InteractiveFly: GeneBrief

Insulin-like peptide 1: Biological Overview | References

BIOLOGICAL OVERVIEW

The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. Eight insulin-like peptides (DILP1-8) have been identified in Drosophila, and several of these are known to regulate growth, metabolism, reproduction, stress responses, and lifespan. However, the functional role of DILP1 is far from understood. Previous work has shown that dilp1/DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. The role of dilp1 in the pupa, as well as in the first week of adult life, was studied, and some comparisons were made to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. Mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. It was next show that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1/dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but the data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage (Liao, 2020).

The insulin/IGF signaling (IIS) pathway plays a central role in nutrient-dependent growth control during development, as well as in adult physiology and aging. More specifically, in mammals, insulin, IGFs, and relaxins act on different types of receptors to regulate metabolism, growth, and reproduction. This class of peptide hormones has been well conserved over evolution and therefore the genetically tractable fly Drosophila is an attractive model system for investigating IIS mechanisms. Eight insulin-like peptides (DILP1-8), each encoded on a separate gene, have been identified in Drosophila. The genes encoding these DILPs display differential temporal and tissue-specific expression profiles, suggesting that they have different functions. Specifically, DILP1, 2, 3, and 5 are mainly expressed in median neurosecretory cells located in the dorsal midline of the brain, designated insulin-producing cells (IPCs). The IPC-derived DILPs can be released into the open circulation from axon terminations in the corpora cardiaca, the anterior aorta, the foregut, and the crop. Genetic ablation of the IPCs reduces growth and alters metabolism, and results in increased resistance to several forms of stress and prolongs lifespan (Liao, 2020).

The functions of the individual DILPs produced by the IPCs may vary depending on the stage of the Drosophila life cycle. Already, the temporal expression patterns hint that DILP1-3 and 5 play different roles during development. Thus, whereas DILP2 and 5 are relatively highly expressed during larval and adult stages, DILP1 and 6 are almost exclusively expressed during pupal stages under normal conditions (Liao, 2020).

DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1 is different from the other insulin-like peptides tested (Liao, 2020).

DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1/DILP1 expression is also high (Liu, 2016). The temporal expression profile of dilp1/DILP1 resembles that of dilp6/DILP6 although the latter peptide is primarily produced by the fat body, not IPCs. Since DILP6 was shown to regulate growth of adult tissues during pupal development, it was asked whether also DILP1 plays a role in growth control. It is known that overexpression of several of the DILPs is sufficient to increase body growth through an increase in cell size and cell number, and especially DILP2 produces a substantial increase in body weight. In contrast, not all single dilp mutants display a decreased body mass. The dilp1, dilp2, and dilp6 single mutants display slightly decreased body weight, whereas the dilp3, dilp4, dilp5, and dilp7 single mutants display normal body weight. However, a triple mutation of dilp2, 3, and 5 causes a drastically reduced body weight, and a dilp1-4,5 mutation results in a further reduction. Note that several of the above studies do not show bona fide effects on cell or organismal growth (e.g., volume or cell numbers/sizes); they only provide body mass data (Liao, 2020).

There is a distinction between how DILPs act in growth regulation. DILPs other than DILP1 and DILP6 promote growth primarily during the larval stages (both feeding and wandering stages) when their expression is high. This nutrient-dependent growth is relatively well-understood and is critical for production of the steroid hormone ecdysone and thereby developmental timing and induction of developmental transitions such as larval molts and pupariation. The growth in the pupal stage, which primarily affects imaginal discs and therefore adult tissues, is far less studied. This study investigated the role of dilp1/DILP1 in growth regulation in Drosophila in comparison to dilp6/DILP6. For this, both bona fide size of body and/or wings were determined and wet weights were provided, and thus it was possible to distinguish between growth and increase of body mass. Mutation of dilp1 diminishes body weight (but not body size), whereas ectopic dilp1 expression promotes organismal growth by increasing both weight and size during the pupal stage, similar to dilp6. Thus, we cannot unequivocally show a role of dilp1 in organismal growth, but it does regulate body mass, suggesting that dilp1 affects metabolism and energy stores. Determination of metabolic rate (MR) and respiratory quotient (RQ) as well as triacylglyceride (TAG) levels during late pupal development provides evidence that dilp1 and dilp6 increase the MR and that the associated increased metabolic cost is fueled by increased lipid catabolism (Liao, 2020).

Since dilp1/DILP1 levels are high the first week of adult life, the role of dilp1 mutation and overexpression on early adult physiology was determined, including metabolism stress resistance and fecundity. Interestingly, the newly eclosed dilp1 mutant flies are less resistant to starvation than controls and dilp2 mutants. Thus, dilp1 acts differently from other dilps for which it has been shown that reduced signaling increases survival during starvation. Also, early egg laying and female fecundity are affected by dilp1 overexpression, and in general, dilp1 manipulations produce more prominent effects in female flies (Liao, 2020).

Taken together, these data suggest that ectopic expression of dilp1/DILP1 promotes growth of adult tissues during the pupal stage, and that this process mainly utilizes stored lipids to fuel the increased MR. The DILP1 signaling also affects the metabolism in the young adult fly, and sex dimorphic effects of altered signaling on stress responses and fecundity were seen (Liao, 2020).

This study shows that dilp1 gain of function stimulates adult tissue growth and increases metabolic rate (MR) during the pupal stage, and also affects adult physiology, especially during the first days of adult life. These stages correspond to the time when dilp1 is normally expressed. The gain of function experiments in this study suggest that the developmental role of ectopic dilp1 could be similar to that of dilp6, namely, to stimulate growth of adult tissues during pupal development. It was furthermore shown that in the adult fly, dilp1 is upregulated during starvation and genetic gain and loss of function of dilp1 signaling diminishes the flies' survival under starvation conditions in a sex-specific manner. These novel findings, combined with previous data that demonstrated high levels of dilp1 during adult reproductive diapause and the role of dilp1 as a pro-longevity factor during aging, suggest a wide-ranging importance of this signaling system. Not only does dilp1 expression correlate with stages of non-feeding (or reduced feeding), these stages are also associated with lack of reproductive activity and encompass the pupa, newly eclosed flies, and diapausing flies. Under normal conditions, the transient expression of dilp1/DILP1 during the first few days of adult life may be associated with a metabolic transition (remodeling from larval to adult fat body) and the process of sexual maturation (gonad growth and differentiation). The data also suggest that dilp1 affects physiology more prominently in young female flies than in males, which might be associated with ovary maturation (Liao, 2020).

It is also interesting to note that the diminished starvation resistance in dilp1 and dilp1/dilp2 mutants is opposite to the phenotype seen after IPC ablation, mutation of dilp1-4, or diminishing IIS by other genetic interventions. Thus, in recently eclosed flies, dilp1 appears to promote starvation resistance rather than diminishing it. Furthermore, the decreased survival during starvation in female dilp1 mutants is the opposite of that shown in dilp6 mutants, indicating that dilp1 action is different from the other insulin-like peptides tested (Liao, 2020).

In Drosophila, the final body size is determined mainly by nutrient-dependent hormonal action during the larval feeding stage. However, some regulation of adult body size can also occur after the cessation of the larval feeding stage, and this process is mediated by dilp6 acting on adult tissue growth in the pupa in an ecdysone-dependent manner. This is likely a mechanism to ensure growth of adult tissues if the larva is exposed to shortage of nutrition during its feeding stage. The current findings suggest that dilp1 can function as another regulator of growth during the pupal stage. Overexpression of dilp1 promotes organismal growth in the pupa, probably at the cost of stored nutrients derived from the larval feeding stage. This is supported by respiratory quotient (RQ) data that clearly show a shift from mixed-energy substrate metabolism in control flies toward almost pure lipid catabolism at the end of pupal development in the dilp1 overexpression flies (also seen for dilp6 gain of function in these experiments). Furthermore, triacylglycerol (TAG) (but not carbohydrate) levels in dilp1 overexpression pupae were clearly decreased, which likely reflects the shift in catabolic energy substrate also seen in the RQ using respirometry. It should be noted that insects predominantly use lipids as fuel during metamorphosis and dilp1 overexpression increases lipid catabolism. This study hence suggests that dilp1 can parallel dilp6 in balancing adult tissue growth and storage of nutrient resources during pupal development. This is interesting since dilp6 is an IGF-like peptide that is produced in the nutrient sensing fat body, whereas the source of the insulin-like dilp1 is the brain IPCs (Liao, 2020).

In contrast to the dilp1 gain of function, the experiments with dilp1 mutant flies did not show a clear effect on adult body growth, only a decrease in weight. Is this a result of compensation by other DILPs? Previous work showed that young adult dilp1 mutant flies display increased dilp6 and vice versa, suggesting feedback between these two peptide hormones in adults. During the pupal stage, this feedback appears less prominent in dilp1 mutants and no effects were detected on (dilp2, dilp3), or dilp6 levels. Furthermore, overexpression of dilp1 in fat body or IPCs has no effect on pupal levels of dilp2 and dilp6. Thus, at present, there is no evidence for compensatory changes in other dilps/DILPs in pupae with dilp1 manipulations. However, under normal conditions (in wild-type pupae), dilp6 levels are far higher than those of dilp1, which could buffer the effects of changes in dilp1 signaling (Liao, 2020).

DILPs and IIS are involved in modulating responses to starvation, desiccation, and oxidative stress in Drosophila. Flies with ablated IPCs or genetically diminished IIS display increased resistance to several forms of stress, including starvation . Conversely, overexpression of dilp2 increases mortality in Drosophila. This study found that young dilp1 mutant flies displayed diminished starvation resistance. In both recently eclosed and 3-day-old flies, mutation of dilp1 decreased survival during starvation (but not in 6- to 7-day-old flies) (Liao, 2020).

Action of dilp1 in the adult fly is also linked to reproductive diapause in females, where feeding is strongly reduced, and both peptide and transcript are upregulated. Related to this, dilp1 mRNA was found to upregulated during starvation in 12-day-old flies. Furthermore, it was shown that expression of dilp1 (dilp1 rescue) increases lifespan in dilp1/dilp2 double mutants, suggesting that loss of dilp2 induces dilp1 as a factor that promotes longevity. Thus, dilp1 activity is beneficial also during adult life, even though its expression under normal conditions is very low. This pro-longevity effect of dilp1 is in contrast to dilp2, 3, and 5 and the mechanisms behind this effect are of great interest to unveil (Liao, 2020).

A previous study showed that in wild-type (Canton S) Drosophila, DILP1 expression in young adults is sex-dimorphic with higher levels in females. In line with this, starvation resistance in young flies is diminished only in female dilp1 mutant and dilp1 overexpression flies. Thus, taken together, previous work showed that dilp1 displays a sex-specific expression and this study shows female-specific function in young adult Drosophila. It is tempting to speculate that the more prominent role of dilp1 in female flies is linked to metabolism associated with reproductive physiology and early ovary maturation, which is also reflected in the dilp1 upregulation during reproductive diapause. In fact, this study shows that egg-laying increased after dilp1 overexpression, and an earlier study demonstrated a decreased egg laying in dilp1 mutant flies. Part of the sex dimorphic effects on body weight of young adults after dilp1 manipulations might be a result of a differential role of dilp1 in water homeostasis (Liao, 2020).

This study shows that IPC-derived dilp1 displays several similarities to the fat body-produced dilp6, including temporal expression pattern, growth promotion, effects on adult stress resistance and lifespan. Additionally, dilp1 may play a role in regulation of nutrient utilization and metabolism during the first few days of adult life, especially in females. At this time, larval fat body is still present and utilized as energy fuel/nutrient store and this source also contributes to egg development. Curiously, there is a change in the action of DILP1 between the pupal and adult stages from being able to stimulate growth (agonist of dInR, like DILP6) in pupae, to acting in a manner opposite to DILP2, DILP6, and other DILPs in adults in regulation of lifespan and stress responses. Only one dInR is known so far (excluding the G protein-coupled receptors for the relaxin-like DILP7 and DILP8). Thus, the mechanisms behind this apparent switch in function of DILP1 signaling remain an open question. One possibility is that DILP1 acts via different signaling pathways downstream the dInR in pupae and adults. An obvious difference between these two stages is the presence of larval-derived fat body in the pupa and during the first few days of adults and its replacement by functional adult fat body in later stages. Perhaps dInR-mediated action differs in these types of fat body when activated by DILP1. Another possibility is stage-specific expression of insulin/IGF-binding proteins such as SDR, ALS, and Imp-L2 that could affect the activity of DILP1 in particular (Liao, 2020).

In the future, it would be interesting to investigate whether DILP1 acts differently on larval/pupal and adult fat body, or act on different downstream signaling in the two stages of the life cycle. Also, the possibility that dilp1 and dilp6 interact to regulate growth and metabolism in Drosophila is worth pursuing (Liao, 2020).

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

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

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

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

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

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

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

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

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

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

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

Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy

The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. This study demonstrates that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0-10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles (Liu, 2016).

Until now the spatiotemporal expression and function of DILP1 in Drosophila was largely unknown. This study shows that under normal rearing conditions both dilp1 transcript and DILP1 peptide are expressed transiently by a set of 14 brain IPCs during pupal stages and the first days of adult life. However, a continuous high dilp1/DILP1 expression can be seen in IPCs of female flies that have entered reproductive diapause. The DILP1 expression, thus, coincides with the non-feeding stages of pupae and the very young fly and the strongly reduced feeding during adult diapause. This temporal expression of DILP1 suggests a specific function of the peptide that may be related to development, growth or energy homeostasis characteristic of states of minimal nutrient intake (Liu, 2016).

The arrest of feeding at the end of the larval life is programmed and continues throughout pupal development. Thus, cell growth and proliferation during this stage occurs without global growth or gain of mass, using stored nutrients and supplies derived from histolysis of obsolete larval tissues. The fat-body-derived DILP6 is known to promote tissue growth during non-feeding stages and dilp6 is induced by ecdysone in the wandering third instar larva. Possibly DILP1 is functionally accessory to DILP6 during this non-feeding period, although a very minor growth promoting role was detected. An additional possibility is a link to stages with no reproductive activity. Characteristic of Drosophila pupae, newly eclosed flies and diapausing adults, is the immature ovaries and lack of vitellogenesis. Thus, DILP1 down-regulation could correlate with both the metabolic transition during the first days of adult life and the onset of sexual maturation. Indeed, this study found that dilp1 mutant flies display a reduced early oviposition, suggesting a role in egg development. This is consistent with the importance of intact systemic insulin signaling in the control of ovarian germ line stem cells (Liu, 2016).

To further understand DILP1 function. an analysis was undertaken of factors regulating its expression and effects of manipulations of DILP1 production. It has been shown that loss of certain DILPs can be compensated by upregulation of others. The effect of loss of dilp2, 3, 5 and dilp6 using mutant flies was examined; the dilp1 transcript was elevated in both mutants. Conversely, w a slight but significant increase in dilp6 expression and a reduced dilp5 expression in the body were detected, but not heads, of 1-week-old dilp1 mutant flies. These findings suggest that loss of dilp1 may affect dilp5 transcription in ovaries or renal tubules and dilp6 in adipocytes of the body. Apparently the loss of dilp1 did not affect dilp2 and dilp3 levels (or dilp5 in the head), suggesting that in the IPCs there are no detectable compensatory transcriptional mechanisms for lack of dilp1 in adult flies. The upregulation of dilp1/DILP1 after loss of dilp2, 3, 5 or dilp6 may be reminiscent of the situation during diapause where the dilp1 transcript is elevated but systemic insulin signaling appears to be generally downregulated, probably due to diminished DILP release (Liu, 2016).

Previous studies have shown that a few neuropeptides and neurotransmitters act on the IPCs to alter their activity and/or production of DILPs. One of the peptides acting on IPCs is sNPF, released from DLP neurons, that triggers increased levels of dilp2 and dilp5 transcripts in the adult brain. In another study sNPF was ectopically targeted to sensory neurons of larvae using the MJ94-Gal4 and it was shown that dilp1 and dilp2 transcripts were elevated. When sNPF was targeted to MJ94 neurons there was an increase in DILP1 immunolabeling of the IPCs of one-week-old flies, but no effect on peptide levels was noted when targeting DLP neurons. However, measuring dilp1 transcript a slight decrease was noted after sNPF overexpression in DLP neurons. These findings suggest that sNPF signaling is involved in regulation of DILP1/dilp1 expression. Earlier work has suggested multiple functional roles of sNPF, including regulation of food intake, modulation of food odor responses, fine control of locomotor behavior and learning and memory (Liu, 2016).

There might, hence, be a link between the role of sNPF in feeding and food odor processing and its action on DILP1 production and/or release in the IPCs. This regulation can perhaps be extended to taste inputs since flies lacking gustatory bristles due to a null mutation in the gene Pox neuro (Poxn) were shown to live longer and in females display increased levels of dilp1, dilp3 and dilp6. The MJ94-Gal4 line used in this study includes gustatory receptors and may, thus, account for the effect of sNPF overexpression on dilp1 levels. The study of Poxn mutants revealed a female-specific upregulation of dilp1, dilp3 and dilp6 and a male downregulation of dilp3 and dilp5, suggesting a sex-specific influence of taste on lifespan mediated by different DILPs (Liu, 2016).

An intriguing finding in this study is that flies kept in reproductive diapause display a sustained high dilp1/DILP1 expression in IPCs until the dormancy is interrupted. Adult diapause is characterized by halted reproduction, a drastic reduction of food intake, accompanied by a shift in energy metabolism towards increased nutrient stores, as well as increased stress tolerance and upregulated immune defense. In Drosophila diapause can only be triggered in newly eclosed flies before 10 h of adult life. Flies at this stage share several features with the late pupae in addition to the lack of food intake and high expression of DILP1/dilp1. For instance, both pupae and adult flies that are less than 24 h old display presence of fat body cells derived from the larva. These dissociated adipocytes constitute an energy and nutrient store during pupal development and also serve the newly eclosed adult until it is ready for flight, food search and feeding, which requires wing expansion and tanning of cuticle. The presence of larval fat body cells is extended somewhat in diapausing flies. Thus, this study tested whether genetic blocking of apoptosis in larval fat body in non-diapausing flies has an effect on DILP1 expression. Similar to a previous study the presence of larval adipocytes could only be extended a few days, but DILP1 expression was increased in 5d old flies compared to controls (Liu, 2016).

The sustained DILP1 expression during diapause is not likely to be due only to the presence of larval fat body since this is no longer detectable after 3 weeks of diapause. Since ovaries do not mature during diapause we tested whether some ovary-derived factor might be involved in the regulation of dilp1/DILP1 expression. By treating newly eclosed flies with precocene 1, an inhibitor of juvenile hormone biosynthesis, ovary maturation was halted and a dose-dependent increase in DILP1 expression was seen in young flies. However, since no clear evidence was obtained for long term effects of larval fat body or ovary maturation in keeping the DILP1 expression high, it is suggested that during diapause the DILP1 level in IPCs remains high due to the altered metabolic state and endocrine signaling (including diminished general IIS) characteristic of dormancy It can be noted that loss of dilp1 does not result in drastically altered diapause incidence, probably due to redundant functions of other DILPs. However, dilp1 mutant flies display reduced egg-laying, indicating a role of the peptide in ovary development, maybe accessory to DILP2 which was shown to control germ line stem cells in Drosophila ovaries (Liu, 2016).

In this work it was found that dilp1 mutant male flies display increased median lifespan under normal conditions and also reduced resistance to starvation. This is similar to the effects of deletion of the IPCs, which renders flies more long-lived, but the reduced starvation resistance in dilp1 mutants is opposite of findings for IPC ablation and dilp6 mutants2,19. The experiments herein do not provide any clues to this sex dimorphism in the role of dilp1. It is noteworthy that deletion of single dilps, except dilp2, has little effect on fly physiology. Thus, dilp1 mutants display unexpectedly strong effects on adult male physiology, in spite of the dilp1/DILP1 expression peaking during pupal stages. One possible explanation is that loss of dilp1 seems to have very limited effects on compensatory expression of dilp2, 3 and 5 in IPCs and has rather small effects on dilp5 and 6 transcripts in the body. In other words, compensatory interactions between dilp1 and other dilps work primarily one-way whereby dilp1 levels are affected by knockout of dilp2, 3, 5 and dilp6. This is different for dilp2 mutants, which display increased dilp3 and dilp5 transcript. Another aspect of interest is that dilp1/DILP1 expression is very weak in flies older than one week (kept at 25°C) and yet effects on lifespan and stress responses are seen in older male dilp1 mutant flies. Therefore it is possible that the adult phenotypes are consequences of developmental effects induced by lack of DILP1 signaling in the pupa and/or first 1-2 days of adult life, similar to findings for DILP6 (Liu, 2016).

In summary, this study is the first to determine the spatial-temporal expression pattern of DILP1. The expression appears to be primarily in the brain IPCs where DILP1 is colocalized with DILP2, 3, 5 and the CCK-like peptide drosulfakinin (DSK). In contrast to DILP2, 3, 5, 6 and 7, it was found that under normal conditions DILP1 is produced by only in the pupa and first few days of adult life. However during adult reproductive diapause DILP1 levels remain high. These findings indicate that DILP1 has a unique function that may be relevant in stages where food intake is low or absent and where sexual maturity has not been reached. Analysis of dilp1 mutant flies reveals that the peptide is important in formation of adult male responses to starvation and general longevity and in female ovary maturation (Liu, 2016).

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

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

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

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

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

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

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

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

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

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

Search PubMed for articles about Drosophila Ilp1

Lee, K. S., Kwon, O. Y., Lee, J. H., Kwon, K., Min, K. J., Jung, S. A., Kim, A. K., You, K. H., Tatar, M. and Yu, K. (2008). Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling. Nat Cell Biol 10(4): 468-475. PubMed ID: 18344986

Liao, S., Post, S., Lehmann, P., Veenstra, J. A., Tatar, M. and Nassel, D. R. (2020). Regulatory roles of Drosophila Insulin-Like Peptide 1 (DILP1) in metabolism differ in pupal and adult stages. Front Endocrinol (Lausanne) 11: 180. PubMed ID: 32373064

Liu, Y., Liao, S., Veenstra, J. A. and Nassel, D. R. (2016). Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy. Sci Rep 6: 26620. PubMed ID: 27197757

Post, S., Liao, S., Yamamoto, R., Veenstra, J. A., Nassel, D. R. and Tatar, M. (2018). Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2. Aging Cell: e12863. PubMed ID: 30511458

date revised: 28 December 2023

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