InteractiveFly: GeneBrief

Ecdysone-inducible gene L2: Biological Overview | References

BIOLOGICAL OVERVIEW

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 (Hwa, 1999). 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 (Yamanaka, 1997). 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 (Osterbur, 1998; Natzle, 1986). 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 (Yamanaka, 1997). 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 (Sloth Anderson, 2000; Honegger, 2008).

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 (Sloth Anderson, 2000). 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 (Boisclair, 2001). 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 (Boisclair, 2001). 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 (Britton, 2002), 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 (Britton, 2002). Upon starvation, the expression of dilp3 and dilp5 is suppressed at the transcriptional level in the m-NSCs (Ikeya, 2002). 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).

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).

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).

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).

Muscle mitohormesis promotes longevity via systemic repression of insulin signaling

Mitochondrial dysfunction is usually associated with aging. An emerging area of investigation focuses on characterizing the molecular mechanisms of the adaptive cytoprotective responses to low levels of stress, in particular, oxidative stress in the mitochondrion (also referred to as mitohormesis). To systematically characterize the compensatory stress signaling cascades triggered in response to muscle mitochondrial perturbation, this study analyzed a Drosophila model of muscle mitochondrial injury. Mild muscle mitochondrial distress was found to preserve mitochondrial function, impede the age-dependent deterioration of muscle function and architecture, and prolong lifespan. Strikingly, this effect is mediated by at least two prolongevity compensatory signaling modules: one involving a muscle-restricted redox-dependent induction of genes that regulate the mitochondrial unfolded protein response (UPR^mt) and another involving the transcriptional induction of the Drosophila ortholog of insulin-like growth factor-binding protein 7 Ecdysone-inducible gene L2), which systemically antagonizes insulin signaling and facilitates mitophagy. Given that several secreted IGF-binding proteins (IGFBPs) exist in mammals, this work raises the possibility that muscle mitochondrial injury in humans may similarly result in the secretion of IGFBPs, with important ramifications for diseases associated with aberrant insulin signaling (Owusu-Ansah, 2013).

This study has shown that forced expression of genes that regulate the UPR^mt is sufficient to retard age-dependent mitochondrial and muscle functional impairment, and overexpression of antioxidant enzymes abolishes the protective effects of muscle mitohormesis due to complex I perturbation. It is noteworthy that exercise physiologists have long acknowledged that interventions to reduce the supposed redox damage in muscles following a bout of physical exercise may actually result in unfavorable alterations to the expression of cytoprotective genes. The current results indicate that a plausible explanation for this effect is that antioxidant treatment dampens the extent of activation of ROS-mediated signaling cascades, culminating in lower levels of mitochondrial repair/ maintenance genes required for reestablishing muscle homeostasis following exercise. This work, coupled with observations in other systems, strengthens the emerging concept that ROS serve as signaling molecules that engage specific signal transduction cascades (Owusu-Ansah, 2013).

A striking finding of this study is that muscle mitochondrial distress upregulates the insulin-antagonizing peptide (ImpL2) which nonautonomously represses insulin signaling. Under adverse environmental conditions, different organs must have the capacity to mount a coordinated adaptive response to the stressor. For instance, the recently discovered exercise-induced Irisin can increase energy expenditure to improve glucose homeostasis. It is interesting that ImpL2 secretion in response to muscle mitochondrial distress also acts in an adaptive manner by stimulating lysosome biogenesis, possibly to enhance prosurvival autophagy, an event that is critical for survival under stress. Notably, in an analogous situation in the heart, sublethal ischemia is known to trigger the release of cardiomyokines that act in an adaptive manner to preserve myocardial tissue health (Owusu-Ansah, 2013).

The idea that mitochondrial respiratory chain deficiency in one tissue can alter events in another tissue has also been observed in C. elegans, where RNAi-mediated knockdown of cytochrome c oxidase-1 subunit Vb in neurons activates the UPR^mt autonomously in neurons and nonautonomously in the gut. As a plausible explanation for the nonautonomous effect of mitochondrial respiratory chain deficiency, the authors hypothesized that a specific signal(s) may be secreted in response to mitochondrial dysfunction in one tissue, to subsequently propagate the mitochondrial stress signal to other tissues. Current studies in Drosophila show that in addition to the UPR^mt other mitochondrial stress responses (in this case, insulin repression) can be transmitted between different organs as well. Accordingly, there may be multiple mechanisms and longevity-promoting signals required for propagating mitochondrial stress responses between organs/tissues (Owusu-Ansah, 2013).

It has been hypothesized that the various compensatory signaling modules activated in response to mitochondrial dysfunction are not necessarily independent processes but, rather, are a part of a complex mitochondrial regulatory network with multiple axes operating in unison to enhance survival under stress. Interestingly, the data support this notion; at least two mitochondrial quality control processes - the UPR^mt and mitophagy - are concurrently active in complex I-disrupted muscles. Importantly, whereas forced expression of UPR^mt genes recapitulates many of the phenotypes associated with the preservation of mitochondrial function, lifespan increase was less robust, when compared to the effect of ImpL2 overexpression. Thus, it appears that the UPR^mt and ImpL2-dependent pathways regulate different facets of an intricate mitochondrial stress response network: induction of the UPR^mt serves primarily to preserve or restore mitochondrial function, whereas ImpL2 induction increases lysosome biogenesis that will enhance the clearance of damaged mitochondria through mitophagy. Interestingly, by selectively culling dysfunctional mitochondria from an otherwise normal pool, mitophagy may augment lifespan by ensuring the propagation of mitochondria with optimum function. The mechanism(s) triggering the longevity-enhancing effect of ImpL2 is likely to extend beyond mitophagy because ImpL2 may help remove misfolded protein aggregates, as has been shown for FOXO/4E-BP signaling. In addition, 4E-BP overexpression is sufficient to increase lifespan and is required for regulating metabolism under stress; accordingly, it may play a role in this context as well. Undoubtedly, future studies should help resolve the full breadth of cytoprotective processes that contribute to the lifespan-promoting effect of ImpL2 (Owusu-Ansah, 2013).

In summary, this study has uncovered a mechanism by which mitochondrial perturbation in muscles can cause systemic effects. Given that the human ortholog of ImpL2 (IGFBP7) has recently been shown to bind to the IGF-1 receptor and blocks activation of insulin-like growth factors, it is enticing to speculate that muscle mitochondrial injury in humans could also lead to upregulation of IGFBP7 (or other IGFBPs) to cause systemic repression of insulin signaling. Such an event will have implications for the known association between mitochondrial dysfunction and diseases associated with aberrant insulin signaling, such as type 2 diabetes. Interestingly, circulating IGFBP7 levels are elevated in patients with type 2 diabetes; whether this elevation is due partly to muscle mitochondrial dysfunction remains to be tested. Unquestionably, future studies in Drosophila and other systems to identify additional signaling molecules elevated in response to mitochondrial perturbation are likely to open up therapeutic opportunities for many metabolic diseases (Owusu-Ansah, 2013).

Systemic organ wasting induced by localized expression of the secreted Insulin/IGF antagonist ImpL2

Organ wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. A model for organ wasting in adult Drosophila is described, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, this study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism (Kwon, 2015).

This study describes the unexpected observation that the overproliferating midgut due to aberrant Yki activity in ISCs induces the bloating syndrome and systemic organ wasting. Additionally, the overproliferating midgut perturbs organismal metabolism, resulting in an increase of hemolymph trehalose and depletion of glycogen and triglyceride storage. Strikingly, it was shown that the accumulation of hemolymph trehalose and organ-wasting processes are dependent on the antagonist of insulin/IGF signaling, ImpL2, which is specifically upregulated in the proliferating midgut. This study provides strong genetic evidence supporting that systemic organ wasting associated with the aberrant activation of Yki in ISCs cannot be explained solely by the perturbation of general gut function. Based on these findings, it is proposed that ImpL2 is a critical factor involved in systemic organ wasting in Drosophila (Kwon, 2015).

An accompanying paper (Figueroa-Clarevega, 2015) shows that transplantation of scrib¹/Ras^V12 disc tumors into wild-type flies induces the bloating syndrome phenotype and systemic organ wasting, affecting ovaries, fat bodies, and muscles. That study also identified ImpL2 as a tumor-driven factor that plays a critical role in the organ-wasting process. These results are consistent with earlier findings and indicate that the bloating syndrome and organ-wasting phenotypes are not associated specifically with perturbation of gut function. Interestingly, Figueroa-Clarevega and Bilder observe that disc tumors derived by the expression of yki^S/A (an active form of yki that is less potent than yki^act used in this study) did not cause organ wasting, which can be explained by the low level of ImpL2 induction in the ^ykiS/A tumors as compared to scrib1/RasV12 tumors (Kwon, 2015).

The current results do not rule out the existence of an additional factor(s) contributing to the bloating syndrome and organ-wasting phenotypes. Indeed, the partial rescue of the bloating syndrome and organ-wasting phenotypes by depletion of ImpL2 in esg^ts>yki^act midguts suggests the existence of an additional factor(s). Moreover, this study observed that ectopic expression of ImpL2 in ECs was not sufficient to reduce whole-body triglyceride and glycogen levels, although it caused hyperglycemia, reduction of Akt1 phosphorylation, and increase of hemolymph volume. Thus, given the involvement of diverse factors in the wasting process in mammals, it is likely that in addition to ImpL2, another factor(s) contributes to systemic organ wasting in Drosophila (Kwon, 2015).

This study shows that the bloating syndrome caused by esg^ts>yki^act is associated with ImpL2, as depletion of ImpL2 from esg^ts>yki^act midguts significantly rescues the bloating phenotype. Given the observation that elevated expression of ImpL2 from esg^ts>yki^act midgut induces hyperglycemia, it is speculated that the accumulation of trehalose in hemolymph is a factor involved in bloating, because a high concentration of trehalose can cause water influx to adjust hemolymph osmolarity to physiological levels. Interestingly, recent findings have shown that disruption of l(2)gl in discs activates yki, suggesting that the bloating syndrome observed in flies with transplanted l(2)gl mutant discs may be due to aberrant yki activity (Kwon, 2015).

The current findings are reminiscent of a previous study showing that in Drosophila, humoral infection with the bacterial pathogen Mycobacterium marinum (closely related to Mycobacterium tuberculosis) causes a progressive loss of energy stores in the form of fat and glycogen—a wasting-like phenotype. Similar to the current observation, the previous study found that infection with M. marinum caused a downregulation of Akt1 phosphorylation. Given the observation that ImpL2 produced from esg^ts>yki^act affects systemic insulin/IGF signaling, it will be of interest to test whether ImpL2 expression is increased upon infection with M. marinum and mediates the effect on the loss of fat and glycogen storage (Kwon, 2015).

yki plays critical roles in tissue growth, repair, and regeneration by inducing cell proliferation, a process requiring additional nutrients to support rapid synthesis of macromolecules including lipids, proteins, and nucleotides. In particular, increased aerobic glycolysis metabolizing glucose into lactate is a characteristic feature of many cancerous and normal proliferating cells. Interestingly, the aberrant activation of yki in ISCs caused a disparity in the gene expression of glycolytic enzymes and the activity of insulin/IGF signaling between the proliferating midgut and other tissues, such as muscle and ovaries. Thus, it is speculated that this disparity favors Yki-induced cell proliferation by increasing the availability of trehalose/glucose to the proliferating midgut, which presumably requires high levels of trehalose/glucose. Additionally, it will be of interest to test whether activation of Yki during tissue growth, repair, and regeneration alters systemic metabolism in a similar manner (Kwon, 2015).

Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues

Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on losing access to these signals. This study demonstrates that differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot properly differentiate. An insulin peptide produced by somatic cells immediately outside of the stem cell niche was found to act locally to promote somatic differentiation through Insulin receptor (InR) activation. These results indicate that there is a local 'differentiation' niche which upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, it was demonstrated that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, this study shows that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that local production of positive and negative InR signals regulate the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation is actively induced by signaling (Amoyel, 2016).

This study shows that PI3K/Tor activity is required for the differentiation of somatic stem cells in the Drosophila testis. Additionally, a 'differentiation' niche was identified immediately adjacent to the stem cell niche that, through the local production of Dilps, leads to the upregulation of PI3K/Tor activity in early CySC daughters and to their commitment to differentiation. The secretion of ImpL2 by CySCs antagonizes the initiation of differentiation in CySCs by blocking available Dilps in the stem cell niche. As a result, CySCs receive little free Dilp ligands. However, as their daughters move away from the hub, they encounter increasing levels of Dilps and decreasing levels of ImpL2, which leads to the upregulation of PI3K/Tor signaling and proper somatic cell differentiation. The fact that ImpL2 is upregulated by the main self-renewal signal (i.e., JAK/STAT) in CySCs leads to a model accounting for the spatial separation of the stem cell niche and the differentiation niche (Amoyel, 2016).

The results are consistent with a model in which autocrine or paracrine production of Dilp6by early cyst cells serves as a differentiation niche in the testis, defining where in the tissue upregulation PI3K/Tor signaling - a prerequisite for differentiation - occurs. This differentiation niche is critical for somatic development because stem cell markers like Zfh1 are maintained in the absence of signals like PI3K/Tor. Notably, JAK/STAT activity is not expanded outside of the niche upon somatic loss of PI3K/Tor signaling, suggesting that differentiation signals play a critical role in downregulating stem cell factors. Intriguingly, recent studies in the Drosophila ovary have identified a differentiation niche in this tissue: autocrine Wnt ligands produced by somatic support escort cells regulate escort cell function, proliferation and viability. Taken together, these studies reveal that at least in Drosophila gonads, there is a defined region immediate adjacent to the stem cell niche where autocrine production of secreted factors induces the differentiation of somatic cells, which in turn promote development of the germ line (Amoyel, 2016).

Several studies have examined the role of insulin signaling in gonadal stem cells. In both testes and ovaries, systemic Dilps have been shown to affect stem cell behavior. In both tissues, nutrition through regulation of systemic insulin controls the proliferation rate of GSCs. The current data showing that Akt1, Dp110 or Tor mutant CySC clones proliferate poorly are consistent with these findings and indicate that basal levels of insulin signaling are required for the proliferation and/or survival of both stem cell pools in the testis. This work also demonstrates that production of a secreted Insulin binding protein ImpL2 by CySCs reduces available Dilps in the stem cell niche, and ImpL2 in the niche milieu should reduce insulin signaling in GSCs and CySCs. While these data seemingly contradict the results that insulin is required for GSC maintenance, a model is suggested in which low constitutive levels of insulin signaling are required for stem cell proliferation and that higher levels are required to induce stem cell differentiation. (Amoyel, 2016).

Prior reports have found that both male and female flies with reduced Insulin or Tor activity are sterile, and the results presented in this study suggest that this is due at least in part to a lack of somatic cell differentiation. The results indicate that Dilp6, the IGF homolog, plays a local role in CySC differentiation, but acts redundantly with other presumably systemic factors, suggesting that both constitutive and nutrient-responsive inputs control CySC differentiation. Indeed, this study shows that in addition to controlling the proliferation of stem cells, systemic insulin is required for their differentiation, as the poorly proliferative Akt1, Dp110 or Tor mutant CySC clones do not differentiate and eventually die by apoptosis. This combination of reduced proliferation and increased apoptosis may explain why other studies suggest that Tor is required for self- renewal in GSCs; indeed prior reports indicate that while Tor mutant GSCs are lost, hyper-activation of Tor leads to faster loss of GSCs through differentiation and recent work indicates that lineage-wide Tor loss blocks the differentiation of GSCs. The use of hypomorphic alleles enabled a genetic separation of the proliferative effects and differentiation requirements of PI3K and Tor in CySCs. Finally, there is evidence that PI3K/Tor activity promotes differentiation of stem cells in gonads in mammals, suggesting that these findings may reflect a conserved role of Tor activity in promoting germ cell differentiation, both through autonomous and non- autonomous mechanisms involving somatic support cells. Moreover, it seems likely that Tor activity may be a more general requirement for the differentiation of many stem cell types, as increased PI3K or Tor has been shown to induce differentiation in many instances. In particular, mouse long term hematopoietic stem cells are lost to differentiation when the PI3K inhibitor Pten is mutated, while Drosophila intestinal stem cells differentiate when Tor is hyperactive due to Tsc1/2 complex inactivation. Moreover, inhibition of Tor activity by Rapamycin promotes cellular reprogramming to pluripotency, while cells with increased Tor activity cannot be reprogrammed, suggesting a conserved role for Tor signaling in promoting differentiated states (Amoyel, 2016).

Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting

Tumors kill patients not only through well-characterized perturbations to their local environment but also through poorly understood pathophysiological interactions with distant tissues. This study uses a Drosophila tumor model to investigate the elusive mechanisms underlying such long-range interactions. Transplantation of tumors into adults induced robust wasting of adipose, muscle, and gonadal tissues that were distant from the tumor, phenotypes that resembled the cancer cachexia seen in human patients. Notably, malignant, but not benign, tumors induced peripheral wasting. The study identified the insulin growth factor binding protein (IGFBP) homolog ImpL2, an antagonist of insulin signaling, as a secreted factor mediating wasting. ImpL2 was sufficient to drive tissue loss, and insulin activity was reduced in peripheral tissues of tumor-bearing hosts. Importantly, knocking down ImpL2, specifically in the tumor, ameliorated wasting phenotypes. The study proposes that the tumor-secreted IGFBP creates insulin resistance in distant tissues, thus driving a systemic wasting response (Figueroa-Clarevega, 2005).

Cachexia remains a major obstacle to cancer treatment, in part because the molecular mechanisms that drive it remain uncertain. This study describes a fly model that mimics certain aspects of human cachexia and utilize this model to identify a specific cachectic mediator. The tumor-induced wasting describe in flies resembles cancer cachexia in its independence from food consumption, its target tissues, its progressive nature, and its induction by certain but not all types of tumors. The fly model does not parallel all features associated with the human condition; for instance, only slight upregulation of putative fly orthologs of mammalian regulators implicated in muscle catabolism. Human cancer cachexia is clearly a heterogeneous and multifactorial condition, and this complexity has impeded progress in its understanding. This work used a reductionist system to identify a single tumor-derived factor that can drive the robust deterioration of peripheral tissues (Figueroa-Clarevega, 2015).

Insulin signaling is a central regulator of tissue mass in both flies and humans. These data demonstrate that ImpL2, a secreted insulin antagonist produced by malignant tumors, is a major mediator that is both necessary and sufficient for wasting. In an accompanying paper in the issue of Developmental Cell, Kwon, (2015) shows that ImpL2 is also a systemic wasting factor in a different fly tumor model. Reduced insulin signaling is further responsible for wasting induced by mycobacterial infection of flies; whether ImpL2 is the relevant mediator in this case is not known. ImpL2 is the single fly homolog of mammalian IGFBPs and can bind to systemic insulin-like ligands to antagonize insulin signaling. By this mechanism, the tumor effectively induces insulin resistance in peripheral tissues (Figueroa-Clarevega, 2015).

Insulin resistance is a frequent feature of both cachectic patients and rodent cachexia models; indeed, some evidence suggests that exogenous insulin can ameliorate tissue loss in these contexts. The seven mammalian IGFBPs are variously upregulated or downregulated in different tumors, but they have been evaluated in cancer, primarily with respect to their affects on tumor growth. These data motivate assessments of whether highly cachectogenic human tumors, such as pancreatic and gastric cancers, display elevated expression of IGFBPs and how therapies designed to correct insulin resistance might be used to treat such tumors (Figueroa-Clarevega, 2015).

ImpL2 joins the list of effectors induced by neoplastic transformation in fly tumors, including mitogens and pro-invasive factors. Recent work shows that the Upd3 mitogen is upregulated by dual activity of JNK and Hippo signaling. The ImpL2 regulatory region, like that of Upd3, contains evolutionarily conserved binding sites for AP-1 and Sd transcription factors, suggesting that it may also be synergistically regulated by these pathways that monitor epithelial integrity. Despite the reduced insulin signaling in neoplastic tumors themselves (e.g., 4EBP levels are elevated ∼21-fold, and they are hypersensitive to PI3K reduction, the tumors nevertheless robustly proliferate. How ImpL2-upregulating tumors escape insulin resistance remains an unanswered question, although metabolic changes suggested by transcriptome alterations may be a possible mechanism (Figueroa-Clarevega, 2015).

While tumor-specific inhibition of ImpL2 causes a significant amelioration of the wasting phenotype, rescue is not complete, suggesting that other aspects of tumor-host interaction remain to be uncovered. A fly homolog of IL-6 was found, a molecule implicated in several rodent cachexia models, was not sufficient to induce wasting, while partial ablation of host innate immune cells did not qualitatively alter wasting phenotypes; however, contributing roles for these factors have not been ruled out. Future work will analyze other tumor-produced factors, including metabolites generated by anabolic and catabolic alterations in the tumor, to evaluate their involvement as well. The manipulability of the simple model developed here, including the ability to rapidly assess fully defined combinations of host and tumor genotypes, opens the door to candidate as well as forward genetic approaches to identify additional factors mediating tumor-host interactions (Figueroa-Clarevega, 2015).

MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells

Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR-8, functions in the fat body to non-autonomously regulate organismal growth, suggesting that fat body-derived humoral factors are regulated by imiR-8. This study found that several putative peptide hormones known to have mitogenic effects are regulated by imiR-8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase-related growth factors are up-regulated in the absence of imiR-8. Drosophila insulin-like peptide 6 (Dilp6) and Imaginal morphogenesis protein-late 2 (Imp-L2), a binding partner of Dilp, are also up-regulated in the fat body of miR-8 null mutant larvae. The fat body-specific reintroduction of miR-8 into the miR-8 null mutants revealed six peptides that showed fat-body organ-autonomous regulation by miR-8. Amongst them, only Imp-L2 was found to be regulated by U-shaped, the miR-8 target for body growth. However, a rescue experiment by knockdown of Imp-L2 indicated that Imp-L2 alone does not account for miR-8's control over the insect's growth. These findings suggest that multiple peptide hormones regulated by miR-8 in the fat body may collectively contribute to Drosophila growth (Lee, 2014).

Male-specific splicing of the silkworm Imp gene is maintained by an autoregulatory mechanism

Sexual differentiation in the silkworm Bombyx mori is controlled by sex-specific splicing of Bmdsx, in which exons 3 and 4 are skipped in males. B. mori insulin-like growth factor II mRNA-binding protein (Imp) is a factor involved in the male-specific splicing of Bmdsx. This study found that the male-specific Imp mRNA is formed as a result of the inclusion of exon 8 and the promoter-distal poly(A) site choice, whereas non-sex-specific polyadenylation occurs at the promoter-proximal poly(A) site downstream of exon 7. Recent studies revealed that Drosophila Sxl, tra in several dipteran and hymenopteran insects, and femM in Apis mellifera, play a central role in sex determination and maintain their productive mode of expression via an autoregulatory function. To determine whether Imp protein is required for the maintenance of the male-specific splicing of its own pre-mRNA, endogenous Imp was knocked down in male cells, and the male-specific splicing of an exogenous Imp minigene was assessed. Knockdown of endogenous Imp inhibited the male-specific splicing of the Imp minigene transcript. In contrast, overexpression of Imp in female cells induced the male-specific splicing of the Imp minigene transcript. Moreover, deletion of adenine-rich (A-rich) sequences located downstream of the proximal poly(A) site repressed the male-specific splicing of the Imp minigene transcript. Finally, gel shift analysis demonstrated that Imp binds to the A-rich sequences. These data suggest that Imp binds to the A-rich sequences in its own pre-mRNA to induce the male-specific splicing of its pre-mRNA (Suzuki, 2014).

Roles of PINK1 in regulation of systemic growth inhibition induced by mutations of PTEN in Drosophila

The maintenance of mitochondrial homeostasis requires PTEN-induced kinase 1 (PINK1)-dependent mitophagy, and mutations in PINK1 are associated with Parkinson's disease (PD). PINK1 is also downregulated in tumor cells with PTEN mutations. However, there is limited information concerning the role of PINK1 in tissue growth and tumorigenesis. This study shows that the loss of pink1 caused multiple growth defects independent of its pathological target, Parkin. Moreover, knocking down pink1 in muscle cells induced hyperglycemia and limited systemic organismal growth by the induction of Imaginal morphogenesis protein-Late 2 (ImpL2). Similarly, disrupting PTEN activity in multiple tissues impaired systemic growth by reducing pink1 expression, resembling wasting-like syndrome in cancer patients. Furthermore, the re-expression of PINK1 fully rescued defects in carbohydrate metabolism and systemic growth induced by the tissue-specific pten mutations. These data suggest a function for PINK1 in regulating systemic growth in Drosophila and shed light on its role in wasting in the context of PTEN mutations (Han, 2021).

This study reports that PINK1 regulated systemic growth in a non-autonomous manner. The loss of PINK1 in muscle cells specifically upregulated the insulin/IGF signaling antagonist ImpL2, thus inhibiting organ growth by dysregulating carbohydrate metabolism and insulin signaling. Furthermore, strong genetic evidence is provided that PINK1 activity is dispensable for cell growth but absolutely required for the induction of tissue wasting following the knockdown of the tumor suppressor gene PTEN. This effect is mediated through systemic insulin signaling. In sharp contrast to wild-type cells, PTEN mutant cells retain high levels of membrane PIP3 independent of InR/PI3K, thus maintaining high cellular insulin signaling and a high rate of cell growth despite low circulatory insulin activities. Thus, mutations in PTEN enhance cell growth by maintaining high levels of insulin signaling, but they also inhibit the growth of other tissues in a non-autonomous manner through the transcriptional repression of PINK1 (Han, 2021).

Although induced by the tumor suppressor PTEN, PINK1 does not inhibit cell growth in a number of cancer cell lines. Instead, PINK1 has been shown to promote cell growth and proliferation in various models. This study demonstrated that in the fly fat body, wing, and eye, the loss of PINK1 impaired cell growth. This is the opposite of PTEN, which leads to overgrowth when mutated. Moreover, despite the fact that the expression of PINK1 is reduced by mutations in pten, ectopic expression of PINK1 does not affect the growth of either wild-type or pten mutant cells, indicating that PINK1 may not simply function as a regulator of cellular growth, particularly in pten-associated tumors. Interestingly, knocking down pten or pink1 in specific tissues reduces the insulin signaling and cell growth of peripheral tissue and leads to hyperglycemia and weight loss. Moreover, re-expression of PINK1 completely rescued the insulin resistance and systemic growth inhibition caused by tissue-specific mutations of pten. Supporting these findings, pten mutant clones have been shown to reduce the insulin signaling and growth potential of surrounding tissues. Furthermore, this study found that the secreted insulin/IGF antagonist impl2 is highly expressed upon the loss of both pink1 and pten and is reduced to wild-type levels by the re-expression of PINK1. Importantly, the systemic growth defects resulting from the tissue-specific knockdown of pten or pink1 were completely rescued by reducing impl2 to wild-type levels. These data suggest that reducing PINK1 expression may mediated cachexia and systemic metabolic dysfunction induced by tumors associated with PTEN mutations (Han, 2021).

Much like PTEN mutations, tumors induced in the adult Drosophila midgut by expressing the oncogene Yorkie (Yki) in intestine stem cells, or by transplanting tumors that express RasV12 in cells mutant for scribble, also secrete high levels of ImpL2, thereby reducing systemic insulin/IGF signaling and inducing organ wasting. It was established that the overexpression of ImpL2 in specific tissues in flies led to wasting in surrounding tissues. ImpL2 is a major mediator that is both necessary and sufficient for wasting. Together with the current results, these data suggest that a common mechanism underlying tumor cachexia may be the disruption of systemic insulin/IGF signaling that then results in hyperglycemia, insulin resistance, and organ wasting. There are seven IGF-binding proteins (IGFBPs) in mammals, which are dysregulated in different kinds of tumors. In the context of cancer, analyses of IGFBPs have largely focused on their effects on tumor growth. Recently, IGFBP-3 was found to be upregulated in human pancreatic cancer, supporting a potential role for insulin-binding peptides in the induction of tumor cachexia. Moreover, IGFBP-7 expression is associated with tumor growth and functions as a tumor suppressor in the colon, rectum, breast, and thyroid by either directly inducing apoptosis or inhibiting tumor growth via the deregulation of p16, p21, p53, and ERK signaling. In glioblastoma, however, IGFBP-7 plays an oncogenic role and stimulates tumor cell proliferation. It is possible that besides functions in tumor cells, IGFBP-7 may play a role in tumor cachexia as ImpL2 in flies (Han, 2021).

The InR/PI3K cascades in tumor cells with expression of Yki and RasV12 may also be shut down by reduced circulatory insulin/IGF signaling. This suggests that other mechanisms of organ wasting may exist. It has recently been demonstrated that Yki-associated tumors produce the epidermal growth factor receptor (EGFR) ligand, Vein, to trigger autonomous EGFR signaling and produce the platelet-derived growth factor (PDGF)- and vascular endothelial growth factor (VEGF)-related factor 1 (Pvf1) ligand to non-autonomously activate PVR (PDGF and VEGF receptor-related) signaling and wasting in peripheral cells. For PTEN mutant tumors, PI3K/AKT signals remain high regardless of circulatory ImpL2 level. This is explained by the function of PTEN as a negative regulator of AKT downstream of InR/PI3K. The re-expression of impl2 completely abolished the systemic growth defects without affecting the overgrowth of pten mutant cells. Therefore, it is suggested that the induction of ImpL2 expression by mutations in pten through PINK1 is both necessary and sufficient for peripheral organ wasting. The results, together with previous studies, highlight the importance of dissecting metabolic responses to cancer. The focus of cancer research is gradually expanding, from cancer cells to the tumor microenvironment to the system as a whole. In addition, it will be interesting to test whether some malignant tumors that harbor PTEN and/or PINK1 mutations (e.g., glioblastoma) display the elevated expression of IGFBPs, which alter systemic metabolism in a manner similar to Drosophila. Furthermore, these findings may provide a new strategy for treating such tumors (Han, 2021).

PTEN is endowed with both lipid and protein phosphatase activities. Because PTEN functionally antagonizes the lipid kinase PI3K, the depletion of PTEN results in high levels of the lipid second messenger PIP3, resulting in the increased membrane recruitment and activation of Akt. This leads to enhanced cellular growth, proliferation, and survival. PINK1 promotes cell growth and prevents cell death through its cytosolic downstream targets, such as mTORC2 and p53. However, PINK1 has also been shown to function inside mitochondria to promote the activity of complex I of the mitochondrial respiratory chain, and a recent study using three-dimensional (3D) structured illumination super-resolution microscopy suggested that under physiological conditions, PINK1 localizes to the cristae membrane and intracristae space. Moreover, endogenous PINK1 localizes to both cytosolic and mitochondrial fractions. Consistent with the localization of PINK1 to the IMM, this study found that targeting PINK1 to the IM fully restored complex I activity and rescued the growth defects of pink1 mutants. By contrast, OMM-localized PINK1 did not affect growth associated with pink1 mutation, but completely rescued muscle degeneration phenotypes of pink1 mutants associated with the loss of mitophagy. These data suggest that PINK1 activity is required inside mitochondria but not in the cytosol to promote complex I activity and cell growth. As a protein kinase, PINK1 kinase activity is required to maintain complex I activity in mitochondria, and PINK1 kinase activity in cytosol protects neurons. Inside mitochondria, PINK1 directly phosphorylates the NDUFA10/ND42 subunit to promote complex I activity. However, the overexpression of both wild-type and phosphorylation-mimic ND42 is not able to suppress the defective growth of pink1 mutants, indicating that multiple physiological targets of PINK1 may exist. It has been recently reported that PINK1 could phosphorylate the IMM protein MIC60 to promote complex I assembly and maintain oxidative phosphorylation. Nevertheless, PINK1 promotes growth when inside the mitochondria and protects cells against stress by initiating mitophagy when anchored to the OMM (Han, 2021).

Similar to knocking down pink1 in muscle cells, the disruption of complex I activity by muscle-specific knockdown of NDUFS1/ND75 reduces insulin signaling non-autonomously by inducing ImpL2 expression. Although the mechanisms are unclear, this work, together with the observations of other groups, strongly implies that the knockdown of pink1 disrupts the function of complex I, thus upregulating the expression of ImpL2 and inhibiting cell growth non-autonomously. These findings describe key functional associations between PINK1 and PTEN in tumorgenesis and systemic growth. This study shows that impaired PINK1-associated mitochondrial function plays a key role in wasting-like syndrome and reveal that protecting mitochondrial function in cancer patients may provide tremendous benefit (Han, 2021).

A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities

Insulin and related peptides are key hormones for the regulation of growth and metabolism. A novel high affinity insulin-related peptide-binding protein (IBP) is described that is secreted from cells of the insect Spodoptera frugiperda. This IBP is composed of two Ig-like C2 domains, has a molecular mass of 27 kDa, binds human insulin with an affinity of 70 pm, and inhibits insulin signaling through the insulin receptor. The binding protein also binds insulin-like growth factors I and II, proinsulin, mini-proinsulin, and an insulin analog lacking the last 8 amino acids of the B-chain (des-octa peptide insulin) with high affinity, whereas an insulin analog with a Asp-B10 mutation bound with only 1% of the affinity of human insulin. This binding profile suggests that IBP recognizes a region that is highly conserved in the insulin superfamily but distinct from the classical insulin receptor binding site. The closest homologue of the Spodoptera frugiperda binding protein is the essential gene product IMP-L2, found in Drosophila, where it is implicated in neural and ectodermal development (Garbe, 1993: Development 119, 1237-1250). This study shows that the IMP-L2 protein also binds insulin and related peptides, offering a possible functional explanation to the IMP-L2 null lethality (Andersen, 2000. Full text of article).

IMP-L2: an essential secreted immunoglobulin family member implicated in neural and ectodermal development in Drosophila.

The Drosophila IMP-L2 gene was identified as a 20-hydroxyecdysone-induced gene encoding a membrane-bound polysomal transcript. IMP-L2 is an apparent secreted member of the immunoglobulin superfamily. Deficiencies that remove the IMP-L2 gene were used to demonstrate that IMP-L2 is essential in Drosophila. The viability of IMP-L2 null zygotes is influenced by maternal IMP-L2. IMP-L2 null progeny from IMP-L2+ mothers exhibit a semilethal phenotype. IMP-L2 null progeny from IMP-L2 null mothers are 100% lethal. An IMP-L2 transgene completely suppresses the zygotic lethal phenotype and partially suppresses the lethality of IMP-L2 null progeny from IMP-L2 null mothers. In embryos, IMP-L2 mRNA is first expressed at the cellular blastoderm stage and continues to be expressed through subsequent development. IMP-L2 mRNA is detected in several sites including the ventral neuroectoderm, the tracheal pits, the pharynx and esophagus, and specific neuronal cell bodies. Staining of whole-mount embryos with anti-IMP-L2 antibodies shows that IMP-L2 protein is localized to specific neuronal structures late in embryogenesis. Expression of IMP-L2 protein in neuronal cells suggests a role in the normal development of the nervous system but no severe morphological abnormalities have been detected in IMP-L2 null embryos (Garbe, 1993. Full text of article).

Search PubMed for articles about Drosophila Imp-L2

Alic, N., Hoddinott, M. P., Vinti, G. and Partridge, L. (2011). Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor. Aging Cell 10: 137-147. PubMed ID: 21108726

Amoyel, M., Hillion, K. H., Margolis, S. R. and Bach, E. A. (2016). Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues. Development 143(21):3914-3925 PubMed ID: 27633989

Andersen, A. S., Hansen P. H., Schaffer, L. and Kristensen, C. (2000). A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities. J. Biol. Chem. 275(22): 16948-53. PubMed ID: 10748036

Arquier, N., et al. (2008). Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides. Cell Metab. 7(4): 333-8. PubMed ID: 18396139

Boisclair, Y. R., Rhoads, R. P., Ueki, I., Wang, J. and Ooi, G. T. (2001). The acid-labile subunit (ALS) of the 150 kDa IGF-binding protein complex: an important but forgotten component of the circulating IGF system. J. Endocrinol. 170: 63-70. PubMed ID: 11431138

Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. and Edgar, B. A. (2002). Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. PubMed ID: 11832249

Brogiolo, W., et al. (2001). An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr. Biol. 11: 213-221. PubMed ID: 11250149

Colombani, J., et al. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed ID: 14505573

Figueroa-Clarevega, A. and Bilder, D. (2015). Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting. Dev Cell 33: 47-55. PubMed ID: 25850672

Flatt, T., et al. (2008). Drosophila germ-line modulation of insulin signaling and lifespan. Proc. Natl. Acad. Sci. 105(17): 6368-73. PubMed ID: 18434551

Garbe, J. C., Yang, E. and Fristrom, J. W. (1993). IMP-L2: an essential secreted immunoglobulin family member implicated in neural and ectodermal development in Drosophila. Development 119(4): 1237-50. PubMed ID: 8306886

Han, Y., Zhuang, N. and Wang, T. (2021). Roles of PINK1 in regulation of systemic growth inhibition induced by mutations of PTEN in Drosophila. Cell Rep 34(12): 108875. PubMed ID: 33761355

Honegger, B., et al. (2008). Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance. J. Biol. 7(3): 10. PubMed ID: 18412985

Hwa, V., Oh, Y. and Rosenfeld, R. G. (1999). The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr. Rev. 20: 761-787. PubMed ID: 10605625

Ikeya, T., Galic, M., Belawat, P., Nairz, K. and Hafen, E (2002). Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12: 1293-1300. PubMed ID: 12176357

Kwon, Y., Song, W., Droujinine, I. A., Hu, Y., Asara, J. M. and Perrimon, N. (2015). Systemic organ wasting induced by localized expression of the secreted Insulin/IGF antagonist ImpL2. Dev Cell 33: 36-46. PubMed ID: 25850671

Lee, G. J., Jun, J. W. and Hyun, S. (2014). MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells. Insect Mol Biol. PubMed ID: 25492518

Natzle, J. E., Hammonds, A. S. and Fristrom, J. W. (1996). Isolation of genes active during hormone-induced morphogenesis in Drosophila imaginal discs. J. Biol. Chem. 261: 5575-5583. PubMed ID: 3007512

Osterbur, D. L., et al. (1988). Genes expressed during imaginal discs morphogenesis: IMP-L2, a gene expressed during imaginal disc and imaginal histoblast morphogenesis. Dev. Biol. 129: 439-448. PubMed ID: 2843403

Owusu-Ansah, E., Song, W. and Perrimon, N. (2013). Muscle mitohormesis promotes longevity via systemic repression of insulin signaling. Cell 155: 699-712. Abstract

Rulifson, E. J., Kim, S. K. and Nusse, R. (2002). Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296: 1118-1120. PubMed ID: 12004130

Sarraf-Zadeh, L., Christen, S., Sauer, U., Cognigni, P., Miguel-Aliaga, I., Stocker, H., Kohler, K. and Hafen, E. (2013). Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions. Dev Biol 381: 97-106. PubMed ID: 23773803

Sloth Andersen, A., et al. (2000). A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities. J. Biol. Chem. 275: 16948-16953. PubMed ID: 10748036

Suzuki, M. G., Kobayashi, S. and Aoki, F. (2013). Male-specific splicing of the silkworm Imp gene is maintained by an autoregulatory mechanism. Mech. Dev. [Epub ahead of print]. PubMed ID: 24231282

Wajapeyee, N., et al. (2008). Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 132: 363-374. PubMed ID: 18267069

Yamanaka, Y., Wilson, E. M., Rosenfeld, R. G. and Oh, Y. (1997): Inhibition of insulin receptor activation by insulin-like growth factor binding proteins. J. Biol. Chem. 272: 30729-30734. PubMed ID: 9388210

Biological Overview

date revised: 25 April 2024

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