Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance
© Honegger et al.; licensee BioMed Central Ltd. 2008
Received: 21 July 2007
Accepted: 13 March 2008
Published: 15 April 2008
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.
Here, we show 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.
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, we suggest that their common precursor was an ancestral insulin-binding protein.
Insulin/insulin-like growth factor (IGF) signaling (termed IIS) is involved in the regulation of growth, metabolism, reproduction and longevity in mammals [1–3]. 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 [7, 8], 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 [6, 9–13]. 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 .
IIS is astonishingly well conserved in invertebrates. In Drosophila, IIS acts primarily to promote cellular growth, but it also affects metabolism, fertility and longevity [15, 16]. 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 [17–19]. Ablation of the m-NSCs causes a developmental delay, growth retardation and elevated carbohydrate levels in the larval hemolymph [18, 19], reminiscent of the phenotypes of starved or IIS-impaired flies.
The Drosophila genome does not encode an obvious homolog of the IGFBPs. Furthermore, genetic analyses of IIS in Drosophila and Caenorhabditis elegans have not revealed a functional insulin-binding protein so far. Here, we report 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.
Genetic screen to identify negative regulators of IIS
Imp-L2 has previously been shown to be upregulated 8–10 hours after ecdysone treatment [20, 21]. 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 (Figure 1g). 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 .
Overexpression of Imp-L2 impairs growth non-autonomously
Next, we assessed the effect of Imp-L2 overexpression on phosphatidylinositol(3,4,5)trisphosphate (PIP3) levels 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 (Additional data file 1, Figure S1A,B). Co-overexpression of Imp-L2 together with dInR reduced the PIP3 levels (Additional data file 1, Figure S1D), similar to the effect caused by PTEN (Additional data file 1, Figure S1C), a negative regulator of IIS. Therefore, Imp-L2 inhibits PI 3-kinase/PKB signaling upstream of PIP3, without affecting dInR levels (Additional data file 1, Figure S1B',D').
Size increase in Imp-L2mutants
We used two strategies to generate loss-of-function mutations in Imp-L2. First, we performed an ethylmethane-sulfonate (EMS) reversion screen in which we selected mutated chromosomes carrying EP5.66 that no longer suppressed the dInR overexpression phenotype (Figure 1a). One allele (Imp-L2MG2) containing a point mutation resulting in a premature stop at amino acid 232 was identified in this way (Figure 1e,f). This truncation destroys the conserved cysteine bridge of the second Ig domain (Figure 1g). Overexpression of the truncated Imp-L2 version had no inhibitory effect on size (Figure 1e), suggesting that Imp-L2MG2 is a functional null allele.
Second, we generated additional Imp-L2 alleles by imprecise excision of GE24013 (GenExel), a P-element located 349 bp upstream of the ATG start codon of the Imp-L2-RB transcript (Figure 1f). We obtained Imp-L2 deletions (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 (Figure 2d,e). Introducing one copy of a genomic rescue construct (Figure 1f)  into homozygous mutant flies reverted the weight to the level of Imp-L2+/- flies, which were already heavier (+14% in males, +44% in females, Figure 2e) 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 (Figure 2e). 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.
The weight difference was more pronounced in mutant females than in males, although the increases in wing area and cell number were similar (Figure 2e and data not shown). This differential effect was caused by enlarged ovaries in Imp-L2 mutant females (data not shown).
Imp-L2 binds to and antagonizes Dilp2
It has previously been shown that Imp-L2 can bind human insulin and insulin-related peptides . To address whether Imp-L2 binds Dilp2, we constructed a Flag-tagged version of Dilp2, which is functional (data not shown). Using in vitro translated, 35S-labeled Imp-L2 together with Flag-Dilp2 extracted from stably transfected S2 cells, we could show that Imp-L2 binds Dilp2 in vitro (Figure 3e). A truncated form of Imp-L2 lacking a functional second Ig domain (like that produced by the MG2 allele) failed to bind Dilp2 (Figure 3e).
Imp-L2 is essential under adverse nutritional conditions
The dampening of IIS upon starvation could be achieved either by enhanced secretion of stored Imp-L2 or by an upregulation of Imp-L2 production. Indeed, expression profiling revealed a slight upregulation of Imp-L2 after 12 hours complete starvation . We could not detect a change in Imp-L2 protein expression in the brain, the ring gland or the gut after complete starvation for 24 hours (data not shown). However, Imp-L2 was induced in fat body cells, where it appeared in vesicle-like structures (Figure 4d). Thus, under adverse nutritional conditions, Drosophila larvae weaken IIS by upregulating Imp-L2 expression in the fat body.
IIS signaling has evolved in animals to regulate growth and metabolism in accordance with environmental conditions. Appropriate IIS activity is ensured at several levels, including the controlled expression of binding partners of the extracellular ligands. Surprisingly, the well-characterized vertebrate IGFBPs have no obvious homologs in lower organisms. Here, we used a genetic strategy to search for negative regulators of IIS in Drosophila. Our approach led to the identification of Imp-L2 as a functional insulin-binding protein and antagonist of IIS.
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. Our analysis of IIS activity (by means of the tGPH reporter in vivo) shows that Imp-L2 functions to downregulate IIS. We further show 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 .
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 (although this binding could not be detected in a different assay ). We speculate 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.
Interestingly, Dilp2 and Imp-L2 are found in a complex with dALS (acid-labile subunit ). 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.
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 . Our 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.
Our study provides the first functional characterization of an insulin-binding protein in invertebrates. We have identified Imp-L2 as a secreted antagonist of IIS in Drosophila. Given the sequence homology of their Ig domains, we propose 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.
Materials and methods
The following fly stocks and transgenes have been used: y w; w1118; arm-Gal4; Act5C-Gal4; UAS-GFP; UAS-lacZ (all from the Bloomington Drosophila stock center); GMR-Gal4 (a gift of M. Freeman); ppl-Gal4 (a gift of M. Pankratz); UAS-dInR ; Df(3L)AC1 ; tGPH ; GMR>w+>Gal4 ; UAS-dPTEN ; UAS-dilp2 ; GE24013 (GenExel). All crosses were performed at 25°C unless stated otherwise.
EP screen and isolation of Imp-L2alleles
The EP screen that led to the identification of Imp-L2 will be described elsewhere (F.W., W.B., H.S., D. Nellen, K. Basler and E.H., unpublished work). A double-headed EP element (containing ten Gal4-binding sites at each end) suppressing the GMR-Gal4, UAS-InR big eye phenotype was identified in the Imp-L2 locus. Plasmid rescue of EP5.66 revealed that it was inserted 6,969 bp upstream of the first exon of the Imp-L2-RB (CG15009-RB) transcript.
To obtain loss-of-function alleles of Imp-L2, we performed an EMS mutagenesis screen in which we selected mutated chromosomes carrying EP5.66 that could no longer suppress the dInR overexpression phenotype in the eye. EP5.66 males were fed with 25 mM EMS and subsequently crossed to GMR-Gal4, UAS-dInR virgins. 39,000 F1 flies were screened for a reversion of the suppressive effect of EP5.66 on the growth phenotype caused by GMR-Gal4, UAS-dInR. Only one of the identified reversion lines, Imp-L2MG2, could be confirmed. Sequencing the genomic DNA of Imp-L2MG2 revealed a point mutation that resulted in a truncation (Trp232Stop).
In order to generate additional Imp-L2 mutants, the P-element GE24013 (marked with white+) inserted 102 bp upstream of the first exon of the Imp-L2-RC transcript was mobilized by supplying Δ2–3 transposase. Jump starter males were mated with balancer females, and single F1 w- males were recrossed to balancer virgins. Stocks (350) were established and molecularly tested for deletions by single-fly PCR using several primer pairs, leading to the identification of the alleles Imp-L2Def42, Imp-L2Def20, Imp-L2Def35, Imp-L2Def223 and Imp-L2Def29.
Construction of plasmids
In order to generate the UAS-Imp-L2 construct, a BglII/XhoI fragment of Imp-L2 was excised from the Imp-L2-RB containing cDNA clone LP06542 and inserted into pUAST . To obtain UAS-s.Imp-L2, the second and third exons of Imp-L2 were amplified by PCR from genomic DNA. The fragment was subcloned into pCRII-Topo (Invitrogen). The insert was then excised with EcoRI and cloned into pUAST . Because of the lack of the first exon of the Imp-L2-RB transcript (containing three upstream open reading frames), UAS-s.Imp-L2 has a stronger phenotype than UAS-Imp-L2. The EP element contains ten UAS sites, whereas the UAS transgenes contain only five.
For the generation of the genomic rescue construct, the genomic fragment L2G314 (kindly provided by J. Natzle) was used. The fragment (5 kb of genomic sequence upstream of the first exon of the Imp-L2-RB transcript and 1 kb downstream of the third exon) was excised with BamHI and Asp718 and inserted into the pCaSpeR-4 transformation vector .
The Flag-dilp2 construct was created by PCR amplification of the dilp2 coding sequence without the signal peptide sequence from the full-length cDNA clone, EST GH11579 (obtained from Research Genetics). The resulting PCR product was then equipped with the hemagglutinin signal peptide sequence and a Flag tag and inserted into pUAST .
Drosophila embryonic S2 cells were grown at 25°C in Schneider's Drosophila medium (Gibco/Invitrogen) supplemented with 10% heat-inactivated fetal-calf serum (FCS), penicillin and streptomycin.
For the construction of the stably expressing Flag-dilp2 cell line, S2 cells were co-transfected with UAS-Flag-dilp2, Act-Gal4 and a third vector containing a blasticidin-resistance gene, using effectene transfection reagent (Qiagen). Two days after the transfection, the selection medium (Schneider's containing 10% FCS and 25 μg/ml blasticidin) was added to the cells. After 10 days the selection medium was replaced by Schneider's containing 10% FCS and 10 μg/ml blasticidin.
In vitropulldown assay
S2 cells expressing Flag-dilp2 were grown to confluence in 175 cm2 culture flasks, washed with ice-cold PBS and extracted in immunopreciptiation (IP) buffer (120 mM NaCl, 50 mM Tris pH 7.5, 20 mM NaF, 1 mM benzamidine, 1 mM EDTA, 6 mM EGTA, 15 mM Na4P2O7, 0.5% Nonidet P-40, 30 mM β-glycerolphosphate, 1× Complete Mini protease inhibitor (Roche)). After incubation for 15 min on an orbital shaker at 4°C, solubilized material was recovered by centrifugation at 13,000 rpm for 15 min and supernatants were collected. Anti-Flag antibody (5 μg, Sigma M2, F3165) was added and incubated over night at 4°C while rotating. Protein G sepharose beads (Amersham Biosciences) were added for 2 h and the beads were washed four times with IP buffer. Cell lysate from native S2 cells was subjected to the same procedure and the resulting beads were used as control. To verify the immunoprecipitation, a fraction of the beads was incubated with SDS loading buffer (62.5 mM Tris-HCl pH 6.8, 20 mM DTT, 2% SDS, 25% glycerol, 0.02% bromophenol blue) for 5 min at 90°C and the proteins were separated by SDS-PAGE. The presence of Flag-Dilp2 was confirmed by immunoblotting.
For the in vitro translation the Imp-L2-RC cDNA (SD23735) was cloned into pCRII.1 (Invitrogen) downstream of the SP6 polymerase promoter. As a control, the point mutation encoding a non-functional, truncated version of Imp-L2 (identified in the EMS reversion mutagenesis) was inserted into Imp-L2-RC (in pCRII.1 see above) using the Quick-Change site-directed mutagenesis protocol (Stratagene). Both the Imp-L2 and the Imp-L2MG2 constructs were translated in vitro using the TNT Quick coupled transcription/translation system (Promega) according to the manufacturer's protocol. Briefly, 2 μg of DNA was incubated with 20 μCi [35S]methionine and 20 μl TNT Quick Master Mix in a total volume of 25 μl for 90 min at 30°C. The product (2.5 μl) was used in the in vitro pulldown assay together with Flag-Dilp2 bound to beads or with control beads in IP buffer containing 0.05% NP-40. The reaction was rotated overnight at 4°C, the beads were washed six times with IP buffer (0.05% NP-40) and incubated with SDS loading buffer containing 100 mM DTT for 10 min at 80°C. The dissociated proteins were separated using SDS-PAGE and detected by autoradiography.
Freshly eclosed flies were collected, separated according to sex, placed on normal fly food for 3 days and anesthetized for 1 min with ether before weighing. Weight was determined using a Mettler Toledo MX5 microbalance. Wing size was analyzed as described . ImageJ 1.32j software was used to determine the pixels of the wing area. Scanning electron microscope pictures were taken from adult flies that were critical-point dried and coated with gold.
Heat-shock induced overexpression clones (y, w, hs-Flp; GMR>w+>Gal4) were induced 24–48 h after egg-laying by a 1 h heat shock at 37°C. Tangential sections of adult eyes were generated as described .
For all starvation experiments, eggs were collected for 2 h on apple agar plates supplemented with yeast. After 72 h, larvae were quickly washed in PBS and transferred either to a new apple agar plate with yeast (normal food, called 'yeast' henceforth), a solution containing 20% glucose in PBS, or a filter paper soaked with 1% glucose in PBS or PBS only. After 24 h, dead larvae were counted.
For the tGPH reporter analysis under starvation, the 'PBS' or 'yeast' conditions were used (see above). After 4 h starvation, larvae were dissected in PBS, fixed and stained with Hoechst. Pictures were taken using a Leica SP2 confocal laser scanning microscope.
Immunohistochemistry and in situhybridization
The antibody against Imp-L2 was described earlier  and kindly provided by J. Natzle (Department of Molecular and Cellular Biology, University of California, Davis, USA). Antibody staining against Imp-L2 was performed using the following dilutions: rat anti-Imp-L2 (1:500), donkey anti-rat-FITC (1:200, Jackson). Other antibodies used were: anti-β-galactosidase (1:2,000, polyclonal, rabbit), an antibody against the carboxyl terminus of dInR (INRcT, 1:10,000) . Nuclei were either stained with 4',6-diamidino-2-phenylindole (DAPI) or Hoechst. Pictures were taken using a Leica SP2 confocal laser scanning microscope.
RNA in situ hybridization using digoxigenin-labeled probes was performed as described . The probes against Imp-L2 were derived from s.Imp-L2 in a pBluescript SK+ vector.
Additional data files
The following file is available: Additional data file 1 contains three figures. Figure S1 shows that the overexpression of Imp-L2 results in reduced PIP3 levels in vivo. In Figure S2, the dynamic expression pattern of Imp-L2 during development is shown. Figure S3 demonstrates that a reduction in Dilp levels enhances the growth-inhibitory effect of Imp-L2.
We thank P. Léopold for openly communicating results before publication, J. Natzle for the Imp-L2 antibody and the plasmid used for the genomic rescue construct, Ch. Hugentobler, A. Baer, A. Straessle, P. Gast and B. Bruehlmann for technical support, J. Reiling for critical reading of the manuscript, E. Brunner and the members of the Hafen lab for helpful discussions and valuable suggestions, and GenExel and the Bloomington stock center for fly stocks. This work was supported by grants from the Swiss National Science Foundation and the Kanton of Zürich.
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