InteractiveFly: GeneBrief

Ecdysone-inducible gene L2: Biological Overview | References

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

Insulin/insulin-like growth factor (IGF) signaling (termed IIS) is involved in the regulation of growth, metabolism, reproduction and longevity in mammals . The activity of IIS is regulated at multiple levels, both extracellularly and intracellularly: the production and release of the ligands is regulated, and normally IGFs are also bound and transported by IGFBPs in extracellular cavities of vertebrates (Hwa, 1999). IGFBPs not only prolong the half-lives of IGFs, but they also modulate their availability and activity. Besides the classical IGFBPs (IGFBP1-6), a related protein called IGFBP-7 (or IGFBP-rP1, Mac25, TAF, AGM or PSF) has been identified as an insulin-binding protein. Although the reported binding of IGFBP-7 to insulin awaits confirmation, it can compete with insulin for binding to the insulin receptor (InR) and inhibit the autophosphorylation of InR (Yamanaka, 1997). Furthermore, IGFBP-7 is suspected to be a tumor suppressor in a variety of human organs, including breast, lung and colon. A recent publication demonstrates that IGFBP-7 induces senescence and apoptosis in an autocrine/paracrine manner in human primary fibroblasts in response to an activated BRAF oncogene (Wajapeyee, 2008; Honegger, 2008 and references therein).

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

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

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

Imp-L2 has previously been shown to be upregulated 8-10 hours after ecdysone treatment (Osterbur, 1998; Natzle, 1986). It encodes a secreted member of the immunoglobulin (Ig) superfamily containing two Ig C2-like domains. Whereas several orthologs of Imp-L2 are present in invertebrates such as arthropods and nematodes, the homology in vertebrates is confined to the second Ig C2-like domain, which is homologous to the carboxyl terminus of human IGFBP-7. The carboxy-terminal part of IGFBP-7 differs considerably from the other IGFBPs, possibly accounting for the affinity of IGFBP-7 for insulin (Yamanaka, 1997). Interestingly, Imp-L2 has been shown to bind human insulin, IGF-I, IGF-II and proinsulin, and its homolog in the moth Spodoptera frugiperda, Sf-IBP, can inhibit insulin signaling through the insulin receptor (Sloth Anderson, 2000; Honegger, 2008).

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

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

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

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

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

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

It has been shown that Imp-L2 can bind human insulin and insulin-related peptides (Sloth Anderson, 2000). To address whether Imp-L2 binds Dilp2, a Flag-tagged version of Dilp2 was constructed, that is functional. Using in vitro translated, ³⁵S-labeled Imp-L2 together with Flag-Dilp2 extracted from stably transfected S2 cells, it was shown that Imp-L2 binds Dilp2 in vitro. A truncated form of Imp-L2 lacking a functional second Ig domain (like that produced by the MG2 allele) failed to bind Dilp2 (Honegger, 2008).

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

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

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

Interestingly, Dilp2 and Imp-L2 are found in a complex with dALS (acid-labile subunit (Arquier, 2008). In vertebrates, most of the circulating IGFs are part of ternary complexes consisting of an IGF, IGFBP-3 and ALS (Boisclair, 2001). These ternary complexes prolong the half-lives of the IGFs and restrict them to the vascular system, because the 150 kDa complexes cross the capillary barrier very poorly. IGFs can also be found in binary complexes of about 50 kDa with several IGFBP species but there is only little (< 5%) free circulating IGF (Boisclair, 2001). Thus, it will be interesting to analyze the composition and bioactivities of Dilp2/Imp-L2/ALS complexes in Drosophila (Honegger, 2008).

IIS coordinates nutritional status with growth and metabolism in developing Drosophila. It has been shown that IIS regulates the storage of nutrients in the fat body (Britton, 2002), an organ that resembles the mammalian liver as the principal site of stored glycogen. Even under adverse nutritional conditions, fat body cells with increased IIS activity continue stockpiling nutrients, thereby limiting the amount of circulating nutrients, which induces hypersensitivity to starvation of the larva (Britton, 2002). Upon starvation, the expression of dilp3 and dilp5 is suppressed at the transcriptional level in the m-NSCs (Ikeya, 2002). This study reveals an additional layer of IIS regulation. Whereas Imp-L2 is not expressed in the fat body of fed larvae, starved animals induce Imp-L2 expression in the fat body to systemically dampen IIS activity. A lack of this control mechanism is lethal under unfavorable nutritional conditions, as Imp-L2 mutant larvae fail to cope with starvation (Honegger, 2008).

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

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

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

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

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

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

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

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

Drosophila germ-line modulation of insulin signaling and lifespan

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Search PubMed for articles about Drosophila Imp-L2

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Biological Overview

date revised: 21 March 2008

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