InteractiveFly: GeneBrief

Insulin-related peptide: Biological Overview | References

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Search PubMed for articles about Drosophila Dilp2

Araki, E., et al. (1994). Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186-190. 7526222

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 Citation: 18396139

Bauer, J. H., et al. (2005). Neuronal expression of p53 dominant-negative proteins in adult Drosophila melanogaster extends life span. Curr. Biol. 15: 2063-2068. PubMed Citation: 16303568

Bauer, J. H., et al. (2007). Expression of dominant-negative Dmp53 in the adult fly brain inhibits insulin signaling. Proc. Natl. Acad. Sci. 104(33): 13355–60. PubMed Citation: 17686972

Belgacem, Y. H. and Martin, J. R. (2006). Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila. J. Neurobiol. 66(1): 19–32. PubMed Citation: 16187303

Bonkowski, M. S., et al. (2006). Targeted disruption of growth hormone receptor interferes with the beneficial actions of calorie restriction. Proc. Natl. Acad. Sci. 103: 7901–7905. PubMed Citation: 16682650

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 Citation: 11250149

Broughton, S. J., et al. (2005). Longer lifespan, altered metabolism and stress resistance in Drosophila from ablation of cells making insulin-like ligands. Proc. Natl. Acad. Sci. 102(8): 3105–10. PubMed Citation: 15708981

Broughton, S., et al. (2008). Reduction of DILP2 in Drosophila triages a metabolic phenotype from lifespan revealing redundancy and compensation among DILPs. PLoS ONE 3(11): e3721. PubMed Citation: 19005568

Butler, A. A. and Roith, D. L. (2001). Control of growth by the somatropic axis: Growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63: 141-164. 11181952

Chen, Q., Ma, E., Behar, K. L., Xu, T. and Haddad, G. G. (2002). Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster. J. Biol. Chem. 277(5): 3274–9. PubMed Citation: 11719513

Clancy, D. J., et al. (2001). Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292: 104–106. PubMed Citation: 11292874

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

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 Citation: 18412985

Houthoofd, K., et al. (2003). Life extension via dietary restriction is independent of the Ins/IGF-1 signaling pathway in Caenorhabditis elegans. Exp. Gerontol. 38: 947–954. PubMed Citation: 12954481

Hwangbo, D. S., Gersham, B., Tu, M. P., Palmer, M. and Tatar, M. (2004). Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429: 562–566. PubMed Citation: 15175753

Ikeya, T., et al. (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. 12176357

LaFever. L. and Drummond-Barbosa, D. (2005). Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila. Science 309: 1071-1073. Medline abstract: 16099985

Lee, K. S., et al. (2008). Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling. Nat. Cell Biol. 10(4): 468-75. PubMed Citation: 18344986

Libert, S., et al. (2007). Regulation of Drosophila life span by olfaction and food-derived odors. Science 315: 1133–1137. PubMed Citation: 17272684

Mattaliano, M. D., et al. (2007). The Drosophila ARC homolog regulates behavioral responses to starvation. Mol. Cell Neurosci. 36(2): 211–21. PubMed Citation: 17707655

Min, K.-J., Yamamoto, R., Buch, S., Pankratz, M. and Tatar, M. (2008). Drosophila lifespan control by dietary restriction independent of insulin-like signaling. Aging Cell 7(2): 199–206. PubMed Citation: 18221413

Panowski, S. H. et al. (2007). PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature 447: 550–555. PubMed Citation: 17476212

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. 12004130

Tamemoto, H., et al. (1994). Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372(6502): 182-6. 7969452

Tatar, M. (2007). Diet restriction in Drosophila melanogaster: design and analysis. Interdiscip. Top. Gerontol. 35: 115–136. PubMed Citation: 17063036

Tiessen, J., Underwood, L. and Ketelslegers, J. (1999) Regulation of insulin growth factor-1 in starvation and injury. Nutr. Rev. 57: 167-176. 10439629

Walker, G., et al. (2005). Dietary restriction in C. elegans: from rate-of-living effects to nutrient sensing pathways. Mech. Ageing Dev. 26: 929–937. PubMed Citation: 15896824

Wang, M. C., Bohmann, D. and Jasper, H. (2005). JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell 121(1): 115–25. PubMed Citation: 15820683

Withers, D. J., et al. (1998). Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391: 900-904. 9495343

Wu, Q., Zhang, Y., Xu, J. and Shen, P. (2005). Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila. Proc. Natl. Acad. Sci. 102(37): 13289-94. 16150727

Xu, G. G. and Rothenberg, P. L. (2001). Insulin receptor signaling in the beta-cell influences insulin gene expression and insulin content: evidence for autocrine beta-cell regulation. Diabetes 47: 1243-1252. 9703324

date revised: 10 December 2009

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