that there is functional redundancy between DILPs and that there was insufficient reduction in overall DILP levels for these phenotypes to appear. Consistent with redundancy, while it was shown that DILP2 levels are not limiting for the hemolymph carbohydrate phenotype, over-expression of dilp2 alone can rescue the growth and hemolymph carbohydrate phenotypes due to early ablation of the mNSCs (Rulifson, 2002). Alternatively DILP2 may not be involved in producing these phenotypes when the mNSCs are ablated, other than the increased whole body trehalose. To determine which interpretation is correct would require independent manipulation to varying degrees of the different dilps (Broughton, 2008).

The increases in dilp3 and 5 transcripts when dilp2 is knocked down suggest that there may be compensatory increases in DILP3 and 5 proteins. It is interesting in this respect that the data show that dilp3 is the only dilp in the mNSCs whose expression is sensitive to reduced insulin signaling in these cells, implicating an autocrine feedback loop. The other two dilps may be regulated in response to other signals, such as nutritional status in the case of dilp5 (Min, 2008). Hence, different dilps may be produced in response to different intrinsic/extrinsic stimuli but once produced function redundantly (Broughton, 2008).

A specific role for DILP2 in trehalose metabolism raises the possibility of distinct roles for DILPs 3 and 5 in one or more of the phenotypes of the ablated flies, including lifespan. Although all seven DILPs are capable of promoting growth (Brogiolo, 2001) and thus acting redundantly in this circumstance, the phenotypes resulting from a lowering of a subset of the DILPs despite the persistence of the remaining ligands suggests at least some functional specificity among them. This notion is supported by the finding that the abolition of sexual dimorphism in locomotor behaviour, which is a consequence of mNSC ablation in males, unlike growth or hemolymph sugars, cannot be rescued by injection or over-expression of DILP2 alone (Belgacem, 2006). In addition, Drosophila p70/S6 kinase in the mNSCs mediates hunger regulation of feeding behavior in larvae, and over-expression of DILPs 2 and 4, but not DILP3, suppresses this hunger-driven behavior (Wu, 2005). The specificity of the DILPs may be determined by their biochemical properties, regulation of their synthesis as well as their sites of release (Broughton, 2008).

The physiological role of the regulation of trehalose metabolism is not known. Increased whole-body trehalose has been correlated with resistance to anoxia (Chen, 2002). No such resistance was apparent in the DILP2-knock-down lines or the mNSC-ablated flies. The increased trehalose in the dilp2RNAi/d2GAL flies correlated with a slight increase in resistance to starvation, indicating that these trehalose stores play, if any, only a minor part in starvation tolerance. Furthermore, this observation also indicates that the starvation resistance of the mNSC-ablated flies does not stem from the increased whole-body trehalose. These data are consistent with a recent finding that Drosophila ARC protein, which is expressed in the dilp-producing mNSCs, is a regulator of behavioural responses to starvation but is not a general regulator of insulin signalling (Mattaliano, 2007).. Mutants are starvation resistant likely due to their loss of normal starvation induced hyperlocomotion. It is therefore possible that the mNSC-ablated flies are starvation resistant predominantly because of a reduction in ARC, and the slight effect on starvation resistance following DILP2 knock down in the dilp2RNAi flies may be due to an alteration of metabolic rates and the consumption and distribution of energy sources, of which increased whole body trehalose may be a sign (Broughton, 2008).

Further investigation of putative specific roles of the individual DILPs, which awaits production of specific mutants or effective RNAi against dilps 3 and 5, may shed light on the links between the different aspects of fly physiology they control. However, the finding that DILP2 levels are not limiting for lifespan, fecundity and stress resistance clearly demonstrates that thinking needs to be changed about how dilps regulate lifespan and other traits, and experimental manipulation needs to be directed to address this issue (Broughton, 2008).

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. This study has identified a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (Dilp2) and the other extends to Adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and Dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. It is proposed that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of Dilp2 and suppressing the release of AKH when haemolymph glucose levels are high (Oh, 2019).

Glucose-sensing neurons respond to glucose or its metabolites, which act as signalling cues to regulate their neuronal activity. According to the glucostatic hypothesis proposed in 1953, feeding and related behaviours are regulated by neurons in the brain that sense changes in glucose levels in the blood. Despite the discovery of glucose-sensing neurons in the hypothalamus through electrophysiological methods more than ten years later, the physiological role of these neurons remained unclear until recently, when a population of glucose-excited neurons in the Drosophila brain were determined to function as an internal nutrient sensor to mediate the animal's consumption of sugar (Dus, 2015). A large number of glucose-sensing neurons appear to be present in animals; it is speculated that these neurons mediate physiological functions that are critical for the wellbeing of the animal, including glucose homeostasis. This study reports the identification of a pair of glucose-excited neurons in the Drosophila brain that maintain glucose homeostasis by coordinating the activity of the two key hormones involved in the process: insulin and glucagon (Oh, 2019).

To identify neurons that respond to sugar on the basis of its nutritional value, a two-choice assay was used to screen Vienna tiles (VT)-Gal4 Drosophila lines that had been crossed to UAS-Kir2.1, tub-Gal80ts flies (inward-rectifier potassium ion channel allele Kir2.1 with tubulin-temperature-sensitive Gal80) for defects in their ability to select nutritive D-glucose over non-nutritive L-glucose. Two independent Gal4 lines, VT58471 and VT43147-Gal4, were isolated that failed to select D-glucose after periods of starvation and appeared to contain dorsolateral cells that resemble those that are labelled by the corazonin (Crz)-Gal4 line. Flies in which Crz-Gal4-expressing neurons had been inactivated failed to select D-glucose even when starved. These results suggest that the dorsolateral neurons labelled by Crz-Gal4 and two candidate Gal4 lines mediate the behavioural response to sugar (Oh, 2019).

A Crz antibody was used to confirm the identity of the dorsolateral neurons. A previous study demonstrated that a subset of Crz-expressing neurons also express short neuropeptide F (sNPF). Immunolabelling revealed that the dorsolateral neurons expressing Crz indeed express sNPF. On the basis of these findings, these Crz+sNPF+ neurons were named CN neurons. To restrict Gal4 expression to a few cells that include the dorsolateral neurons, T58471-Gal4 was crossed to choline acetyltransferase (ChAT)-Gal80, generating CN-Gal4, which unambiguously labelled a pair of CN neurons when crossed to UAS-mCD8::GFP. Flies in which these dorsolateral neurons were inactivated using CN-Gal4 failed to select D-glucose when starved. Each CN cell body projects an axon that bifurcates to form two major branches. One branch (axon 1) projects to the pars intercerebralis (PI) region of the brain and the other branch (axon 2) projects ventrolaterally towards the corpora cardiaca (CC). An intersectional approach was used to define these projections further, thereby validating that axon 1 innervates the PI and axon 2 projects to the CC. This approach was also used to induce the expression of tetanus toxin (TNT) to silence a pair of CN neurons. These flies failed to choose D-glucose even after starvation when CN neurons were inactivated. This provided further evidence of the contribution of the pair of the dorsolateral CN neurons to glucose-evoked behaviour (Oh, 2019).

Attempts were made to determine whether CN neurons respond to glucose and other sugars. Calcium-imaging studies using ex vivo brain preparations of flies carrying the calcium indicator UAS-GCaMP6s14 and CN-Gal4 revealed that CN neurons were robustly activated by D-glucose with substantial calcium oscillations. CN neurons also responded to D-trehalose and D-fructose, which are found in the haemolymph, but failed to respond to (1) the non-nutritive sugar L-glucose; (2) the non-haemolymph sugar sucrose; and (3) the non-sugar nutrients amino acids. D-Glucose and D-trehalose are key sugars in the haemolymph, although D-trehalose stimulates the activity of CN neurons only after a substantial delay (about 12 min), possibly because it requires additional metabolic steps to be converted to glucose. D-Fructose applied at 20 mM activated CN neurons, although the concentration of D-fructose in the haemolymph is much lower (<2 mM). These findings suggest that the pair of CN neurons responds only to D-glucose under normal physiological conditions (Oh, 2019).

It was next determined whether activation of CN neurons by D-glucose requires glucose metabolism inside the cell. Exposing the brain to D-glucose mixed with 2DG, phlorizin or nimodipine, which inhibits glycolysis, glucose transport or voltage-gated calcium channels, respectively, blunted the glucose-induced stimulation of CN neurons. In the presence of pyruvate (an end product of glycolysis), the CN neurons demonstrated activity similar to that seen in the presence of other haemolymph sugars. Application of the ATP-sensitive potassium channel (KATP)16 blocker glibenclamide resulted in activation of CN neurons (Fig. 2b, c). Furthermore, glucose-induced calcium transients of these neurons were not abrogated by the application of the sodium-channel blocker tetrodotoxin (TTX). Using RNA-mediated interference (RNAi) lines, it was also determined that glucose transporter 1 (Glut1), hexokinase C (Hex-C), a subunit of the KATP channel (SUR1) and the voltage-gated calcium channel are required in CN neurons for the two-choice behaviour. Consistent with the behavioural results, the glucose-induced calcium response of CN neurons requires Glut1, SUR1 and a voltage-gated calcium channel, further supporting the role of the intracellular glucose metabolic pathway in stimulating CN neuronal activity (Oh, 2019).

The calcium-dependent nuclear import of LexA (CaLexA) system was used to measure cellular activity in CN neurons in intact flies; GFP signal driven by the CaLexA system in starved flies was significantly reduced compared to the signal in fed flies, and the signal was restored when starved flies were refed D-glucose. These results suggest that the activity of CN neurons is stimulated by the increase in glucose levels observed under fed conditions. In addition to the altered CaLexA signals, the effect of glucose on the number and intensity of synaptotagmin (Syt)-GPF+ puncta in fed, starved and refed animals. The Syt-GPF+ signals decreased significantly in axon 1 in starved animals and returned to normal levels after the flies were fed with D-glucose. However, this nutrient-dependent plasticity was not observed in Crz-Gal4-labelled axonal processes that did not originate from the dorsolateral CN neurons (Oh, 2019).

Next attempts were made to determine whether CN neurons are coupled with IPCs20 at the synaptic level. A modified GFP reconstitution across synaptic partners (GRASP) method was used, and the GRASP signals were found to be visible around the synapse between CN neurons and insulin-producing cells (IPCs), indicating physical coupling between CN neurons and IPCs (Oh, 2019).

To determine whether the coupling between CN neurons and IPCs is functional, ATP-gated P2X2 purine receptors were used in CN neurons and the calcium indicator GCaMP6s14 in IPCs, and then CN neurons were stimulated using ATP while recording from the IPCs. ATP-induced CN-neuron activity was accompanied by a significant increase in the amplitude of GCaMP signals in the IPCs in fed flies; this effect was reduced in starved flies. This finding supports the hypothesis that the nutrient-dependent synaptic changes observed between CN neurons and IPCs have functional consequences. The CN neurons did not appear to be functionally coupled to glucose-excited diuretic hormone 44 (Dh44) neurons. Furthermore, whether CN neuronal activity is required for Dilp2 secretion from IPCs was tested. A significant reduction in the intensity of Dilp2 immunoreactivity was observed in the IPCs of fed control flies, but not in fed flies in which CN neurons had been inactivated. These results suggest that an excitatory signal from the CN neurons contributes to the secretion of Dilp2 from IPCs in response to increased glucose levels. Using mass spectrometry and dot blot assay, it was further validated that the flies carrying CN-Gal4 and UAS-Kir2.1 had lower Dilp2 levels circulating in the haemolymph than wild-type flies, in contrast to the higher dilp2 levels found in IPCs (Oh, 2019).

To further clarify the role of CN neurons in mediating glucose-evoked activity in IPCs, CN neurons were inactivated by expressing TNT13, and then the responsiveness of IPCs to glucose was evaluated. The amplitude of calcium signals in IPCs that had been exposed to D-glucose was significantly reduced when the CN neurons were inactivated. Furthermore, it was found that IPCs harbour at least three subpopulations of neurons with distinct responses to glucose or KATP channel blocker. These findings suggest that CN neuronal activity is required for the majority of IPCs to respond to glucose (Oh, 2019).

To determine whether nutrient-dependent plasticity also occurs in axon 2 of the CN neurons, the number and intensity of Syt-GFP+ puncta was monitored before and after feeding flies with D-glucose. A significant reduction was observed in these parameters in starved flies, and a restoration to normal levels was found after refeeding starved flies with D-glucose. This raised the possibility of coupling between CN neurons and AKH-producing cells. Using a modified GRASP method, GRASP fluorescent signals were observed around AKH-producing cells. To determine whether there is any functional connectivity between these cells, the CN neurons were activated while monitoring the activity of AKH-producing cells, and calcium transients found in the AKH-producing cells appeared to decrease during activation of CN neurons (Oh, 2019).

To probe this observation further, the Arclight receptor, which increases fluorescent signals when cells become hyperpolarized, was expressed in AKH-producing cells, and P2X2 receptors were expressed in CN neurons. When the CN neurons were activated using ATP, the Arclight fluorescence intensity in fed flies increased significantly compared with that in starved flies, validating the occurrence of nutrient-dependent changes in the synapses between CN neurons and AKH-producing cells. Notably, when CN neurons were inactivated, the intracellular AKH levels decreased significantly compared with controls. Using mass spectrometry and dot blot assay, significantly higher levels of AKH were expressed in haemolymph of flies carrying CN-Gal4 and UAS-Kir2.1 compared with those in control flies. These findings suggest that CN neuronal activity inhibits the release of AKH from the CC and the increase of AKH levels in haemolymph (Oh, 2019).

Next the identities were investigated of the key neurotransmitters in axon 1 and axon 2 for regulating the functionally opposing synaptic activities. The role of Crz and sNPF was tested in the two-choice behaviour using RNAi lines, and sNPF in CN neurons and sNPF receptor in the postsynaptic IPCs, but not Crz or its receptor, were found to be important. sNPF but not Crz levels in CN neurons were significantly reduced when CN neurons were exposed to D-glucose. Approximately a half of the IPCs that had responded to glucose failed to respond glucose when the dominant-negative allele of sNPF receptor was expressed in IPCs. Furthermore, it was observed that intracellular AKH levels remained high in AKH-producing cells in fed control flies, but declined significantly in fed flies in which the function of sNPF receptor was inhibited in AKH-producing cells (Oh, 2019).

Finally, whether sNPF alters activity of IPCs and/or the CC was determined. The activity of IPCs was significantly stimulated by the application of sNPF26, whereas CC activity was significantly inhibited by sNPF. These functionally opposing effects of sNPF are probably mediated by Gq in IPCs and by Gi/o in AKH-producing cells via the sNPF receptor, which is a G-protein-coupled receptor. Exposing the brain to U73122, a PLC inhibitor that inhibits the Gq pathway, eliminated the glucose-evoked activation of IPCs, but had no effect on sNPF-induced inhibition of AKH-expressing cells. Conversely, exposing the brain to pertussis toxin, a Gi inhibitor, blunted the sNPF-induced inhibition of AKH-producing cells, but had no effect on the glucose-evoked activation of IPCs. These results indicate that axon 1 and axon 2 can have opposing synaptic activities through a mechanism involving the same neurotransmitter and receptor but with distinct downstream factors coupled with opposing outputs (Oh, 2019).

To determine whether CN neuronal activity can alter circulating sugar levels in flies, circulating concentrations of glucose and trehalose in haemolymph were monitored; they were significantly increased in flies in which CN neurons were inactivated compared with controls. This finding illustrates that dysfunctional CN neuronal input to IPCs and AKH-producing cells results in a defect in glucose homeostasis (Oh, 2019).

This study identified and characterized a pair of glucose-sensing neurons in the Drosophila brain that have an essential role in maintaining glucose homeostasis. This was achieved by counterbalancing the activities of Drosophila equivalents of insulin- and glucagon-producing cells. When food consumption leads to a rise in haemolymph sugar levels, CN neurons excite the IPCs through sNPF and its receptor, which appear to be coupled to the Gq signalling cascade to induce the secretion of Dilp2, while suppressing the release of AKH by using the same sNPF receptor, which in this case is coupled with Gi signalling pathway. It is speculated that precise control of these opposing functions is facilitated because the nutrient-dependent plastic changes arise from a single cell (Oh, 2019).

This study demonstrates how the activity of the two key endocrine systems is coordinated in metazoans and that their coordination is under the direct control of glucose-sensing neurons. Such coordination has been proposed to occur in mammals via the sympathetic and parasympathetic nerves that connect the pancreatic islets with glucose-sensing neurons in the hypothalamus and hindbrain. The finding that a large proportion of IPCs respond to glucose through CN neurons in insects raises an intriguing possibility that both direct and indirect mechanisms control endocrine function in mammals. Finally, this work may shed light on the function of glucose-sensing neurons. Further research is needed to understand how these regulatory processes are affected by excessive nutrition and other metabolic disturbances, including obesity (Oh, 2019).

Antagonistic regulation by insulin-like peptide and activin ensures the elaboration of appropriate dendritic field sizes of amacrine neurons

Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. This study reports that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. DILP2 is expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. It is suggested that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields (Luo, 2020).

This study has shown that insulin/TOR/SREBP signaling positively regulates dendritic development. Insulin-like peptide DILP2 from L5 axons signals through insulin receptors on Dm8 dendrites to promote dendritic expansion via the canonical TOR pathway and SREBP. The roles of the insulin/TOR signaling pathway in cell growth and differentiation have been very well established. However, this is the first study to definitively demonstrate that insulin/TOR/SREBP signaling regulates dendritic field sizes during development. In Xenopus tadpoles, insulin receptor is expressed in retina and optic tectum, where it regulates synapse density and visual circuit function. However, the source of insulin or insulin-like growth factor (IGF) and the signaling pathway have not been described in developing Xenopus tectum. In vitro studies in cultured cells suggest that insulin/IGF signaling can not only regulate dendrite spine morphology and neurite outgrowth, but also function as a synaptic component at the postsynaptic density (PSD). It would be interesting to determine whether afferent-derived insulin/IGF is also utilized for dendritic development in vertebrate nervous systems (Luo, 2020).

The PI3K/AKT/TOR signaling pathway is known to mediate a broad spectrum of functions through two major complexes, TORC1 and TORC2, which act through different and varied effectors. In cultured hippocampal neurons, the PI3K-TOR pathway regulates dendrite size and branching patterns, presumably by interacting with the microtubule plus-end-tracking protein CLIP-170 to enhance crosstalk between CLIP-170 and the actin-binding protein IQGAP1 (Swiech, 2011). Dm8 dendritic development appears to involve the TORC1 complex and one of its downstream targets, SREBP. Removing the TORC2 component, Rictor, or TORC1 targets other than SREBP had no significant effect on Dm8 dendritic field size. In contrast, SREBP mutant Dm8 neurons had a reduced dendritic field size, and overexpression of SREBP rescued the dendritic phenotype of Tor mutants. While the possibility cannot be ruled out that other TOR targets play an additional or minor role, the results suggest that TOR works primarily through SREBP to regulate Dm8 dendritic development. Recent studies showed that SREBP enhances the transcription of cholesterol and fatty acid synthesis enzymes to regulate dendritic development of Da neurons in the peripheral nervous system. Thus, mounting evidence suggests that lipid synthesis may be a key regulatory point for controlling dendritic development in both central and peripheral nervous systems (Luo, 2020).

Using a novel receptor-based activity-dependent GRASP (R-synGRASP) technique, this study found synaptic defects between R7 photoreceptors and mutant Dm8 neurons that lack Insulin/TOR pathway signaling components. While the R-GRASP method revealed that Tor or chico mutant Dm8 dendrites formed membrane contacts with R7 axonal terminals in numbers that correlated with the dendritic field sizes, the results from the R-synGRASP method suggested these contacts do not constitute active synapses. Notably, Pten mutant Dm8 neurons appear to form more active synapses with peripheral R7 photoreceptors than central R7 photoreceptors, in contrast to wild types. At this stage of inquiry, the nature of such defects remains unclear. The presynaptic structures of R7 photoreceptors appear to be unaltered, as judged by the localization of the active zone marker, Bruchpilot. The level of histamine receptors on dendrites is significantly reduced in Tor- but not chico mutant Dm8 neurons, suggesting that reduced receptor levels might only partially account for the lack of R-synGRASP signal. It has been shown that insulin signaling regulates protein trafficking in various neuronal models, and mutations in SREBP and its upstream regulator easily shocked (eas) have been associated with excitability defects. Further studies will be required to resolve the nature of the synaptic defects in Tor and chico mutant Dm8 neurons (Luo, 2020).

Antagonistic regulation of Dm8 dendritic expansion by afferent-derived factors ensures robust size control A previous study showed that R7-derived Activin negatively regulates the expansion of Dm8 dendritic fields (Ting, 2014). This action is countered by positive regulation by L5-derived DILP2 revealed in this study. Despite the antagonistic effects on Dm8 dendritic expansion, both Activin and DILP2 are derived from afferents, delivered to the growth cones, and likely act on dendrites at close range. Both L3 and L5 lamina neurons express DILP2, but RNAi-mediated knockdown of dlip2 in L5 neurons alone is sufficient to reduce Dm8 dendritic expansion. This result suggests that L3-derived DILP2 is not necessary for normal development of Dm8 dendrites, even though the axonal terminals of L3 and L5 are only few micrometers apart. Similarly, Activin derived from R7 and R8 photoreceptors acts respectively on Dm8 and Tm20 neurons, even though the R7 and R8 axonal terminals are also only separated by a few micrometers (Ting, 2014). Furthermore, both Activin and DILP2 appear to have multiple functions in the developing optic lobe. In addition to its function on Dm8 dendritic development, R7-derived Activin functions in an autocrine fashion to control R7 axonal tiling. Glia-derived DILPs regulate lamina neuron differentiation, while L3 and L5 neurons express DILP2, which also affects photoreceptor axonal tiling. It is thus tempting to speculate that afferent-derived factors tend to function at short range on multiple targets during distinct developmental stages (Luo, 2020).

The results from genetic interaction experiments further argue that Activin and DILP2 signal in developing Dm8 dendrites through parallel pathways. Interestingly, the antagonistic regulation of Dm8 dendritic expansion by two afferent-derived factors is required for elaboration of appropriately large and robust Dm8 dendritic fields - removing both signaling events rendered highly variable sizes of Dm8 dendritic fields. While antagonistic regulation has been shown to control a wide range of biological processes, including gene and protein expression, this work provides the first example of antagonistic regulation for robust control of dendritic field sizes (Luo, 2020).

How does antagonistic regulation by insulin and Activin signaling ensure appropriate and robust sizes of Dm8 dendritic fields? Several lines of evidence suggest the involvement of spatiotemporal restriction of insulin signaling. Strong insulin receptor expression was detected in Dm8 neurons at early pupal stages (20-40 hr APF), when the cells had just begun to expand their dendritic arbors, but expression disappeared by late pupal stages (60 hr APF), before the dendrites expanded to adult sizes. Furthermore, the insulin receptors in developing Dm8 neurons were largely restricted to the central dendritic field, corresponding to one single column. Thus, insulin signaling likely occurs only over a short developmental period in response to insulin signal from one or a few L5 neurons to transiently promote Dm8 dendritic expansion during the early pupal stages (Luo, 2020).

To further understand how antagonistic regulation might control dendritic field sizes, simulations were carried out of Dm8 dendritic development using a kinetic Monte Carlo method. The variance of dendritic field sizes correlated with the difference between two key dendritic development parameters, branching and terminating rates: high branching and low terminating rates favor dendritic field expansion at the cost of high variability, while low branching and high terminating rates lead to small but robust dendritic field sizes. This study found that elaboration of large and consistent dendritic fields could be achieved by spatial and/or temporal segregation of growing and terminating phases. A previous study using in vivo imaging of dendritic dynamics showed that dendrites are elaborated at different rates according to the developmental stage. Thus, it is speculated that spatiotemporal restriction of insulin signaling, coupled with Activin-mediated negative regulation, allows Dm8 neurons to elaborate a large and consistent dendritic field. The large and robust sizes of Dm8 dendritic fields not only ensure a consistent receptive field for approximately 14 R7 photoreceptor inputs, but also can receive negative feedback signals to generate a center-surround signal structure (Luo, 2020).

Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2

The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. This study finds that dilp1 is highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Dilp1 and dilp2 single and double mutants were used to describe interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. dilp1 and dilp2 interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. Repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. The data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide (Post, 2018).

Based on mutational analyses of the insulin receptor (daf-2, InR) and its associated adaptor proteins and signaling elements, numerous studies in C. elegans and Drosophila established that decreased insulin/IGF signaling (IIS) extends lifespan. Studies on how reduced IIS in Drosophila systemically slows aging also reveal systems of feedback where repressed IIS in peripheral tissue decreases DILP2 production in brain insulin-producing cells (IPC), which may then reinforce a stable state of longevity assurance. This study finds that expression of dilp1 is required for loss of dilp2 to extend longevity. This novel observation contrasts with conventional interpretations where reduced insulin ligand is required to slow aging: Elevated dilp1 is associated with longevity in dilp2 mutants, and transgene expression of dilp1 increases longevity (Post, 2018).

dilp1 and dilp2 are encoded in tandem, likely having arisen from a duplication event. Perhaps as a result, some aspects of dilp1 and dilp2 are regulated in common: Both are expressed in IPCs, are regulated by sNPF, and have strongly correlated responses to dietary composition. Nonetheless, the paralogs are differentially expressed throughout development. While dilp2 is expressed in larvae, dilp1 expression is elevated in the pupal stage when dilp2 expression is minimal. In reproductive adults, dilp1 expression decreases substantially after eclosion and dilp2 expression increases (Post, 2018).

Furthermore, DILP1 production is associated with adult reproductive diapause. IIS regulates adult reproductive diapause in Drosophila, a somatic state that prolongs survival during inclement seasons. DILP1 may stimulate these diapause pro-longevity pathways, while expression in nondiapause adults is sufficient to extend survival even in optimal environments (Post, 2018).

The current data suggest a hypothesis whereby dilp1 extends longevity in part through induction of adipokinetic hormone (AKH), which is also increased during reproductive diapause and acts as a functional homolog of mammalian glucagon. Critically, AKH secretion has been shown to increase Drosophila lifespan and to induce triacylglycerides and free fatty acid catabolism. Here, it is noted that dilp1 mutants were more sensitive to starvation than wild-type and dilp2 mutants, as might occur if DILP1 and AKH help mobilize nutrients during fasting and diapause. Mammalian insulin and glucagon inversely regulate glucose storage and glycogen breakdown, while insulin decreases glucagon mRNA expression. It is propose that DILP2 in Drosophila indirectly regulates AKH by repressing dilp1 expression, while DILP1 otherwise induces AKH (Post, 2018).

A further connection between dilp1 and diapause involves juvenile hormone (JH). In many insects, adult reproductive diapause and its accompanied longevity are maintained by the absence of JH. Furthermore, ablation of JH-producing cells in adult Drosophila is sufficient to extend lifespan, and JH is greatly reduced in long-lived Drosophila insulin receptor mutants. In each case, exogenous treatment of long-lived flies with a JH analog (methoprene) restores survival to the level of wild-type or nondiapause controls. JH is a terpenoid hormone that interacts with a transcriptional complex consisting of Met (methoprene tolerant), Taimen, and Kruppel homolog 1 (Kr-h1). As well, JH induces expression of kr-h1 mRNA, and this serves as a reliable proxy for functionally active JH. This study finds that dilp2 mutants have reduced kr-h1 mRNA, while the titer of this message is similar to that of wild-type in dilp1 - dilp2 double mutants. DILP1 may normally repress JH activity, as would occur in diapause when DILP1 is highly expressed. Such JH repression may contribute to longevity assurance during diapause as well as in dilp2 mutant flies maintained in laboratory conditions (Post, 2018).

Does DILP1 act as an insulin receptor agonist or inhibitor? Inhibitory DILP1 could directly interact with the insulin receptor to suppress IIS, potentially even in the presence of other insulin peptides. Such action could induce programs for longevity assurance that are associated with activated FOXO. Alternatively, DILP1 may act as a typical insulin receptor agonist that induces autophosphorylation and represses FOXO. In this case, to extend lifespan, DILP1 should stimulate cellular responses distinct from those produced by other insulin peptides such as DILP2 or DILP5. Through a third potential mechanism, DILP1 may interact with binding proteins such as IMPL2 or dALS to indirectly inhibit IIS output. These distinctions may be resolvednin a future study using synthetic DILP1 applied to cells in culture (Post, 2018).

A precedent exists from C. elegans where some insulin-like peptides are thought to function as antagonists. In genetic analyses, ins-23 and ins-18 stimulate larval diapause and longevity, while ins-1 promotes Dauer formation during development and longevity in adulthood. Moreover, C. elegans ins-6 acts through DAF-2 to suppress ins-7 expression in neuronal circuits to affect olfactory learning, where ins-7 expression inhibits DAF-2 signaling. These studies propose that additional amino acid residues of specific insulin peptides contribute to their distinct functions, and notably, the B-chain of DILP1 has an extended N-terminus relative to other DILP sequences (Post, 2018).

While dFOXO and DAF-16 are intimately associated with how reduced IIS regulates aging in Drosophila and C. elegans, in the current work, the behavior of FOXO does not correspond with how longevity is controlled epistatically by dilp1 and dilp2. Mutation of dilp2 did not impact FOXO activity, as measured by expression of target genes InR and 4eBP, and interactions with dilp1 did not modify this result. Some precedence suggests only a limited role for dfoxo as the mediator of reduced IIS in aging, as dfoxo only partially rescues longevity benefits of chico mutants, revealing that IIS extends lifespan through some FOXO-independent pathways. On the other hand, dilp1 expression from a transgene in the dilp1-2 double mutant background did induce FOXO targets. Differences among these results might arise if whole animal analysis of dFOXO targets obscures its role when IIS regulates aging through actions in specific tissues. In this vein, this study found that dilp2 controls thorax ERK signaling but not AKT, suggesting that dilp2 mutants may activate muscle-specific ERK/MAPK anti-aging programs (Post, 2018).

Dilp1 and dilp2 redundantly regulate glycogen levels and blood sugar, while these dilp loci interact synergistically to modulate dilp5 expression and starvation sensitivity. In contrast, dilp1 and dilp2 interact in a classic epistatic fashion to modulate longevity and AKH. Such distinct types of genetic interactions may reflect unique ways DILP1 and DILP2 stimulate different outcomes from their common tyrosine kinase insulin-like receptor, along with outcomes based on cell-specific responses. Understanding how and what is stimulated by DILP1 in the absence of dilp2 will likely reveal critical outputs that specify longevity assurance (Post, 2018).

Insulin-like peptides regulate feeding preference and metabolism in Drosophila

Fruit flies have eight identified Drosophila insulin-like peptides (DILPs) that are involved in the regulation of carbohydrate concentrations in hemolymph as well as in accumulation of storage metabolites. This study investigated diet-dependent roles of DILPs encoded by the genes dilp1-5, and dilp7 in the regulation of insect appetite, food choice, accumulation of triglycerides, glycogen, glucose, and trehalose in fruit fly bodies and carbohydrates in hemolymph. The wild type and the mutant lines demonstrate compensatory feeding for carbohydrates. However, mutants on dilp2, dilp3, dilp5, and dilp7 showed higher consumption of proteins on high yeast diets. High nutrient diets led to a moderate increase in concentration of glucose in hemolymph of the wild type flies. Mutations on dilp genes changed this pattern. The dilp2 mutation led to a drop in glycogen levels independently of diet. Lack of dilp3 led to dramatic increase in circulating trehalose and glycogen levels, especially at low protein consumption. Lack of dilp5 led to decreased levels of glycogen and triglycerides for all diets, whereas knockout on dilp7 caused increase in glycogen levels and simultaneous decrease in triglyceride levels at low protein consumption. Fruit fly appetite was influenced by dilp3 and dilp7 genes. These data contribute to the understanding of Drosophila as a model for further studies of metabolic diseases (Semaniuk, 2018).

This study has found that fruit flies consume more food on diets with low rather than higher percentages of carbohydrates, indicative of compensatory feeding for carbohydrates. DILPs are known to be connected with signaling pathways that control appetite, and diminution of insulin signaling leads to increased consumption of unpalatable food by fruit flies or their larvae. In this study, the lack of a functional DILP4, DILP5, or DILP7 led to significantly increased food consumption on almost all diets. However, the lack of either DILP2 or DILP3 led to increased food consumption only on high protein diets. Notably, dilp7 mutants had the most pronounced increase in food intake on low sucrose concentrations. These mutants also showed compensatory responses to changes in concentration of the yeast solution, for example consuming a substantially greater volume of food than wild type flies on the 3S-3Y (3% sucrose and 3% yeast or yeast autolysate) and the 3S-6Y diets. DILP7 is believed to be a relaxin-like peptide in Drosophila, regulating egg laying decisions and thus it would have a major role to play for the female flies used in the present study. It is notable that protein and carbohydrate intake are essential for reproduction, with protein appetite being tightly coupled to mating and egg production. Interestingly, human relaxins have been shown to be orexigenic. Partial co-expression of dilp7 with short neuropeptide F is involved in feeding behavior and, additionally, DILP7 has been reported to be involved in the regulation of feeding behavior by other authors. Interestingly, the effect was obtained by inactivation of Dilp7 producing neurons. Increased appetite is also characteristic of other DILP mutants, though to a lesser degree. This may suggest that other DILPs also function at least partially as relaxin-like peptides. However, this poses the question as to whether specific DILPs, especially DILP7, may bind to receptors other than the Drosophila insulin receptor (dInR). Human relaxins and insulin-like peptides are known to bind to specific G-protein coupled receptors distinct from the tyrosine kinase type insulin receptor (Semaniuk, 2018).

It was shown recently that responses to a continuous lack of protein and carbohydrates are mediated by distinct dopaminergic neurons. Moreover, activation of protein feeding simultaneously inhibited carbohydrate feeding. These results add complexity to these data, suggesting that the response is additionally mediated by DILPs (Semaniuk, 2018).

The only DILP mutation which led to substantial increase in the level of hemolymph glucose was DILP2. Mutation of all the other DILPs resulted in a slight decrease in hemolymph glucose on some diet treatments and a slight increase on others (e.g., dilp5 mutant). The regulation of hemolymph glucose levels by DILP2 has also been confirmed by other authors, and its physiological proximity to insulin is supported by the similarity of amino acid sequences of the two peptides. The dependence of hemolymph glucose on protein intake by dilp3, dilp5, and dilp7 mutants suggests that DILPs may also be involved in regulation of distinct branches of glucose metabolism such as glycolysis, gluconeogenesis, glycogenesis, and the pentose phosphate pathway. Particularly, the level of circulating glucose may depend on the balance between cellular influx and efflux of glucose, glycogen synthesis and breakdown, synthesis of glucose from organic or amino acids via gluconeogenesis, as well as on glucose catabolism via glycolysis and/or pentose phosphate pathways. In fruit flies, glucose can also be used for trehalose synthesis. However, circulating trehalose levels showed an inverse relationship to circulating glucose in the dilp3 mutant. This suggests that a lack of DILP3 may result in conversion of glycogen stores into trehalose instead of glucose. This possible conversion was inhibited by an increased intake of protein. Therefore, the current data imply a role of DILP3 in regulating trehalose concentration in hemolymph. This finding is not consistent with previous data, which showed that a knockdown of the dilp2 gene but not dilp3 gene led to an increase in trehalose content in whole flies. However, the diet used in that study was more concentrated than most of the diets in the current study. Notably, it has been shown that trehalose promotes selective secretion of DILP3. Interestingly, dilp3 mutants also had high levels of glucose in their bodies. A similar pattern of diet-dependent accumulation of trehalose in the body was observed for dilp7 mutants although this accumulation was not reflected in an increase in hemolymph trehalose levels, suggesting that dilp7 is not involved in regulation of this branch of metabolism (Semaniuk, 2018).

Using nutritional geometry, this study has shown that both dilp2 and dilp5 are involved in regulation of glycogen synthesis in fruit flies, a function that has not been reported previously for these DILPs. Glycogen, along with proteins, contributes to fly weight, what is reflected in this study where dilp2 and dilp5 mutants were significantly lighter than controls on a high carbohydrate diet. Earlier, it was shown that dilp5 expression was dependent on dietary carbohydrate. On the other hand, in a previous study no significant change was found in dilp5 expression with an increase in sugar concentration in the larval diet. However, dilp5 expression was dependent on the type of carbohydrate: increased glucose concentration did not change dilp5 expression, but fructose suppressed steady-state levels of dilp5 transcripts (Semaniuk, 2018).

The current data show that TAG and glycogen accumulation were most pronounced on high sucrose diets in wild type flies. Lack of DILP5 led to a decrease in TAG content for all treatments. Hence, DILP5 seems to be especially necessary for nutrient sensing on high carbohydrate and high yeast diets, and also for converting ingested food into carbohydrate and lipid storage on these diets. Recent data on dilp5 expression confirm these suggestions. In particular, expression of dilp5 is dependent on both consumed carbohydrate and proteinin, and was triggered by yeast in larvae in another study (Semaniuk, 2018).

The role of Dilp5 in regulation of glycogen and TAG synthesis/breakdown has not been reported previously. This study design was notable in two respects. First, flies were allowed to choose between protein and carbohydrate sources. This choice takes into account the impact of a dilp mutation on nutrient sensing. Indeed, dilp expression and release of the peptides from neurosecretory cells is dependent on availability of particular nutrients. The sensory responses to particular nutrients which are transduced to insulin-producing cells was shown to be mediated by biogenic amines and food odors. Secondly, conditions were created where flies were relatively free to fly and otherwise move around, thereby expending energy and creating a demand for glycogen and TAG catabolism (Semaniuk, 2018).

As compared with control flies, lower levels of TAG were also found in the dilp7 mutants. The effects of dilp7 knockout on lipid metabolism were also indirectly confirmed by recent data showing that down-regulation of insulin signaling led to reduced lipid accumulation. In previous studies, ablation of insulin-producing cells either resulted in no change in lipid content or slightly increased it (Semaniuk, 2018).

The present study explored the influence of dilp knockouts on (1) food intake and (2) levels of stored and circulating metabolites. The functions of DILPs in Drosophila resemble those of insulin in mammals, namely the lowering of glucose levels in blood (hemolymph in insects) by increasing glucose uptake by cells and directing it into production of storage metabolites, including glycogen and lipids. The current data showed that the balance of dietary macronutrients (proteins and carbohydrates) influences the outcomes of an insulin-like peptide deficiency. Moreover, this study has shown that knockouts of specific insulin-like peptides of Drosophila affect feeding behavior. It was clearly shown that different DILPs mediate regulation of particular aspects of diet-dependent metabolism. From the current data, this study showed that DILP2 and DILP5 can be involved in diet-independent accumulation of glycogen. DILP3 was shown to influence trehalose synthesis and/or release into the hemolymph, whereas DILP5 and DILP7 mainly affected TAG synthesis on high carbohydrate diets. These specific roles for DILPs are likely conferred by nutrient-dependent DILP release by neurosecretory cells and specific target cells for DILPs. The specific connection between feeding behavior and DILPs, found in the current study, may reflect an impact of nutrient cues on signaling effects of DILPs. In other words, specific ratios of macronutrients can evoke DILP release and subsequent regulation of metabolism in particular tissues (Semaniuk, 2018).

Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor

Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. This study identified Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. Using genetic and ex vivo organ culture experiments, it was shown that the Mth ligand Stunted is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients (Delanoue, 2016).

Environmental cues, such as dietary products, alter animal physiology by acting on developmental and metabolic parameters like growth, longevity, feeding, and energy storage or expenditure. The systemic action of this control suggests that intermediate sensor tissues evaluate dietary nutrients and trigger hormonal responses. Previous work in Drosophila melanogaster established that a specific organ called the fat body translates nutritional information into systemic growth-promoting signals. The leptinlike Janus kinase-signal transducers and activators of transcription (JAK-STAT) ligand unpaired 2 and the CCHamid2 peptide are produced by fat cells in response to both sugar and fat and trigger a metabolic response. Dietary amino acids activate TORC1 signaling in fat cells and induce the production of relay signals that promote the release of insulin-like peptides (Dilps) by brain insulin-producing cells (IPCs). Two fat-derived peptides (GBP1 and GBP2) activate insulin secretion in response to a protein diet, although their receptor and neural targets remain uncharacterized. To identify critical components of this organ crosstalk, a genetic screen was conducted in Drosophila larvae. The gene methuselah (mth), which encodes a heterotrimeric GTP-binding protein (G protein)-coupled receptor belonging to the subfamily of the secretin-incretin receptor subfamily came out as a strong hit. Impairing mth function in the IPCs reduces larval body growth, whereas silencing mth in a distinct set of neurons or in the larval fat body had no impact on pupal volume. Larvae in which expression of the mth gene is reduced by RNA interference (RNAi), specifically in the IPCs (hereafter, dilp2>mth-Ri), present an accumulation of Dilp2 and Dilp5 in the IPCs, whereas dilp2 gene expression remains unchanged, a phenotype previously described as impaired Dilp secretion. Indeed, forced depolarization of the IPCs rescues pupal volume and Dilp2 accumulation upon IPC-specific mth depletion. Therefore, Mth is required for Dilps secretion and larval body growth (Delanoue, 2016).

Two peptides encoded by the stunted (sun) gene, SunA and SunB, serve as bona fide ligands for Mth and activate a Mth-dependent intracellular calcium response. Silencing sun in fat cells, but no other larval tissue, of well-fed larvae mimics the mth loss-of-function phenotype with no effect on the developmental timing. Conversely, overexpression of sun in the larval fat body (lpp>sun) partially rescues the systemic growth inhibition observed upon feeding larvae a diet low in amino acids or upon 'genetic starvation' [silencing of the slimfast (slif) gene in fat cells. This growth rescue is abolished in mth¹ homozygous mutants. This shows that Sun requires Mth to control growth. However, sun overexpression has no effect in animals fed a normal diet. A modification of sun expression does not prevent fat body cells from responding to amino acid deprivation as seen by the level of TORC1 signaling, general morphology, and lipid droplet accumulation but affects the ability of larvae to resist to starvation (Delanoue, 2016).

Dilp2-containing secretion granules accumulate in the IPCs following starvation and are rapidly released upon refeeding. Mth is required in the IPCs to promote Dilp secretion after refeeding, and forced membrane depolarization of IPCs using a bacterial sodium channel (dilp2>NaChBac) is dominant over the blockade of Dilp2 secretion in dilp2>mth-Ri animals. This dominance indicates that Mth acts upstream of the secretion machinery. In addition, Dilp2 secretion after refeeding is abrogated in lpp>sun-Ri animals, and overexpression of sun in fat cells prevents Dilp2 accumulation upon starvation. Altogether, these findings indicate that Mth and its ligand Sun are two components of the systemic nutrient response controlling Dilp secretion (Delanoue, 2016).

Hemolymph from fed animals triggers Dilp2 secretion when applied to brains dissected from starved larvae. This insulinotropic activity requires the function of Mth in the IPCs and the production of Sun by fat body cells. Conversely, overexpressing sun in the fat body (lpp>sun) is sufficient to restore insulinotropic activity to the hemolymph of starved larvae. A 2-hour incubation with a synthetic peptide corresponding to the Sun isoform A (Sun-A) is also sufficient to induce Dilp secretion from starved brains. A similar effect is observed with an N-terminal fragment of Sun (N-SUN) that contains the Mth-binding domain but not with a C-terminal fragment (C-SUN) that does not bind Mth. The insulinotropic effect of N-SUN is no longer observed in brains from larvae of the mth allele, mth¹ . This absence of effect indicates that N-SUN action requires Mth in the brain. In addition, preincubation of control hemolymph with antiserum containing Sun antibodies specifically suppresses its insulinotropic function. These results indicate that Sun is both sufficient and necessary for insulinotropic activity in the hemolymph of protein-fed animals (Delanoue, 2016).

To directly quantify the amount of circulating Sun protein, Western blot experiments wee performed on hemolymph using antibodies against Sun. A 6-kD band was detected in hemolymph collected from fed larvae, and size was confirmed using Schneider 2 (S2) cell extracts. The band intensity was reduced upon sun knockdown in fat body cells but not in gut cells. Therefore, circulating Sun peptide appears to be mostly contributed by fat cells, as suggested by functional experiments. The levels of circulating Sun are strongly reduced upon starvation. In line with this, sun transcripts are drastically reduced after 4 hours of protein starvation and start increasing after 1 hour of refeeding, whereas expression of the sun homolog CG31477 is not modified. sun transcription is not affected by blocking TORC1, the main sensor for amino acids in fat body cells. However, adipose-specific TORC1 inhibition induces a dramatic reduction of circulating Sun, indicating that TORC1 signaling controls Sun peptide translation or secretion from fat cells. PGC1-Spargel is a transcription activator, the expression of which relies on nutritional input. PGC1 was found to be required for sun transcription, and fat body silencing of PGC1 and sun induce identical larval phenotypes. Although PGC1 expression is strongly suppressed upon starvation, blocking TORC1 activity in fat cells does not reduce PGC1 expression. Conversely, knocking down PGC1 does not inhibit TORC1 activity. This finding suggests that PGC1 and TORC1 act in parallel. Therefore, Sun production by fat cells in response to nutrition is controlled at two distinct levels by PGC1 and TORC1 (Delanoue, 2016).

The Sun peptide is identical to the ε subunit of the mitochondrial F₁F₀-adenosine triphosphatase (F₁F₀-ATPase) synthase (complex V). Indeed, both endogenous Sun and Sun labeled with a hemagglutinin tag (Sun-HA) colocalize with mitochondrial markers in fat cells , and the Sun peptide cofractionates with mitochondrial complex V in blue native polyacrylamide gel electrophoresis. In addition, silencing sun in fat cells decreases mitochondrial Sun staining and the amounts of adenosine triphosphate (ATP). However, recent evidence indicates that an ectopic (ecto) form of the F₁F₀-ATP synthase is found associated with the plasma membrane in mammalian and insect cells. In addition, coupling factor 6, a subunit of complex V, is found in the plasma. Therefore, Stunted could participate in two separate functions carried by distinct molecular pools. To address this possibility, a modified form of Stunted carrying a green fluorescent protein (GFP) tag at its N terminus (GFP-Sun), next to the mitochondria-targeting signal (MTS), was used. When expressed in fat cells, GFP-Sun does not localize to the mitochondria, contrarily to a Sun peptide tagged at its C-terminal end (Sun-GFP). This suggests that addition of the N-terminal tag interferes with the MTS and prevents mitochondrial transport of Sun. However, both GFP-Sun and Sun-GFP are found in the hemolymph and rescue pupal size and Dilp2 accumulation in larvae fed a low-amino acid diet as efficiently as wild-type Sun (wt-Sun) and do so in a mth-dependent manner. This indicates that the growth-promoting function of Sun requires its secretion but not its mitochondrial localization and suggests the existence of one pool of Sun peptide located in the mitochondria devoted to F₁F₀-ATP synthase activity and ATP production and another pool released in the hemolymph for coupling nutrient and growth control. In this line, although fat body levels of Sun are decreased upon starvation, its mitochondrial localization is not reduced. This finding indicates that starvation affects a nonmitochondrial pool of Sun. In support of this, starved fat bodies contain normal levels of ATP and lactate, indicating that mitochondrial oxidative phosphorylation is preserved in fat cells in poor nutrient conditions. Last, other subunits from complex V (ATP5a) or complex I (NdufS3) were not detected in circulating hemolymph. Therefore, the release of Sun in the hemolymph relies on a specific mechanism (Delanoue, 2016).

In conclusion, this study has provided evidence for a molecular cross-talk between fat cells and brain IPCs involving the ligand Stunted and its receptor Methuselah. Stunted is a moonlighting peptide present both in the mitochondria as part of the F₁F₀-ATP synthase complex and as an insulinotropic ligand circulating in the hemolymph. The mechanism of Stunted release remains to be clarified. The beta subunit of the ectopic form of F₁F₀-ATP synthase is a receptor for lipoproteins, which serve as cargos for proteins and peptides. In addition, Drosophila lipid transfer particle-containing lipoproteins were shown to act on the larval brain to control systemic insulin signaling in response to nutrition. This suggests that Sun could be loaded on lipoproteins for its transport. Given the role of insulin-insulin-like growth factor (IGF) signaling in aging, the current findings could help in understanding the role of Sun/Mth in aging adult flies (Delanoue, 2016).

The same genetic screen previously identified the fly tumor necrosis factor α Eiger (Egr) as an adipokine necessary for long-term adaptation to protein starvation, and recent work pointed to other adipose factors, illustrating the key role of the larval fat body in orchestrating nutrient response. The multiplicity of adipose factors and their possible redundancy could explain the relatively mild starvation-like phenotype obtained after removal of only one of them. Overall, these findings suggest a model whereby partially redundant fat-derived signals account for differential response to positive and negative valence of various diet components, as well as acute versus long-term adaptive responses (Delanoue, 2016).

Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila

Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. This study investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. These findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans (Metaxakis, 2014).

Sleep syndromes are highly prevalent in elderly humans and, with a continuing increase in life expectancy and a greater proportion of elderly people worldwide, effective treatments with fewer side effects are becoming increasingly needed. Sleep in flies shares striking similarities with sleep in humans, including an age-related reduction in sleep quality. This study used Drosophila to examine age-related sleep pathologies and to suppress these pathologies through genetic and pharmacological perturbation of insulin/IGF and TOR signaling (Metaxakis, 2014).

This study has shown that the highly conserved IIS pathway, with roles in growth and development, metabolism, fecundity, stress resistance, and lifespan, also affects sleep patterns in Drosophila. Reduced IIS increases and consolidates night sleep, while decreasing day sleep and inducing day activity. Interestingly, dilp2-3 double mutant flies as well as flies with neuron or fat-body-specific down-regulation of IIS showed no obvious or only mild sleep phenotypes in a previous study, suggesting that a strong and/or systemic reduction in IIS activity may be necessary to induce the activity and sleep phenotypes. Consistently, dilp2-3 double mutant flies have very mild growth, lifespan, and metabolic phenotypes compared to the dilp2-3,5 triple mutant flies used in this study. Reduced IIS activity resulted in increased sleep consolidation in young flies. Importantly, reduced IIS ameliorated the age-related decline in sleep consolidation seen in wild-type flies, thus showing that it is malleable. Contrary to the increased sleep consolidation with reduced IIS, high calorie diets have been reported to accelerate sleep fragmentation. Furthermore, dietary sugar affects sleep pattern in flies. Taken together, these findings reveal a role of nutrition and metabolism in sleep regulation and age-related sleep decline in flies (Metaxakis, 2014).

In humans, several studies suggest a link between nutrition and sleep. The amino acid tryptophan can promote sleep, possibly by affecting synthesis of the sleep regulators serotonin and melatonin. Also, the carbohydrate/fat content of the diet seemingly affects sleep parameters. However, most of these studies are based on correlational methods and small sample size, and it is not yet clear how diet affects sleep. Interestingly, sleep duration can affect metabolism, risk for obesity and diabetes, and even food preference. These findings associate sleep and metabolism; thus, manipulation of nutrient-sensing pathways, such as IIS and TOR signalling, may affect activity and sleep in humans (Metaxakis, 2014).

The transcription factor FoxO is an important downstream mediator of IIS. In C. elegans all aspects of IIS are dependent on daf-16, the worm ortholog of foxO. In contrast, in Drosophila IIS-mediated lifespan extension is dependent on dfoxo, whereas several phenotypes of reduced IIS are dfoxo-independent. Activity and sleep were unaffected by the loss of dfoxo in wild-type flies. In contrast, under low IIS conditions loss of dfoxo specifically affected daytime behaviour, with night time behaviours unaffected. Reduced IIS therefore affects day and night sleep and activity through distinct mechanisms. It also uncouples the effects of IIS on lifespan and on night sleep consolidation, since dfoxo is essential for extended longevity of flies with reduced IIS. dFOXO has been previously shown to increase neuronal excitability, possibly via transcription of ion channel subunits or other (Metaxakis, 2014).

It is suggested that a possible such regulator could be octopaminergic signalling, known to promote arousal in Drosophila. Octopamine, the arthropod equivalent of noradrenaline, regulates several behavioural/physiological processes, including glycogenolysis and fat metabolism, as well as synaptic and behavioural plasticity. Moreover, octopamine can affect sleep by acting on insulin-producing cells in the fly brain, thus linking IIS and sleep/activity. Indeed, this study found that IIS mutants have increased octopamine levels and, importantly, pharmacological inhibition of octopaminergic signalling reverted the increased day activity of IIS mutants. Noteworthy, mRNA expression of octopamine biosynthetic enzymes was not changed, but tyramine levels were significantly reduced, suggesting that increased translation, reduced degradation, or increased activity of the tyramine-β hydroxylase regulates octopamine levels in IIS mutants. In contrast to day activity, increased lifespan of IIS mutants was not affected by pharmacological inhibition of octopaminergic signalling, thus separating longevity from the day activity phenotype (Metaxakis, 2014).

The effect of reduced IIS on day sleep/activity was mediated through AKH, the equivalent of human glucagon, an antagonist of. In flies, AKH coordinates the response to hunger through mobilizing energy stores and increasing food intake, as well as inducing a starvation-like hyperactivity. Loss of AKH receptor (AkhR) abrogated the increased activity of IIS mutants without affecting night sleep. These results demonstrate that day and night phenotypes of IIS mutants can be uncoupled, suggesting that the increased night sleep of IIS mutants is not just a compensatory consequence of increased day activity (Metaxakis, 2014).

dilp2-3,5 mutants have increased octopamine levels, and loss of AkhR in the dilp2-3,5 mutant background reduced their octopamine level back to wild-type levels, suggesting that AkhR-mediated regulation of octopamine controls day hyperactivity in IIS mutants. In support of these findings, octopaminergic cells mediate the increased activity effect of AKH in other insects. Flies lacking dFOXO did not respond to chemically induced AKH release, suggesting that AKH affects activity through dFOXO. Therefore, it is suggested that dFOXO and AkhR act through overlapping mechanisms to enhance octopaminergic signalling and induce activity (Metaxakis, 2014).

In flies, AkhR is highly expressed in fat body and its loss alters lipid and carbohydrate store levels. Therefore, AkhR might indirectly enhance octopaminergic signalling through alterations in lipid and carbohydrate metabolism. In support of this idea, lipid metabolism affects sleep homeostasis in flies. Additionally, AkhR expression in octopaminergic cells could regulate octopamine synthesis and release in flies. Interestingly, expression of AkhR is altered in dfoxo mutants, thus implicating dFOXO in AkhR regulation. Both are highly expressed in fat body, an important organ for metabolism in flies, and fat-body-specific insulin receptor may regulate AkhR function through dFOXO activation (Metaxakis, 2014).

In larval motor neurons, dFOXO increases neuronal excitability and octopamine increases glutamate release, suggesting there is at least a spatial functional link between the two. Thus, together with a possible role in AkhR synthesis, dFOXO could act downstream of octopamine to increase activity (Metaxakis, 2014).

To determine the mechanism underlying the IIS-dependent amelioration of age-related sleep decline, downstream components and genetic interactors of IIS were investigated. One such interactor that affects health and ageing is TORC1. TORC1 is a major regulator of translation, through S6K, 4E-BP, and of autophagy, through ATG1. Inhibiting TOR signalling, and thus translation, by rapamycin treatment in wild-type flies recapitulated the sleep features of IIS mutants, even in old flies. This rescue of sleep quality was blocked by ubiquitous expression of activated S6K, suggesting that reduced S6K activity is required for the rescue. These findings, together with previous results showing S6K to regulate hunger-driven behaviours, highlight the importance of S6K as a regulator of behaviour in flies. Thus, manipulating TOR signalling can improve sleep quality through S6K (Metaxakis, 2014).

In mammals, rapamycin treatment has beneficial effects on behaviour throughout lifespan. Although complete block of TOR activity is detrimental for long-term memory, a moderate decrease through rapamycin treatment can improve cognitive function, abrogate age-related cognitive deterioration, and reduce anxiety and depression. Moreover, increased TOR activity throughout development is detrimental for neuronal plasticity and memory. In flies, rapamycin prevents dopaminergic neuron loss in mutants with parkinsonism. Although the role of TOR in brain function has not been well studied in flies, the advantageous effect of rapamycin in both mammalian brain function and sleep in flies may be mediated through common neurophysiological mechanisms (Metaxakis, 2014).

Gene expression studies have suggested that protein synthesis is up-regulated during sleep, which may be an essential stage in macromolecular biosynthesis. Consistent with this, inhibiting protein synthesis in specific brain domains prolongs sleep duration in mammals, suggesting that sleep is maintained until specific levels of biosynthesis occur and aids in explaining the ubiquitously conserved need for sleep. Brief cycloheximide treatment has been shown to prolong night sleep and increased consolidation in flies, indicating an evolutionarily conserved role for protein synthesis inhibition on sleep regulation. Contrary to reduced IIS, cycloheximide reduced day activity, possibly due to the global effect of cycloheximide on protein synthesis or due to toxic defects in flies' physiology. Decreased protein synthesis rates may enhance the necessity for increased sleep duration, to allow sufficient synthesis of proteins and other macromolecules during sleep, allowing organisms to be healthy and functional during the day (Metaxakis, 2014).

Alternatively, the effect of protein synthesis inhibition on night sleep could be the result of reduced expression of specific sleep regulators. This study found that DopR1 and dilp2-3,5 mutants share night phenotypes and that rapamycin did not affect sleep of DopR1 mutants, suggesting that TOR acts on dopaminergic signalling to affect night sleep. Reduced IIS elevated expression of DopR1, independently of dFOXO, in accordance with data from mammals. This effect may be feedback caused by down-regulation of dopaminergic signalling in IIS mutants, although not through direct regulation of DopR. Under normal physiological conditions, dopamine signalling is determined by the level of extracellular dopamine and the rate of DAT-mediated dopamine clearance from the synaptic cleft. The rate of dopamine clearance is dependent on the turnover rate of DAT and the number of functional transporters at the plasma membrane. This study found that reduced IIS and rapamycin treatment induced increased expression of DAT, suggesting an increased rate of dopamine clearance from the synaptic cleft, and thus a reduction in the amplitude of dopamine signalling, without changes in total dopamine levels. DAT function and IIS have recently been linked in mammals. DAT function increases upon insulin stimulation and is diminished on insulin depletion, through alterations in DAT membrane localization. However, IIS-dependent regulation of DAT subcellular localization in Drosophila has not yet been demonstrated. The current data suggest down-regulating dopaminergic signalling, either by loss of DopR1 or increasing DAT levels, is beneficial for sleep quality. In agreement with this it was shown that artificially increasing dopaminergic signalling, through short-term methamphetamine treatment, increases both day and night activity and reduces night sleep, and reverts the beneficial effect of reduced IIS on night behaviours. In mammals, cocaine administration, which enhances dopaminergic signalling, increases TOR activity. Also, rapamycin blocks cocaine-induced locomotor sensitization. Interestingly, cocaine stimulates S6K phosphorylation in rat brains, and this effect is blocked by rapamycin. Taken together, these results show that in flies and mammals dopaminergic and IIS/TOR signalling may interact in similar ways (Metaxakis, 2014).

In conclusion, reduced IIS extends lifespan in diverse organisms. This study has have shown that it can also ameliorate age-related sleep fragmentation, but that the mechanisms by which it does so are distinct from those by which it extends lifespan. Reduced IIS affected day activity and sleep phenotypes through increased octopaminergic signalling, but enhanced octopaminergic signalling did not increase lifespan. Similarly, in Drosophila increased lifespan from reduced IIS requires dfoxo, but the night sleep phenotypes of IIS mutants were independent of this transcription factor. Reduced IIS thus acts through multiple pathways to ameliorate different aspects of loss of function during ageing. IIS links metabolism and behaviour through its components, such as S6K and dFOXO, which act through different neuronal circuits and neurons to affect sleep. The strong evolutionary conservation of these circuits and their functions suggests that pharmacological manipulation of IIS effectors could be beneficial in treatments of sleep syndromes in humans (Metaxakis, 2014).

The nutrient-responsive hormone CCHamide-2 controls growth by regulating insulin-like peptides in the brain of Drosophila melanogaster

The coordination of growth with nutritional status is essential for proper development and physiology. Nutritional information is mostly perceived by peripheral organs before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and the failure of endocrine regulation in humans can cause diseases including obesity and diabetes. In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. This study shows that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet β-cells; however, the role of Brs3 in insulin regulation remains elusive. This genetic approach in Drosophila melanogaster provides the first evidence that bombesin receptor signaling with its endogenous ligand promotes insulin production (Sano, 2015)

Organisms need to coordinate growth and metabolism with their nutritional status to ensure proper development and the maintenance of homeostasis. In multicellular animals, nutritional information is mostly perceived by peripheral organs. It is subsequently relayed to other peripheral organs or to the central nervous system (CNS), which generates appropriate physiological and behavioral responses. Endocrine systems ensure this type of organ-to-organ communication via hormonal signals secreted from specialized glandular cells. For example, mammalian insulin is secreted from pancreatic β-cells in response to high blood glucose levels; insulin is then received by its receptor in the liver as well as in many other tissues to promote glucose uptake and anabolism, thereby reducing blood sugar levels. In a similar manner, leptin secreted from adipose tissues is received by the hypothalamus, where it acts to alter energy expenditure and food intake. Caloric restriction reduces the secretion of leptin, leading to both an increase in appetite and a decrease in energy expenditure, which is known to be an adaptive response to starvation. These findings demonstrate the significance of peripheral tissues in the maintenance of homoeostasis. However, only a few peripheral hormones have been identifie, and the mechanisms by which they regulate an organism's development or physiology in response to external stimuli remain elusive (Sano, 2015)

It has been reported that the endocrine system of Drosophila allows adipose tissue, known as the fat body, to communicate with the CNS in a manner similar to that observed in mammals. This signaling depends on nutritional conditions and ultimately couples growth and metabolism with nutritional status. To date, two pathways have been described. In one pathway described from larvae, the fat body-specific down-regulation of either the Slimfast (Slif) amino acid transporter or the Target of Rapamycin (TOR) nutrient-sensing pathway affects systemic growth, suggesting that a hitherto unidentified amino acid-dependent signal(s) is secreted by the fat body for proper growth control. In a second pathway that was identified in adults, Unpaired-2 (Upd2), which is a functional analogue of leptin, was identified as another fat body-derived growth regulator. The expression of upd2is both sugar- and lipid-sensitive and is apparently independent of the amino acid-activated TOR pathway. Although no signaling molecules that act downstream of the Slif/TOR pathway have been identified yet, these fat body-derived signals ultimately regulate the production of insulin-like peptides (Drosophila insulin-like peptides; Dilps) secreted from the brain (Sano, 2015)

Dilps are evolutionarily conserved peptide hormones with functions similar to those of mammalian insulin/insulin-like growth factor (IGF), including the control of tissue growth and blood sugar levels in response to nutritional conditions. Eight dilp genes exist in the Drosophila melanogaster genome. Unlike mammalian insulin, which is secreted from the pancreas, the major Dilps (Dilp2, -3, and -5) are specifically expressed in bilateral clusters of neurosecretory cells [insulin-producing cells (IPCs)] located in the anteromedial region of the brain hemispheres. With regard to the regulation of insulin-like peptides, the knockdown of the Slif/TOR pathway or upd2 in the larval fat body results in the down-regulation of Dilp2 secretion. Upd2, a type-I cytokine, activates the JAK/STAT pathway through its receptor Domeless (Dome). Dome is expressed in the GABAergic neurons juxtaposed to the IPCs in the adult brain. Activation of Dome by Upd2 blocks GABAergic inhibition of the IPCs and thereby facilitates Dilp secretion. Therefore, signaling from peripheral tissues to the brain appears to be essential for the regulation of organismal growth and metabolism in response to nutrition availability in Drosophila melanogaster (Sano, 2015)

This study has investigated the roles of CCHa2 and its receptor in growth control in Drosophila. CCHa2 was identified as a bioactive peptide that activates a G protein-coupled receptor (GPCR) encoded by CG14593 (now named CCHa2-R). Strong expression of CCHa2 in the larval fat body and gut motivated an examination of the roles of CCHa2 and its receptor in nutrient sensing and growth control. By generating mutants of CCHa2 and CCHa2-R, this study has shown that CCHa2/CCHa2-R signaling from the periphery to the CNS can control the synthesis and secretion of Dilps. These results demonstrate that CCHa2 is a novel hormone derived from peripheral tissues and that CCHa2/CCHa2-R form an additional afferent hormonal signaling pathway that coordinates systemic growth with nutrition availability (Sano, 2015)

A previous study suggested the existence of an amino acid-sensitive Dilp regulator(s) in larvae. This as-yet-unidentified Dilp regulator(s) is regulated by the Slif/TOR pathway, and leucine and isoleucine, positive regulators of TOR signaling, are sufficient to promote the secretion of Dilp2 in both in vivo and ex vivo co-cultures of brain and fat bodies. The current results demonstrate that the TOR pathway is required for CCHa2 expression during the larval stages. However, feeding with amino acids, including leucine and isoleucine, was insufficient to promote CCHa2 expression. CCHa2 expression was, however, induced by feeding with glucose. Therefore, unlike predicted amino acid-dependent Dilp regulator(s), CCHa2 was found to be primarily sensitive to glucose. Some biological substances are produced by the metabolism of specific nutrients. For example, pyrimidine or purine bases are synthesized from amino acids. Therefore, it is possible that CCHa2 is down-regulated when glucose is abundant but other nutrients are not available, to limit growth in inhospitable environments. The reduction of CCHa2 mRNA in TOR-pathway knockdown larvae may recapitulate this scenario (Sano, 2015)

In addition to CCHa2, Upd2 was reported to be a glucose-sensitive Dilp regulator expressed in the fat body. The expression of upd2 in adult flies is up-regulated by feeding with a high-glucose or high-lipid diet. CCHa2 and Upd2, however, responded differently when the TOR pathway was disturbed: whereas CCHa2 expression was down-regulated in TOR-pathway-knockdown larvae, upd2 was up-regulated by the inhibition of the TOR pathway in adults. Furthermore, the time course of CCHa2/CCHa2-R signaling is distinct from that of Upd2/Dome signaling. Disruption of upd2 down-regulated animals' growth from larval to adult stages, whereas CCHa2-R mutations reduced growth until late-L3 stages, after which growth was recovered, leading to adults of normal size. This growth recovery resulted from up-regulation of dilp6 expression, which appears to be a consequence of dysregulated brain Dilps. The lack of growth recovery in upd2 -knockdown animals in spite of abnormal Dilp production remains unexplained. Nevertheless, these results indicate that Drosophila melanogaster possesses multiple insulin regulators that have different nutrient sensitivities. Multi-input Dilp regulation might be advantageous under the imbalanced nutritional conditions that arise in the wild, and this could represent a general strategy for animal growth regulation (Sano, 2015)

In mammals, different hormones are secreted in response to long-term or short-term metabolic changes. For instance, gut-derived cholecystokinin, glucagon-like peptide-1, and PYY3-36, as well as stomach-derived ghrelin, all of which control feeding behavior, are secreted in response to food ingestion. These hormones respond to acute metabolic changes and immediately signal to the feeding center in the brain. On the other hand, the synthesis or secretion of leptin and adiponectin is affected by the amount of lipid stored in adipocytes, suggesting that leptin and adiponectin respond to long-term changes in metabolic status. The expression of CCHa2 responds to yeast and glucose within 6 hours, indicating that CCHa2 mediates relatively rapid changes in metabolic status. Thus, it appears that CCHa2 functions as a short-acting metabolic regulator analogous to the mammalian gut- or stomach-derived hormones described above, and that Drosophila melanogaster CCHa2 might have an important role in the maintenance of energy homeostasis under volatile nutritional conditions (Sano, 2015)

The results from the calcium imaging experiments using brain explants and IPC-specific CCHa2-R knockdown strongly suggest that CCHa2 crosses the blood-brain barrier (BBB) to regulate the IPCs, although the underlying mechanism remains elusive. The Drosophila BBB consists of two different glial cell layers composed of either the perineurial glia (PG) or the subperineurial glia (SPG). The SPG cell layer, which is adjacent to the neurons of the brain, forms septate junctions, which function as a barrier to separate the humoral space and the brain, analogously to the mammalian tight junctions formed between endothelial cells. Although several studies have identified important molecules involved in the formation of these septate junctions, little is known about functional aspects of the BBB. CCHa2 could provide an ideal model for the study of BBB function as well as drug delivery across the BBB (Sano, 2015)

These experiments also show that peripheral tissue-derived CCHa2 directly activates IPCs in the brain. In mammals, direct sensing of blood glucose levels by pancreatic β-cells is a major trigger for insulin secretion. In these cells, glucose metabolism inhibits the ATP-dependent potassium channel (K_ATP channel) and opens voltage-dependent calcium channels (VDCCs), resulting in the exocytosis of insulin-containing granules. The K_ATP channel also seems to be involved in insulin secretion in Drosophila IPCs. Interestingly, a group of Gαs- and Gαq/11-coupled GPCRs can also activate the insulin secretion pathway in mammals. The closest mammalian homologues of CCHa2-R-the Bombesin-related receptor subtypes 3, 1, and 2 (also known as gastrin-releasing-peptide receptor)-signal through Gαq/11. The slow rise in [Ca2+] in the IPCs in response to CCHa2 application is consistent with CCHa2-R's mediation of Dilp release through the same pathway (Sano, 2015)

In contrast to Dilp2, dilp5 is also regulated by CCHa2/CCHa2-R signaling at the transcriptional level. Although the expression of dilp5 in the IPCs is activated by the conserved transcription factors Dachshund and Eyeless, whether CCHa2-R regulates these factors in IPCs remains unknown (Sano, 2015)

Overexpression of CCHa2-R in IPCs using the GAL4/UAS system displayed inhibitory effects on dilp5 expression, which prevented investigation of whether direct CCHa2-R activation in IPCs is sufficient for Dilp regulation. CCHa2-R expression in the brain is not specific to IPCs but occurs in other central neurons. Therefore, although it was shown that CCHa2-R expression in the IPCs is required for full dilp5 expression, it is possible that there may also be additional indirect pathways by which CCHa2 may up-regulate the Dilps. Although BBB glial cells are proposed to receive as-yet-unidentified signal(s) from the fat body and re-activate neural stem cells in the brain by secreting Dilp6],CCHa2-R nlsGFP was undetectable in the BBB glial cells. Thus BBB cells are unlikely to receive CCHa2 signals or to relay the signals to the IPCs (Sano, 2015)

The closest mammalian homologue of CCHa2-R is Brs3, an orphan GPCR, which is a member of the bombesin-like peptide receptor family. Brs3-deficient mice develop obesity in association with a reduced metabolic rate and elevated feeding activity. Interestingly, Brs3 is expressed in pancreatic β-cells both in mice and humans. However, its involvement in insulin regulation has been controversial. Only if Brs3 knockout adult mice become obese (especially after 23 weeks old) do their plasma insulin levels increase. Since hyper-insulinemia is generally observed in genetically obese mice, the elevation of insulin is most likely the consequence of the obesity rather than the loss of Brs3 function. On the other hand, a Brs3 agonist promoted insulin secretion in both rodent insulinoma cell lines and in islets isolated from wild-type but not Brs3 mutants. This vigorous genetic approach combined with direct observations of Dilp production in IPCs has provided the first evidence that Bombesin-related receptor signaling activated by its endogenous ligand promotes insulin production (Sano, 2015)

A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion

Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, a genetic tool was developed to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. This system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics (Park, 2014. PubMed ID: 25101872).

Suppression of insulin production and secretion by a Decretin hormone

Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, this study identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency leads to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppresses secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of mammalian Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. Human NMUR1 is expressed in islet &beta; cells, and purified NMU suppressed insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia failed to suppress insulin secretion. The study proposes Lst as an index member of an ancient hormone class called decretins, which suppress insulin output. (Alfa, 2015).

The coupling of hormonal responses to nutrient availability is fundamental for metabolic control. In mammals, regulated secretion of insulin from pancreatic b cells is a principal hormonal response orchestrating metabolic homeostasis. Circulating insulin levels constitute a dynamic metabolic switch, signaling the fed state and nutrient storage (anabolic pathways) when elevated, or starvation and nutrient mobilization (catabolic path ways) when decreased. Thus, insulin secretion must be precisely tuned to the nutritional state of the animal. Increased circulating glucose stimulates b cell depolarization and insulin secretion. In concert with glucose, gut-derived incretin hormones amplify glucose-stimulated insulin secretion (GSIS) in response to ingested carbohydrates, thereby tuning insulin output to the feeding state of the host (Alfa, 2015).

While the incretin effect on insulin secretion during feeding is well-documented, counter-regulatory mechanisms that suppress insulin secretion during or after starvation are incompletely understood. Classical starvation experiments in humans and other mammals revealed that sustained fasting profoundly alters the dynamics of insulin production and secretion, resulting in impaired glucose tolerance, relative insulin deficits, and 'starvation diabetes'. Remarkably, starvation-induced suppression of GSIS was not reverted by normalizing circulating glucose levels, suggesting that the dampening effect of starvation on insulin secretion perdures and is uncoupled from blood glucose and macronutrient concentrations. Based on these observations, it has been postulated that hormonal signals induced by fasting may actively attenuate insulin secretion suggested that enteroendocrine 'decretin' hormones may constrain the release of insulin to prevent hypoglycemia. This concept is further supported by recent studies identifying a G protein that suppresses insulin secretion from pancreatic b cell. Thus, after nutrient restriction, decretin hormones could signal through G protein-coupled receptors (GPCRs) to attenuate GSIS from b cells (Alfa, 2015).

The discovery of hormonal pathways regulating metabolism in mammals presents a formidable challenge. However, progress has revealed conserved mechanisms of metabolic regulation by insulin and glucagon-like peptides in Drosophila, providing a powerful genetic model to address unresolved questions relevant to mammalian metabolism. Similar to mammals, secretion of Drosophila insulin-like peptides (Ilps) from neuroendocrine cells in the brain regulates glucose homeostasis and nutrient stores in the fly. Ilp secretion from insulin-producing cells (IPCs) is responsive to circulating glucose and macronutrients and is suppressed upon nutrient withdrawal. Notably, recent studies have identified hormonal and GPCR-linked mechanisms regulating the secretion of Ilps from IPCs, suggesting further conservation of pathways regulating insulin secretion in the fly (Alfa, 2015).

In mammals, the incretin hormones gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells following a meal and enhance glucose-stimulated insulin production and secretion from pancreatic b cells. Thus, It was postulated that a decretin hormone would have the 'opposite' hallmarks of incretins. Specifically, a decretin (1) derives from an enteroendocrine source that is sensitive to nutrient availability, (2) is responsive to fasting or carbohydrate deficiency, and (3) suppresses insulin production and secretion from insulin-producing cells. However, like incretins, the action of decretins on insulin secretion would be manifest during feeding, when a stimulus for secretion is present (Alfa, 2015).

This study identifed a secreted hormone, Limostatin (Lst), that suppresses insulin secretion following starvation in Drosophila. lst is regulated by starvation, and flies deficient for lst display phenotypes consistent with hyperinsulinemia. Lst production was shown to be localized to glucose-sensing, endocrine corpora cardiaca (CC) cells associated with the gut, and show that lst is suppressed by carbohydrate feeding. Using calcium imaging and in vitro insulin secretion assays, a 15-aa Lst peptide (Lst-15) was identified that is sufficient to suppress activity of IPCs and Ilp secretion. An orphan GPCR was identified in IPCs as a candidate Lst receptor. Moreover, Neuromedin U (NMU) is likely a functional mammalian ortholog of Lst that inhibits islet b cell insulin secretion. These results establish a decretin signaling pathway that suppresses insulin output in Drosophila (Alfa, 2015).

Limostatin is a peptide hormone induced by carbohydrate restriction from endocrine cells associated with the gut that suppresses insulin production and release by insulin-producing cells. Thus, Drosophila Lst fulfills the functional criteria for a decretin and serves as an index member of this hormone class in metazoans. Results here also show that Lst signaling from corpora cardica cells may be mediated by the GPCR encoded by CG9918 in insulin-producing cells. In addition, the results reveal cellular and molecular features of a cell-cell signaling system in Drosophila with likely homologies to a mammalian entero-insular axis (Alfa, 2015).

Reduction of nutrient-derived secretogogues, like glucose, is a primary mechanism for attenuating insulin output during starvation in humans and flies. Consistent with this, it was found that circulating Ilp2HF levels were reduced to a similar degree in lst mutant or control flies during prolonged fasting. Therefore, lst was dispensable for Ilp2 reduction during fasting. However, lst mutants upon refeeding or during subsequent ad libitum feeding had enhanced circulating Ilp2HF levels compared to controls, findings that demonstrate a requirement for Lst to restrict insulin output in fed flies. Thus, while induced by nutrient restriction, Lst decretin function was revealed by nutrient challenge. This linkage of feeding to decretin regulation of insulin output is reminiscent of incretin regulation and action (Campbell, 2013; Alfa, 2015 and references therein).

Recent studies have demonstrated functional conservation in Drosophila of fundamental hormonal systems for metabolic regulation in mammals, including insulin, glucagon, and leptin (Rajan, 2012). This study used Drosophila to identify a hormonal regulator of insulin output, glucose, and lipid metabolism without an identified antecedent mammalian ortholog -- emphasizing the possibility for work on flies to presage endocrine hormone discovery in mammals. Gain of Lst function in these studies led to reduced insulin signaling, and hyperglycemia, consistent with prior work. By contrast, loss of Lst function led to excessive insulin production and secretion, hypoglycemia, and elevated triglycerides, phenotypes consistent with the recognized anabolic functions of insulin signaling in metazoans, and with the few prior metabolic studies of flies with insulin excess (Alfa, 2015).

Prior studies show that somatostatin and galanin are mammalian gastrointestinal hormones that can suppress insulin secretion. Somatostatin-28 (SST-28) is a peptide derivative of the pro-somatostatin gene that is expressed widely, including in gastrointestinal cells and pancreatic islet cells. Islet somatostatin signaling is thought to be principally paracrine, rather than endocrine, and serum SST-28 concentrations increase post-prandially. Galanin is an orexigenic neuropeptide produced throughout the CNS and in peripheral neurons and has been reported to inhibit insulin secretion. Unlike enteroendocrine-derived hormones that act systemically, galanin is secreted from intrapancreatic autonomic nerve terminals and is thought to exert local effects. In addition, Galanin synthesis and secretion are increased by feeding and dietary fat. Thus, like incretins, output of SST- 28 and galanin are induced by feeding, but in contrast to incretins, these peptides suppress insulin secretion. Further studies are needed to assess the roles of these peptide regulators in the modulation of insulin secretion during fasting (Alfa, 2015).

While sequence-based searches did not identify vertebrate orthologs of Lst, this study found that the postulated Lst receptor in IPCs, encoded by CG9918, is most similar to the GPCRs NMUR1 and NMUR2. In rodents, NMU signaling may be a central regulator of satiety and feeding behavior, and this role may be conserved in other organisms. In addition, NMU mutant mice have increased adiposity and hyperinsulinemia, but a direct role for NMU in regulating insulin secretion by insulin-producing cells was not identified. In rodents, the central effects of NMU on satiety are thought to be mediated by the receptor NMUR2; however, hyperphagia, hyperinsulinemia, and obesity were not reported in NMUR2 mutant mice. Together, these studies suggest that a subset of phenotypes observed in NMU mutant mice may instead reflect the activity of NMU on peripheral tissues like pancreatic islets, but this has not been previously shown. Notably, humans harboring the NMU R165W allele displayed obesity and elevated insulin C-peptide levels, without evident hyperphagia -- further suggesting that the central and peripheral effects of NMU reflect distinct pathways that may be uncoupled. This study has shown that NMU is produced abundantly in human foregut organs and suppresses insulin secretion from pancreatic b cells, supporting the view that NMU has important functions outside the CNS in regulating metabolism. Thus, like the incretin GLP-1, NMU may have dual central and peripheral signaling functions in the regulating metabolism. Demonstration that NMU is a mammalian decretin will require further studies on NMU regulation and robust methods to measure circulating NMU levels in fasting and re-feeding. In summary, these findings should invigorate searches for mammalian decretins with possible roles in both physiological and pathological settings (Alfa, 2015).

The Drosophila insulin degrading enzyme restricts growth by modulating the PI3K pathway in a cell autonomous manner

Mammalian Insulin Degrading Enzyme (IDE) cleaves insulin among other peptidic substrates but its function in insulin signaling remains elusive. This study used the Drosophila system to define the function of IDE in the regulation of growth and metabolism. Either loss- or gain of function of Drosophila IDE (dIDE) can restrict growth in a cell autonomous manner by affecting both cell size and cell number. dIDE can modulate dILP2 levels, thereby restricting activation of the PI3K pathway and promoting activation of FOXO. Larvae reared in high sucrose exhibit delayed developmental timing due to insulin resistance. dIDE loss of function was shown to exacerbate these phenotypes, and mutants display increased levels of circulating sugar, along with augmented expression of a lipid biosynthesis marker. It is proposed that dIDE is a modulator of insulin signaling, and that its loss of function favors insulin resistance, a hallmark of diabetes mellitus type II (Galagovsky, 2014).

p53- and ERK7-dependent ribosome surveillance response regulates Drosophila Insulin-Like peptide secretion

Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. This study uncovered a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. Impairment of ribosome biogenesis specifically in the IPCs was shown to strongly inhibits dILP secretion, consequently leading to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, evidence is provided that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, it is concluded that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions (Hasygar, 2014).

This study reports a novel cell-autonomous control mechanism for dILP secretion. Specifically, it is concluded that 1) inhibition of ribosome biogenesis in the IPCs at any level tested, including ribosomal gene expression (Myc), ribosome maturation (Rio1, Rio2, Tsr1) or by introducing imbalance of ribosomal components (Rpl35A), triggers a response to inhibit dILP secretion, 2) this inhibitory response is dependent on p53, a known surveillance factor for ribosome biogenesis, 3) a downstream effector of this ribosome surveillance pathway is protein kinase ERK7, as erk7 mRNA levels are elevated upon inhibited ribosome biogenesis and p53 activation and erk7 is essential to inhibit dILP secretion in both conditions, 4) the ribosome surveillance mechanism discovered in this study likely contributes to starvation-induced inhibition of dILP secretion. These findings significantly broaden the view about the regulatory functions of the ribosome surveillance pathways, which have been mainly explored at the level of proliferating cells. Ribosome biogenesis serves as the key determinant of cell autonomous growth control and it is finely tuned to match with the cellular nutrient and energy status. Coupling dILP secretion to the ribosome biogenesis pathway is an elegant mechanism for multicellular animals to synchronize the hormonal growth control with the local cell autonomous regulation of growth. Comparable to the finding in the IPCs, inhibition of ribosome biogenesis in the fat body leads to a block of dILP secretion in the IPCs through an unknown humoral mechanism. Linking ribosome biogenesis to growth control through parallel mechanisms likely provides a robust regulatory network to tune down systemic growth signals whenever any region of the body experiences nutrient deprivation. This synchronization is likely important to maintain balanced growth throughout the spectrum of dietary conditions. Future studies should be aimed to explore the possible interrelationship between the fat body-derived signals and the cell-autonomous mechanism discovered here (Hasygar, 2014).

These findings highlight that the p53-mediated ribosome surveillance pathway can serve highly cell type-specific functions in vivo. This is interesting when considering human ribosomopathies, genetic diseases caused by impaired ribosome biogenesis manifesting with a wide spectrum of tissue-specific defects. One of the ribosomopathies, Shwachman-Diamond syndrome (SDS), is manifested with a failure in pancreatic function. A mouse model of SDS displays general preservation of ductal and endocrine compartments, but reduced amount of zymogen granules. Moreover, SDS mutant mice have reduced glucose tolerance, suggesting compromised endocrine function. It will be interesting to learn whether p53 and ERK7 act as mediators of the secretion-related defects observed in SDS (Hasygar, 2014).

Compared to other members of the MAP kinase family, ERK7 has remained relatively poorly characterized. Earlier studies in mammalian and Drosophila cells have shown that ERK7 protein levels are actively regulated at the level of protein degradation. In mammals, an increase in ERK7 levels leads to autophosphorylation and consequent activation. Consistent with the idea that ERK7 is mainly regulated through abundance, it was observed that elevated ERK7 expression had a prominent impact in the function of IPCs. Interestingly, earlier studies have linked ERK7 function to growth regulation by showing that ERK7 protein is stabilized by serum and amino acid starvation. The data provides evidence that impaired ribosome biogenesis as well as starvation increases the expression of erk7 mRNA revealing a novel regulatory level for ERK7 function. In addition to the conditions explored here, ERK7 expression levels increase towards the end of larval development when growth is ceased. It will be interesting to learn further how ERK7 expression is regulated and whether ERK7 has a function in tissue growth control beyond its role in the IPCs. Earlier studies in cell culture have shown evidence that ERK7 regulates cancer cell proliferation and autophagy, suggesting that ERK7 may have a broader role in the regulation of tissue growth (Hasygar, 2014).

bantam miRNA promotes systemic growth by connecting insulin signaling and ecdysone production

During the development of multicellular organisms, body growth is controlled at the scale of the organism by the activity of long-range signaling molecules, mostly hormones. These systemic factors coordinate growth between developing tissues and act as relays to adjust body growth in response to environmental changes. In target organs, long-range signals act in concert with tissue-autonomous ones to regulate the final size of a given tissue. In Drosophila, the steroid hormone ecdysone plays a dual role: peaks of secretion promote developmental transitions and maturation, while basal production negatively controls the speed of growth. The antagonistic action of ecdysone and the conserved insulin/insulin growth factor (IGF) signaling pathway regulate systemic growth and modulate final body size. This study has unraveled an unexpected role of bantam microRNA in controlling body size in Drosophila. The data reveals that, in addition to its well-characterized function in autonomously inducing tissue growth, bantam activity in ecdysone-producing cells promotes systemic growth by repressing ecdysone release. Evidence is provided that the regulation of ecdysone production by insulin signaling relies on the repression of bantam activity. These results identify a molecular mechanism that underlies the crosstalk between these two hormones and add a new layer of complexity to the well-characterized role of bantam in growth control (Boulan, 2013).

Because ban and ecdysone affect systemic growth in an opposite manner, it is likely that ban acts in PG cells by preventing ecdysone production. The circulating levels of the active form of ecdysone (20E) were measured in in P0206>ban animals at two developmental time points: at the beginning of the wandering phase (early L3w), and just before the larva/ pupa transition (late L3w). To do so, wandering larvae were precisely staged by monitoring gut clearance of blue food. As expected, 20E levels were already high in early L3w (blue gut) control larvae and further peaked in late L3w (clear gut). In contrast, P0206>ban larvae showed lower circulating 20E levels than controls at both stages, and the amplitude of the peak was strongly reduced. Ecdysone signaling in target tissues was also reduced in late larval development upon ban overexpression in the PG (in phm>banD animals) as measured by the expression levels of E75A and Broad-Complex (BR-C), two targets of EcR. Furthermore, the phantom (phm), disembodied (dib), and shade (sad) genes, which are specifically expressed in the PG and encode enzymes required for ecdysteroid biosynthesis, showed reduced expression in phm>ban animals. Consistent with the reduced levels of 20E production and signaling in phm>ban larvae, an increase in 20E levels, produced by feeding the animals ecdysone- supplemented medium, rescued pupa formation. These findings indicate that targeted expression of ban in ecdysone-producing cells has a negative impact on 20E production (Boulan, 2013).

banΔ1 mutant larvae displayed high lethality in late larval development. Thus, ecdysone signaling was assessed in these larvae during earlier stages. Two different developmental points precisely staged with respect to the transition from second (L2) to third (L3) larval instar were selected: 2 hr and 20 hr after ecdysis to the third instar (AL3E). No changes in the expression of BR-C were detected, most probably because at that time, 20E levels had not yet reached the minimum threshold to activate this target. However, the quantification of E75A mRNA levels revealed higher expression in banΔ1 larvae when compared to controls, as did the quantification of dib, phm, and sad mRNA levels. Ni predicted ban target site was found in the 3' UTR of these genes, suggesting that this repression is not direct (Boulan, 2013).

Ecdysone signaling negatively regulates body growth. To test whether the undergrowth phenotype observed in ban mutants is a result of abnormally high ecdysone levels, whole-mount ecdysone signaling was reduced by removing one copy of EcR or impaired ecdysone synthesis by depleting the levels of Sad and Phm in the PG of banΔ1 animals. In all cases, the size of banΔ1 pupae was largely rescued. Collectively, these results suggest that ban participates in reducing ecdysone production in PG cells and corroborate the hypothesis that the systemic growth defects observed in ban mutants are caused by increased ecdysone levels. Consistent with the cell-autonomous growth-promoting role of ban, the PG was larger in P0206>ban animals (that produce less ecdysone) than in controls, whereas it was much smaller in ban mutants. Thus, the impact of ban on ecdysone production is not a consequence of changes in PG size (Boulan, 2013).

Despite displaying higher levels of ecdysone, banΔ1 mutant animals reached metamorphosis with a delay. It has been reported that a strong reduction in larval growth rates can affect developmental timing as a result of a delay in the attainment of critical size for metamorphosis. In order to address whether banΔ1 animals are delayed as a consequence of their reduced growth rates, as a simple proxy of critical size, the time at which the minimal viable size for metamorphosis was achieved in was determined banΔ1 mutant and wild-type animals. Larvae were synchronized at the second (L2) to third (L3) instar transition and then starved at fixed time points to assess survival and capacity to enter into metamorphosis. Remarkably, banΔ1 larvae reached the threshold of 50% of survival with a delay when compared to wild-type animals. These data, together with the fact that targeted expression of ban in the ring gland largely rescued growth rates and developmental delay of banΔ1 animals, support the proposal that the developmental delay is at least in part a consequence of reduced growth rates. Other activities of ban, such as reduced growth of the imaginal tissues or impaired dendrite development, might also affect the timing of metamorphosis (Boulan, 2013).

The production of ecdysone is tightly controlled during larval development. Under normal conditions, ecdysone levels are low during the growth period, thereby allowing optimal body growth rates, and peak at the end of the third-instar larval stage to induce entry into metamorphosis. To monitor whether ban activity levels are also dynamically regulated in the PG, use was made of a ban sensor that expresses GFP under control of a ubiquitously active tubulin promoter and carries two perfect ban fixation sites in its 3' UTR thus making it repressed in the presence of the miRNA. A control sensor lacking the fixation sites showed high GFP expression both in early and late larval PGs. ban sensor levels, however, were low in the PG of second- and early third-instar larvae and considerably increased in wandering third-instar larvae. This observation leads to the proposal that high ban activity in young larvae contributes to the maintenance of low ecdysone titers and the promotion of systemic growth, whereas reduced activity in late PGs contributes to the generation of the ecdysone peak, the cessation of growth, and entry into metamorphosis (Boulan, 2013).

What is the upstream signal that regulates ban activity? The conserved insulin/insulin growth factor (IGF) signaling pathway directly promotes growth in target tissues and is the main relay to couple body growth to nutritional state. In young feeding larvae, insulin signaling in the PG also promotes the basal production of ecdysone, which in turn inhibits body growth. This buffering mechanism, based on the antagonistic action of insulin and ecdysone, modulates final body size in response to nutritional changes. Interestingly, ban activity levels were strongly reduced in early PGs expressing different transgenes that activate the insulin pathway. Increased levels of circulating Dilp2 also reduced ban activity in early PGs, as monitored by increased expression of the ban sensor. Thus, insulin signaling represses ban activity in ecdysone-producing cells (Boulan, 2013).

Thanks to a nutrient-sensing mechanism in the fat body, the equivalent to the vertebrate liver, food conditions control the secretion or expression of brain-derived Dilps, which are the main systemic supply of this hormone during the growth period. Consistent with the repression caused by increased insulin signaling, young feeding larvae growing on amino acid-rich medium showed a clear decrease in ban activity in PG cells. This reduction depended on the enhanced activity of Dilps, because the inhibition of insulin signaling in the PG was sufficient to restore ban activity to normal levels (Boulan, 2013).

In order to address whether ban mediates the action of insulin in regulating ecdysone production, genetic interactions were performed in gain- and loss-of-function conditions. Remarkably, the reduced body size phenotype obtained by enhanced insulin signaling in the PG via several transgenes was completely rescued by simultaneously increasing ban levels in these cells. Given the fact that this rescue implies a much greater effect on body size than the overexpression of UAS-ban transgene alone, it was concluded that the effects of ban and insulin signaling are not additive but rather epistatic. This conclusion is further supported by the observation that modulation of insulin signaling in the PG no longer affected body size in a banΔ1 mutant background. Altogether, these results indicate that the regulation of ecdysone production by insulin signaling relies on the modulation of ban activity in PG cells (Boulan, 2013).

So far, these results unravel a novel role of ban in promoting larval body growth by reducing ecdysone production. In contrast, ban was initially identified by its capacity to induce organ growth in a cell-autonomous manner. That finding prompted an exploration of the contribution of the systemic and cell-autonomous activities of ban to organ growth. The imaginal discs of Drosophila are epithelial sacs that grow in feeding larvae to give rise after metamorphosis to the ectodermal structures of the adult flies, such as legs, wings, or eyes. In banΔ1 mutant larvae, the size of the wing imaginal discs was strongly reduced when compared to control animals. Targeted expression of ban in the PG partially rescued the wing growth defects observed in ban mutant larvae. This result supports the proposal that both the systemic and cell-autonomous activities of ban are required to promote organ growth (Boulan, 2013).

In conclusion, these results establish that ban promotes systemic growth by inhibiting the synthesis of the steroid hormone ecdysone. During the growth period, ban mediates the insulin-dependent regulation of ecdysone production and therefore acts as a buffering mechanism to adjust final body size in response to nutrient availability. Such a crosstalk between insulin and steroid hormones and its impact on the modulation of growth and developmental decisions are also observed in Caenorhabditis elegans. Depending on environmental conditions, the juvenile form of C. elegans either enters maturation to give rise to an adult worm or arrests development to form a dauer larva, a state that is specifically adapted for survival. This decision is determined by the levels of the steroid hormone dafachronic acid (DA). Mutations that enhance insulin signaling, thereby mimicking a favorable environment, increase DA levels and cause animals to become incapable of forming dauer larvae. Remarkably, the deletion of ban orthologs in C. elegans (the mir-58 family) causes severe growth defects and prevents entry into the dauer state under environmental stress (Alvarez-Saavedra, 2010). On the basis of these observations, it is proposed that the role of ban in preventing the production of steroid hormones in function of insulin and nutrient levels might be conserved in other organisms in order to regulate body growth and maturation (Boulan, 2013).

Developmental ethanol exposure leads to dysregulation of lipid metabolism and oxidative stress in Drosophila

Ethanol exposure during development causes an array of developmental abnormalities, both physiological and behavioral. In mammals, these abnormalities are collectively known as Fetal Alcohol Effects (FAE) or Fetal Alcohol Spectrum Disorder (FASD). This study has established a Drosophila melanogaster model of FASD; it was previously shown that developmental ethanol exposure in flies leads to reduced expression of insulin like peptides (dILPs) and their receptor. This work linked that observation to dysregulation of fatty acid metabolism and lipid accumulation. Further, it was shown that developmental ethanol exposure in Drosophila causes oxidative stress, that this stress is a primary cause of the developmental lethality and delay associated with ethanol exposure, and, finally, that one of the mechanisms by which ethanol increases oxidative stress is through abnormal fatty acid metabolism. These data suggest a previously uncharacterized mechanism by which ethanol causes the symptoms associated with FASD (Logan-Garbisch, 2014).

The sleeping beauty: How reproductive diapause affects hormone signaling, Metabolism, immune response and somatic maintenance in Drosophila melanogaster

Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. Diapause was induced in D. melanogaster and effects were monitored over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagon- and insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, Drosomycin and Cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. This study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy (Kubrak, 2014: 25393614).

Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. This study examined whether increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. It was demonstrated that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. These findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging (Alic, 2011).

Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila

Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, 2011).

This study found that CNS progenitors are able to continue growing at their normal rate under fasting conditions severe enough to shut down all net body growth. Jeb/Alk signaling was identified as a central regulator of this brain sparing, promoting tissue-specific modifications in TOR/PI3K signaling that protect growth against reduced amino acid and Ilp concentrations. These findings highlight that a 'one size fits all' wiring diagram of the TOR/PI3K network should not be extrapolated between different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk signaling confers brain sparing is discussed, and how they may be integrated into an overall model for starvation-resistant CNS growth (Cheng, 2011).

One mechanism by which Alk spares the CNS is by suppressing the growth requirement for amino acid sensing via Slif, Rheb, and TORC1 components in neuroblast lineages. An important finding of this study is that in the presence of Alk signaling Tor has no detectable growth input (evidence from Tor clones), but in its absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to its typical role as a positive regulator of both growth and proliferation. The growth requirement for Slif/TORC1 is clearly much less in the CNS than in other tissues such as the wing disc but a low-level input cannot be ruled out due to possible perdurance inherent in any clonal analysis. Although Slif, Rheb, Tor, and Raptor mutant neuroblast clones attain normal volume, this reflects increased cell numbers offset by reduced average cell size. Atypical suppression of proliferation by TORC1 has also been observed in wing discs, where partial inhibition with rapamycin advances G2/M progression (Cheng, 2011).

Alk signaling in neuroblast lineages does not override the growth requirements for all TOR pathway components. The downstream effectors S6k and 4E-BP retain functions as positive and negative growth regulators, respectively. 4E-BP appears to be particularly critical in the CNS as mutant animals have normal mass, but mutant neuroblast clones are twice their normal volume. In many tissues, 4E-BP is phosphorylated by nutrient-dependent TORC1 activity. In CNS progenitors, however, 4E-BP phosphorylation is regulated in an NR-resistant manner by Alk, not by TORC1. Hence, although the pathway linking Alk to 4E-BP is not yet clear, it makes an important contribution toward protecting CNS growth during fasting (Cheng, 2011).

A second mechanism by which Alk spares CNS growth is by maintaining PI3K signaling during NR. Alk suppresses or overrides the genetic requirement for InR in PI3K signaling, which may or may not involve the direct binding of intracellular domains as reported for human ALK and IGF-IR (Shi, 2009). Either way, in the CNS, glial Jeb expression stimulates Alk-dependent PI3K signaling and thus neural growth at similar levels during feeding and NR. In contrast, in tissues such as the salivary gland, where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides concentrations during NR strongly reduce growth (Cheng, 2011).

The finding that Alk signals via PI3K during CNS growth differs from the Ras/MAPK transduction pathway described in Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling during growth is well documented in humans, both in glioblastomas and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with regard to Alk ligands, as there is no clear Jeb ortholog and human ALK can be activated, directly or indirectly, by the secreted factors Pleiotrophin and Midkine (Cheng, 2011).

A comparison of these results with those of previous studies indicates that CNS super sparing only becomes fully active at late larval stages. Earlier in larval life, dietary amino acids are essential for neuroblasts to re-enter the cell cycle after a period of quiescence. This nutrient-dependent reactivation involves a relay whereby Slif-dependent amino acid sensing in the fat body stimulates Ilp production from a glial cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps activate InR and PI3K/TOR signaling in neuroblasts thus stimulating cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb from the glial cell niche may change in line with the developmental transition of neuroblast growth from high to low nutrient sensitivity (Cheng, 2011).

The results of this study suggest a working model for super sparing in the late-larval CNS. Central to the model is that Jeb/Alk signaling suppresses Slif/ RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains S6k and PI3K activation, thus freeing CNS growth from the high dependence upon amino acid sensing and Ilps that exists in other organs. The CNS also contrasts with other spared diploid tissues such as the wing disc, in which PI3K-dependent growth requires a positive Tor input but is kept in check by negative feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient (in the salivary gland) to promote organ growth during fasting. However, both Alk manipulations produce organ-sparing percentages intermediate between the 2% salivary gland and the 96% neuroblast values, arguing that other processes may also contribute. For example, some Drosophila tissues synthesize local sources of Ilps that could be more NR resistant than the systemic supply from the IPCs. In mammals, this type of mechanism may contribute to brain sparing as it has been observed that IGF-I messenger RNA (mRNA) levels in the postnatal CNS are highly buffered against NR. It will also be worthwhile exploring whether mammalian neural growth and brain sparing involve Alk and/or atypical TOR signaling. In this regard, it is intriguing that several studies show that activating mutations within the kinase domain of human ALK are associated with childhood neuroblastomas. In addition, fetal growth of the mouse brain was recently reported to be resistant to loss of function of TORC1. Finally, a comparison between the current findings and those of a cancer study, highlights that insulin/IGF independence and constitutive PI3K activity are features of NR-resistant growth in contexts as diverse as insect CNS development and human tumorigenesis (Cheng, 2011).

Drosophila lifespan control by dietary restriction independent of insulin-like signaling

Reduced insulin/insulin-like growth factor (IGF) signaling may be a natural way for the reduction of dietary nutrients to extend lifespan. While evidence challenging this hypothesis is accumulating with Caenorhabditis elegans, for Drosophila melanogaster it is still thought that insulin/IGF and the mechanisms of dietary restriction (DR) might as yet function through overlapping mechanisms. This study aimed to understand this potential overlap. It was found that over-expression of dFOXO in head fat body extends lifespan and reduces steady-state mRNA abundance of insulin-like peptide-2 under conditions of high dietary yeast, but not when yeast is limiting. In contrast, conditions of DR that increase lifespan change only insulin-like peptide-5 (ilp5) mRNA abundance. Thus, reduction of ilp5 mRNA is associated with longevity extension by DR, while reduction of insulin-like peptide-2 is associated with the diet-dependent effects of FOXO over-expression upon lifespan. To assess whether reduction of ilp5 is required for DR to extend lifespan, its diet-dependent change with was blocked by RNAi. Loss of the ilp5 dietary response did not diminish the capacity of DR to extend lifespan. Finally, the capacity was assessed of DR to extend lifespan in the absence of dFOXO, the insulin/IGF-responsive transcription factor. As with the knockdown of ilp5 diet responsiveness, DR was equally effective among genotypes with and without dFOXO. It is clear from many Drosophila studies that insulin/IGF mediates growth and metabolic responses to nutrition, but no evidence was found that this endocrine system mediates the interaction between dietary yeast and longevity extension (Min, 2008).

Dietary restriction and insulin/insulin-like growth factor (IGF) signaling (IIS) affect Drosophila lifespan, and in many animals insulin signaling is the prominent endocrine response to nutrient status. From these relationships, it was anticipated that mechanisms of Drosophila longevity extension by DR might overlap with those of IIS. This study evaluated this idea in several ways, but found no evidence to support the hypothesis (Min, 2008).

Over-expression of dFOXO in adult fat bodies modulates lifespan and leads to reduction in mRNA of insulin-like ligand ilp2 (Hwangbo, 2004). Medial secretory neurons also produce ilp3 and ilp5, but neither of these change when dFOXO extends lifespan. A role for ilp2 in longevity control has also been suggested in the analysis of aging mediated by JNK, and this effect required dFOXO (Wang, 2005). If DR modulates aging through insulin-like signals, it might expected from these observations that ilp2 would be reduced in flies when DR extends longevity. However, this was found not the case because ilp5 was the only insulin-like mRNA repressed by yeast-restricted diets (Min, 2008).

Thus, while ilp2 appears to be associated with longevity control downstream of dFOXO and JNK, the otherwise static ilp5 is correlated with the effect of DR upon longevity. Accordingly, whether diet-induced reduction of ilp5 was required for DR to extend lifespan was investigated. Expression of RNAi in the MSN reduced mRNA of ilp2, ilp3 and ilp5 by approximately 50%. Importantly, this treatment eliminated the nutrient responsiveness of ilp5, permitting determination of wheter this impaired the ability of DR to extend lifespan. The survival of more than 3800 adults distributed in three genotypes was simultaneously analyzed among four diets. Relative to fully fed adults, DR increased lifespan with equal efficiency with and without diet-responsive change in ilp5. It is conclude that reduction of ilp5 is not sufficient to extend lifespan, although it cannot be ruled out that diet-responsive change in ilp5 may as yet be necessary for DR to modulate aging (Min, 2008).

To investigate if a component of IIS distal to ligand synthesis contributes to DR, whether dFOXO was required for restricted diet to extend lifespan was evaluated. Longevity extension was compared across four diets using 4100 flies representing confirmed, heterozygote and wild-type dfoxo genotypes. DR was found to extend lifespan with equal efficiency with and without dFOXO (Min, 2008).

Despite the apparent independence of insulin/IGF and DR in the control of Drosophila lifespan, it was found that dFOXO over-expressed from fat body extends lifespan in a nutrient-dependent manner. dFOXO expressed from the head fat body extends lifespan only upon high-yeast diets, while dFOXO expressed from visceral fat body extends lifespan best upon a restricted diet. At face value, longevity extension induced by dFOXO over-expression in fully fed flies would be consistent with expectations if DR functions through IIS: dFOXO would be intrinsically induced on restricted diets, and over-expression on full diets copies this effect. This explanation, however, is not supported by analysis with dFOXO loss-of-function genotypes. Some insight is provided by the association of reduced ilp2 and lifespan when dFOXO is expressed in different fat bodies. Reduction of ilp2 may increase lifespan independent of nutrition, but head and visceral fat bodies have unique conditions from which they repress brain synthesis of ilp2 (Min, 2008).

The observations are consistent with current perspectives from C. elegans where DR efficiently extends lifespan in mutants of the insulin/IGF signaling pathway (Walker, 2005; Houthoofd, 2007; Panowski, 2007). Analysis of the interaction between DR and insulin/IGF was also studied in the context of the pituitary development mutations of mice. The Ames dwarf has deficient pituitary production of prolactin, growth hormone and thyroid-stimulating hormone, and these animals consequently have reduced circulating IGF and insulin. Because DR extends lifespan to the same extent in Ames and wild-type mice, it is thought that the mechanisms of Ames longevity assurance are independent of those induced by restricted diet. However, mice that only lack the growth hormone response because of knockout of the growth hormone receptor locus appear to be refractory to the effect of DR upon longevity (Bonkowski, 2006). The results are consistent with the observations from C. elegans and the initial conclusion from mice, but differ from the sole report of insulin/IGF and DR interactions with Drosophila (Clancy, 2002) where wild-type and homozygous mutants of chico were aged on a series of sugar–yeast diets. Lifespan of chico females was optimal on a diet of 8% sugar and yeast, while the longevity of wild-type flies was highest upon a diet of 6.5% sugar and yeast. In both genotypes, survival was reduced as nutrients were diluted below these optima. Importantly, the plots of longevity versus diet were parallel in the range of nutrients at concentrations greater than each genotype's optimum. These results were interpreted to represent overlap in the mechanism of insulin/IGF and DR (Clancy, 2002). However, DR occurs in the range of diets where nutrient dilution increases lifespan, not where lifespan is reduced by malnutrition. The parallelism in the range where restricted diet increases lifespan suggests that DR is equally efficient in these genotypes (Tatar, 2007), as was found for mutants of dfoxo and for inhibition of ilp5 dynamics. Overall, these data suggest that the mechanisms of DR function may be independent of insulin/IGF in Drosophila. This argument is also consistent with recent work on diet and olfaction where sensory modulation of Drosophila longevity was not associated with changes in ilp mRNA (Libert, 2007). The basic problem still exists: how does reduced nutrient intake modulate systems that extend fly longevity (Min, 2008)?

Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance

Insulin and insulin-like growth factors (IGFs) signal through a highly conserved pathway and control growth and metabolism in both vertebrates and invertebrates. In mammals, insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and modulate their mitogenic, anti-apoptotic and metabolic actions, but no functional homologs have been identified in invertebrates so far. This study shows that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2) binds Drosophila insulin-like peptide 2 (Dilp2) and inhibits growth non-autonomously. Whereas over-expressing Imp-L2 strongly reduces size, loss of Imp-L2 function results in an increased body size. Imp-L2 is both necessary and sufficient to compensate Dilp2-induced hyperinsulinemia in vivo. Under starvation conditions, Imp-L2 is essential for proper dampening of insulin signaling and larval survival. It is concluded that Imp-L2, the first functionally characterized insulin-binding protein in invertebrates, serves as a nutritionally controlled suppressor of insulin-mediated growth in Drosophila. Given that Imp-L2 and the human tumor suppressor IGFBP-7 show sequence homology in their carboxy-terminal immunoglobulin-like domains, it is suggested that their common precursor was an ancestral insulin-binding protein (Honegger, 2008).

Insulin/insulin-like growth factor (IGF) signaling (termed IIS) is involved in the regulation of growth, metabolism, reproduction and longevity in mammals . The activity of IIS is regulated at multiple levels, both extracellularly and intracellularly: the production and release of the ligands is regulated, and normally IGFs are also bound and transported by IGFBPs in extracellular cavities of vertebrates. IGFBPs not only prolong the half-lives of IGFs, but they also modulate their availability and activity. Besides the classical IGFBPs (IGFBP1-6), a related protein called IGFBP-7 (or IGFBP-rP1, Mac25, TAF, AGM or PSF) has been identified as an insulin-binding protein. Although the reported binding of IGFBP-7 to insulin awaits confirmation, it can compete with insulin for binding to the insulin receptor (InR) and inhibit the autophosphorylation of InR. Furthermore, IGFBP-7 is suspected to be a tumor suppressor in a variety of human organs, including breast, lung and colon. A recent publication demonstrates that IGFBP-7 induces senescence and apoptosis in an autocrine/paracrine manner in human primary fibroblasts in response to an activated BRAF oncogene (Wajapeyee, 2008; Honegger, 2008 and references therein).

IIS is astonishingly well conserved in invertebrates. In Drosophila, IIS acts primarily to promote cellular growth, but it also affects metabolism, fertility and longevity. Seven insulin-like peptides (Dilp1-7) homologous to vertebrate insulin and IGF-I have been identified as putative ligands of the Drosophila insulin receptor (dInR). These Dilps are expressed in a spatially and temporally controlled pattern, including expression in median neurosecretory cells (m-NSCs) of both brain hemispheres. The m-NSCs have axon terminals in the larval endocrine gland and on the aorta, where the Dilps are secreted into the hemolymph. Ablation of the m-NSCs causes a developmental delay, growth retardation and elevated carbohydrate levels in the larval hemolymph (Ikeya, 2002; Rulifson, 2002), reminiscent of the phenotypes of starved or IIS-impaired flies (Honegger, 2008).

The Drosophila genome does not encode an obvious homolog of the IGFBPs. Furthermore, genetic analyses of IIS in Drosophila and C. elegans have not revealed a functional insulin-binding protein so far. This study reports the identification of the secreted protein Imp-L2 as a binding partner of Dilp2. Imp-L2 is not essential under standard conditions, but flies lacking Imp-L2 function are larger. Under adverse nutritional conditions, Imp-L2 is upregulated in the fat body and represses IIS activity in the entire organism, allowing the animal to endure periods of starvation (Honegger, 2008).

It was reasoned that the overexpression of a Dilp-binding protein that impinges on the ligand-receptor interaction should counteract the effects of receptor overexpression. dInR overexpression during eye development (by means of a GMR-Gal4 strain, in which the Gal4 protein is overexpressed in photoreceptor neurons, and a UAS-dInR, which expresses dInR when activated by Gal4) results in hyperplasia of the eyes, a phenotype that is sensitive to the levels of the Dilps (Brogiolo, 2001). A collection of enhancer-promoter (EP) elements, which allow the overexpression of nearby genes, was screened for suppressors of the dInR-induced hyperplasia. A strong suppressor (EP5.66) carried an EP element 8.5 kb upstream of the Imp-L2 coding sequence. Two different UAS transgenes, both containing the Imp-L2 coding sequence but varying in strength, confirmed that the suppression was caused by Imp-L2. Whereas the weaker UAS-Imp-L2 (containing 5' sequences with three upstream open reading frames) only partially suppressed the dInR-induced overgrowth, UAS-strong.Imp-L2 (UAS-s.Imp-L2, lacking the 5' sequences) completely reversed the phenotype. In addition, a point mutation in the Imp-L2 coding sequence abolished the suppressive effect of EP5.66. Imp-L2 is therefore a potent antagonist of dInR-induced growth (Honegger, 2008).

Imp-L2 has previously been shown to be upregulated 8-10 hours after ecdysone treatment. It encodes a secreted member of the immunoglobulin (Ig) superfamily containing two Ig C2-like domains. Whereas several orthologs of Imp-L2 are present in invertebrates such as arthropods and nematodes, the homology in vertebrates is confined to the second Ig C2-like domain, which is homologous to the carboxyl terminus of human IGFBP-7. The carboxy-terminal part of IGFBP-7 differs considerably from the other IGFBPs, possibly accounting for the affinity of IGFBP-7 for insulin. Interestingly, Imp-L2 has been shown to bind human insulin, IGF-I, IGF-II and proinsulin, and its homolog in the moth Spodoptera frugiperda, Sf-IBP, can inhibit insulin signaling through the insulin receptor (Honegger, 2008 and references therein).

To further assess the function of Imp-L2 as a secreted inhibitor of insulin signaling, Imp-L2 was ectopically expressed using various Gal4 drivers. Strong ubiquitous over-expression of Imp-L2 by Act-Gal4 led to lethality with both UAS transgenes. Whereas driving UAS-s.Imp-L2 by the weaker ubiquitous arm-Gal4 driver also resulted in lethality, driving UAS-Imp-L2 generated flies that were decreased in size and weight (-15% in males and -29% in females) but eclosed at the expected ratio and had wild-type appearance. By generating clones of cells that over-express Imp-L2, it was confirmed that cell specification and patterning were normal in Imp-L2-overexpressing ommatidia. However, a reduction of cell size was observed in the clones. This reduction seemed to be non-autonomous because wild-type ommatidia close to the clone were also reduced in size. Given the convex nature of the eye it was not possible to quantify the effects of Imp-L2 overexpression on more distantly located ommatidia. Eye-specific overexpression of both UAS-Imp-L2 and UAS-s.Imp-L2 by GMR-Gal4 led to a strong reduction in eye size. Whereas the GMR-Gal4, UAS-Imp-L2 flies were of normal size, body weight was reduced by 38.3% and development was delayed by one day in GMR-Gal4, UAS-s.Imp-L2 male flies. Next, the ppl-Gal4 driver was used to over-express Imp-L2 in the fat body, a tissue that can be expected to produce and secrete Imp-L2 more efficiently than the eye. Driving UAS-s.Imp-L2 by ppl-Gal4 was lethal, whereas ppl-Gal4, UAS-Imp-L2 flies showed a pronounced reduction in body size and were delayed by 2 days. Both the size decrease and the developmental delay are characteristic phenotypes of reduced IIS such as in chico mutants, supporting the hypothesis that Imp-L2 acts as a secreted negative regulator of this pathway (Honegger, 2008).

Next, the effect of Imp-L2 overexpression on phosphatidylinositol(3,4,5)trisphosphate (PIP₃) levels was assessed using a green fluorescent protein-pleckstrin homology domain fusion protein (tGPH) that specifically binds PIP₃ and serves as a reporter for PIP₃ levels in vivo. The amount of membrane-bound tGPH reflects signaling activity in the phosphoinositide 3-kinase/protein kinase B (PI 3-kinase/PKB) pathway. Overexpression of dInR resulted in a severe increase of membrane PIP₃ levels. Co-overexpression of Imp-L2 together with dInR reduced the PIP₃ levels, similar to the effect caused by PTEN, a negative regulator of IIS. Therefore, Imp-L2 inhibits PI 3-kinase/PKB signaling upstream of PIP₃, without affecting dInR levels (Honegger, 2008).

Two strategies were used to generate loss-of-function mutations in Imp-L2. (1) An ethylmethane-sulfonate (EMS) reversion screen was performed in which mutated chromosomes carrying EP5.66 were selected that no longer suppressed the dInR overexpression phenotype. One allele (Imp-L2^MG2) containing a point mutation resulting in a premature stop at amino acid 232 was identified in this way. This truncation destroys the conserved cysteine bridge of the second Ig domain. Overexpression of the truncated Imp-L2 version had no inhibitory effect on size, suggesting that Imp-L2^MG2 is a functional null allele (Honegger, 2008).

(2) Additional Imp-L2 alleles were generated by imprecise excision of GE24013 (GenExel), a P-element located 349 bp upstream of the ATG start codon of the Imp-L2-RB transcript. Imp-L2 deletions were obtained (Def20, Def42) lacking the entire coding sequence. Heteroallelic combinations of the mutant alleles increased body size: whereas mutant males showed a 27% increase in body weight, mutant females were 64% heavier. Introducing one copy of a genomic rescue construct into homozygous mutant flies reverted the weight to the level of Imp-L2^+/- flies, which were already heavier (+14% in males, +44% in females) than the controls. By measuring the cell density in the wing, the size increase could be attributed primarily to an increase in the number of cells, because cell size was only slightly affected. Apart from the size increase, the flies lacking Imp-L2 appeared completely normal, eclosed with the expected frequency and were not delayed. Thus, under standard conditions, Imp-L2 loss-of-function dominantly increases growth by augmenting cell number without perturbing patterning, developmental timing or viability (Honegger, 2008).

The weight difference was more pronounced in mutant females than in males, although the increases in wing area and cell number were similar. This differential effect was caused by enlarged ovaries in Imp-L2 mutant females (Honegger, 2008).

The facts that Imp-L2 is a secreted protein and that removal of Imp-L2 function did not rescue either chico or PI3K mutant phenotypes are consistent with the hypothesis that Imp-L2 acts upstream of the intra-cellular IIS cascade at the level of the ligands. Immunohistochemistry in larval tissues revealed that, besides strong expression in corpora cardiaca (CC) cells, Imp-L2 protein was also weakly expressed in the seven m-NSCs that produce Dilp1, Dilp2, Dilp3 and Dilp5 and project their axons directly to the subesophageal ganglion, the CC, the aorta and the heart. Thus, Imp-L2 potentially interacts with some of the Dilps directly at their source. Therefore tests were performed for genetic interactions of Imp-L2 with the dilp genes. A deficiency (Df(3L)AC1) uncovering dilp1-5 not only dominantly suppressed the dInR-mediated big eye phenotype, but also dominantly enhanced the small eye phenotype caused by eye-specific overexpression of Imp-L2. dilp2 is the most potent growth regulator of all dilp genes. Weak ubiquitous overexpression of dilp2 by arm-Gal4 caused an increase in body and organ size, and this phenotype was dominantly enhanced by heterozygosity for Imp-L2. In homozygous Imp-L2 mutants, expression of dilp2 under the control of arm-Gal4 caused lethality, reminiscent of strong dilp2 expression. Expressing Imp-L2 and dilp2 individually at high levels in the fat body also caused lethality, but coexpression resulted in viable flies of wild-type size. Thus, Imp-L2 decreases the sensitivity to high insulin levels and is sufficient to rescue the lethality resulting from dilp2-induced hyperinsulinemia (Honegger, 2008).

It has been shown that Imp-L2 can bind human insulin and insulin-related peptides. To address whether Imp-L2 binds Dilp2, a Flag-tagged version of Dilp2 was constructed, that is functional. Using in vitro translated, ³⁵S-labeled Imp-L2 together with Flag-Dilp2 extracted from stably transfected S2 cells, it was shown that Imp-L2 binds Dilp2 in vitro. A truncated form of Imp-L2 lacking a functional second Ig domain (like that produced by the MG2 allele) failed to bind Dilp2 (Honegger, 2008).

Despite being a potent inhibitor of Dilp2 action, Imp-L2 is not essential under standard conditions. Hyperactivation of the dInR pathway leads to increased accumulation of nutrients in adipose tissues, precluding them from circulating and thus resulting in starvation sensitivity at the organismal level (Britton, 2002). Therefore whether Imp-L2 functions as an inhibitor of IIS under stress conditions was tested. Wild-type and Imp-L2 mutant early third instar larvae were exposed to various starvation conditions and scored for survival. Larvae lacking Imp-L2 showed a massive increase in mortality rate when exposed to 1% glucose or PBS for 24 hours. To test whether the inability of the mutant larvae to cope with starvation was due to a failure in adjusting IIS, PIP₃ levels were monitored under these conditions. Whereas control flies showed a decrease of PIP₃ levels when exposed to complete starvation for 4 hours, Imp-L2 mutant larvae still contained PIP₃ levels that were comparable to those of control larvae reared on normal food, suggesting that Imp-L2 is necessary to adjust IIS under starvation conditions. The fact that PIP₃ levels were also slightly reduced in Imp-L2 mutants upon starvation could be attributed to the downregulation of dilp3 and dilp5 at the transcriptional level (Honegger, 2008).

It is concluded that Imp-L2 encodes a secreted peptide containing two Ig C2-like domains. Consistent with its secretion, the effects of Imp-L2 overexpression are non-autonomous. Tissue-specific over-expression of Imp-L2, for example in the larval fat body, results in a systemic response, and the entire animal is impaired in its capacity to grow. Conversely, the loss of Imp-L2 function produces larger animals. Analysis of IIS activity (by means of the tGPH reporter in vivo) shows that Imp-L2 functions to downregulate IIS. This study further showed that wild-type Imp-L2 (but not a truncated version lacking the second Ig C2-like domain) binds Dilp2, consistent with previous findings that Imp-L2 binds human insulin, IGF-I, IGF-II and proinsulin (Honegger, 2008).

Thus, despite lacking any clear ortholog of the classical IGFBPs with their characteristic amino-terminal IGFBP motifs, invertebrates such as flies can regulate IIS activity at the level of the ligands as a result of Imp-L2 expression. Orthologs of Imp-L2 are present in C. elegans, Apis mellifera, Anopheles gambiae, Spodoptera frugiperda and Drosophila pseudoobscura. Importantly, the second Ig C2-like domain of Imp-L2 also has sequence homology to the carboxyl terminus of IGFBP-7, which is the only IGFBP that, besides binding to IGFs, also binds insulin. It is speculated that Imp-L2 resembles an ancestral insulin-binding protein and that IGFBP-7 evolved from such an ancestor molecule by replacing the amino-terminal Ig C2-like domain with the IGFBP motif (Honegger, 2008).

Interestingly, Dilp2 and Imp-L2 are found in a complex with dALS (acid-labile subunit (Arquier, 2008). In vertebrates, most of the circulating IGFs are part of ternary complexes consisting of an IGF, IGFBP-3 and ALS. These ternary complexes prolong the half-lives of the IGFs and restrict them to the vascular system, because the 150 kDa complexes cross the capillary barrier very poorly. IGFs can also be found in binary complexes of about 50 kDa with several IGFBP species but there is only little (< 5%) free circulating IGF. Thus, it will be interesting to analyze the composition and bioactivities of Dilp2/Imp-L2/ALS complexes in Drosophila (Honegger, 2008).

IIS coordinates nutritional status with growth and metabolism in developing Drosophila. It has been shown that IIS regulates the storage of nutrients in the fat body, an organ that resembles the mammalian liver as the principal site of stored glycogen. Even under adverse nutritional conditions, fat body cells with increased IIS activity continue stockpiling nutrients, thereby limiting the amount of circulating nutrients, which induces hypersensitivity to starvation of the larva. Upon starvation, the expression of dilp3 and dilp5 is suppressed at the transcriptional level in the m-NSCs. This study reveals an additional layer of IIS regulation. Whereas Imp-L2 is not expressed in the fat body of fed larvae, starved animals induce Imp-L2 expression in the fat body to systemically dampen IIS activity. A lack of this control mechanism is lethal under unfavorable nutritional conditions, as Imp-L2 mutant larvae fail to cope with starvation (Honegger, 2008).

This study provides the first functional characterization of an insulin-binding protein in invertebrates. Imp-L2 is a secreted antagonist of IIS in Drosophila. Given the sequence homology of their Ig domains, it is proposed that Imp-L2 is a functional homolog of vertebrate IGFBP-7. Because both Imp-L2 and IGFBP-7 are potent inhibitors of growth and Imp-L2 is essential for the endurance of periods of starvation, it is likely that the original function of the insulin-binding molecules was to keep IIS in check when nutrients were scarce. Thus, in accordance with several reports suggesting that IGFBP-7 acts as a tumor suppressor, loss of IGFBP-7 may provide tumor cells with a growth advantage under conditions of local nutrient deprivation, such as in prevascularized stages of tumorigenesis (Honegger, 2008).

Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides

In metazoans, factors of the insulin family control growth, metabolism, longevity, and fertility in response to environmental cues. In Drosophila, a family of seven insulin-like peptides, called Dilps, activate a common insulin receptor. Some Dilp peptides carry both metabolic and growth functions, raising the possibility that various binding partners specify their functions. This study identifies ALS, the fly ortholog of the vertebrate insulin-like growth factor (IGF)-binding protein acid-labile subunit (ALS), as a Dilp partner that forms a circulating trimeric complex with one molecule of Dilp and one molecule of Imp-L2, an IgG-family molecule distantly related to mammalian IGF-binding proteins (IGFBPs). Drosophila ALS antagonizes Dilp function to control animal growth as well as carbohydrate and fat metabolism. These results lead to the proposal of an evolutionary perspective in which ALS function appeared prior to the separation between metabolic and growth effects that are associated with vertebrate insulin and IGFs (Arquier, 2008).

CG8561 has been previously identified as a candidate gene encoding a putative Drosophila ortholog of the vertebrate ALS protein, which has been called dALS (Colombani, 2003). The dALS protein contains a series of 21 leucine-rich repeats (LRRs) that also form the core of the vertebrate ALS. Based on sequence similarity and the presence of LRRs, two additional related sequences were found in the Drosophila genome. The expression levels of all three genes was examined in larval tissues and in normally fed or starved animals. CG8561 is exclusively expressed in two larval tissues that play important roles in growth and metabolic regulation: the 14 IPCs in the brain, and the fat body (FB), a larval tissue that shares some functions with the vertebrate liver and fat (Colombani, 2003). Remarkably, dALS expression in the FB is suppressed under amino acid restriction, a finding reminiscent of the strong downregulation of the vertebrate ALS gene observed in the liver under starvation (Colombani, 2003). The two other related genes did not show clear expression in any of the larval tissues, nor did they show nutrition-regulated expression. Therefore the analysis focused on CG8561 (Arquier, 2008).

This work provides strong evidence for the formation of a trimeric complex involving Dilp2, ALS, and Imp-L2, a molecule with Dilp-binding protein function in Drosophila. No binding was observed between ALS and Dilp2 in the absence of Imp-L2, suggesting that, as with the trimeric IGF-1 complexes circulating in mammalian blood, the binding of ALS requires prior formation of a dimeric Dilp/Imp-L2 complex. Dilp5, another member of the ILP family in Drosophila, is also capable of forming a complex with ALS in cultured cells. Interestingly, the binding of Dilp5 and ALS is suppressed by excess Imp-L2, suggesting that one or more other Dilp-BPs produced in S2 cells compete with ALS binding for the formation of Dilp5 complexes. It is proposed that ALS may function as a common scaffold protein for different Dilp/Dilp-BP complexes in the hemolymph, with specific Dilp-BPs participating in the specialization of Dilp functions. At present, the technical difficulty of measuring the levels of endogenous Dilps in the hemolymph of Drosophila larvae precludes a detailed analysis of the types and amounts of circulating Dilp/Dilp-BP/ALS complexes (Arquier, 2008).

No abnormal phenotypes were observed upon ALS overexpression or silencing in the brain IPCs. This could be due to a lack of sensitivity in the method, as it was found that expressing ALS^M in the 14 IPCs leads to very low accumulation of ALS^M in the hemolymph as compared to its expression in the FB. Conversely, silencing ALS in the IPCs does not reduce global ALS transcript levels, possibly because an important ALS transcription from FB cells is masking this effect. It was also noticed that, when expressed in the IPCs, ALS^M is not present in the same vesicular structures as Dilp2, suggesting that the two molecules are not found in a preassembled complex before being released into the hemolymph. Determination of the function of IPC-produced ALS will require further examination (Arquier, 2008).

The results point to a dual effect of ALS in the control of IIS that depends on nutritional status. This dual effect is interpreted in light of the complex functions of IGFBPs and ALS in mammals. Under optimal nutritional conditions, Dilps are not limiting, and overexpression of ALS can induce the recruitment of more Dilps into stable but inactive trimeric complexes. If the release of active Dilp molecules is limited by the amounts of the various proteases that break apart the trimeric complexes, the net effect of ALS overexpression will be growth inhibition, as observed in vivo. In contrast, fasting leads to a general inhibition of IIS that may reveal a positive function for ALS: Dilp molecules becoming limiting, and ALS overexpression may increase the half-life of circulating Dilps and thereby enhance Dilp signaling (as long as the proteases are not limiting). Along these lines, the severe downregulation of ALS transcription observed under limited nutrient conditions (Colombani, 2003) suggests that ALS participates in the adaptation of IIS to limited nutrition and the necessity of slowing down growth rate as well as carbohydrate and fat metabolism. Alternatively, the opposing results observed in starved versus fed conditions could be explained by the differential regulation of Dilp/ALS complexes involved in distinct regulations of IIS in response to nutritional conditions (Arquier, 2008).

It has been proposed that in vertebrates, the formation of trimeric IGF/IGFBP/ALS complexes contributes to the functional separation between insulin and IGFs. This study has provided evidence that such complexes are required for both the growth and metabolic functions carried out by the Dilps in Drosophila. The work suggests an alternative scenario in which ALS, Imp-L2, and possibly additional Dilp-BPs participate in an ancestral function used for both metabolism and growth control (Arquier, 2008).

Remote control of insulin secretion by fat cells in Drosophila

Insulin-like peptides (ILPs) couple growth, metabolism, longevity, and fertility with changes in nutritional availability. In Drosophila, several ILPs called Dilps are produced by the brain insulin-producing cells (IPCs), from which they are released into the hemolymph and act systemically. In response to nutrient deprivation, brain Dilps are no longer secreted and accumulate in the IPCs. The larval fat body, a functional homolog of vertebrate liver and white fat, couples the level of circulating Dilps with dietary amino acid levels by remotely controlling Dilp release through a TOR/RAPTOR-dependent mechanism. Ex vivo tissue coculture was used to demonstrate that a humoral signal emitted by the fat body transits through the hemolymph and activates Dilp secretion in the IPCs. Thus, the availability of nutrients is remotely sensed in fat body cells and conveyed to the brain IPCs by a humoral signal controlling ILP release (Géminard, 2009).

Due to the lack of immunoassay, the study of the regulation of Dilp levels in Drosophila has been limited so far to the analysis of their expression level in response to nutritional conditions. This study presents evidence that the secretion of Dilp2 and Dilp5 as well as a GFP linked to a signal peptide (secGFP) is controlled by the nutritional status of the larva. The data also indicate that the IPCs have the specific ability to couple secretion with nutritional input. This suggests that all Dilps produced in the IPCs could be subjected to a common control on their secretion that could therefore override differences in their transcriptional regulation. It was further shown that the regulation of Dilp secretion plays a key role in controlling Dilp circulating levels and biological functions, since blocking neurosecretion in the IPCs led to growth and metabolic defects, and conversely, expression of Dilp2 in nonregulated neurosecretory cells is lethal upon starvation. Interestingly, previous reports suggest that Dilp release could also be controlled in the adult IPCs, raising the possibility that this type of regulation contributes to controlling metabolic homeostasis, reproduction, and aging during adult life (Géminard, 2009).

Dilp release is not activated by high-carbohydrate or high-fat diets, but rather depends on the level of amino acids and in particular on the presence of branched-chain amino acids like leucine and isoleucine. This finding is consistent with the described mechanism of TOR activation by leucine in mammalian cells (Avruch, 2009: Nicklin, 2009). In particular, it was recently shown that Rag GTPases can physically interact with mTORC1 and regulate its subcellular localization in response to L-leucine (Sancak, 2008). Interestingly, the present work indicates that amino acids do not directly signal to the IPCs, but rather they act on fat-body cells to control Dilp release. TOR signaling has been previously shown to relay the nutritional input in fat-body cells. Tor signaling is required for the remote control of Dilp secretion, since inhibition of Raptor-dependent TOR activity in fat cells provokes Dilp retention. Surprisingly, activation of TOR signaling in fat cells of underfed larvae is sufficient to induce Dilp release, indicating that TOR signaling is the major pathway relaying the nutrition signal from the fat body to the brain IPCs. In contrast, inhibition of PI3K activity in fat cells does not appear to influence Dilp secretion in the brain. This result is in line with previous in vivo data showing that reduction of PI3K levels in fat cells does not induce systemic growth defects. Altogether, this suggests that the nutritional signal is read by a TOR-dependent mechanism in fat cells, leading to the production of a secretion signal that is conveyed to the brain by the hemolymph (Géminard, 2009).

Ex vivo brain culture experiments demonstrate that hemolymph or dissected fat bodies from fed larvae constitute an efficient source for the Dilp secretion factor. This signal is absent in underfed animals, suggesting that it could be identified by comparative analysis of fed and underfed states. The nature of the secretion signal is unknown. It is produced and released in the hemolymph by fat cells, and its production relies on TORC1 function. Given the role of TORC1 in protein translation, one could envisage that the secretion factor is a protein or a peptide for which translation is limited by TORC1 activity and relies on amino acid input in fat-body cells. In mammals, fatty acids and other lipid molecules have the capacity to amplify glucose-stimulated insulin secretion in pancreatic β cells. The fly fat body carries important functions related to lipid metabolism, and a recent link has been established between TOR signaling and lipid metabolism in flies (Porstmann, 2008), leaving open the possibility that a TOR-dependent lipid-based signal could also operate in this regulation. Interestingly, carbohydrates do not appear to contribute to the regulation of insulin secretion by brain cells in flies. This finding is reminiscent of the absence of expression of the Sur1 ortholog in the IPCs and suggests that global carbohydrate levels are controlled by the glucagon-like AKH produced by the corpora cardiaca cells (Géminard, 2009).

These experiments demonstrate that Dilp secretion is linked to the polarization state of the IPC membrane, suggestive of a calcium-dependent granule exocytosis, like the one observed for insulin and many other neuropeptides. The nature of the upstream signal controlling membrane depolarization is not known. Recent data concerning the function of the nucleostemin gene ns3 in Drosophila suggest that a subset of serotonergic neurons in the larval brain act on the IPCs to control insulin secretion (Kaplan, 2008). Therefore, it remains to be known whether the IPCs or upstream serotonergic neurons constitute a direct target for the secretion signal. So far, no link has been established between the serotonergic stimulation of IPC function and the nutritional input (Géminard, 2009).

In 1998, J. Britton and B. Edgar presented experiments where starved brain and fed fat bodies were cocultured, allowing arrested brain neuroblasts to resume proliferation in the presence of nutrients (Britton and Edgar, 1998). From these experiments, the authors proposed that quiescent neuroblasts were induced to re-enter the cell cycle by a mitogenic factor emanating from the fed fat bodies. The present data extend these pioneer findings and suggest the possibility that the factor sent by the fed fat bodies is the secretion factor that triggers Dilp release from the IPCs, allowing neuroblasts to continue their growth and proliferation program through paracrine Dilp-dependent activation (Géminard, 2009).

In conclusion, this work combines genetic and physiology approaches on a model organism to decipher key physiological regulations and opens the route for a genetic study of the molecular mechanisms controlling insulin secretion in Drosophila (Géminard, 2009).

Nutrition-responsive glia control exit of neural stem cells from quiescence

The systemic regulation of stem cells ensures that they meet the needs of the organism during growth and in response to injury. A key point of regulation is the decision between quiescence and proliferation. During development, Drosophila neural stem cells (neuroblasts) transit through a period of quiescence separating distinct embryonic and postembryonic phases of proliferation. It is known that neuroblasts exit quiescence via a hitherto unknown pathway in response to a nutrition-dependent signal from the fat body. This study has identified a population of glial cells that produce insulin/IGF-like peptides in response to nutrition, and shows that the insulin/IGF receptor pathway is necessary for neuroblasts to exit quiescence. The forced expression of insulin/IGF-like peptides in glia, or activation of PI3K/Akt signaling in neuroblasts, can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblasts from systemic control (Chell, 2010).

A transcriptome analysis comparing VNCs from newly hatched larvae and VNCs from larvae at the end of the first instar suggested that the expression of dILP6 and dILP2 increases in the VNC during neuroblast reactivation. The seven dILPs are expressed in distinct spatiotemporal patterns during development. dILP6 is reported to be expressed in the larval gut and the pupal fat body , whereas dILP2 is known to be expressed in the IPC neurons of the brain (along with dilps 1, 3, and 5). To determine whether dILP6 is also expressed in the CNS, a dilp6-GAL4 line was generated. dilp6-GAL4 drives expression in a subset of the surface glia that wraps the CNS. Strong expression was evident by mid first instar and was maintained throughout neuroblast reactivation. The expression of dILP2 was assayed by immunohistochemistry; it too was expressed in the same surface glial population. The glial cells labeled by dilp6-GAL4 are located above the neuroblasts and underneath the surrounding basement membrane. They are stellate in appearance, with several processes radiating from the central cell body. Thus, dILPs, expressed by glial cells, are ideally positioned to activate the dInR pathway in neuroblasts during reactivation (Chell, 2010).

Drosophila neuroblasts in the central brain and thoracic ventral nerve cord (tVNC) are quiescent for 24 hours between their embryonic and larval phases of proliferation. Quiescent neuroblasts are easily identifiable and are amenable to genetic manipulation, making them a potentially powerful model with which to study the transition between quiescence and proliferation. However, the mechanisms regulating the exit from quiescence, either intrinsic or extrinsic, are not well established. Genetic studies found that Drosophila FGF, in concert with Drosophila Perlecan, promotes the neuroblast transition from quiescence to proliferation, but this effect is indirect (Barrett, 2008). Exit from quiescence is physiologically coupled to larval growth and development via a nutritional stimulus (Britton, 1998). The Drosophila fat body performs many of the storage and endocrine functions of the vertebrate liver and acts as a sensor, coupling nutritional state to organismal growth. In response to dietary amino acids, the fat body secretes a mitogen that acts on the CNS to bring about neuroblast proliferation (Britton, 1998). This fat body-derived mitogen (FBDM) initiates cell growth in quiescent neuroblasts and promotes (or at least permits) cell-cycle re-entry (Britton, 1998). Yet the identity of the FBDM, the cell type upon which it acts, and the downstream pathway activated in neuroblasts have remained unknown (Chell, 2010).

Neuroblast entry into quiescence is governed intrinsically by the same transcription factor cascade that controls neuroblast temporal identity. This study has identified a population of surface glial cells that respond to the nutrition-dependent stimulus by expressing dILPs, and showns that the dInR/PI3K pathway is required by neuroblasts to exit quiescence in response to nutrition. Forced expression of dILPs in glia or activation of PI3K/Akt signaling in neuroblasts can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblast reactivation from systemic nutritional control (Chell, 2010).

Cell growth and division are not strictly coupled in neuroblasts. In Drosophila Perlecan (dPerlecan) loss-of-function mutants, the majority of neuroblasts appear to increase in size but then remain G1 arrested. This suggested that a dedicated mitogen might exist to promote cell-cycle progression. Drosophila Activin-like peptides (ALPs; Zhu, 2008) are required for normal levels of neuroblast division in the larval brain and appear to be one such dedicated mitogen (Chell, 2010).

Perlecan is expressed by glia and forms part of the basement membrane that enwraps the CNS. Perlecan was proposed to modulate Drosophila FGF [Branchless (Bnl)], allowing it to act as a mitogen for neuroblasts. However, it now appears that the action of Bnl is indirect via a still to be identified cell type (Barrett, 2008). One possibility is that Bnl acts on glia to modulate the expression of other proteins, such as dILPs or ALPs, which then in turn act on neuroblasts directly. This study shows that expression of dILPs by glia leads to neuroblast reactivation in the absence of dietary protein; however, the number of mitoses falls short of that seen under normal dietary conditions. This could be explained by the absence of another nutritionally dependent mitogen. It will be of interest to see whether the glial expression of ALPs, like that of dILPs, relies on dietary protein (Chell, 2010).

In the larval CNS, neuroblasts and their progeny are completely surrounded by glial cell processes. If the interaction between neuroblasts and surrounding glia is disrupted by expression of a dominant-negative form of DE-cadherin, the mitotic activity of neuroblasts is severely reduced (Dumstrei, 2003). In the mammalian brain, glial cells are involved in a wide variety of processes, including axon guidance, synapse formation, and neuronal specification. Glial cells, with the extracellular matrix and vasculature, also make up the adult neural stem cell niche. Astrocytes have been shown to promote neural stem cell proliferation in culture and can express proproliferative factors such as FGF-2 and IGF-I. Thus, astrocytes are thought to be a key component of the niches that dynamically regulate neural stem cell proliferation in the adult brain (Chell, 2010).

This study has shown that Drosophila surface glia can transduce systemic signals and, by expressing dILP2 and dILP6, control neuroblast exit from quiescence. Glial cells also express dPerlecan and ana and are the source of the Activin-like peptides that have a direct mitogenic effect on neuroblasts. Thus, much like mammalian glial cells, Drosophila glial cells perform a number of the functions that define a niche and control the proliferation of neural stem cells (Chell, 2010).

Recent results suggest a role for IGF-1 in the control of neural stem cell division (Mairet-Coello et al., 2009). IGF-1 injection into rat embryonic brain results in a 28% increase in DNA content postnatally as a consequence of increased DNA synthesis and entry into S phase. Conversely, DNA synthesis and entry into S phase are decreased when the PI3K/Akt pathway is blocked. Furthermore, the loss of PTEN, the tumor suppressor and PI3K antagonist, enhances the exit from G0 of neural stem cells cultured from mouse embryonic cortex. It was suggested that a concomitant increase in cell size may push the cells to enter G1 (Chell, 2010).

This study shows, in vivo, that glial expression of insulin-like peptides activates the dInR/PI3K/Akt pathway in Drosophila neural stem cells and is responsible for their exit from quiescence. This pathway promotes cell growth and the transition from G0 to G1 and is also sufficient to promote G1-S and mitosis. Given that IGF-1 and the PI3K/Akt pathway can promote cell-cycle progression in vertebrate neural stem cells, this same pathway may regulate vertebrate neural stem cell reactivation in the same way as has been shown in this study for Drosophila (Chell, 2010).

The identity of the proposed FBDM, secreted by the fat body in response to dietary protein, remains unknown. However, explant CNS culture experiments demonstrated that the FBDM can act directly on the CNS to bring about neuroblast reactivation (Britton, 1998). This study has identified the surface glia as a key relay in the nutritional control of neuroblast proliferation. If the receptor protein(s) that controls glial dILP expression/secretion can be identified, then, by extension, it might be possible to identify the FBDM and approach a comprehensive understanding of how neural stem cell proliferation is coupled to nutrition and organism-wide growth (Chell, 2010).

Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila

Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. This study provides in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. Nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. These findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour (Sousa-Nunes, 2011).

In fed larvae, Drosophila neuroblasts exit quiescence from the late first instar (L1) stage onwards. This reactivation involves cell enlargement and entry into S phase, monitored in this study using the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). Reactivated neuroblast lineages (neuroblasts and their progeny) reproducibly incorporated EdU in a characteristic spatiotemporal sequence: central brain --> thoracic --> abdominal neuromeres. Mushroom-body neuroblasts and one ventrolateral neuroblast, however, are known not to undergo quiescence and to continue dividing for several days in the absence of dietary amino acids. This indicates that dietary amino acids are more than mere 'fuel', providing a specific signal that reactivates neuroblasts. However, explanted central nervous systems (CNSs) incubated with amino acids do not undergo neuroblast reactivation unless co-cultured with fat bodies from larvae raised on a diet containing amino acids. Therefore the in vivo requirement for a fat-body-derived signal (FDS) in neuroblast reactivation was tested by blocking vesicular trafficking and thus signalling from this organ using a dominant-negative Shibire dynamin (SHI^DN). This strongly reduced neuroblast EdU incorporation, indicating that exit from quiescence in vivo requires an FDS. One candidate tested was Ilp6, known to be expressed by the fat body, but neither fat-body-specific overexpression nor RNA interference of this gene significantly affected neuroblast reactivation. Fat-body cells are known to sense amino acids via the cationic amino-acid transporter Slimfast (SLIF), which activates the TOR signalling pathway, in turn leading to the production of a systemic growth signal. Fat-body-specific overexpression of the TOR activator Ras homologue was shown to be enriched in brain (RHEB), or of an activated form of the p110 PI3K catalytic subunit, or of the p60 adaptor subunit, had no significant effect on neuroblast reactivation in fed animals or in larvae raised on a nutrient-restricted diet lacking amino acids. In contrast, global inactivation of Tor, fat-body-specific Slif knockdown or fat-body-specific expression of the TOR inhibitors Tuberous sclerosis complex 1 and 2 (Tsc1/2) all strongly reduced neuroblasts from exiting quiescence. Together, these results show that a SLIF/TOR-dependent FDS is required for neuroblasts to exit quiescence and that this may be equivalent to the FDS known to regulate larval growth (Sousa-Nunes, 2011).

Next, the signalling pathways essential within neuroblasts for their reactivation were investigated. Nutrient-dependent growth is regulated in many species by the interconnected TOR and PI3K pathways. In fed larvae, it was found that neuroblast inactivation of TOR signalling (by overexpression of TSC1/2), or PI3K signalling (by overexpression of p60, the Phosphatase and tensin homologue PTEN, the Forkhead box subgroup O transcription factor FOXO or dominant-negative p110), all inhibited reactivation. Conversely, stimulation of neuroblast TOR signalling (by overexpression of RHEB) or PI3K signalling [by overexpression of activated p110 or Phosphoinositide-dependent kinase 1 (PDK1)] triggered precocious exit from quiescence. RHEB overexpression had a particularly early effect, preventing some neuroblasts from undergoing quiescence even in newly hatched larvae. Hence, TOR/PI3K signalling in neuroblasts is required to trigger their timely exit from quiescence. Importantly, neuroblast overexpression of RHEB or activated p110 in nutrient-restricted larvae, which lack FDS activity, was sufficient to bypass the block to neuroblast reactivation. Notably, both genetic manipulations were even sufficient to reactivate neuroblasts in explanted CNSs, cultured without fat body or any other tissue. Together with the previous results this indicates that neuroblast TOR/PI3K signalling lies downstream of the amino-acid-dependent FDS during exit from quiescence (Sousa-Nunes, 2011).

To identify the mechanism bridging the FDS with neuroblast TOR/PI3K signalling, the role of the Insulin-like receptor (InR) in neuroblasts was tested. Importantly, a dominant-negative InR inhibited neuroblast reactivation, whereas an activated form stimulated premature exit from quiescence. Furthermore, InR activation was sufficient to bypass the nutrient restriction block to neuroblast reactivation. This indicates that at least one of the potential InR ligands, the seven ILPs, may be the neuroblast reactivating signal(s). By testing various combinations of targeted Ilp null alleles and genomic Ilp deficiencies, it was found that neuroblast reactivation was moderately delayed in larvae deficient for both Ilp2 and Ilp3 (Df(3L)Ilp2-3) or lacking Ilp6 activity. Stronger delays, as severe as those observed in InR³¹ mutants, were observed in larvae simultaneously lacking the activities of Ilp2, 3 and 5 [Df(3L)Ilp2-3, Ilp5] or Ilp1-5 [Df(3L)Ilp1-5]. Despite the developmental delay in Df(3L)Ilp1-5 homozygotes, neuroblast reactivation eventually begins in the normal spatial pattern -- albeit heterochronically -- in larvae with L3 morphology. Together, the genetic analysis shows that Ilp2, 3, 5 and 6 regulate the timing but not the spatial pattern of neuroblast exit from quiescence. However, as removal of some ILPs can induce compensatory regulation of others, the relative importance of each cannot be assessed from loss-of-function studies alone (Sousa-Nunes, 2011).

Brain median neurosecretory cells (mNSCs) are an important source of ILPs, secreted into the haemolymph in an FDS-dependent manner to regulate larval growth. They express Ilp1, 2, 3 and 5, although not all during the same development stages. However, this study found that none of the seven ILPs could reactivate neuroblasts during nutrient restriction when overexpressed in mNSCs. Similarly, increasing mNSC secretion using the NaChBac sodium channel or altering mNSC size using PI3K inhibitors/activators, which in turn alters body growth, did not significantly affect neuroblast reactivation under fed conditions. Surprisingly, therefore, mNSCs are not the relevant ILP source for neuroblast reactivation. Nonetheless, Ilp3 and Ilp6 messenger RNAs were detected in the CNS cortex, at the early L2 stage, in a domain distinct from the Ilp2⁺ mNSCs. Two different Ilp3-lacZ transgenes indicate that Ilp3 is expressed in some glia (Repo⁺ cells) and neurons (Elav⁺ cells). An Ilp6-GAL4 insertion indicates that Ilp6 is also expressed in glia, including the cortex glia surrounding neuroblasts and the glia of the blood-brain barrier (BBB) (Sousa-Nunes, 2011).

Next the ability of each of the seven ILPs to reactivate neuroblasts when overexpressed in glia or in neurons was assessed. Pan-glial or pan-neuronal overexpression of ILP4, 5 or 6 led to precocious reactivation under fed conditions. Each of these manipulations also bypassed the nutrient restriction block to neuroblast reactivation, as did overexpression of ILP2 in glia or in neurons, or ILP3 in neurons. In all of these ILP overexpressions, and even when ILP6 was expressed in the posterior Ultrabithorax domain, the temporal rather than the spatial pattern of reactivation was affected. Importantly, experiments blocking cell signalling with SHI^DN indicate that glia rather than neurons are critical for neuroblast reactivation. Interestingly, glial-specific overexpression of ILP3-6 did not significantly alter larval mass. Thus, in contrast to mNSC-derived ILPs, glial-derived ILPs promote CNS growth without affecting body growth (Sousa-Nunes, 2011).

Focusing on ILP6, CNS explant cultures were used to demonstrate directly that glial overexpression was sufficient to substitute for the FDS during neuroblast exit from quiescence. In vivo, ILP6 was sufficient to induce reactivation during nutrient restriction when overexpressed via its own promoter or specifically in cortex glia but not in the subperineurial BBB glia, nor in many other CNS cells that were tested. Hence, cortex glia possess the appropriate processing machinery and/or location to deliver reactivating ILP6 to neuroblasts. Ilp6 mRNA is known to be upregulated rather than downregulated in the larval fat body during starvation and, accordingly, Ilp6-GAL4 activity is increased in this tissue after nutrient restriction. Conversely, it was found that Ilp6-GAL4 is strongly downregulated in CNS glia during nutrient restriction. Thus, dietary nutrients stimulate glia to express Ilp6 at the transcriptional level. Consistent with this, an important transducer of nutrient signals, the TOR/PI3K network, is necessary and sufficient in glia (but not in neurons) for neuroblast reactivation. Together, the genetic and expression analyses indicate that nutritionally regulated glia relay the FDS to quiescent neuroblasts via ILPs (Sousa-Nunes, 2011).

This study used an integrative physiology approach to identify the relay mechanism regulating a nutritional checkpoint in neural progenitors. A central feature of the fat-body --> glia --> neuroblasts relay model is that glial insulin signalling bridges the amino-acid/TOR-dependent fat-body-derived signal (FDS) with InR/PI3K/TOR signalling in neuroblasts. The importance of glial ILP signalling during neuroblast reactivation is also underscored by an independent study, published while this work was under revision (Chell, 2010). As TOR signalling is also required in neuroblasts and glia, direct amino-acid sensing by these cell types may also impinge upon the linear tissue relay. This would then constitute a feed-forward persistence detector, ensuring that neuroblasts exit quiescence only if high amino-acid levels are sustained rather than transient. This study also showed that the CNS 'compartment' in which glial ILPs promote growth is functionally isolated, perhaps by the BBB, from the systemic compartment where mNSC ILPs regulate the growth of other tissues. The existence of two functionally separate ILP pools may explain why bovine insulin cannot reactivate neuroblasts in CNS organ culture, despite being able to activate Drosophila InR in vitro. Given that insulin/PI3K/TOR signalling components are highly conserved between insects and vertebrates, it will be important to address whether mammalian adipose or hepatic tissues signal to glia and whether or not this involves an insulin/IGF relay to CNS progenitors. In this regard, it is intriguing that brain-specific overexpression of IGF1 can stimulate cell-cycle re-entry of mammalian cortical neural progenitors, indicating utilization of at least part of the mechanism identified by this study in Drosophila (Sousa-Nunes, 2011).

Regulation of insulin-producing cells in the adult Drosophila brain via the tachykinin peptide receptor DTKR

Drosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophila the regulation of DILP production and release in adult fruit flies is poorly understood. This study investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR (Tachykinin-like receptor at 99D) and NKD (Naked cuticle), in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, DTK-immunoreactive axon terminations were shown close to the presumed dendrites of the IPCs, and DTKR immunolabeling was demonstrated in these cells. Second, targeted RNA interference was used to knock down expression of the DTK receptor, DTKR, in IPCs and the effects the on Dilp transcript levels were monitored in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. Trehalose and lipid levels were measured as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, IPCs were targeted with RNAi or ectopic expression of the other DTK receptor, NKD, but no effect was found on survival at starvation. These results suggest that DTK signaling, via DTKR, regulates the brain IPCs (Birse, 2011).

This study investigated the effects of DTK signaling to IPCs in the Drosophila brain by monitoring Dilp transcript levels and survival at starvation, as well as trehalose and lipid levels in fed and starved flies. The brain IPCs are presumed to release DILP2, DILP3 and DILP5, orthologs of mammalian insulins. Since these insulin-like peptides have been shown to play a significant role in lifespan, in nutritional stress responses and in metabolic regulation, the DTK signaling onto these cells may be of significance for the regulation of vital physiological functions. However, the IPCs are also known to regulate feeding behavior, locomotor activity, sleep-wakefulness and ethanol sensitivity, and they may do so independent of the insulin signaling pathway. Thus, activation or inhibition of signaling in the IPCs may result in actions that are non-insulin mediated, either via other messengers released by the same cells or indirectly by the action of DILPs on specific neurons (see Root, 2011; Birse, 2011 and references therein).

The most direct evidence that DTK signaling affects the brain IPCs is that knock down of the receptor DTKR in IPCs leads to altered expression levels of Dilp2 and Dilp3 transcripts in fed flies and that Dilp3 RNA drops drastically in knockdown flies after 24 h starvation whereas Dilp2 levels increase. Interestingly, the Dilp5 transcript is not affected by DTKR-RNAi, but is the only one that seems affected by starvation both in controls and knockdown flies. It is known that restricted diet conditions in adult (control) flies alter the transcript level of Dilp5, but not Dilp2 and Dilp3 (Broughton, 2010), corroborating the current findings. These data are the first to quantify Dilp transcripts at complete starvation in adults, but an earlier report monitored Dilp transcripts by in situ hybridization of fed and starved third instar larvae. That study noted decreased Dilp3 and Dilp5 transcripts but unaffected levels of Dilp2. This difference could be either dependent on a difference in larval and adult functions of the IPCs or could be due to the difference in techniques used for monitoring transcript levels. Certainly the feeding behavior and metabolism differs greatly between larvae and adults in Drosophila. It should be noted here that insulin expression/signaling also involves autocrine or paracrine feedbacks so that DILP3 may act in stimulatory regulation of expression of DILP2 and DILP5 in the IPCs (Broughton, 2008; Grönke, 2010) whereas DILP6 released from the fat body may negatively regulate the IPCs (Birse, 2011).

Unfortunately there are no reports that unequivocally demonstrate the release of DILPs into the circulation of Drosophila in a quantitative fashion. Thus, indirect measurements, such as Dilp transcript or DILP peptide-immunofluorescence levels in cell bodies of IPCs, have to be matched against the physiological effects seen after manipulations of IPCs. A few indicators of altered insulin signaling have been used here: levels of carbohydrate and lipids as well as effects on lifespan at starvation. As one of the functions of DILPs is to stimulate uptake of circulating blood sugar and thereby decreasing trehalose levels in the circulation, this study monitored whole-body trehalose levels in fed and starved flies after DTKR knockdown in IPCs. Knockdown of DTKR in IPCs had no effect on trehalose in fed flies, but induced an acute drop in trehalose after 5 h starvation, compared with controls, suggesting an increase in insulin signaling. Analysis of Dilp mutants or knockdown indicated that trehalose levels are regulated by DILP2 one of the peptides whose transcripts was indeed altered by DTKR knockdown at starvation. In the current experiments lipid levels were not affected by manipulations of DTKR on IPCs in fed or starved flies. Lipid metabolism may be regulated by multiple DILPs, including Dilp6 (Grönke, 2010), or by compensatory AKH signaling, and this may explain the lack of effect after manipulating only IPC activity (Birse, 2011).

Diminishment of DTKR expression on IPCs results in flies that display a shortened lifespan at starvation. This would also indicate increased insulin signaling, as deletion of IPCs or knocking down combinations of DILPs produce the opposite phenotype, and overexpression of Dilp2 in IPCs resulted in decreased resistance to starvation (Enell, 2010). A similar reduction of lifespan at starvation was seen after knock down of the inhibitory GABA_B receptor on IPCs (Enell, 2010). It is not clear which of the DILPs regulates the lifespan at dietary restriction or starvation, but DILP2 has been suggested as a candidate (Birse, 2011).

The second known DTK receptor, designated NKD, does not seem to play a role in the regulation of IPCs. NKD can be activated only by one of the DTKs, the N-terminally extended DTK-6, which has not been detected in the Drosophila brain, in contrast to the DTK-1-5 known to activate DTKR (Birse, 2011).

It can be mentioned that an earlier report shows that DTKR is expressed in renal (Malpighian) tubules where it regulates DILP5 signaling. It is proposed that this regulation is mediated by DTKs released hormonally from endocrine cells of the midgut. DTKs circulating locally act on DTKR expressed in principal cells of the renal tubules, resulting in a local activation of DILP5 signaling. The DTKR-regulated DILP5 signaling in renal tubules does not affect trehalose levels in fed or starved flies, but seems to be part of the defense against oxidative stress. Thus, this DTK-controlled DILP5 signaling in the tubules is probably independent of the paracrine DTK-mediated IPC regulation in the brain, but further studies of gut-derived DTK action are required to confirm this (Birse, 2011).

In summary these results indicate that in wild-type flies the activated DTKR inhibits insulin signaling in the brain IPCs, and knockdown of the receptor therefore leads to increased insulin signaling. This can be seen in the decreased lifespan and a considerable decrease in trehalose levels during short-term starvation compared with controls, similar to what is expected at increased DILP signaling. The most direct evidence that DTKR is involved in IPC regulation is the effect on Dilp2 and Dilp3 transcript levels seen after receptor knockdown in the IPCs in fed and starved flies. However, it is important for the future to develop a sensitive assay for quantifying hemolymph levels of individual DILPs to monitor how their release is affected by the DTKR signaling to IPCs (Birse, 2011).

Delivery of circulating lipoproteins to specific neurons in the brain regulates systemic insulin signaling

The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. This study demonstrates a function for the Drosophila lipoprotein LTP (FlyBase term: Apolipoprotein lipid transfer particle) in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in blood brain barrier (BBB) glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content (Brankatschk, 2014).

Nutrient sensing by the central nervous system is emerging as an important regulator of systemic metabolism in both vertebrates and invertebrates. Little is known about how nutrition-dependent signals pass the blood brain barrier to convey this information. Like the vertebrate BBB, the BBB of Drosophila forms a tight barrier to passive transport, and is formed by highly conserved molecular components. Its simple structure and genetic accessibility make it an ideal model to study how nutritional signals are communicated to the CNS. Insulin and Insulin-like growth factors are conserved systemic signals that regulate growth and metabolism in response to nutrition. Although fruit flies do not have a single pancreas-like organ, they do produce eight distinct Drosophila Insulin/IGF-like peptides (Dilps) that are expressed in different tissues. A set of three Dilps (dILP2,3,5), released into circulation by Dilp-producing cells (IPCs) in the brain, have particularly important functions in regulating nutrition-dependent growth and sugar metabolism; ablation of IPCs in the CNS causes diabetes-like phenotypes, slows growth and development, and produces small, long-lived adult flies. Systemic Insulin/IGF signaling (IIS) increases in response to dietary sugars, proteins and lipids. Sugars act on IPCs directly to promote Dilp release, but other nutrients are sensed indirectly through signals from the fat body (an organ analogous to vertebrate liver/adipose tissue) (Brankatschk, 2014).

The Drosophila fat body produces two major types of lipoprotein particles: Lipophorin (LPP; Retinoid- and fatty acid-binding glycoprotein), the major hemolymph lipid carrier, and Lipid Transfer Particle (LTP). LTP transfers lipids from the intestine to LPP. These lipids include fatty acids from food, as well as from endogenous synthesis in the intestine. LTP also unloads LPP lipids to other cells (Van Heusden, 1989; Canavoso, 204; Parra-Peralbo, 2011). LPP crosses the BBB and accumulates throughout the brain. It is required for nutrition-dependent exit of neural stem cells from quiescence (Brankatschk, 2010). This study investigated possible functions of LTP in the brain (Brankatschk, 2014).

This work demonstrates a key requirement for lipoproteins in conveying nutritional information across the BBB to specific neurons in the brain. As particles that carry both endogenously synthesized and diet-derived lipids, lipoproteins are well-positioned to perform this function. The data suggest that transport of LTP across the BBB to Dilp2-recruiting neurons (DRNs) influences communication between DRNs and the Dilp-producing IPCs, increasing the release of Dilp2 into circulation. Since the IPCs also deliver Dilp2 to the DRNs, this indicates that these two neuronal populations may communicate bidirectionally. How might LTP affect the function of DRNs? One possibility is that it acts to deliver a signaling lipid to the DRNs. It could do so either directly, or indirectly by promoting lipid transfer from LPP, which is present throughout the brain. LTP enrichment on specific neurons may increase lipid transfer to these cells (Brankatschk, 2014).

This work highlights a key function for BBB cells in transmitting nutritional information to neurons within the brain. Feeding with yeast food increases free calcium in BBB glia, which then increases transport of LTP to DRNs. How might BBB cells detect the difference between yeast and plant food? The data suggest differences in the lipid composition of yeast and plant-derived foods are responsible. Previous work has shown that the lipids in these foods differ in their fatty acid composition. Yeast food has shorter and more saturated fatty acids than plant food (24). How could these nutritional lipids affect the activity of BBB glia? Interestingly, differences in food fatty acid composition are directly reflected in the fatty acids present in membrane lipids of all larval tissues including the brain. Thus, it is possible that the bulk membrane properties of BBB glia are different on these two diets. Membrane lipid composition is known to affect a variety of signaling events. Alternatively, yeast food may influence the specific fatty acids present in signaling lipids that activate BBB glia (Brankatschk, 2014).

This study demonstrates an unexpected functional specialization of the BBB glial network, which permits specific and regulated LTP transport to particular neurons. How this specificity arises is an important question for the future. It is noted that a subset of glial cells within the brain also accumulates LTP derived from the fat body. Could these represent specific transport routes from the BBB (Brankatschk, 2014)?

An alternative possibility is that transport depends on neuronal activity. Mammalian LRP1 promotes localized transfer of IGF in response to neuronal activity. Could LTP delivery by LRP1 and LRP2 (Megalin) in the Drosophila brain depend on similar mechanisms? The remarkable specificity of LTP trafficking in the Drosophila CNS provides a novel framework for understanding information flow between the circulation and the brain (Brankatschk, 2014).

To what extent might this be relevant to vertebrate systems? While it is clear that the vertebrate brain (unlike that of Drosophila) does not depend on lipoproteins to supply it with bulk sterols, this does not rule out possible functions for these particles in nutrient sensing. The vertebrate cerebrospinal fluid is rich in many types of HDL particles, including those containing ApoA-1, which is not expressed in the brain - this suggests that at least some lipoprotein particles in the brain may derive from the circulation. Consistent with this idea, ApoA-I can target albumin-containing nanoparticles across the BBB in rodents. Recent work suggests that lipoproteins may be the source of specific Long Chain Fatty Acids that signal to the hypothalamus to regulate glucose homeostasis, since neuronal lipoprotein lipase is required for this process. Thus, it would be interesting to investigate whether circulating mammalian lipoproteins might reach a subset of neurons in the hypothalamus (Brankatschk, 2014).

It has been known for some time that increasing the amount of yeast in the diet of lab grown Drosophila melanogaster increases the rate of development and adult fertility, but reduces lifespan. This study shows that flies have evolved specific mechanisms to increase systemic IIS in response to yeast, independently of the number of calories in the diet or its proportions of sugars proteins and fats. What pressures could have driven the evolution of such mechanisms? In the wild, Drosophila melanogaster feed on rotting plant material and their diets comprise both fungal and plant components. Drosophila disperse yeasts and transfer them to breeding sites during oviposition improving the nutritional resources available to developing larvae. Yeast that are able to induce more rapid development of the agents that disperse them may propagate more efficiently. On the other hand, it has been noted that Drosophila species that feed on ephemeral nutrient sources like yeasts or flowers have more rapid rates of development than other species. It may be that, even within a single species, the ability to adjust developmental rate to the presence of a short-lived resource is advantageous. Humans subsist on diets of both plant and animal materials that during most of evolution have differed in their availability. It would be interesting to investigate whether Insulin/IGF signaling in humans might respond to qualitative differences in the lipid composition of these nutritional components (Brankatschk, 2014).

Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. This study shows that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2 - FlyBase name: Ecdysone-inducible gene L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together these results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions (Sarraf-Zadeh, 2013).

In higher organisms, the duration of the juvenile stage needs to be variable to ensure the development of a healthy and fertile adult. Environmental stresses, such as adverse nutritional conditions, can delay development until a critical weight is reached. Additional checkpoints ensure that increased growth rates, induced by ideal nutritional conditions, do not lead to a premature passage to the adult stage. In Drosophila, the juvenile growth stage is terminated by pupae formation at the end of the third larval instar. Larval/pupal transition is induced by a pulse of the steroid hormone ecdysone produced by the PG (Sarraf-Zadeh, 2013).

Genetic manipulations of the Drosophila PG revealed the requirements of the IIS, Target of Rapamycin (TOR) and PTTH pathways to control ecdysone production . Recently, IIS dependent growth of the PG has been identified as an additional factor controlling ecdysone production. Overexpression of PI3K, a positive regulator of IIS, leads to premature, increased ecdysone production resulting in a shortened L3 stage and early pupariation. By contrast, overexpression of negative regulators of IIS in the PG delays pupariation caused by lowered and delayed ecdysone production. Reduction of whole organism IIS activity does not change critical weight but delays its attainment. In contrast, ablation of PTTH neurons induces a severe shift in critical weight, suggesting that these neurons play an important role in setting this parameter. When larvae reach the critical weight, PTTH is released on the PG and induces transcription of genes involved in ecdysone production. However, PTTH expression is not modified upon nutritional restriction, indicating that PTTH signaling does not mediate starvation induced developmental delay. Signaling via TOR, the downstream kinase of IIS, links nutritional information to ecdysone production, since starvation induced developmental delay can partially be rescued by upregulating TOR activity in the PG. This suggests that downregulating TOR signaling upon starvation desensitizes the PG for PTTH signals, resulting in delayed ecdysone production. The present study shows that increased IIS activity in the PG due to Imp L2 LOF rescues the delay caused by malnutrition to a large extent, indicating that low IIS also renders the PG irresponsive to the PTTH signal. Whether the effects of low IIS in the PG are mediated by TOR or whether the two pathways act independently remains to be elucidated (Sarraf-Zadeh, 2013).

Evidence is presented for a number of Imp L2 expressing neurons to act as possible regulators of IIS activity in the PG. High Imp L2 levels in the hemolymph can be excluded as possible inhibitors of IIS signaling in the PG, since increasing hemolymph levels of Imp L2 failed to reduce size and IIS activity of PG cells, but resulted in a strong size decrease of the whole organism. On the other hand, increasing Imp L2 levels in Imp L2 positive neurons targeting the PG causes a massive decrease in PG size and lowers IIS activity within PG cells. These results support the idea that the PG does not receive information about the nutritional state of the organism through the hemolymph but rather from Imp L2 expressing neurons. Thus, this work reveals a novel local function of the negative growth regulator Imp L2 in controlling IIS activity and ecdysone production in the PG. This finding reveals a novel mechanism for the spatial regulation of IIS: through locally restricted effects of Imp L2, diverse tissues can be effectively subjected to different levels of IIS (Sarraf-Zadeh, 2013).

Interestingly, the ability of IIS to coordinate growth with development seems to be conserved throughout evolution. In humans, the onset of puberty is linked to the nutritional state, leading to early puberty in well fed western societies. In contrast, juvenile females suffering from type I diabetes mellitus display a notable delay in menarche, indicating that decreased IIS also delays maturation in humans. Moreover, in Caenorhabditis elegans, malnutrition during the first larval stage leads to developmental arrest by inducing dauer formation, which is a larval stage best adapted for survival under adverse environmental conditions. Mutations reducing IIS pathway activity lead to dauer formation independent of the nutritional state. Hence, different phyla developed similar strategies to cope with adverse nutritional conditions during the juvenile state. When IIS activity is below a certain threshold, development is attenuated until sufficient nutrients are available, to ensure the formation of healthy and fertile adults. In Drosophila larval malnutrition leads to delayed pupariation, due to decreased IIS activity in the PG which in turn delays the production of the steroid hormone ecdysone (Sarraf-Zadeh, 2013).

Steroid hormones also play an important role in human development. In cases of human hypogonadism, puberty is prolonged, which can lead to abnormally tall adults if not treated with steroid substitutes. Referring the current data to the human system, the putative Imp L2 homolog IGFBP 7 (also known as IGFBP rP1) also displays a very diverse protein expression pattern, indicating a specialized function in different organs. Amongst other tissues, IGFBP 7 is expressed in different regions of the human brain, leading to the speculation that it might act as a local regulator of steroid production as well (Sarraf-Zadeh, 2013).

In summary, the data provides novel insights into the coupling of developmental cues to nutritional state. Since IIS and steroid hormones play evolutionarily conserved roles in regulating growth and development, the findings on the local function of the insulin binding protein Imp L2 in controlling ecdysone production might be of general interest (Sarraf-Zadeh, 2013).

Direct sensing of systemic and nutritional signals by haematopoietic progenitors in Drosophila

The Drosophila lymph gland is a haematopoietic organ in which progenitor cells, which are most akin to the common myeloid progenitor in mammals, proliferate and differentiate into three types of mature cell -- plasmatocytes, crystal cells and lamellocytes -- the functions of which are reminiscent of mammalian myeloid cells. During the first and early second instars of larval development, the lymph gland contains only progenitors, whereas in the third instar, a medial region of the primary lobe of the lymph gland called the medullary zone contains these progenitors, and maturing blood cells are found juxtaposed in a peripheral region designated the cortical zone. A third group of cells referred to as the posterior signalling centre functions as a haematopoietic niche. Similarly to mammalian myeloid cells, Drosophila blood cells respond to multiple stresses including hypoxia, infection and oxidative stress. However, how systemic signals are sensed by myeloid progenitors to regulate cell-fate determination has not been well described. This study shows that the haematopoietic progenitors of Drosophila are direct targets of systemic (insulin) and nutritional (essential amino acid) signals, and that these systemic signals maintain the progenitors by promoting Wingless (WNT in mammals) signalling. It is expected that this study will promote investigation of such possible direct signal sensing mechanisms by mammalian myeloid progenitors (Shim, 2012).

It is known that metabolic dysfunction in mammals causes abnormal inflammatory responses in the blood system. However, how metabolic stresses impinge on haematopoiesis is still unclear. This study found that starvation of Drosophila larvae leads to blood cell phenotypes. The most striking effect is acceleration of blood cell differentiation both in time and number of cells affected in the lymph gland. Following 24h of starvation, cells occupying the medullary zone begin to express differentiation markers such as Peroxidasin (Hml) normally restricted to the cortical zone. Corresponding to this increase, a substantial reduction of Domeless (Dome) marking the progenitor population is also evident. The protein Eater, normally expressed at very low levels in the progenitors and at high levels in differentiated cells, is expressed at high levels in all cells on starvation (Shim, 2012).

The starvation experiments were carried out on either PBS-soaked Whatman paper or a 1% agar plate. Aseptic conditions to control against indirect effects due to bacterial infection were also used. In all controlled experimental conditions, starvation reduced the progenitor population and caused an increase in the number of differentiating cells, without an obvious alteration in the size of the haematopoietic organ, or the apoptotic profile of its cells (Shim, 2012).

Similarly to metabolically induced inflammation in mammals, starvation in Drosophila larvae activates NFkappaB-like transcription factors, assessed by the expression of the reporter D4–LacZ and antimicrobial peptides in circulating haemocytes and within the lymph gland. Starvation also causes an increase in the number of circulating blood cells arising from the embryonic head mesoderm, infiltration of Pxn⁺ plasmatocytes into the fat body, the Drosophila equivalent of the mammalian liver and adipose tissue, and differentiation of lamellocytes, another hallmark of inflammatory response, in both the lymph gland and in the circulating blood cell population. Finally, starvation induces the rupture of crystal cells, a process known to release coagulation and melanization enzymes. This rupture depends on JNK signalling. Thus, starvation alters the homeostatic balance between progenitors and differentiating blood cells through extensive progenitor differentiation, and also activates mature blood cells in a manner that is reminiscent of mammalian inflammatory response (Shim, 2012).

In Drosophila, the systemic level of glucose is regulated by insulin-like peptides (Dilps) that are produced and secreted by neuroendocrine cells in the brain, much like insulin production by pancreatic β-cells of mammals. As in mammals, insulin signalling in Drosophila plays a conserved role in regulating metabolism and growth, and the levels of nutrients, such as amino acids, regulate secretion of Dilps. This study finds the effects of starvation on Drosophila blood particularly interesting given the connection between myeloid cell function and insulin signalling in human metabolic diseases. The mechanisms are delineated by which a systemic signal, namely insulin signalling, controls maintenance and differentiation of progenitors in the haematopoietic organ (Shim, 2012).

The insulin-producing cells (IPCs) were specifically ablated by inducing cell death with the expression of the pro-apoptotic genes hid and rpr, and robust differentiation of blood cells was found in the lymph gland similar to that seen on starvation. Although several larval dilp genes, including dilp2, 3 and 5, are produced by IPCs, further analysis showed that deficiency mutants containing a dilp2 lesion or a specific deletion of the dilp2 gene using the null dilp2 mutant allele cause blood cell differentiation. Depletion of any of the other dilp genes, including dilp6 or 7, does not cause this phenotype. No Dilp2 expression was detected in the lymph gland cells, and it is proposed that the ligand source is the IPC neurons in the brain. Consistent with previous findings, it was found that starvation blocks Dilp2 release from the IPCs. Furthermore, forced depolarization of the IPCs by expressing the bacterially derived voltage-gated sodium channel (NaChBac), which will cause an increase in Dilp secretion, suppresses blood cell differentiation under both well-fed and starved conditions. Finally, overexpression of Dilp2 using the neuronal driver elav–Gal4 causes suppression of differentiation of mature blood cells. Taken together, it is concluded that Dilp2 expression from the IPC neurons is essential for progenitor maintenance and loss of Dilp2 release during starvation results in excessive differentiation of blood cells (Shim, 2012).

The loss-of-function, heteroallelic combination InR^E19/InR^GC25 for the Drosophila insulin receptor (InR) is viable and larvae from this genotype also exhibit extensive differentiation of the progenitor population. As Dilp2 is a secreted protein, its target receptor could, in principle, be functional in any tissue that then, in turn, signals to the haematopoietic organ using a secondary pathway. However, disrupting InR function directly in the lymph gland with the use of the lymph-gland-specific driverHHLT-Gal4 was found to cause precocious differentiation of the progenitors from an earlier stage in development than is seen in wild type. As HHLT–Gal4 is also expressed in the heart (dorsal vessel), the possible involvement of cardiogenic cells was examined by disrupting InR using the heart-specific driver Mef2–Gal4; this does not induce abnormal differentiation in the lymph gland, indicating that the HHLT–Gal4-driven phenotype is due to its expression in the haematopoietic system. Within the lymph gland, downregulation of InR in the progenitor cell population (using dome–Gal4) causes their robust differentiation, whereas loss of InR in the already differentiating cells of the cortical zone (using hml–Gal4) or the niche cells of the posterior signalling centre (using antp–Gal4) does not affect the progenitor population. Consistent with these findings, high levels of InR transcript were detected in the progenitors, and mutant clones of InR (InR^E19/InR^E19) within the medullary zone region induce precocious differentiation. Furthermore, downregulation of chico, a downstream effector of insulin receptor signalling, in the progenitor population recapitulates the InR^RNAi phenotype, whereas activation of PI(3)K kinase inhibits differentiation of blood cells. These results establish that the haematopoietic progenitor directly responds to brain IPC-derived Dilp2 by activating InR signalling, which serves to maintain the progenitor cell population within the lymph gland (Shim, 2012).

Next the function of AKT, which acts downstream of InR as a protein kinase, was examined by downregulating its expression in the progenitors, and this too promotes progenitor differentiation, identical to loss of InR and dilp2. Likewise, loss of the TORC1 components dTOR (using the dominant-negative mutant protein dTOR^DN or Raptor (using RNA-mediated interference (RNAi), which together function downstream of AKT, causes loss of progenitors due to their differentiation. Feeding rapamycin, which blocks dTOR function also phenocopies this effect. Consistent with a role for this pathway in progenitor maintenance, overexpression of rheb, which activates dTOR, strikingly inhibits differentiation of blood cells under both normally fed and starved conditions. Interestingly, mammalian haematopoietic stem cells also respond to mTOR signalling. Overall, it is evident that the canonical Dilp–InR and Rheb–dTOR signalling pathways play a critical role in the maintenance of haematopoietic progenitors, and this maintenance role is overridden during metabolic stress caused by starvation (Shim, 2012).

Dilp2 levels rise during early instars and then gradually decrease during the third instar of larval development, indicating a possible mechanism for maintaining InR signalling through the third instar in well-fed larvae. To determine whether InR signalling is modulated during normal development, levels of phospho-AKT (pAKT) were assessed at different developmental stages in the lymph gland. Using this approach, two distinct phenomena were found. First, pAKT expression in progenitor cells is high during the second instar and gradually decreases in these cells during the third instar. Second, pAKT is low, relative to progenitor levels, in differentiating cells at all stages when they are present. These observations indicate that during the course of normal development, InR signalling is modulated in progenitors, thereby differentially promoting maintenance at different stages. It is also apparent that once cells are committed to differentiate, little, if any, InR signalling occurs, consistent with lower levels of InR expression and the lack of a phenotype associated with InR loss-of-function in these cells (Shim, 2012).

In mammals, glucose levels control insulin secretion. This is less clear in Drosophila, but it is well established that amino-acid levels are sensed by the fat body through the mediation of the amino-acid transporter protein Slimfast (Slif) and that the fat body indirectly controls insulin secretion from the brain IPCs. As expected, it was found that slif^Anti expressed in the fat body mimics the starvation phenotype in the lymph gland, probably owing to decreased Dilp2 secretion from the brain. More interestingly, however, knocking down slif expression directly in the lymph gland, but not in the dorsal vessel, and specifically in the progenitor population within the lymph gland, accelerates differentiation of mature cells similar to that seen with starvation. As with insulin signalling, this result shows that the haematopoietic progenitors themselves directly sense amino-acid levels to maintain their stem-like fate. Taken together, these findings indicate a dual control of haematopoietic homeostasis by systemic levels of insulin and amino acids. Amino acids are sensed by the fat body, which then controls insulin secretion from the brain. Insulin is then directly sensed by the blood progenitors. Amino acids are also directly sensed by the blood progenitors to maintain their undifferentiated state (Shim, 2012).

Supplementation of essential amino acids (EAAs) partially restores the progenitor population during an otherwise starved condition, whereas neither sucrose nor non-essential amino acid (NEAA) supplementation rescues the progenitors from differentiation. Loss of slif in the lymph gland prevents progenitor maintenance despite EAA supplementation, further establishing that the progenitors directly sense EAA and use the signal to promote their maintenance (Shim, 2012).

In wild-type lymph glands, the progenitors (expressing Dome; Dome⁺) rarely overlap with the maturing cells (expressing Pxn; Pxn⁺); however, downregulation of InR or expression of dominant-negative TOR (TOR^DN) in the progenitors causes a significant increase in the number of double-positive cells (Dome⁺ and Pxn⁺) that are in transition towards differentiation. An increase in this particular cell type is reminiscent of the phenotype seen on downregulation of the wingless (wg) signalling pathway, which has previously been linked to the process of progenitor maintenance. Wingless (Wg) is dynamically expressed in the lymph gland with higher levels at earlier stages that then decrease during the third instar. Wg is expressed at high levels by progenitors at these stages and is withdrawn from differentiating cells, which is reminiscent of pAKT staining patterns and the expression of InR in the third instar. Downregulation of InR or Slif in the progenitors was found to causes a significant decrease in Wg expression, whereas Rheb overexpression significantly increases Wg levels when compared with that seen in wild type, indicating that Dilp2–dTOR and Slif–dTOR activities positively regulate the expression of Wg within the progenitors. Importantly, overexpression of Wg restores the progenitor population in both starvation conditions and in the presence of reduced InR levels, demonstrating that Wg is likely to be the most direct downstream target of Dilp2–InR signalling, which maintains progenitors within the progenitors. However, the studies do not rule out either direct or indirect involvement of additional pathways downstream of InR–dTOR in this process (Shim, 2012).

A model describing the systemic and nutritional control of myeloid-like progenitors by insulin and amino acids is presented. The results demonstrate that metabolic changes are perceived by blood progenitors and this causes alteration of their cell-fate determination program. A major consequence of reduced InR and amino acid levels is the reduction of Wg expression in the lymph gland, which functions to promote progenitor maintenance. In addition to accelerated differentiation of myeloid progenitors, starvation also causes a response similar to the inflammatory response typically associated with metabolic disorders. These responses indicate that metabolically induced inflammatory responses in mammals have an ancestral origin that arose to balance an organism's ability to withstand an unfavourable environment and the normal development of myeloid cells. Nutrient/insulin signalling has been linked to the homeostatic control of various Drosophila stem cell populations. Given the highly conserved nature of the blood system in flies and mammals, and the known functional role of metabolism and insulin signalling in myeloid cells, it will be important to determine whether the direct metabolic and nutritional regulation mechanisms uncovered in these studies might also be relevant for the mammalian common myeloid progenitors. Such studies will probably yield insights into chronic inflammation and the myeloid cell accumulation seen in patients with type II diabetes, and other metabolic disorders (Shim, 2012).

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

Genetic studies in Drosophila reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches this study tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-β (GSK3β) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. This study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level it was found that both these pathways converge at GSK3β to control Myc protein stability, while genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia (Parisi, 2011).

Previous studies in vertebrates have indicated a critical function for Myc downstream of growth factor signaling including insulin-like growth factor, insulin and TOR pathways. In Drosophila, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways, no clear molecular mechanisms linking these pathways to Myc have been elucidated yet (Parisi, 2011).

It has been demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway. This study further unravels the pathways that control Myc protein stability and shows that signaling by insulin and TOR induce Myc protein accumulation by regulating GSK3β activity in S2 cells. GSK3β is a constitutively active kinase that is regulated by multiple signals and controls numerous cellular processes. With the biochemical data it is proposed that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (see Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways). In particular, activation of insulin signaling was shown to induce activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize. Interestingly, insulin-induced Myc protein accumulation, when GSK3β activity was blocked by the presence of LiCl or by expression of GSK3β-KD, was similar to that obtained with insulin alone. Since it was shown that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, it would be expected that activation of insulin signaling concomitantly with the inhibition of GSK3β activity would result in a higher level of Myc than that obtained with insulin or LiCl alone. The results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone, supporting the hypothesis that GSK3β and insulin signaling, at least in the current experimental condition, depend on each other in the mechanism that regulates Myc protein stability (Parisi, 2011).

In a similar biochemical approach, the effect of AAs was analyzed on Myc protein stability and how TOR signaling is linked to mechanisms that inactivate GSK3β to stabilize Myc protein in S2 cells. In these experiments it was possible to demonstrate that treatment with amino acids (AAs) increased Myc protein stability, and it was also shown that treatment with rapamycin, an inhibitor of TORC1, reduced insulin-induced Myc upregulation. The reduction of Myc protein accumulation by rapamycin was blocked by inhibition of the proteasome pathway, linking TOR signaling to the pathway that controls Myc protein stability. TORC1 is a central node for the regulation of anabolic and catabolic processes and contains the central enzyme Rheb-GTPase, which responds to amino acids by activating TOR kinase to induce phosphorylation of p70-S6K and 4E-BP1. Analysis of the molecular mechanisms that act downstream of TOR to regulate Myc stability shows that AA treatment induces p70-S6K to phosphorylate GSK3β on Ser 9, an event that results in its inactivation and accumulation of Myc protein (Parisi, 2011).

Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels. Adding back AAs lead to a substantial increase in Myc protein levels, which did not further increase when AAs where added to cells in the presence of the GSK3β inhibitor LiCl. These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments it is concluded that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability. However, it has to be pointed out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone, the experiments also suggest that Myc protein stability by TOR signaling is not solely directed through the inhibition of GSK3β activity, but other events and/or pathways contribute to Myc regulation. In conclusion, the biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however it cannot be excluded that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells (Parisi, 2011).

Reduction of insulin and TOR signaling in vivo reduces cell size and proliferation, and clones mutant for chico, the Drosophila orthologue of IRS1-4, or for components of TOR signaling, are smaller due a reduction in size and the number of cells. The experiments showed that reducing insulin signaling by expression of PTEN or using TOR^TED, a dominant negative form of TOR, decreased Myc protein levels in clones of epithelial cells of the wing imaginal discs, while the opposite was true when these signals were activated using Dp110 or Rheb^AV4 . Those experiments suggested that the mechanism of regulation of Myc protein by insulin and TOR pathways was conserved also in vivo in epithelial cells of the larval imaginal discs (Parisi, 2011).

During these experiments it was also noted that Myc protein was induced in the cells surrounding and bordering the clones (non-autonomously), particularly when clones where positioned along the dorsal-ventral axis of the wing disc. This upregulation of Myc protein was not restricted to components of the insulin signaling pathway since it was also observed in cells surrounding the clones mutant for components of the Hippo pathway or for the tumor suppressor lethal giant larvae (lgl), which upregulates Myc protein cell-autonomously. It is suspected that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. It can be speculated that clones with different growth rates, caused by different Myc levels, might secrete factors to induce Myc expression in neighboring cells. As a consequence, these Myc-expressing cells will speed up their growth rate in an attempt to maintain proliferation and tissue homeostasis. Further analysis is required to identify the mechanisms responsible for this effect (Parisi, 2011).

In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, a genetic analysis was performed. The ability to induce growth and proliferation was measured in the eye by measuring the size and number of the ommatidia from animals expressing members of the insulin and TOR pathways in different dm genetic background (dm⁺, dm^P0 and dm⁴). The data showed that Dp110 increased the size and number of the ommatidia, however only the alteration in the total number was dependent on dm levels. These data suggest that Myc is required downstream of insulin pathway to achieve the proper number of ommatidia. However, when insulin signaling was reduced by PTEN, a significant decrease in the size of ommatidia was seen and it was dependent on dm expression levels, suggesting that Myc activity is limiting for ommatidial size and number. Activation of TOR signaling induces growth, and the genetic analysis showed that Myc significantly contributes to the size of the ommatidial cells thus suggesting that Myc acts downstream of TOR pathway to control growth (Parisi, 2011).

Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway, implying that they may share common targets. In support of this observation, mosaic analysis with a repressible cell marker (MARCM) experiments in the developing wing disc showed that overexpression of Myc partially rescues the growth disadvantage of clones mutant for the hypomorphic Rheb^7A1 allele, further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones (Parisi, 2011).

The genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K. A possible explanation for this difference could lie in the fact that S6K is not capable of auto-activation of its kinase domain unless stimulated by TOR kinase. TOR activity is dependent on its upstream activator Rheb; consequently the enzymatic activity of the Rheb/GTPase is the limiting factor that influences S6K phosphorylation and therefore capable of maximizing its activity (Parisi, 2011).

Interestingly, these experiments also showed that activation of TOR signaling has a negative effect on the number of ommatidia, and this correlates with the ability of Rheb^AV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dm^P0 mutant background, which leading to the speculation that 'excessive' protein synthesis, triggered by overexpression of TOR signaling, could elicit a Myc-dependent stress response, which induces apoptosis. Alternatively, high protein synthesis could result in an enrichment of misfolded proteins that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process (Parisi, 2011).

This analyses provide novel genetic and biochemical evidences supporting a role for Myc in the integration of the insulin and TOR pathway during the control of growth, and highlights the role of GSK3β in this signaling. It was found that insulin signaling inactivates GSK3β to control Myc protein stability, and a similar biochemical regulation is also shared by activation of the TOR pathways. In support of this data, a recent genomic analysis in whole larvae showed a strong correlation between the targets of Myc and those of the TOR pathway; however, less overlap was found between the targets of Myc and those of PI3K signaling (Parisi, 2011).

Statistical analysis applied to the genetic interaction experiments revealed that, in the Drosophila eye, proliferation induced by activation of the insulin pathway is sensitive to variations in Myc levels, while a significant interaction was seen mostly when TOR increased cell size. The data therefore suggests that there is a correlation between Myc and the InR signaling and it is expected that the InR pathway also shares some transcriptional targets with Myc. Indeed, an overlap was found between the targets induced by insulin and Myc in Drosophila S2 cells and these targets have also been reported in transcriptome analyses in the fat body upon nutritional stress, suggesting that Myc acts downstream of InR/PI3K and TOR signaling and that this interaction might be specific to some tissues or in a particular metabolic state of the cell (Parisi, 2011).

Insulin/IGF signaling drives cell proliferation in part via Yorkie/YAP

The insulin/IGF signaling (IIS) pathway is a potent inducer of cell proliferation in normal development and in cancer. The mechanism by which this occurs, however, is not completely understood. The Hippo signaling pathway regulates cell proliferation via the transcriptional co-activator Yorkie/YAP, however the signaling inputs regulating Hippo activity are not fully elucidated. This study presents evidence linking these two conserved, oncogenic pathways in Drosophila and in mammalian cells. It was found that activation of IIS and of Yorkie signaling correlate positively in hepatocellular carcinoma. IIS activates Yorkie in vivo, and that Yorkie plays an important role in the ability of IIS to drive cell proliferation. Interestingly, the converse was also found -- that Yorkie signaling activates components of the insulin/TOR pathway. In sum, this crosstalk between IIS and Yorkie leads to coordinated regulation of these two oncogenic pathways (Strassburger, 2012).

This study provides evidence that the insulin/IGF signaling (IIS) pathway and the Hpo/Yki signaling pathway are intricately interlinked in normal development and in cancer. In addition to observing a correlation in activation of the two pathways in hepatocellular carcinoma, mechanistic studies suggest that IIS activates Yki signaling and vice-versa. IIS appears to activate Yki via two mechanisms, a minor one involving Akt and a second major one via another target of PDK1. Both appear to function via Hpo repression. A previous report found that Akt can phosphorylate and inactivate MST1 in HeLa cells in the context of pro-apoptotic signaling. Whether a direct phosphorylation of Hpo by Akt might explain the growth effects observed was tested in this this study. However, to Akt-mediated phosphorylation of Hpo was observed either by in vitro kinase assay or by isoelectric focusing of cell extracts treated in the presence or absence of insulin. Furthermore, by expressing phospho-mimetic or alanine mutant versions of Hpo in vivo in the fly, no effect on Hpo activity was observed. In sum, no data was found supporting the idea that the growth-promoting effect of IIS occurs via direct phosphorylation of Hpo by Akt, in agreement with the finding that the major mechanism by which IIS affects Yki is Akt-independent . In addition, a previous report found that Akt might also directly phosphorylate and inhibit YAP, which is surprising given that both Akt and YAP have similar - and not antagonistic - effects on cell growth and proliferation. Another study was not able to confirm this finding leaving this an open issue. Likewise, the connections from Yki to IIS appear to be multiple, as transcriptional regulation of a large number of components of the IIS pathway was seen upon Yki activation. These interconnections lead to coordinated activation of the two pathways (Strassburger, 2012).

The main finding of this study is that insulin/IGF signaling drives proliferation partly via Hpo/Yki signaling. It was found that IIS regulates Yki activity in vivo, both at the biochemical level, and in terms of Yki transcriptional activity. Although analysis of Yki phosphorylation and of four Yki targets (DIAP1, merlin, expanded and cyclin E) consistently indicate that IIS activates Yki, surprisingly no increase was seen in expression of another yorkie target, bantam, upon expression of Dp110-CAAX, consistent with previous findings. Since bantam is an outlier compared to all other yorkie targets tested, this likely suggests bantam has other transcriptional regulatory inputs. Since the growth caused by Dp110-CAAX expression is abrogated in the absence of Hpo and highly sensitive to Yki gene dosage, this indicates that Yki is an important and limiting effector mechanism by which IIS drives tissue growth. Since the regulation of Yki/YAP by IIS is conserved from flies to humans, this mechanism likely represents an ancestral mechanism by which IIS drives tissue growth (Strassburger, 2012).

Hpo appears to be a central hub for integrating numerous inputs that affect tissue growth. The overgrowth caused by removal of upstream regulators of Hpo, such as Merlin, Expanded, Fat, Dachsous, Dco, Kibra and Crumbs, are mild in comparison to the overgrowth caused by loss of Hpo, suggesting these upstream components function in a combinatorial and additive manner to regulate Hpo. The work described in this study provides an additional input into Hpo signaling. Since IIS is responsive to nutrient conditions in addition to growth factor signaling, this links nutrient status to Hpo/Yki signaling and animal growth. It is tempting to speculate that nutrient overload and hyperactivation of the IIS pathway over an extended time in an adult organism might, via this mechanism, potentially contribute towards hyperplasia (Strassburger, 2012).

Recently, the YAP signaling pathway was found to be linked to several other oncogenic pathways including TGF-beta, beta-catenin and EGF signaling. In each case, however, the link is that YAP affects signaling through the other pathway. For instance, YAP interacts with β-catenin to regulate a subset of Wnt target genes, and YAP upregulates expression of the EGFR ligand amphiregulin to activate EGF signaling. The findings presented in this study are the first in which the link also goes in the other direction, with IIS regulating Hpo and Yki signaling, thereby adding growth factor signaling as an input into Hpo/Yki (Strassburger, 2012).

Thus far, Yki has been found to regulate expression of genes affecting mainly cell proliferation (e.g. CycE) and apoptosis (e.g. diap1). The data presented in this study suggest a mechanism by which Yki can induce tissue growth by also affecting TOR activity, thus activating both cell proliferation and cell growth in Drosophila. This further extends and is in line with recently published data that show that YAP overexpression can drive IIS in mouse cardiomyocytes (Strassburger, 2012).

In sum, the data presented in this study reveal one molecular mechanism by which IIS drives proliferation, they identify a novel input into Hpo signaling, and they elucidate cross-talk between two established oncogenic pathways of relevance for cancer development (Strassburger, 2012).

Neuronal Cbl controls biosynthesis of insulin-like peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. This study shows that in Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. dCbl is highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increases sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl results in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulates the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolishes the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increases insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells (Yu, 2012).

Drosophila germ-line modulation of insulin signaling and lifespan

Ablation of germ-line precursor cells in Caenorhabditis elegans extends lifespan by activating DAF-16, a forkhead transcription factor (FOXO) repressed by insulin/insulin-like growth factor (IGF) signaling (IIS). Signals from the gonad might thus regulate whole-organism aging by modulating IIS. To date, the details of this systemic regulation of aging by the reproductive system are not understood, and it is unknown whether such effects are evolutionarily conserved. This study reports that eliminating germ cells (GCs) in Drosophila increases lifespan and modulates insulin signaling. Long-lived germ-line-less flies show increased production of Drosophila insulin-like peptides (dilps) and hypoglycemia but simultaneously exhibit several characteristics of IIS impedance, as indicated by up-regulation of the Drosophila FOXO (dFOXO) target genes 4E-BP and l (2)efl and the insulin/IGF-binding protein IMP-L2. These results suggest that signals from the gonad regulate lifespan and modulate insulin sensitivity in the fly and that the gonadal regulation of aging is evolutionarily conserved (Flatt, 2008).

Ectopic misexpression of bam ⁺ in the female germ line, by using the binary GAL4>UAS system or heat shock-induction, eliminates GCs. Previous data suggest that the lost GCs are germ-line stem cells (GSCs): heat shock-induced bam ⁺ expression causes GC loss, but GCs that were not GSCs at the time of heat shock develop normally. Although grandchildless-like mutants lack pole cells and cannot form primordial GCs, heat shock-induced bam ⁺ overexpression eliminates female GSCs in the third larval instar (L3) or later but not before the L3 stage. When driving constitutive overexpression of UASp-bam ⁺ with the germ-line-specific nanos (nos)-GAL4::VP16 driver, it was found that GC loss continues in adult females, after the ovary has completed development. Females initially have the capacity to lay a small number of eggs but become fully sterile by day 7. Similarly, in males, bam ⁺ overexpression induced GC depopulation in the L3 stage or later. Moreover, bam ⁺ overexpression caused a dramatic expansion of somatic cells in ovaries and testes, reminiscent of the enlarged somatic gonads of agametic grandchildless-like mutants. Thus, grandchildless-like mutants and flies misexpressing bam ⁺ have expanded somatic gonads but complete GC loss at different times (Flatt, 2008).

GC loss induced by misexpression of bam ⁺ significantly increased lifespan in females and males, in several independent experiments. Lifespan was increased by 31.3% and 50% in females and 21% and 27.8% in males by GC ablation in a y w background by driving y w;UASp-bam ⁺ with nos-GAL4::VP16; effects are relative to a coisogenic control (y w;UASp-bam ⁺; control 1) and a control with a heterozygous background (y w/w¹¹¹⁸; nos-GAL4::VP16; control 2). Longevity was also extended when UASp-bam ⁺ was driven by nos-GAL4::VP16 in an independent background (w¹¹¹⁸) lacking one copy of genomic bam. The capacity for GC ablation to extend lifespan was likewise effective with the germ-line driver nos-GAL4-tubulin (NGT-GAL4) in the y w and w¹¹¹⁸ backgrounds. Thus, bam ⁺ misexpression in the germ line is sufficient to force GC loss and to increase lifespan in multiple genetic backgrounds and with different germ-line drivers. Because the failure of grandchildless-like mutants to develop GCs has no consistent major effects on lifespan, it was hypothesize that GC loss during late development or in the adult might promote longevity because GCs associate and interact with somatic cells before loss (Flatt, 2008).

If the germ line produces a signal that shortens lifespan or represses a somatic signal that extends lifespan, GC overproliferation should decrease lifespan. To test this prediction, a sterile heteroallelic null mutant of bam was examined in which mitotically active, nondifferentiating GSCs overproliferate. Thus, eliminating GC proliferation slows aging, whereas GC overproliferation shortens lifespan in the fly, as in the nematode. However, the possibility cannot be completely excluded that the longevity effects of bam are independent of its effects on GCs (Flatt, 2008).

Germ-line loss might slow aging simply by abolishing the survival costs of producing gametes. To rule out that egg production is required for GCs to shorten lifespan, a female-sterile mutant of egalitarian (egl) was examined. Mutants of egl prevent differentiation of cystoblasts into oocytes. Consequently, flies produce eggs with 16 rather than 15 nurse cells, and egg chambers degenerate before they acquire yolk. Lifespan of sterile egl mutant females (egl^PR29/egl^wu50) was reduced compared with fertile controls, suggesting that oogenesis per se might not be sufficient for reproduction to shorten lifespan. This result adds to a growing number of cases showing that the tradeoff between reproduction and survival can be decoupled (Flatt, 2008).

In C. elegans, lifespan extension by GC loss requires the FOXO transcription factor DAF-16; FOXO activity is normally repressed by IIS. Because reduced IIS slows Drosophila aging [by mutations disrupting IIS, constitutive activation of Drosophila FOXO (dFOXO), or ablation of insulin-producing cells, it was reasoned that GC loss might extend lifespan by down-regulating IIS. Accordingly, message abundance was measured for the three Drosophila insulin-like peptides (dilps) produced by median neurosecretory cells (mNSCs), the major insulin-producing cells (IPCs) in the brain of the adult. Rather than reduced message from the dilp2, dilp3, and dilp5 loci, it was found that these transcripts were induced upon GC loss by 1.8- to 26-fold relative to controls, in two independent genetic backgrounds (Flatt, 2008).

Previous attempts to quantify DILPs by Western blot analysis have failed because of low ligand abundance, and current technology does not permit detection of circulating DILPs in the hemolymph. However, several observations suggest that increased dilp message in GC-ablated flies might be biologically meaningful. Immunostaining of brains with DILP antibody indicated that the IPCs of GC-less flies produced as much and, in some cases, more DILP protein than controls, and DILP⁺ staining of IPC axonal projections was strong, suggesting functional DILP transport. Furthermore, neural DILPs homeostatically regulate sugar levels in the hemolymph, and GC-less flies had reduced amounts of stored and circulating carbohydrates (Flatt, 2008).

The hyperinsulinism of GC-less flies is a paradox because lifespan should not be extended in the face of increased DILPs. Because high DILP levels should activate IIS in peripheral tissues and repress dFOXO, transcripts were measured of two major dFOXO targets from body tissue, the translational regulator thor (encoding 4E-BP), and the small heat shock protein l (2)efl, which are normally induced when IIS is repressed and dFOXO is activated. Message levels of both dFOXO targets were up-regulated in GC knockout flies. Although it cannot be ruled out that these targets have transcriptional inputs other than dFOXO, flies with GC loss, despite elevated DILPs, express markers consistent with active dFOXO and reduced IIS (Flatt, 2008).

Because reduced IIS causes dephosphorylation and nuclear translocation of dFOXO, nuclear accumulation of dFOXO can be used to assess IIS pathway activity. To confirm that dFOXO is active in GC-less flies, its localization was examined with immunostaining in peripheral fat body, a major site of IIS activity, and by Western blotting analysis with cell fractionation in whole-body tissue. As expected, dFOXO was predominantly nuclear in GC flies, indicating that dFOXO is active. Yet, despite differential up-regulation of dFOXO targets, GC-less and control flies did not differ in nuclear dFOXO localization, which suggests that GC loss might affect dFOXO activity independent of its subcellular localization, as recently found in C. elegans (Flatt, 2008).

There are many mechanisms by which IIS can be impeded between the site of insulin production and FOXO-dependent responses of peripheral tissues: at the level of insulin secretion or transport and at many steps within intracellular IIS of target tissues. To initiate an understanding of IIS impedance in GC-less flies, whether GC loss might change transcript abundance of two DILP cofactors, dALS and IMP-L2, was assessed. In mammals, circulating IGFs form a complex consisting of IGF-1, IGF-binding proteins (IGF-BPs), and the liver-secreted scaffold protein acid labile substrate (ALS); by creating a pool of circulating IGFs, this ternary complex limits ligand availability. The Drosophila homolog of ALS (dALS) is expressed in DILP-expressing IPCs and the fat body and is up-regulated in dFoxo null mutants. Consistent with the model that dALS functions as a DILP cofactor, dALS forms a circulating trimeric complex containing DILP2 and IMP-L2, an Ig-like homolog of IGF-BP7. Binding of dALS requires prior formation of a dimeric complex containing DILP2 and IMP-L2. In cell culture experiments, IMP-L2 binds mammalian insulin and IGF-1/-2, and fall army worm (Spodoptera frugiperda) IMP-L2 inhibits IIS through the human insulin receptor. Because overexpression of dALS and IMP-L2 can systemically antagonize DILP function and IIS in Drosophila in vivo, message abundance of dALS and IMP-L2 was measured upon GC loss. Although dALS levels did not change, IMP-L2 message was increased 7-fold in GC-less flies. Although this observation is correlational, it might suggest a potential explanation for why IIS might be impeded in GC-less flies in the face of elevated DILP production. It will be of major interest to determine whether GC loss can modulate DILP availability and IIS by affecting IMP-L2 (Flatt, 2008).

Together, these results show that GCs regulate aging and modulate IIS in the fly. Although future work is required to fully characterize IIS state upon GC loss, it was observed that GC-less flies exhibit characteristics of both increased and decreased IIS. Increased DILPs and hypoglycemia are suggestive of increased IIS, but GC-less flies also have markers of IIS impedance. The induction of dFOXO targets is consistent with the finding that lifespan extension by GC loss in the nematode requires FOXO/DAF-16. In the worm, GC loss induces nuclear translocation of DAF-16 and activates DAF-16 targets, but nuclear accumulation is also observed in worms that lack the entire gonad and have normal lifespan. Similarly, it was found that GC-less and control flies differ in dFOXO target activation, but not dFOXO localization, suggesting that IIS can affect aging by modulating FOXO/DAF-16 activity independent of subcellular localization. Indeed, dietary restriction in C. elegans extends longevity by activating AMP-activated protein kinase (AMPK), which phosphorylates and activates DAF-16 but does not promote DAF-16 nuclear translocation (Flatt, 2008).

Because extended longevity by GC loss is associated with up-regulation of DILPs, GC loss might impede IIS downstream of DILP production. In humans, compensatory hyperinsulinemia is a hallmark of severe insulin resistance, and mutations in the tyrosine kinase domain of the insulin receptor can cause hyperinsulinemic hypoglycemia coupled with insulin resistance. Recent studies with fly and mouse also suggest that lifespan can be extended despite hyperinsulinemia. In Drosophila target-of-rapamycin (dTOR) mutants, longevity extension is associated with elevated DILP2 and hypoglycemia, and brain-specific insulin receptor substrate-2 (Irs-2) knockout mice are hyperinsulinemic but insulin-resistant and long-lived. Clearly, further experiments are needed to unravel the mechanisms by which insulin production can be uncoupled from IIS sensitivity and modulation of lifespan (Flatt, 2008).

The finding that GC loss affects neural DILP production also adds to growing evidence suggesting evolutionary conservation of endocrine feedback between brain and gonad. In Drosophila, neural DILPs bind to the insulin-like receptor (dINR) on GSCs to regulate GC proliferation, and neuronal InR knockout (NIRKO) mice show impaired spermatogenesis and ovarian follicle maturation. Conversely, in rats, ovariectomy decreases IGF-1 receptor density in the brain but increases circulating IGF-1 levels. Together with progress made in the worm and mouse, the Drosophila system will allow dissection of the mechanisms underlying the fundamental and intricate relationship among IIS, reproduction, and aging (Flatt, 2008).

Insulin signalling regulates remating in female Drosophila

Mating rate is a major determinant of female lifespan and fitness, and is predicted to optimize at an intermediate level, beyond which superfluous matings are costly. In female Drosophila melanogaster, nutrition is a key regulator of mating rate but the underlying mechanism is unknown. The evolutionarily conserved insulin/insulin-like growth factor-like signalling (IIS) pathway is responsive to nutrition, and regulates development, metabolism, stress resistance, fecundity and lifespan. This study shows that inhibition of IIS, by ablation of Drosophila insulin-like peptide (DILP)-producing median neurosecretory cells, knockout of dilp2, dilp3 or dilp5 genes, expression of a dominant-negative DILP-receptor (InR) transgene or knockout of Lnk, results in reduced female remating rates. IIS-mediated regulation of female remating can occur independent of virgin receptivity, developmental defects, reduced body size or fecundity, and the receipt of the female receptivity-inhibiting male sex peptide. These results provide a likely mechanism by which females match remating rates to the perceived nutritional environment. The findings suggest that longevity-mediating genes could often have pleiotropic effects on remating rate. However, overexpression of the IIS-regulated transcription factor dFOXO in the fat body-which extends lifespan-does not affect remating rate. Thus, long life and reduced remating are not obligatorily coupled (Wigby, 2011).

The effects of IIS on female remating can - at least to some extent - act independently of SP, the major male-derived molecular effector of female receptivity. This finding is consistent with the lack of interaction effects between nutrition and SP on female mating rate found by Fricke (2010). These two major regulators of female remating, IIS and SP, are likely to signal the normal requirement for remating in response to factors that limit female reproduction, namely nutrients required to produce eggs and sperm required for fertilization. This dual mechanism for controlling remating, via IIS and SP, may enable female mating rate to most effectively match reproductive opportunities while avoiding costly superfluous matings (Wigby, 2011).

Females may benefit unconditionally from their first mating as they need to obtain sperm to fertilize eggs. Thus, the lack of effect of IIS on virgin receptivity may be because sexually mature females gain from a rapid first mating - and there is no benefit to delaying mating -- whatever may be the nutritional conditions. However, in D. melanogaster, as in many insects, a single mating fails to provide sufficient sperm to fertilize all the eggs produced over a lifetime, meaning that females must remate to replenish sperm stores. A tighter calibration of nutrition with remating rate may be beneficial following the first mating, because nutrition affects female fecundity and the rate of sperm use such that, under poor nutritional conditions, females will need to replenish stored sperm (i.e. mate) less frequently. Hence, the regulation of female remating receptivity in response to nutritional status is likely to be key for female fitness (Wigby, 2011).

The sexual behaviour of IIS mutant females broadly mimics that of females on a poor diet, which is consistent with the hypothesis that reduced IIS partly (though not wholly) mimics dietary restriction. Like reduced IIS, restriction of dietary nutrients can result in increased lifespan and decreased mating rates. Manipulating components of the IIS pathway, as performed in this study, could generate a mismatch between the perceived and real nutritional environment, resulting in potentially sub-optimal mating rates for a given rate of egg-laying. However, it is clear that there is no obligatory link between egg-laying and mating rate, because females that lack the ability to produce eggs display normal mating and remating behaviours. Moreover, this study shows that females can possess normal fecundity but show reduced mating rates under IIS suppression (Wigby, 2011)

Lifespan can be extended by genetic manipulations that reduce IIS, including several mutants used in this study (MNC-ablated; dilp2 and dilp2-3; InRDN; Lnk). However, lifespan can also be extended by reducing mating frequency. The results therefore highlight the importance of controlling mating rates in studies that investigate the genetics of ageing, to avoid confounding effects of differential sexual activity on lifespan. The discovery that several IIS manipulations that increase lifespan also increase the inter-mating interval raises an important potential confound regarding the conclusions of ageing studies in which flies are maintained in mixed sex groups. Reduced mating rates in experimental mutant lines could potentially confound ageing studies because females might live longer owing to reduced mating rates rather than as a direct effect of the genetic manipulations themselves. The solution to this potential confound is to control mating rates in lifespan studies in order to test for direct effects on lifespan. However, the results from the dFOXO experiment show that it is also possible to uncouple the regulation of female sexual behaviour and the regulation of lifespan, in accordance with the uncoupling of lifespan and fecundity. Thus, both behavioural and physiological aspects of reproduction can be uncoupled from lifespan extension under certain conditions (Wigby, 2011).

The effects of single dilp mutants on remating were, surprisingly, only marginally weaker than the effects of MNC ablation or dilp2-3 double mutants, despite the apparently weaker genetic intervention. However, ablation of the MNCs is incomplete, and DILP levels are reduced rather than abolished in the flies that were used. Moreover, there is compensation and synergism between DILPs such that knockouts of single dilp genes can affect the expression of one or more of the other dilps. For example, dilp2 and dilp2-3 mutant flies exhibit increased expression of dilp5, while dilp3 mutants exhibit reduced levels of dilp2 and dilp5 expression. Such effects could explain the relatively strong phenotypes of the single dilp knockouts in comparison with the dilp2-3 knockout and MNC-ablated females (Wigby, 2011).

The extracellular DILPs, the InR and the intracellular IIS component, Lnk, all regulate female remating rate, but it is currently unclear which downstream molecules are involved. A major downstream target of the IIS pathway is the transcription factor dFOXO, but no effect of fat body dFOXO expression was found on female mating. One possibility is that dFOXO mediates the effect of reduced IIS on remating rates in tissues other than the fat body. Another possibility is that the effect of IIS on remating rate occurs via the target of rapamycin (TOR) pathway. The TOR pathway senses amino acids and runs parallel to, and interacts with, IIS. The IIS and TOR pathways interact to control growth, and TOR signalling, like IIS, has been shown to regulate lifespan. Moreover, recent work shows that the TOR pathway is involved in mating-induced changes in diet choice, supporting the idea that TOR functions in the coordination of behavioural responses to mating and the nutritional environment. It will be important to investigate the mating behaviour of TOR-pathway mutants to determine whether this pathway is involved in the regulation of mating and whether the effects of IIS on female remating are mediated through TOR signalling. It will also be important to determine through which tissues IIS regulates remating (Wigby, 2011).

This work shows that components of the IIS pathway modulate sexual behaviour by significantly altering the receptivity of mated female D. melanogaster. Thus, a likely molecular basis is provided for the link between nutrition and sexual behaviour in insects, which is an important step in understanding the mechanisms underlying life-history traits and trade-offs. Reproduction and nutrition are linked across a broad range of taxa, including mammals, and many of the effects of IIS (e.g. on lifespan and fecundity) are highly evolutionarily conserved. It is concluded that the regulation of mating behaviour via IIS could be common among animals (Wigby, 2011).

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. This study shows that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. These effects are mediated through activation of extracellular signal-related kinase (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan (Lee, 2008).

Neuropeptides regulate a wide range of animal behaviours related to nutrition. In particular, mammalian NPY produced in the hypothalamus of the brain controls food consumption. NPY injection in the hypothalamus of rats produces hyperphagia and obese phenotypes. The Drosophila orthologue of NPY is sNPF. This peptide is expressed in the nervous system and it regulates food intake and body size; overexpression of sNPF produces bigger and heavier flies. Likewise, the G-protein-coupled receptor of sNPF (sNPFR1) is expressed in neurons and shows significant similarity with vertebrate NPY receptors. In mammals, however, little is known about how NPY and sNPF systemically modulate growth, metabolism and lifespan. This study shows that these neuropeptides control expression of insulin-like peptides and subsequently affect insulin signalling in target tissues (Lee, 2008).

Initially the effects of sNPF and sNPFR1 on body size were characterized by measuring the length of flies from head to abdomen. The body size of sNPF hypomorphic Drosophila mutants (sNPF^c00448) was 23% of that of the wild-type, whereas overexpressing two copies of the sNPF in the sensory neurons and sensory structures of the nervous systems by MJ94-Gal4 (MJ94>2XsNPF) increased body size by 24%. Similar changes were seen in the overall size of adult wings, which resulted from changes in both cell size and number. Effects on body size were associated with sNPF expression levels: relative to wild type, sNPF levels were 3.5-fold higher in MJ94>2XsNPF and less than half of the wild type in sNPF^c00448. In contrast to the effect of sNPF on body size, there was little effect on size from repression or overexpression of the sNPF receptor in MJ94-expressing cells (Lee, 2008).

Drosophila insulin-like peptides (Dilps) modulate growth and adult size; therefore, whether sNPF has a role in insulin-producing neurons was tested. For positive controls, Dilp2 was overexpressed in insulin-producing cells (IPCs) through Dilp2-Gal4, which increased body size, and the IPCs were ablated by expression of Dilp2>reaper to decrease body size. To investigate sNPF signalling, sNPFR1 was overexpressed in the IPCs and a 10% increase in body size was observed. Conversely, expression of the sNPFR1 dominant-negative mutant (Dilp2>sNPFR1-DN) reduced body size by 14%. Manipulation of the sNPF ligand with IPCs expressing Dilp2-Gal4, however, did not affect body size: flies overexpressing sNPF (Dilp2>2XsNPF) or in which sNPF was silenced by RNAi (Dilp2>sNPF-Ri) were of similar size to the wild type. Taken together, these results suggest that sNPF peptide may be secreted from MJ94-expressing sensory neurons and activate sNPFR1 of Dilp2-expressing IPCs (Lee, 2008).

To assess this model, the sNPF ligand and sNPFR1 receptor were visualized in the larval brain. Seven IPCs were detected in each brain hemisphere using the marker Dilp2-Gal4>nGFP. Neurons containing sNPF peptide in the axon terminal and cell body (sNPFnergic neurons) were stained adjacent to these IPCs. As expected, sNPFR1 receptors were localized in the plasma membrane of IPCs marked with Dilp2>DsRed. sNPFR1 was also localized in the neurons of the larval brain hemispheres, sub-oesophagus ganglion, ventral abdominal neurons and descending axons in the ventral ganglion (Lee, 2008).

To study genetic interactions between sNPFR1 and Dilps in IPCs, Dilp1 and Dilp2 interference mutants were generated in the sNPFR1 overexpression background. In contrast to the 10% body size increase by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1), inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri) generated reduced body size by 10% and 15%, respectively. Inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated a reduction in body size of 8% and 13%, indicating that Dilp1 and Dilp2 are downstream genes of sNPFR1 in IPCs for regulating body size (Lee, 2008).

To test whether sNPF regulates Dilp expression in larval IPCs, expression of Dilp1, 2, 3 and 5 were assessed in sNPF mutants. Neuronal overexpression of sNPF (MJ94>2XsNPF) markedly increased expression of Dilp2 in IPCs; it also produced novel Dilp2 expression outside of these cells. As expected, reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2. In common with Dilp2, the expression of Dilp1 was positively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPF^c00448). Consistent with the model, expression of Dilp1 and Dilp2 was increased more than fourfold with overexpression of the receptor in IPCs (Dilp2>sNPFR1) and decreased by half with inhibition of the receptor gene in IPCs (Dilp2>sNPFR1-DN). Larval IPCs also express Dilp3 and Dilp5. Expression of Dilp3 was reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPF^c00448) but expression of Dilp5 was not regulated by any sNPF mutants. There are few functions known to distinguish these various insulin-like peptides. Nutrition-dependent growth regulation is associated with expression of Dilp3 and Dilp5, but not with that of Dilp2. Recent reports show that Dilp2 is reduced in long-lived flies expressing dFOXO or Jun-N-terminal kinase (JNK), whereas Dilp5 is uniquely upregulated upon dietary restriction that increases lifespan (Lee, 2008).

To investigate how Drosophila sNPF regulates Dilp expression, the activation of Drosophila MAP kinase signalling, which includes the action of ERK (encoded by Rolled) and JNK, was measured. sNPF overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor protein sNPFR1 in IPCs also increased pERK. There were no detectable changes in phospho-activated pJNK in these sNPF and sNPFR1 mutants. Next, whether ERK activation in IPCs was sufficient to induce Dilp expression was tested. Expression of a constitutively active ERK in IPCs (Dilp2>rolled^SEM) increased expression of Dilp1 and Dilp2 more than threefold, and both transcripts were repressed less than half by the expression of an ERK inhibitory phosphatase DMKP-3 in IPCs (Dilp2>DMKP-3). In addition, the inhibition of ERK with the sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+DMKP-3) also repressed expression of Dilp1 and Dilp2 compared with that of sNPFR1 overexpression in IPCs (Dilp2>sNPFR1). These results indicate that sNPF and sNPFR1 signalling regulate ERK activation in IPCs, which in turn modulates expression of Dilp1 and Dilp2 (Lee, 2008).

To further examine the effect of sNPF on Dilp, Drosophila CNS-derived neural BG2-c6 cells, which endogenously express sNPFR1 were treated with a synthetic sNPF peptide. Dilp1 and Dilp2 were induced within 15 min, and the elevated transcript persisted for 1 h. Concomitant with this gene expression, sNPF-treated cells activated ERK. Importantly, sNPF did not induce Dilp expression significantly when cells were treated with ERK-specific kinase MEK inhibitor PD98059. To compare the functional conservation of sNPF and NPY in the regulation of insulin expression, similar tests were conduced with rat insulinoma INS-1 cells, which express NPY receptors NPYR1 and NPRY2. When treated with the human NPY peptide, expression of insulin1 and insulin2 and ERK was activated within 15 min. Furthermore, treatment with the MEK inhibitor PD98059 and NPY abolished the induction of insulin1 and insulin2. Together, these findings suggest that the regulation of insulin expression by sNPF or NPY through ERK is evolutionarily conserved in Drosophila and mammals (Lee, 2008).

To verify that sNPF induction of Dilp expression has a physiological consequence, insulin signals at a target tissue, the Drosophila fat body were examined. Fat body cells in flies with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than in the control, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPF^c00448 reduced cell size by 38% and 51% respectively. These differences in size correspond to changes in insulin signal transduction within the cells. Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effect was seen with neuronal inhibition of sNPF (MJ94>sNPF-Ri and sNPF^c00448). Activated Akt represses the transcription factor dFOXO by phosphorylation and subsequent cytoplasmic localization. In wild-type flies, dFOXO localized equally in the cytoplasm and nucleus. As predicted, neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO, whereas inhibition of sNPF (MJ94>sNPF-Ri and sNPF^c00448) yielded fat body cells with dFOXO predominantly localized in the nucleus. Finally, dFOXO induces expression of the translational inhibitor d4E-BP, and, consistent with the current observations, expression of d4E-BP was elevated in animals where sNPF was inhibited (MJ94>sNPF-Ri and sNPF^c00448) and reduced in animals where sNPF was overexpressed (MJ94>2XsNPF) (Lee, 2008).

Besides cell growth, Drosophila insulin-like peptides modulate aspects of metabolism and ageing. For instance, ablation of the IPCs reduces animal size, elevates the level of haemolymph carbohydrates.Therefore trehalose and glucose were assessed in sNPF mutant flies. As predicted, both carbohydrates were reduced upon sNPF overexpression, and both were elevated in sNPF hypomorphs. Also the lifespan of sNPF mutants was measured. As expected, inhibition of sNPF by MJ94>sNPF-Ri increased median lifespan by 16-21%, whereas sNPF overexpression (MJ94>2XsNPF) did not affect lifespan in flies (Lee, 2008).

Overall, the effects on Dilp1 and Dilp2 expression in IPCs regulated by sNPF are associated with cellular, carbohydrate and lifespan responses that are predicted to be caused by changes in the actual level of available insulin peptides. It is concluded that sNPF ultimately regulates insulin secretion from the IPC to affect target tissue insulin/dFOXO signalling and thus modulate growth, metabolism and lifespan (Lee, 2008).

Regulation of food consumption by neuropeptides is a critical step for interventions for managing obesity and metabolic syndromes. Mammalian NPY is known to positively regulate appetite and has thus been thought to promote weight gain primarily by affecting food intake. Thus study revealed a novel physiological role for NPY that is conserved by sNPF of Drosophila. These neuropeptides can affect growth, metabolism and lifespan by modulating ERK-regulated transcription of insulin-like peptides. In Drosophila, sNPFnergic and IPC neurons are adjacent in the brain. This study found, however, that pancreatic β-cells are also responsive to NPY, which is of hypothalamic origin. Although the hypothalamic neurosecretory cells and responding pancreatic endocrine cells are spatially distinct in mammals, recent developmental analysis suggests a parallel developmental pathway for hypothalamic neurosecretory cells and the IPCs of Drosophila, raising the possibility of a common molecular mechanism for β-cell formation. This would suggest that β-cells are not only evolutionarily tied to the hypothalamic neurosecretory cells but also that they retain their functional relationship to their hypothalamic origin by regulating insulin in response to the neuropeptide NPY (Lee, 2008).

Expression of dominant-negative Dmp53 in the adult fly brain inhibits insulin signaling

In Drosophila, p53 (Dmp53) is an important mediator of longevity. Expression of dominant-negative (DN) forms of Dmp53 in adult neurons, but not in muscle or fat body cells, extends lifespan. The lifespan of calorie-restricted flies is not further extended by simultaneously expressing DN-Dmp53 in the nervous system, indicating that a decrease in Dmp53 activity may be a part of the CR lifespan-extending pathway in flies. This report shows that selective expression of DN-Dmp53 in only the 14 insulin-producing cells (IPCs) in the brain extends lifespan to the same extent as expression in all neurons and this lifespan extension is not additive with CR. DN-Dmp53-dependent lifespan extension is accompanied by reduction of Drosophila insulin-like peptide 2 (dILP2) mRNA levels and reduced insulin signaling (IIS) in the fat body, which suggests that Dmp53 may affect lifespan by modulating insulin signaling in the fly (Bauer, 2007).

Expression of DN-Dmp53 constructs in the adult nervous system extends lifespan. This lifespan extension is not additive to CR (Bauer, 2005), suggesting that Dmp53 may be part of the CR pathway of lifespan extension. To understand more about the mechanisms by which neuronal reduction of Dmp53 extends lifespan, the expression of DN-Dmp53 was examined in subsets of neuronal cells. General expression of DN-Dmp53 in the nervous system, using two different, broadly expressing neuronal specific promoters, ELAV (pan-neuronal) or Cha (cholinergic neurons), both lead to significant lifespan extension. When expression of DN-Dmp53 was restricted to smaller subsets of neurons, including dopaminergic neurons, serotoninergic neurons, neurons of the mushroom body, or to IPCs, only expression in the 14 IPCs cells led to lifespan extension. Thus, expression of DN-Dmp53 in only the 14 IPCs is sufficient to extend Drosophila lifespan. When combined with CR, IPC-specific DN-Dmp53 expression did not further extend lifespan, suggesting that reduction of Dmp53 activity in the IPCs may be a component of CR-dependent lifespan extension. Interestingly, IPCs are the functional equivalent of mammalian pancreatic β-cells, but reside in the fly brain. This data suggests that Dmp53 controls insulin secretion from the IPCs. A consequence of inhibition of Dmp53 activity is reduced dILP2 mRNA and subsequent down regulation of IIS in the fat body, Drosophila's major insulin responsive metabolic organ. In this relevant target tissue, two different assays, tGPH and dFoxO subcellular localization, show that IIS is diminished. These data suggest that DN-Dmp53 expression might extend lifespan through modulation of IIS. Interestingly, p53 has been linked to insulin regulation in mammals. Mice over-expressing the shorter p44 isoform of p53, that is thought to resemble Dmp53 more than p53 itself, have elevated levels of IGF-1and IGF-1R. Furthermore, p53 null mice have 75% reduced levels of IGF-1 (Bauer, 2007).

Loss or destruction of IPCs has been postulated to extend lifespan in flies. A loss of the IPCs does not explain the lifespan extension seen with the long-lived flies expressing DN-Dmp53 specifically in IPCs; it was possible to visualize the presence of the IPCs, at least up until day 44, as measured by simultaneous expression of GFP. Evidence was presented suggesting that the IPCs remain functionally active. Of the three dILPs produced in the IPCs, only dILP2 mRNA levels are lowered, whereas dILP3 and dILP5 levels remain unchanged. Furthermore, the fat body cells exhibit a nearly normal insulin response to a short period of starvation and sucrose re-feeding in the long-lived DN-Dmp53- expressing flies. Thus, the lifespan extension induced by expression of DN-Dmp53 in IPCs is not due to loss or damage of the IPCs (Bauer, 2007).

IIS related lifespan extension in the fly is thought to be mediated through alterations in the InR, CHICO (InR substrate), dPTEN and perhaps dFoxO. Interestingly, increasing levels of dPTEN or dFoxO in the fat body, but not the nervous system, extends lifespan. Experiments on another nutrient sensing system, the TOR signaling pathway, also supports the importance of the fat body in lifespan extension; down-regulation of the TOR pathway in the fat body, but not the nervous system leads, to lifespan extension. IIS can modulate TOR signaling via phosphorylation of Tsc2 by protein kinase B/Akt. Thus, down-regulated IIS may lead to suppression of TOR signaling (Bauer, 2007).

The fly thus appears to have at least two different tissues that can influence longevity: one, neuronal in nature, of which Dmp53 is part; the other as part of a larger nutrient-sensing signaling network active in the fat body. Are these two separate systems or is there cross-talk between them? One possible means of connecting these two systems could be through the neuroendocrine system, where alterations in the nervous system, through control of hormonal secretion, could affect the physiology of the fat body. It is thus of considerable interest that the site of production and secretion for three of the major insulin-like peptides, dILP2, dILP3, and dILP5, is the 14 ICPs located in the brain of the fly. The finding that expression of DN-Dmp53 specifically in these IPCs affects IIS in fat body cells and extends lifespan is intriguing. It remains to be determined whether this is one of the cellular sites linking the longevity determining effects of the brain with those associated with the fat body (Bauer, 2007).

It is tempting to speculate even further: The data suggest that the CR and DN-Dmp53 lifespan-extending pathways are related. It may therefore be that the lifespan-extending effects of CR are also accompanied by a down regulation of IIS. Dmp53 might thus be a point of convergence for these two lifespan-extending pathways, but further experiments are needed to clarify this point (Bauer, 2007).

Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila: Potential role of insulin pathway

Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. This study reports on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. Overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppresses the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons leads to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, evidence is provided that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. This study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways (Wu, 2006).

The relatively simple Drosophila larva offers a genetically tractable model to define and characterize different neuronal signaling pathways that constitute a complete central feeding apparatus. Younger third-instar larvae forage actively and use their mouth hooks for food intake. Larvae normally feed on liquid food, and their food ingestion can be quantified by measuring the contraction rate of the mouth hooks. This study examined how food deprivation affects larval feeding response to a liquid (e.g., 10% glucose-agar paste) and less accessible solid food (e.g., 10% glucose agar blocks). To extract embedded glucose from the solid food, larvae have to pulverize the food by scraping agar surface with mouth hooks. Unless stated otherwise, synchronized third-instar larvae (74 h after egg laying) were used for the assays (Wu, 2006).

When fed ad libitum, normal larvae (w¹¹¹⁸) display significant feeding activity in the liquid food with an average mouth-hook contraction frequency of ~30 times in a 30-s test period; in contrast, these larvae declined the solid food. However, larvae withheld from food (on a wet tissue) for 40 or 120 min display increased intake of both liquid and solid foods. For example, larvae fasted for 120 min show a 100% and >500% increase in mouth-hook contraction rate in liquid and solid food, respectively. Thus, deprivation not only enhances feeding rate in a graded fashion, but also triggers motivated foraging on the less accessible food normally rejected by fed larvae. In addition, larvae display virtually identical feeding responses to liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions. Therefore, these paradigms appear to provide a general assessment of larval feeding response (Wu, 2006).

dS6K is a cell-autonomous effector of nutrient-sensing pathways. This study investigated a possible role of neural dS6K in coupling peripheral physiological hunger signals and neuronal activities critical for hunger-driven behaviors. The transcripts of dilp1, dilp2, dilp3, and dilp5 are predominantly expressed in two small clusters of medial neurosecretory cells that project to the ring gland, the fly heart, and the brain lobes. A gal4 driver containing a 2-kb fragment from the dilp2 promoter (dilp2-gal4) was generated that directs the specific expression of a GFP reporter in those cells. Using dilp2-gal4, two transgenes, UAS-dS6K^DN, encoding a dominant negative, and UAS-dS6K^ACT, a constitutively active form of dS6K, were expressed. When fed ad libitum, control larvae (w x UAS-dS6K^DN or UAS-dS6K^ACT) behave like w larvae. However, dilp2-gal4 x UAS-dS6K^DN larvae displayed a 50% increase in the rate of liquid-food intake and significant feeding of the solid food. Conversely, fasted larvae overexpressing dS6K activity (dilp2-gal4 x UAS-dS6K^ACT) showed attenuated feeding response to both liquid and solid foods. These findings reveal that dS6K in DILP neurons mediates hunger regulation of approaching/consumptive behaviors, controlling both quality and quantity of food for ingestion. The body size and the developmental rate of all four groups of larvae were measured, and no significant differences were detected (Wu, 2006).

DILPs act as neurohormones in Drosophila larvae. Down-regulation of dS6K activity in DILP neurons may reduce DILP release, thereby promoting increased food intake that is normally triggered only by hunger. A corollary of this interpretation is that overproduction of DILPs in the nervous system should interfere with hunger response by deprived animals. To test this idea, a neural-specific elav-gal4 driver was used to direct dilp expression in the larval nervous system. Three UAS-dilp lines (UAS-dilp2, UAS-dilp3, and UAS-dilp4) were chosen for the analysis. The elav-gal4 x UAS-dilp2 and UAS-dilp4 larvae displayed normal feeding response when fed ad libitum. However, the same larvae fasted for 120 min displayed significantly attenuated feeding rates, similar to those of dilp2-gal4 x UAS-dS6K^ACT larvae. For example, the comparative analysis of the elav-gal4 x UAS-GFP control and elav-gal4 x UAS-dilp2 and UAS-dilp4 experimental larvae showed that the latter were ~30% and 33–45% lower in the ingestion rate of the liquid and solid food, respectively; surprisingly, elav-gal4 x UAS-dilp3 and UAS-GFP larvae showed virtually identical feeding responses. Therefore, DILP2 and DILP4 negatively regulate hunger-driven feeding activities. Taken together, these results suggest that a high level of dS6K activity in DILP neurons may suppress hunger response by reducing DILP release (Wu, 2006).

Attempts were made to delineate the signaling mechanism that couples the dS6K activity in DILP neurons with its broad impact on hunger-driven feeding activities. A previous study showed that fasted larvae ablated of NPF or its receptor (NPFR1) neurons are deficient in motivated feeding of the less-preferred solid food but normal in feeding of richer liquid food. It was of interest to enquire whether the NPF/NPFR1 neuronal pathway might be one of the downstream effectors of the DILP pathway. To test this hypothesis, the function of three components of the dInR signaling pathway were analyzed in NPFR1 neurons: dInR, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN), and phosphatidylinositol 3-kinase (dPI3K). Five different transgenes were used: UAS-dInR^ACT and UAS-dInR^DN encode a constitutively active and a dominant-negative form of dInR, respectively; UAS-Dp110 and UAS-dPI3K^DN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively; and UAS-dPTEN encodes a functional enzyme. When fed ad libitum, npfr1-gal4 x UAS-dInR^DN, UAS-dPTEN, or UAS-dPI3K^DN larvae display hyperactive feeding of the solid food, similar to w larvae deprived for 40 min. In contrast, fasted larvae overexpressing dInR or dPI3K (npfr1-gal4 x UAS-dInR^ACT or UAS-Dp110) display attenuated feeding response to the solid food. Importantly, larvae with up- or down-regulated dInR signaling in NPFR1 neurons do not exhibit significant changes in the intake rate of the richer liquid food relative to the paired controls. Taken together, these findings suggest that the dInR pathway negatively regulates the activity of NPFR1 neuron and mediates the DILP-regulated change in food preference but not ingestion rate. Furthermore, the results suggest that NPFR1 neurons are the direct targets of DILPs (Wu, 2006).

A possible role of dS6K in hunger regulation of the functioning of NPFR1 neurons was evaluated, by expressing UAS-dS6K^DN and UAS-dS6K^ACT using npfr1-gal4. When fed ad libitum, npfr1-gal4 x UAS-dS6K^DN larvae display hyperactive feeding of the solid food, similar to npfr1-gal4 x UAS-dInR^DN larvae. However, these larvae, unlike dilp2-gal4 x UAS-dS6K^DN animals, display no increases in the ingestion rate of the richer liquid food. Conversely, fasted larvae overexpressing dS6K (npfr1-gal4 x UAS-dS6K^ACT) display attenuated feeding response to the solid food. These findings suggest that dS6K also negatively regulates the activity of NPFR1 neurons in food preference, but does not mediate the regulation of feeding rate by DILP signaling (Wu, 2006).

The food response was evaluated of the solid and liquid food by larvae overexpressing an npfr1 cDNA under the control of an npfr1-gal4 driver. In the presence of the liquid food, both experimental (npfr1-gal4 x UAS-npfr1) and control larvae (e.g., npfr1-gal4 x UAS-ANF-GFP), fed or fasted, show similar intake rates and comparable increases in feeding response to hunger. However, when forced to feed on the solid food, fed experimental larvae exhibit significant intake of the solid food (30 times per 30 s), whereas fed controls rejected the same food. Thus, NPFR1 overexpression selectively promotes change in food preference without increasing ingestion rate. It was also observed that the feeding responses of NPFR1-overexpressing larvae and controls fasted for 120 min were indistinguishable. Thus, the effect of NPFR1 overexpression on food preference is detectable only in fed or mildly fasted larvae, suggesting hunger-activated NPFR1 signaling approaches a plateau in severely fasted animals (Wu, 2006).

npfr1 activity was selectively knocked down by expressing npfr1 dsRNA in the nervous system. The UAS-npfr1^dsRNA lines were previously used to functionally disrupt npfr1 activity. It was found that 120-min fasted larvae expressing npfr1 dsRNA in NPFR1 or the nervous system (npfr1-gal4, elav-gal4, or appl-gal4 x UAS-npfr1^dsRNA) were deficient in motivated feeding of the solid but not liquid food. In contrast, all control larvae, including those expressing npfr1^dsRNA in muscle cells (MHC82-gal4 x UAS-npfr1^dsRNA), showed normal feeding responses. These results indicate that neural NPFR1 mediates hunger regulation of food selection (Wu, 2006).

A potential problem of the previous transgenic studies is that NPF/NPFR1 signaling is likely to be disrupted in a relatively early stage of larval development. Conceivably, the NPF/NPFR1 neuronal pathway could be essential for ad libitum or hunger-driven feeding of richer liquid foods, but such an activity might be masked by some yet-unidentified compensatory mechanism triggered by its early loss. To test this idea, attempts were made to disrupt NPF/NPFR1 neuronal signaling in a temporally controlled manner by expressing a temperature-sensitive allele of shibire (shi^ts1) driven by npf-gal4 or npfr1-gal4. The shi^ts1 allele encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). At the permissive temperature of 23°C, 120-min-fasted experimental larvae (npf-gal4 and npfr1-gal4 x UAS-shi^ts1) and paired controls (y w x UAS-shi^ts1 and npf-gal4 and npfr1-gal4 x w¹¹¹⁸) displayed normal feeding responses to both liquid and solid foods. However, if larvae were incubated at 30°C for 15 min, controls still displayed normal feeding activities, whereas the experimental larvae showed attenuated feeding response to the solid but not liquid food. Therefore, there was no detectable developmental or physiological compensation for the loss of NPF signaling in Drosophila larvae. These results also suggest that the NPF/NPFR1 neuronal pathway is acutely required to initiate and maintain larval hunger response. The foraging activity of the experimental larvae was completely restored when the assay temperature was reduced to 23°C, suggesting that the NPF system can modulate the intensity and duration of feeding response (Wu, 2006).

This study has shown that dS6K regulates different, yet coordinated, behaviors controlling quantitative and qualitative aspects of hunger-adaptive food response. Evidence is provided that dS6K mediates hunger regulation of two opposing insulin- and NPY-like signaling activities, dynamically modifying larval food preference and feeding rate based on the nutritional state. For example, hunger stimuli may cause a reduction of dS6K activity in DILP neurons, resulting in the suppression of DILP signaling that negatively regulates a downstream NPF/NPFR1-dependent and another NPF-independent neuronal pathway. The DILP/NPFR1 neuronal pathway selectively mediates hunger-adaptive change in food preference, possibly by overriding the high threshold of food acceptance set by a separate default pathway, enabling hungry animals to be receptive to less preferred foods. The NPF/NPFR1-independent pathway promotes a general increase in the ingestion rate of preferred/less preferred foods, enabling animals to compete effectively for limited food sources. This study also implicates the presence of a separate default pathway for mediating the selective intake of preferred foods (baseline feeding) in larvae fed ad libitum. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in nondeprived larvae does not affect ad libitum feeding in the liquid food. It is suggested that the conserved S6K pathway may be critical for regulating behavioral adaptation to hunger in diverse organisms, including humans, and its components are potential drug targets for appetite control (Wu, 2006).

The functional differences of DILP1–7 have not been reported previously. In this study, dilp2, dilp3, and dilp4 were shown to be functionally distinct. DILP2 and DILP3 both are produced in the same medial neurosecretory cells. However, unlike DILP2, DILP3 is apparently not involved in suppressing deprivation-motivated feeding. It is still unclear whether the differential activities of DILP2 and DILP3 reflect their structural divergence or are caused by the presence of yet-unidentified dInR isoforms. DILP4 is not expressed within the two medial clusters of DILP neurons. Under acute deprivation, the level of dilp4 transcripts showed a 5-fold reduction in the larval CNS. Thus, it is possible that DILP4 may play a localized role in promoting feeding response inside the CNS (Wu, 2006).

Feeding is a reward-seeking behavior, and deprivation strengthens the reinforcing effect (reward value) of food. These studies suggest a previously uncharacterized role of the DILP/dInR signaling pathway in regulating an animal's perception of food quality. The DILP/NPF neural network may regulate an animal's incentive to acquire lower-quality foods by modifying the reward circuit. This hypothesis is interesting in light of the findings that foods and abused substances may act on the same reward circuit, and highly palatable foods can reduce drug-seeking behaviors. It is also possible that the DILP/NPF system might represent a specialized neural circuit that positively alters the reward value of lesser-quality foods. Conceivably, a better understanding of the action of this signaling system may provide fresh insights into neural mechanisms for controlling eating and drug-seeking behaviors (Wu, 2006).

Given its prominent role in behavioral adaptation to hunger, the insulin/NPY-like neural network is likely of primary importance to animal evolution. In addition, insulin and NPY family molecules have been found in a wide range of animals from humans to worms. Therefore, the insulin/NPY-like network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective lifestyles (Wu, 2006).

Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila

Insulin signaling pathways are implicated in several physiological processes in invertebrates, including the control of growth and life span; the latter of these has also been correlated with juvenile hormone (JH) deficiency. In turn, JH levels have been correlated with sex-specific differences in locomotor activity. This study examined the involvement of the insulin signaling pathway in sex-specific differences in locomotor activity in Drosophila. Ablation of insulin-producing neurons in the adult pars-intercerebralis was found to increase trehalosemia and to abolish sexual dimorphism relevant to locomotion. Conversely, hyper-insulinemia induced by insulin injection or by over-expression of an insulin-like peptide decreases trehalosemia but does not affect locomotive behavior. Moreover, this study also showed that in the head of adult flies, the insulin receptor (InR) is expressed only in the fat body surrounding the brain. While both male and female InR mutants are hyper-trehalosemic, they exhibit similar patterns of locomotor activity. These results indicate that first, insulin controls trehalosemia in adults, and second, like JH, it controls sex-specific differences in the locomotor activity of adult Drosophila in a manner independent of its effect on trehalose metabolism (Belgacem, 2006).

In Drosophila, sexual dimorphism has been reported for the number of activity/inactivity phases (start/stops) during locomotion. Feminizing cells (FCs) of the mid-anterior part of the pars intercerebralis (PI), and JH have been implicated in the control of this dimorphism. This study showed that insulin-producing cells (IPCs) are also located in the mid-anterior part of the PI in adult flies, in the same cluster as, but distinct from, the PI neurons termed feminizing cells (FCs). Second, this study showed by conditional genetic ablation that these cells are involved in the control of sex-specific differences in locomotion: flies without IPCs present the same number of start/stops, such that males have a feminine activity profile. Third, perturbation of insulin pathways by mutations affecting the insulin receptor (InR) also has a similar effect on sex-specific locomotion. This result corroborates the idea that control of the number of start/stops by IPCs is mediated by insulin-like peptides and not by another product from these cells. Indeed, the ablation of IPCs removes the cells and everything in them, like putative unrelated but co-expressed peptides or transmitters. However, the finding that the InR mutation leads to a similar phenotype than the IPCs ablation is in accordance with the statement that the number of start/stop might be mediated by the insulin-like peptides. In contrast, hyper-insulinemia induced by insulin injection or by over-expression of the dilp2 gene (hsdilp2) does not affect the number of start/stops in males and thus is not implicated in this sexual dimorphism (Belgacem, 2006).

Another physiological parameter that was investigated is the carbohydrate level in hemolymph. IPCs control the trehalose level at the organismal level via the secretion of insulin analogues. Indeed, a decrease in the insulin level (caused by ablation of IPCs or disruption of insulin signaling with InR mutations) increases the trehalose level, while augmentation of the insulin level (caused by insulin injection or over-expression of dilp2) reduces it. These results clearly show that insulin has an endocrine function in adult Drosophila, as in mammals. In some other insects, insulin and/or insulin analogues have been shown to influence carbohydrate levels, particularly as hypoglycemic hormones (Belgacem, 2006).

This study also reports that the insulin receptor is expressed at the brain periphery in the fat body (FB), as well as in the corpus allatum, the well established site for the JH synthesis. Surprisingly, no InR was found on neuronal cells of the brain. Perhaps InR is not expressed at all in the central nervous system of adult Drosophila, or it was not detected because a heterologous antibody (anti-human) was used to detect it (Belgacem, 2006).

The antibody used was directed against the α-subunit of human insulin receptor, and precisely from the sequence of the third exon, which encodes the insulin-binding domain. A sequence homology performed between this human InR third exon and Drosophila, reveals 36% of homology over 102 amino-acid residues, suggesting that this domain is well conserved. Moreover, the strong detection of the over-expressed InR in the muscle is in accordance with the specificity of the antibody used. Alternatively and in an independent way, physiological approaches have also shown that injection of heterologous insulin (from bovine) is able to activate its invertebrate homologue, both in blowflies as well as in Drosophila, again suggesting a well-conserved domain between different species. Finally, a third argument in accordance with the specificity of the InR is supported by the similar results obtained from the two independent approaches: immunohistological staining analysis and injection of labeled insulin leading to a similar localization in the brain fat body (Belgacem, 2006).

Although InR is expressed both in the head fat body and in the corpus allatum, physiological and behavioral results suggest that the observed effects on sex-specific differences in locomotor activity probably result from disruption of the insulin pathway in the corpus allatum, rather than in the fat body. Indeed, the disruption of the cc-ca gland, by the ablation of the cc, which leads, in males, to a female-like activity profile supports this assumption. Conversely, it is suspected that altered trehalose metabolism phenotype might be due to the distortion of the insulin pathway in the fat body. However, sex-specific differences in locomotor activity under control of signal arising from the fat body could not yet be totally excluded, since two recent studies have suggested that genetic disruption and/or manipulation of the FB affects behavior. The findings that the InR is specifically expressed in FB cells and that the locomotor activity of males lacking InR function is feminized could also correlate with a role for the FB in a sexually dimorphic behavior. Obviously, further experiments, as for instance, tissue-specific targeting of InR disruption, which will require specific GAL4 drivers either in the corpus allatum or fat body, will be necessary for such fine differential dissection (Belgacem, 2006).

The molecular linkage of the insulin pathway to locomotor activity patterns, which also depend on JH levels, remains to be elucidated. Mammalian hydroxymethylglutaryl-CoA reductase (HMGCR), a key enzyme in cholesterol and sterol synthesis, is transcriptionally regulated by the insulin pathway. Drosophila HMGCR is also a central enzyme in the JH biosynthetic pathway and likely plays an important role in JH regulation. Thus, transcriptional regulation of HMGCR by insulin-dependent regulatory elements may link the JH and insulin pathways (Belgacem, 2006).

In conclusion, this study has shown that the insulin signaling pathway is implicated in both males and females in the regulation of trehalose levels, since hypo- and hyper-insulinemia affects both sexes. Moreover, this pathway is also implicated in sex-specific differences in locomotor activity, since perturbations resulting in hypo-insulinemia feminize the locomotor behavior of males, whereas hyper-insulinemia has no effect. Therefore, this new insulin-dependent effect seems to be distinct from the hormonal role of insulin in trehalose level regulation and is likely mediated by either a different intracellular signaling pathway or under control of different tissues. Finally, the identification of the JH target, and more specifically its receptor, is the next crucial step in understanding how brain structures and neurons differentially control sex-specific aspects of locomotor activity (Belgacem, 2006).

Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila

Stem cells reside in specialized niches that provide signals required for their maintenance and division. Tissue-extrinsic signals can also modify stem cell activity, although this is poorly understood. This study reports that neural-derived Drosophila insulin-like peptides (DILPs) directly regulate germline stem cell division rate, demonstrating that signals mediating the ovarian response to nutritional input can modify stem cell activity in a niche-independent manner. A crucial direct role is demonstrated for DILPs in controlling germline cyst growth and vitellogenesis (LaFever, 2005).

Germline and somatic stem cells support oogenesis throughout adult life in Drosophila. Germline stem cells (GSCs) reside within a specialized niche where they are exposed to a unique combination of signals required for stem cell function. However, GSCs are also controlled by tissue-extrinsic signals, such as Drosophila insulin-like peptides (DILPs), which mediate the ovarian response to nutrition. On a protein-rich diet, germline and somatic stem cells have high division rates, and their progeny exhibit high division and development rates. On a protein-poor diet or under reduced insulin signaling, rates of division and development are reduced, and progression through vitellogenesis is blocked. It remains unclear, however, how DILPs control the response of GSCs in coordination with their differentiating progeny and with follicle cells (LaFever, 2005).

In adult females, DILPs are produced in two clusters of medial neurosecretory cells in the brain, and stage 10 follicle cells express dilp5 mRNA. Ablation of brain DILP-producing cells results in reduced egg production rates and a partial block in vitellogenesis. To examine the role of the brain DILP-producing cells in previtellogenic stages, they were ablated and follicle cell proliferation rates were measured. Females missing brain DILP-producing cells (ablated) have a severely impaired ability to up-regulate follicle cell proliferation in response to a protein-rich diet. The rate of germline development is reduced in coordination with follicle cell divisions because no abnormalities are observed in previtellogenic egg chambers. Ablation of DILP-producing cells reduces the size of eclosed adults and delays development. Ablated females in which these developmental defects are rescued by an hs-dilp2 transgene expressed during larval stages show a reduced follicle cell proliferation rate, comparable to that of nonrescued, ablated females. Thus, the impaired response to a protein-rich diet is not a secondary consequence of the developmental defects. Moreover, the 2.3-fold delay caused by ablation of brain DILP-producing cells is very similar to that caused by blocking reception of DILP signals by the germ line. This indicates that the brain is the major source of DILPs that determine the rate of egg chamber development with little, if any, contribution from dilp5-expressing follicle cells (LaFever, 2005).

To examine whether DILPs control the rate of germline development directly or indirectly, germline cysts unable to respond to DILPs were created by inducing Drosophila insulin receptor (dinr) mutant clones using the flipase (FLP)/FLP-recognition target (FRT) technique. Germline cysts homozygous for dinr³³⁹, a genetic null allele, had normal morphology and correct cell number; however, 83% of these cysts were developmentally delayed, showing markedly decreased size relative to neighboring wild-type egg chambers. Further quantification of these data showed a 2.4-fold delay in the development of dinr³³⁹ cysts. Similar results were obtained for germline cysts homozygous for dinr^E19 and dinr³⁵³, which are viable hypomorphic alleles; 78% and 64% of dinr^E19 and dinr³⁵³ cysts, respectively, were developmentally delayed. These results reveal that dinr function is required cell autonomously for a normal rate of germline cyst development. Thus, the rate of cyst development is regulated by a DILP signal that is received directly (LaFever, 2005).

Progression through vitellogenesis requires DILP signaling; however, it has been unclear whether this role is direct. Reduced juvenile hormone levels are present in homozygous viable dinr mutants, and the block in yolk uptake in these mutants can be partially bypassed by treatment with methoprene, a juvenile hormone analog, suggesting an indirect role for DILPs in promoting vitellogenesis. To specifically address whether direct activation of germline cysts by DILPs is required for vitellogenesis, mosaic ovarioles were examined in which the entire germ line was homozygous dinr mutant for the ability of their egg chambers to undergo vitellogenesis. All egg chambers containing dinr³³⁹ or dinr^E19 homozygous mutant cysts failed to progress through vitellogenesis and degenerated. In the case of dinr³⁵³, the allele with the higher level of dinr activity, only one out of six egg chambers containing homozygous mutant cysts failed to undergo vitellogenesis. These results suggest that the level of insulin signaling within the germ line controls vitellogenesis, revealing a direct role for DILPs in this process. Moreover, complete loss of dinr function in the germ line results in a complete block in vitellogenesis, whereas this block is partial upon ablation of brain DILP-producing cells. Thus, DILP5 expressed in stage 10 follicle cells likely signals in combination with brain DILPs to regulate vitellogenesis (LaFever, 2005).

It was next asked whether DILPs control GSC division rate directly by binding to receptors on their surface (a cell-autonomous requirement for dinr in GSCs) or indirectly by regulating signals produced by niche cells (a non-cell-autonomous requirement). dinr mosaic ovarioles containing one wild-type and one mutant GSC were examined and the number of wild-type versus mutant cystoblasts and cysts present in their germaria was counted. Because each cystoblast or cyst corresponds to one GSC division, the ratio of mutant to wild-type cystoblasts and cysts is a measure of their relative division rates. For dinr³³⁹ homozygous mutant GSCs, a relative division rate of 0.31 was found, whereas, for wild-type GSCs, it was 0.90. Similarly, the relative division rates of dinr³⁵³ and dinr^E19 GSCs were 0.55 and 0.65, respectively. Thus, dinr homozygous mutant GSCs divide more slowly than wild-type GSCs, and GSC division rate appears sensitive to the level of dinr activity. These results demonstrate that GSCs directly receive the DILP signal to regulate their division rate without mediation by the stem cell niche (LaFever, 2005).

Germline and somatic cells respond to nutritional status in a coordinated manner; however, it is unclear whether somatic cells receive the DILP signal directly (a cell-autonomous role of dinr in follicle cell proliferation) as does the germ line, or indirectly through secondary signals (a non-cell-autonomous role). The percentages of dinr mutant and control follicle cells were measured in mosaic ovarioles carrying one wild-type and one dinr mutant somatic stem cell. If follicle cells receive the DILP signal directly, the reduced level of insulin signaling in dinr mutant follicle cells should result in lower rates of proliferation (i.e., fewer mutant than control follicle cells should be observed), whereas if they receive the signal indirectly, the proliferation rates should be similar. In dinr^E19 mosaic ovarioles, 51% of follicle cells were wild-type and 49% were mutant, indicating similar proliferation rates. dinr mutant follicle cells appeared to enter the endoreplicative cycle normally, but pycnotic (degenerating) nuclei and cell death were observed within dinr^E19 and dinr³³⁹ mutant follicle cell clones starting at stage 8. These results reveal that although a reduction in dinr activity delays germline cyst development cell autonomously, it does not cause a cell-autonomous reduction in follicle cell proliferation rate. Furthermore, in ovarioles carrying a fully dinr mutant germ line, excess follicle cells were not observed, showing that proliferation of surrounding wild-type follicle cells remains coordinated with germline growth. These results suggest that follicle cells respond indirectly to increased DILP levels through a secondary signal from the germ line. Similar degrees of coordination between germ line and soma have been observed in the presence of developmentally delayed dMyc mutant germline clones (LaFever, 2005).

These data demonstrate that tissue-extrinsic DILP signals can directly modify GSC proliferative activity, acting in parallel to signals from their niche. Evidence is provided that, in addition to its previously reported indirect roles in Drosophila and mammals through secondary hormonal signals, insulin signaling plays a crucial direct role during Drosophila oogenesis in regulating not only GSC division rate but also germline cyst development rate and progression through vitellogenesis. Insulin may, therefore, have important direct roles in mammalian oogenesis. Finally, the data suggest that the coordinated response of germline and somatic cells to nutrition involves communication between these tissues. These results have broad significance, in light of the long-known effects of nutrition on human fertility and of the high degree of conservation of insulin signaling functions (LaFever, 2005).

Ablation of insulin-producing neurons in flies: Growth and diabetic phenotypes

In the fruit fly Drosophila, four insulin genes are coexpressed in small clusters of cells [insulin-producing cells (IPCs)] in the brain. Ablation of these IPCs causes developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph. All of the defects were reversed by ectopic expression of a Drosophila insulin transgene. On the basis of these functional data and the observation that IPCs release insulin into the circulatory system, it is concluded that brain IPCs are the main systemic supply of insulin during larval growth. It is proposed that IPCs and pancreatic islet beta cells are functionally analogous and may have evolved from a common ancestral insulin-producing neuron. Interestingly, the phenotype of flies lacking IPCs includes certain features of diabetes mellitus (Rulifson, 2002).

The Drosophila genome contains five Drosophila insulinlike peptide genes (dilp1 through -5) with significant homology to mouse and human insulins and two others with far less similarity (dilp6 and -7). These genes are expressed in tissues ranging from early embryonic mesoderm to small clusters of larval brain neurons, ventral nerve cord neurons, salivary gland, and midgut. Using messenger RNA (mRNA) in situ hybridization, it has been established that the most prominent insulin gene expression during larval stages, a period of intensive feeding and rapid growth, is within two bilaterally symmetric clusters of neurosecretory cells in the pars intercerebralis region of the protocerebrum (Rulifson, 2002).

An 859-base pair promoter fragment, comprised of sequences immediately 5' of dilp2, is sufficient to drive gene expression in the small clusters of larval brain neurons that express dilp1, -2, -3, and -5. To assess the role of the brain IPCs as an insulin-producing endocrine system, the brain IPCs were ablated using the dilp2 promoter to express the cell death-promoting factor, Reaper. The IPC ablation results in deficiency of brain neuron-derived insulin only. IPC ablation caused undergrowth phenotypes, developmental delays, and lethality similar to Drosophila insulin receptor (DInR) mutants. To rule out an underlying cause of these phenotypes other than insulin deficiency, such as non-IPC death from Reaper or loss of other essential brain IPC functions, a heat shock-inducible dilp2 transgene was used with ubiquitous expression to reverse the effect of the IPC ablation (Rulifson, 2002).

The defect in growth was quantified by comparing larval length after 120 hours of development, a time at which synchronized cultures of normal larvae will reach wandering third instar and puparium formation. After IPC ablation, larvae attained a mean length only 58% of normal size. Larvae with IPCs ablated but expressing the inducible dilp2 transgene had their mean length rescued to 88% of normal. The developmental time to reach wandering third instar and puparium formation was approximately 5 days in normal larvae but lengthened to 12 days in larvae after IPC ablation, a developmental rate similar to that observed in animals homozygous for loss-of-function mutations in DInR. Larvae with ablated IPCs that expressed the inducible dilp2 transgene require approximately 6 days to reach puparium formation. Thus, systemic DILP2 expression is sufficient to compensate for IPC ablation. The fact that brain IPC ablation is rescued by dilp2 alone suggests that insulin made by brain IPCs may be partially redundant (Rulifson, 2002).

IPC ablation produces small-sized adults of normal proportion. Examination of adult wings revealed reductions in both cell size and number after IPC ablation. Under the strongest condition of IPC ablation, mean wing size was reduced to 61% of normal, whereas wing cell number and size were reduced to 72% and 85% of normal, respectively. Under a less severe regimen of IPC ablation, mean wing size was reduced to 74% of normal, with reductions in cell number and size to 81% and 91% of normal, respectively. As in larval growth, the dilp2 transgene effectively reverses the effects of IPC ablation on wing growth and, in fact, caused a slight overgrowth effect, possibly due to the 20% lengthening of developmental time that allowed more growth. The overall reduction in cell size and number after IPC ablation is similar to that in DInR and IRS1-4 mutants. This, together with the observation that brain IPC-derived insulins can activate the DInR in vitro, suggests that brain IPCs are a key source of insulin for this growth control pathway (Rulifson, 2002).

The role of brain IPCs and insulin in the regulation of carbohydrate metabolism was also investigated. Trehalose is a disaccharide composed of two glucose molecules and is the principal blood sugar in many insects. Using the same heat shock regimen, the combined concentration was examined of glucose and trehalose in the hemolymph of wandering third instar larvae, a brief and discrete developmental stage before puparium formation when feeding has ceased. The IPC-ablated larvae had an average combined glucose and trehalose level of 38% above normal, and these levels returned to normal when DILP2 was provided systemically by the transgene. Elevated hemolymph carbohydrate levels in larvae lacking IPCs indicate that insulin is an essential regulator of energy metabolism in Drosophila. This accumulation of carbohydrate in the blood is reminiscent of that seen in human diabetes mellitus, although it should be noted that carbohydrate levels were measured during development rather than in adults (Rulifson, 2002).

To investigate how central nervous system (CNS)-derived insulin regulates systemic functions, the Drosophila IPC contacts outside the CNS were examined. The morphology of the brain IPCs was examined with the use of the dilp2 promoter to drive expression of mGFP, a membrane bound green fluorescent protein (GFP). The IPC clusters within the pars intercerebralis extend processes that terminate at the lateral protocerebrum and subesophageal ganglion. IPC processes also terminate on the heart and in the corpora cardiaca (CC) compartment of the ring gland, after crossing the midline and exiting the CNS. Labeling of the IPC processes with mGFP and DILP2 antibody revealed localization of DILP2 peptide within the processes that contact the heart and ring gland. DILP2 peptide is concentrated in a graded distribution outside the cells of synthesis on the heart, and colabeling with myosin heavy chain antibody, which labels the columnar heart epithelium, showed that the IPC processes and DILP2 are localized outside the lumen of the heart. These results suggest the heart surface may be the site of insulin release to the openly circulating hemolymph. It is proposed that brain IPCs are essential for organism-wide growth control and carbohydrate homeostasis through release of insulin peptides into circulating hemolymph. These cellular functions are notably similar to those of mammalian pancreatic ß cells (Rulifson, 2002).

In Drosophila and other insects, a fraction of CC cells synthesize adipokinetic hormone (AKH). AKH resembles glucagon in its activation of glycogen phosphorylase through heteromeric GTP-binding protein (G protein) and adenosine 3',5'-monophosphate (cAMP) signaling to elevate blood sugar, and the two proteins have some limited sequence similarity. Double labeling of AKH mRNA and DILP2 peptide shows that IPCs extend processes to the CC and that AKH-expressing cells contain DILP2. CC cells accumulate DILP2 within membrane-bound particles of the perinuclear space, suggesting the possibility that DILP2 is taken up by AKH cells. The possibility that dilp2 is transiently or minutely transcribed by AKH cells cannot be ruled out, although expression of either the dilp2 promoter or dilp1, -2, -3, and -5 mRNAs in the AKH cells has not been detected. Thus, in addition to contacts between IPCs themselves, the primary sites of IPC contact outside the CNS are the heart and the CC. Though they lack strict morphological homology, these intercellular contacts are analogous to the association of pancreatic ß cells with other ß cells, with glucagon-expressing a cells, and with blood vessels in the islets of Langerhans and may reflect underlying evolutionary conservation (Rulifson, 2002).

Thus, there is remarkable similarity of the organ systems underlying conserved insulin function in diptera and mammals. Moreover, the presence of IPCs in the nervous systems of other invertebrate and protochordate species and in primary cell cultures from mammalian fetal brain provides further evidence for a common ancestral insulin-producing organ of neural origin. These results also raise the possibility that common mechanisms of cell specification regulate development of pancreatic a cells and Drosophila brain IPCs (Rulifson, 2002).

Nutrient-dependent expression of Insulin-like Peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila

The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies. This study establishes a causal link between nutrient availability and insulin-dependent growth. In addition to the Drosophila insulin-like peptide 2 (dilp2) gene, overexpression of dilp1 and dilp3-7 is sufficient to promote growth. Three of the dilp genes are expressed in seven median neurosecretory cells (m-NSCs) in the brain. These m-NSCs possess axon terminals in the larval endocrine gland and on the aorta, from which DILPs may be released into the circulatory system. Although expressed in the same cells, the expression of the three genes is controlled by unrelated cis-regulatory elements. The expression of two of the three genes is regulated by nutrient availability. Genetic ablation of these neurosecretory cells mimics the phenotype of starved or insulin signaling mutant flies. These results point to a conserved role of the neuroendocrine axis in growth control in multicellular organisms (Ikeya, 2002).

The insulin/IGF signaling pathway plays a key role in the control of growth in vertebrates and invertebrates. In mammals, the primary role of insulin and the insulin receptor is energy homeostasis by the regulation of blood glucose levels. But mutations in the human insulin receptor gene also cause embryonic growth retardation. The primary growth regulatory function in mammals, however, is mediated by the IGF-1 and IGF-2 growth factors and the IGF-1 receptor. In Drosophila, there is a single insulin-like receptor, and it regulates postembryonic growth, reproduction, and aging. In vertebrates and invertebrates, intracellular signal transduction from the insulin/IGF receptors depends on insulin receptor substrate (IRS) proteins. In mammals, loss of IRS1 function causes severe reduction in embryonic and postembryonic growth (Araki, 1994; Tamemoto, 1994). Loss of IRS2 leads only to a moderate reduction in growth, but mice become hyperglycemic and contain increased body fat, and females are sterile (Withers, 1998). In Drosophila, flies mutant for chico, which encodes the single Drosophila homolog of IRS1-4, are developmentally delayed, have a severely reduced body size and increased fat, and females are sterile. This demonstrates a striking conservation of the role of the insulin/IGF signaling pathway during evolution (Ikeya, 2002 and references therein).

Several lines of evidence suggest a link between the activity of the insulin/IGF signaling pathway and nutrient availability. The female sterility and the growth retardation phenotypes of IRS2^-/- and IRS1^-/- mice, respectively, are similar to those observed in starved mice (Butler, 2001, Tiessen, 1999). In Drosophila, growth retardation, reduced body weight due to fewer and smaller cells, and female sterility are phenotypes not only characteristic of chico mutant flies but also of flies that have been starved during development. In fact, oogenesis is blocked during stem cell divisions and before the onset of vitellogenesis in starved flies and in chico mutant flies. Furthermore, starvation reduces the activity of the insulin signaling pathway in vivo. In nematodes, starvation or mutations in the insulin signaling pathway arrest development at the so-called Dauer stage. Moreover, caloric restriction and mutations in the insulin/IGF1 signaling system extend life span in vertebrates and invertebrates. It is not known, however, whether the link between nutrient availability and the activity of the insulin signaling pathway is direct, and how the nutritional status is translated into insulin/IGF receptor activity in the target tissues (Ikeya, 2002 and references therein).

One obvious hypothesis for a connection between nutrient availability and insulin/IGF activity is that nutrients control the expression of the insulin/IGF growth factors. In mammals, blood glucose levels not only regulate the release of insulin from the pancreatic ß cells, but also regulate the insulin gene expression via an autocrine loop (Xu, 2001). The regulation of IGF-1 levels that control postnatal growth is more complex. Growth hormone synthesized by the pituitary controls IGF-1 expression in the liver, accounting for approximately one-third of the postnatal growth promoting activity. Growth also depends on GH-independent expression of IGF-1 and GH activity that is independent of IGF-1 (Ikeya, 2002 and references therein).

The role of insulin-like growth factors in invertebrates in the regulation of growth and their regulation in response to starvation is less well defined. In Drosophila, the search for insulin-like genes (dilps) in the genome has revealed seven genes that show highly regulated temporal and spatial expression. Ubiquitous overexpression of one of the dilp genes, dilp2, is sufficient to increase body size. This growth-promoting activity of DILP2 is dependent on the insulin receptor signaling pathway. Three dilp genes are expressed in small cell clusters in the central region of the brain. These three genes are expressed in the same median neurosecretory cells (m-NSCs) possessing axon terminals in the ring gland and the corpora cardiaca. Although all three peptides can promote growth when overexpressed, they are regulated differentially in response to starvation. Furthermore, genetic ablation of the m-NSCs produces a phenotype reminiscent of chico mutant flies (Ikeya, 2002).

Expression of dilp2, 3, and 5 genes is detected in the same two clusters of cells. The position of these cell clusters in the pars intercerebralis of the larval brain suggests that they correspond to the m-NSC clusters that stain with an anti-Bombyxin antibody. Indeed, the expression of GFP under the control of the dilp2 cis-regulatory elements permits the visualization of the axonal projections of the dilp expressing cells. Axon projections are seen in the corpora cardiaca of the ring gland and on the aorta, the site from which neuropeptides are released into the hemolymph. Therefore, dilp2, 3, and 5 are coexpressed in the cluster of m-NSCs identified in larger insects and in Drosophila. Although expressed in the same cells, the temporal expression pattern of dilp 2, 3, and 5 in the m-NSCs differs. While dilp2 is expressed already in the first instar stage, dilp2 and dilp5 expression is detectable in the second instar stage, while dilp3 expression starts at the mid to late third instar stage. The successive activation of dilp genes in the m-NSCs correlates with the increasing growth that occurs during the last larval instar (Ikeya, 2002).

The dilp2, 3, and 5 genes are located together with dilp1 and dilp4 in a gene cluster spanning 26 kb on the third chromosome. To search for enhancer elements controlling the expression of the three genes in the m-NSCs, a series of lacZ reporter genes containing various amounts of upstream genomic sequences was constructed. Upstream fragments of 1.0 kb, 1.7 kb, and 450 bp derived from the dilp2, 3, and 5 genes, respectively, are sufficient to drive reporter gene expression specifically in the m-NSC. Thus, each of the three genes possesses its own m-NSC-specific enhancer. A further dissection of these upstream sequences has revealed that a 394 bp fragment located at position -540 to -146 of dilp2 is sufficient to recapitulate the expression of dilp2 in the m-NSCs. This construct, however, is no longer expressed in imaginal discs, suggesting that different enhancer elements control expression in this tissue. The cis-regulatory elements that control dilp3 expression are more complex. The m-NSC-specific expression depends on two separate elements located between -763 to -1167 and between +1 to -165, including the putative transcription start site. The fragment located between the m-NSC enhancers drives expression of dilp3 in gut muscles (Ikeya, 2002).

Surprisingly, sequence comparison of the genomic sequences required for m-NSC expression of dilp2, 3, and 5 did not reveal obvious stretches of similarity that may identify a common m-NSC-specific enhancer element in the different genes. It therefore appears that even within this small group of cells, the three dilp genes are regulated by different combinations of transcription factors (Ikeya, 2002).

A causal link between starvation-induced reduction in growth and reduced insulin receptor activity may involve the regulation of circulating DILP levels by nutrient availability. To test this hypothesis, the expression of dilp2, 3, and 5 was examined in third instar larvae that had been starved for 24 hr. In nonstarved larvae, mRNA transcripts of dilp2, 3 and dilp5 were detected in the m-NSCs. Upon starvation, dilp3 and dilp5 transcript levels are reduced, while dilp2 transcript levels remain unchanged. Similar results were obtained when the dilp5-lacZ reporter construct was used. In these larvae, lacZ mRNA is severely reduced upon starvation, while LacZ activity is still detectable owing to the stability of the LacZ protein. Therefore, the enhancer elements that respond to starvation are located in the 450 bp fragment used to analyze dilp5 expression. These results demonstrate that at least part of the nutrient-dependent regulation of growth is mediated by the regulated expression of the dilp3 and dilp5 genes (Ikeya, 2002).

During larval development, the seven dilp genes are expressed in a variety of different tissues in addition to the m-NSCs. To begin to address the role of DILP production in the m-NSCs, these cells were specifically ablated. The dilp2-Gal4 line, which is exclusively expressed in the m-NSCs starting at the late third instar stage, was used to drive expression of the proapoptotic gene reaper (rpr) in these cells. dilp2:rpr flies are viable but eclose one day later than control flies. Freshly eclosed flies show a slight but significant reduction in body weight. The size difference is also observed in the reduced wing area. Interestingly, the largest difference between control and dilp2:rpr flies is observed in the size of the abdomen of females. This difference becomes further enhanced during the first three days of adult life. During this phase, egg production is stimulated by feeding and mating in wild-type females. Comparison of the ovaries of three-day-old wild-type and dilp2:rpr females revealed a striking difference in ovary size. Each ovary is composed of approximately 15 ovarioles. Ovarioles are oocyte tubes containing stem cells at the tip and oocytes of increasing maturity toward the oviduct. While wild-type females possess multiple vitellogenic oocytes in each ovariole, dilp2:rpr females possess at most a single vitellogenic oocyte. This reduced size of the ovary of dilp2:rpr flies is reflected in the reduced fecundity of these females. While control flies lay on average 60 eggs per day, dilp2:rpr females produce only 10 eggs per day. The dilp2:rpr flies exhibit a developmental delay, reduced body size, and reduced fecundity of females owing to a partial block in production of vitellogenic oocytes. These phenotypes resemble those of weak mutations in the genes coding for components of the insulin signaling pathway. While chico females are almost completely sterile and severely reduced in size, certain combinations of Inr alleles produce females very similar to the dilp2:rpr flies. It is possible that the partially penetrant phenotype of m-NSC ablation is due to the late onset of expression of the dilp2-Gal4 driver line, resulting in only a partial ablation of the m-NSCs. Since dilp5 expression is also observed in the follicle cells of the female ovary, this m-NSC independent source of DILP may provide an alternative explanation for the partially penetrant phenotype of m-NSC ablation (Ikeya, 2002).

Nutrient-dependent regulation of growth and reproduction is observed in all multicellular organisms. The results presented here provide further support for an evolutionary conserved signaling pathway involved in this regulation. In mammals, insulin secretion from the pancreatic ß cells is regulated by the concentration of glucose in the blood, and thereby regulates energy homeostasis in response to food intake. Embryonic and postembryonic growth is regulated by IGF-1 production that in part depends on GH synthesized from the pituitary. In insects, the m-NSCs appear to play a role in both functions. The release of insulin-like peptides from the corpora cardiaca in Bombyx is regulated by carbohydrate levels in the hemolymph in a way analogous to the release of glucose from pancreatic ß cells in mammals. Expression of dilp3 and dilp5 is repressed by food withdrawal. Furthermore, ablation of the m-NSCs results in growth retardation. These results are consistent with a recent study showing that early Reaper-induced ablation of the m-NSC cells using a multimerized dilp2-Gal4 construct severely reduced growth and led to a concomitant increase in glucose and trehalose levels in the hemolymph of these animals (Rulifson, 2002). Given the data from Bombyx and Drosophila together, it is suggested that the m-NSCs possess functions similar to those of the pancreas and the pituitary in mammals (Ikeya, 2002).

In insects, the growth regulatory function of DILPs is 2-fold. (1) Circulating levels of DILPs in the hemolymph activate growth in the target tissues by the activation of the insulin receptor PI3K pathway in each cell. This action is complemented by the local production of DILPs in the target tissues in a manner similar to the expression of IGF-1 in target tissues. (2) DILPs exert their effect on growth indirectly. The m-NSCs project their axon terminals into the ring gland where ecdysone and JH are synthesized. The stimulation of ecdysone synthesis by insulin-like peptides is well documented in many insects. Furthermore, JH levels are reduced in long-lived insulin receptor mutant flies, suggesting that DILPs also regulate JH synthesis. Through the regulation of the levels of one or both of these two hormones, DILPs may regulate growth indirectly. Under starvation conditions, reduced JH levels may result in the premature increase in ecdysone titer in third instar larvae, leading to the precocious initiation of metamorphosis and thus producing flies with fewer cells. Alternatively, a precocious rise in ecdysone titer may be caused by the increase in the local concentration of DILPs in the ring gland due to the increased retention of insulin-like peptides in the corpora cardiaca during starvation (Ikeya, 2002).

In nematodes, nutrient availability regulates the developmental program and fertility without having a direct effect on cell size or cell number. In the absence of food, the larvae enter the immature long-lived Dauer stage. This response is controlled by two pathways, the insulin signaling pathway and the daf-4/TGF-ß pathway. Each of these pathways acts nonautonomously in the nervous system, and they converge on the nuclear hormone receptor daf-12. This implies an intermediate steroid or lipid hormone signal. Indeed, daf-9 that acts genetically between daf-2 and daf-4 signaling encodes a cytochrome P450 enzyme involved in steroid and fatty acid metabolism. It is interesting to note that the two activities of the Prothoracicotropic hormone (PTTH) that regulate ecdysone synthesis in Bombyx involve a member of the TGF-ß superfamily and an insulin-related peptide synthesized in distinct sets of neurosecretory cells in the brain. Therefore, it is likely that the nutrient-dependent growth regulation in nematodes and Drosophila is conserved in spite of the absence of an autonomous requirement of insulin signaling in cell growth in C. elegans (Ikeya, 2002).

Egg maturation is blocked by starvation in many species. In insects, ecdysone produced by the ovary is required for yolk protein production in the fat body and oocyte maturation. Ecdysone production is stimulated by insulin-like peptides in vitro and in vivo. Ablation of m-NSCs significantly slows down oogenesis. In humans, brain-specific knockouts of the insulin receptor or IRS2 also block oocyte maturation by affecting the synthesis of gonadotropins. Furthermore, steroidogenesis in the female gonad is required for oocyte maturation and is regulated by the expression of IGF-1 in different gonadal cells. Interestingly, expression of dilp5 is also detected in the ovarian follicle cells in Drosophila. Local production of DILP5 may stimulate ecdysone production in the female ovary directly. The similar roles of insulin-related peptides in growth regulation, energy homeostasis, and oogenesis in nematodes, insects, and mammals are striking. How far the underlying mechanisms are also conserved remains to be investigated (Ikeya, 2002).

Specific insulin-like peptides encode sensory information to regulate distinct developmental processes

An insulin-like signaling pathway mediates the environmental influence on the switch between the C. elegans developmental programs of reproductive growth versus dauer arrest. However, the specific role of endogenous insulin-like peptide (ILP) ligands in mediating the switch between these programs remains unknown. C. elegans has 40 putative insulin-like genes, many of which are expressed in sensory neurons and interneurons, raising the intriguing possibility that ILPs encode different environmental information to regulate the entry into, and exit from, dauer arrest. These two developmental switches can have different regulatory requirements: this study shows that the relative importance of three different ILPs varies between dauer entry and exit. Not only was one ILP, ins-1, found to ensure dauer arrest under harsh environments, and two other ILPs, daf-28 and ins-6, were found to ensure reproductive growth under good conditions, it was also shown that daf-28 and ins-6 have non-redundant functions in regulating these developmental switches. Notably, daf-28 plays a more primary role in inhibiting dauer entry, whereas ins-6 has a more significant role in promoting dauer exit. Moreover, the switch into dauer arrest surprisingly shifts ins-6 transcriptional expression from a set of dauer-inhibiting sensory neurons to a different set of neurons, where it promotes dauer exit. Together, these data suggest that specific ILPs generate precise responses to dauer-inducing cues, such as pheromones and low food levels, to control development through stimulus-regulated expression in different neurons (Cotnild, 2011).

The unfolded protein response in a pair of sensory neurons promotes entry of C. elegans into dauer diapause

In response to unfavorable environmental conditions such as starvation, crowding, and elevated temperature, Caenorhabditis elegans larvae enter an alternative developmental stage known as dauer, which is characterized by adaptive changes in stress resistance and metabolism. The genetic dissection of the molecular mechanisms of the C. elegans dauer developmental decision has defined evolutionarily conserved signaling pathways of organismal neuroendocrine physiology. This study has identified a mechanism by which a dominant mutation in a neuronal insulin gene, daf-28^sa191, causes constitutive entry into dauer diapause. Expression of the mutant DAF-28 insulin peptide results in endoplasmic reticulum (ER) stress in the ASI pair of chemosensory neurons. The neuronal ER stress does not compromise cellular survival but activates PEK-1, the C. elegans ortholog of the mammalian eIF2alpha kinase PERK, which in turn phosphorylates Ser49 of eIF2alpha, specifically in the ASI neuron pair, to promote entry into dauer diapause. These data establish a novel role for ER stress and the unfolded protein response (UPR) in promoting entry into dauer diapause and suggest that, in addition to cell-autonomous activities in the maintenance of ER homeostasis, the UPR may act in a non-cell-autonomous manner to promote organismal adaptation to stress during larval development (Kulalert, 2013).

Search PubMed for articles about Drosophila Dilp2

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