New Results

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

Stephanie Post, Sifang Liao, Rochele Yamamoto, Jan A. Veenstra, Dick R. Nässel, Marc Tatar
doi: https://doi.org/10.1101/380410

Summary

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 in adults. In contrast, dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause, although we find that dilp1 is also highly expressed in adult dilp2 mutants under non-diapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism and adipokinetic hormone (AKH), a functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1-dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly interact to control circulating sugar, starvation resistance and compensatory dilp5 expression. These later interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in C. elegans and Drosophila, suggesting that dilp2 modulates lifespan via dilp1 and in part by regulating Akh. Whether DILP1 acts as an insulin receptor agonist or inhibitor remains to be resolved.

Introduction

Insulin/IGF signaling (IIS) is a fundamental pathway that regulates aging, development, metabolism, growth and other critical systems. The Drosophila melanogaster genome encodes several insulin-like peptide genes (dilps) that signal through a single insulin-like receptor (InR) and one apparent set of signal transduction elements (Brogiolo et al., 2001; Colombani, Andersen, & Leopold, 2012; Garofalo, 2002; Grönke, Clarke, Broughton, Andrews, & Partridge, 2010). Among their physiological functions, dilps regulate aging: mutation of dilp2 alone is sufficient to extend lifespan, although longevity is somewhat further extended in a dilp2-3,5 triple mutant (Grönke et al., 2010). How specific dilps modulate aging is not understood. Here, we demonstrate that dilp1 is upregulated in the absence of dilp2, that dilp1 expression is required for loss of dilp2 to slow aging, and that exogenous expression of dilp1 in a dilp1-2 mutant is sufficient to extend lifespan.

The dilp1 gene is encoded approximately 1.2kb upstream of dilp2, potentially as a result of a tandem duplication event (Tatar, Bartke, & Antebi, 2003). The two paralogs are expressed in different developmental and life history stages. Dilp2 is initially expressed in embryos and then throughout larval instar stages (Brogiolo et al., 2001; Slaidina, Delanoue, Grönke, Partridge, & Leopold, 2009). Pupae show decreased expression of dilp2, but the ligand is again highly expressed in adults. In contrast, during normal development, dilp1 is only expressed at high levels in the pupal stage, when dilp2 mRNA is minimal (Slaidina et al., 2009). While their timing is distinct, dilp1 and dilp2 are both expressed in median neurosecretory cells of the Drosophila brain, the insulin-producing cells (IPCs) analogous to mammalian pancreatic β-cells (Brogiolo et al., 2001; Broughton et al., 2005; Y. Liu, Liao, Veenstra, & Nässel, 2016; Rulifson, Kim, & R., 2002).

The function of dilps in aging has been best studied for dilp2, dilp3, dilp5 and dilp6 (Bai, Kang, & Tatar, 2012; Broughton et al., 2010; Grönke et al., 2010). Mutation of dilp2 alone, or dilp2, dilp3 and dilp5 together, extend lifespan: the normal function of these ligands appears to promote processes permissive to aging (Grönke et al., 2010). On the other hand, induction of dilp6 in fat body promotes longevity, perhaps because this decreases DILP2 secreted from the IPCs (Bai et al., 2012). Similarly, increased FOXO expression in head fat body and increased JNK activity in IPCs extends lifespan, perhaps again because these manipulations decrease dilp2 expression in the IPCs (Hwangbo, Gershman, Tu, Palmer, & Tatar, 2004; Wang, Bohmann, & Jasper, 2005). Across these studies, there has been no attention to dilp1, which appears to only be produced in the adult IPCs during the state of adult reproductive diapause (Liu et al., 2016). Drosophila diapause, however, is strongly associated with slow or negligible aging (Tatar & Yin, 2001). The positive association between dilp1 and diapause survival suggests that this enigmatic insulin hormone may possess unusual functions in the control of aging.

Understanding how these various dilps regulate aging is complicated by the fact that genetic or RNAi reduction of any one dilp gene induces compensatory expression in other dilp genes. For instance, a dilp2 mutant increases expression of dilp3 and dilp5 and these ligands consequently stimulate insulin/IGF signaling (Grönke et al., 2010). Such compensation makes it difficult to dissect the role of signaling downstream of the fly insulin/IGF receptor in aging. Complex compensation and interaction is also known for C. elegans insulin-like gene paralogs (Fernandes de Abreu et al., 2014). For example, ins-6 is upregulated in an ins-23 mutant, and these two paralogs interact epistatically to regulate lifespan (Fernandes de Abreu et al., 2014). Notably, C. elegans ins-18 and ins-23 are proposed to act as insulin-like receptor antagonists that additively regulate dauer formation and favor longevity (Matsunaga, Matsukawa, Iwasaki, Nagata, & Kawano, 2018). Some interactions are established for Drosophila insulin paralogs, such as the inverse regulation of aging by dilp6 and dilp2 (Bai et al., 2012), but the functional relationships between dilp2 and other dilps have not been characterized.

The relationship between dilp1 and dilp2 may represent in Drosophila a longevity regulatory system similar to that suggested with C. elegans paralogs ins-6 and ins-23 (Fernandes de Abreu et al., 2014). We find that dilp1 is strongly upregulated in dilp2 mutants, consistent with dilp1 serving a role in diapause conditions where it might regulate metabolism and slow aging. To test this model, we generated dilp1-2 double mutants to complement similarly constructed dilp1 and dilp2 single mutants (Gronke et al., 2010). As previously reported, dilp2 mutants are long-lived. We now see that dilp1 mutants have wildtype longevity as do dilp1-dilp2 double mutants; thus loss of dilp1 fully rescues the extended longevity of dilp2 to wildtype. We find that dilp1 is also downstream of dilp2 in the control of Drosophila adipokinetic hormone (AKH), the functional homolog of mammalian glucagon. We confirmed the positive role of dilp1 upon longevity and AKH by transgene dilp1 expression in a dilp1-dilp2 double mutant. In contrast to longevity and AKH, dilp1 and dilp2 do not control other physiological traits (e.g. hemolymph sugar, starvation resistance and glycogen) in this epistatic manner, suggesting that these phenotypes are not regulated through the same mechanisms by which these insulin-like peptides modulate aging. Our data together reveal a novel pathway by which a unique insulin-like ligand, DILP1, positively regulates longevity.

Results

Studies on the control of aging by IIS in Drosophila have measured dilp2, dilp3 and dilp5 mRNA or protein (Alic, Hoddinott, Vinti, & Partridge, 2011; Broughton et al., 2010; Hwangbo et al., 2004). While dilp1 of the adult IPC is not observed in non-diapause conditions, we sought to characterize its expression in dilp mutants known to extend lifespan. In wildtype adult females, dilp1 mRNA is considerably lower than that of dilp2 (Fig 1A). Strikingly, dilp1 mRNA is elevated about 14-fold in dilp2 mutants relative to its expression in wildtype (Fig 1B), while there is little compensatory expression of dilp2 in dilp1 mutants (Fig 1C). Dilp2 appears to repress dilp1, and we propose that dilp1 may function in the absence of dilp2 to regulate metabolism and aging.

Figure 1.
Figure 1. Dilp1 mRNA is induced by depletion of dilp2.

RNA from 7-10 day old female adult flies was assayed by q-RT-PCR. n=6 per genotype. (A) Dilp1 expression is 100-fold lower than dilp2 expression in wildtype flies. (B) Dilp1 expression increases 100-fold in dilp2 mutant flies compared to wildtype flies, t test p<0.001. (C) Dilp2 expression increases 2-fold in dilp1 mutant flies compared to wildtype flies, t test p=0.005.

Epistasis analysis of lifespan

Adult dilp2 mutants have elevated blood sugar and extended lifespan (Grönke et al., 2010). To test if these phenotypes require the expression of dilp1, we generated a dilp1-dilp2 null double mutant by homologous recombination, in parallel with matching dilp1 and dilp2 null single mutant knock-outs (Fig. S1, Supporting Information). If the functions of dilp1 are redundant to those of dilp2, we would expect the dilp1-dilp2 double mutants to have greater longevity and higher blood sugar than either single mutant. Alternatively, if the functions of dilp1 are downstream of dilp2, we expect the dilp1-dilp2 double mutants to have wildtype lifespan and metabolism.

Our new null allele of dilp2 increases lifespan by about 20-30% (Fig 2A, Fig S2D, Supporting Information), confirming previous observations (Grönke et al., 2010). Null mutation of dilp1 has no effect on adult survival, again as previously reported (Grönke et al., 2010). Remarkably, survival of the dilp1-dilp2 double null mutant is indistinguishable from wildtype or the dilp1 mutant (Fig 2A-B), revealing a classic epistatic interaction between dilp1 and dilp2 in the control of longevity.

Figure 2.
Figure 2. A dilp1 mutation suppresses aging and Akh phenotypes of mutant dilp2.

(A) Dilp2 mutants but not double mutants are long-lived, Cox hazard analysis p<0.0001, chi2=201, n=341-365 per genotype. (B) Dilp2 mutants but not double mutants have decreased mortality. (C) Dilp2 mutants but not double mutants have increased Akh expression. RNA from 7-10 day old female adult flies was assayed by q-RT-PCR, n=9 per genotype. Two-way ANOVA dilp1 p=<0.001, dilp2 p=0.921, dilp1 x dilp2 p<0.001. (D) Dilp2 mutants but not dilp1 or double mutants have increased AKH immunolabeling in corpora cardiaca from 6-7 day old female flies. Representative images shown. (E) Quantification of AKH immunolabeling, n=9-14 samples from 3 replicates, ANOVA * p<0.05.

Epistasis analysis of adipokinetic hormone

Dilp1 is only normally expressed in adults during reproductive diapause, a slow-aging stage associated with many metabolic changes, including activity of adipokinetic hormone (AKH), the functional homolog of mammalian glucagon (Kubrak, Kucerova, Theopold, & Nässel, 2014; Kucerova et al., 2016; Y. Liu et al., 2016). Accordingly, we studied how dilp1 and dilp2 affect AKH through genetic analysis of single and double mutants. Akh mRNA is increased in dilp2 mutants, is similar to wildtype in dilp1 mutants, and is restored to wildtype levels in the dilp1-dilp2 double mutants (Fig 2C). Physiologically, insulin regulates glucagon at the level of mRNA and protein (Pearson, Unger, & Holland, 2016). We therefore examined AKH immunostaining in the adult corpora cardiaca (CC). AKH peptide in the CC is increased in dilp2 mutants, and is similar to wildtype in dilp1 and in dilp1-dilp2 double mutants (Fig 2D-E). These data suggest that dilp1 is epistatically downstream of dilp2 in pathways to regulate lifespan and AKH – dilp1 expression is required for dilp2 to modulate these phenotypes.

Epistasis analysis of developmental and metabolic traits

Drosophila insulin, including dilp1 and dilp2, affect many additional traits, including body weight and metabolism (Grönke et al., 2010). Similar to the epistatic interactions observed for lifespan and AKH, body mass was decreased in dilp2 mutants (as previously reported (Grönke et al., 2010)), but similar to wildtype in dilp1 mutants and in dilp1-dilp2 double mutants (Fig 3A). In contrast, hemolymph (blood) sugar concentrations in single mutants of dilp1 and dilp2 are similar to those seen in wildtype, while the dilp1-dilp2 double mutant has elevated hemolymph sugar (Fig 3B): for this trait, these insulin paralogs appear to have parallel, redundant functions (Table 1). On the other hand, glycogen content is equally decreased by both single mutants and the double mutant, indicating that both dilp1 and dilp2 are required to maintain the titer of this energy storage molecule (Fig 3C).

View this table:
Table 1.

Two-way ANOVA p values for dilp1-dilp2 epistasis between single and double mutants

Figure 3.
Figure 3. Dilp1 and dilp2 interact to regulate metabolism, physiology and dilp compensation.

Female flies 7-10 days old were assayed. For two-way ANOVA statistics, refer to Table 1. (A) Body size is decreased in dilp2 mutants, n=5 per genotype, 22-44 flies per replicate. Hemolymph sugar levels (B) and glycogen levels (C) differ significantly between single and double mutants, n=5 per genotype. (D) Starvation sensitivity differs significantly between single and double mutants, n=118-150 flies from 3 replicates, Log-rank (Mantel-Cox) **** p<0.0001. Dilp3 (E) and dilp5 (F) expression compensate for dilp1 and dilp2 differently, n=3-6 per genotype.

Many longevity-extending IIS manipulations increase resistance to starvation (Clancy et al., 2001; Grönke et al., 2010). Unexpectedly, our long-lived dilp2 null genotype has starvation survival similar to wildtype, while dilp1 mutants and dilp1-dilp2 double mutants are more sensitive to starvation (Fig 3D). These data suggest that dilp1, which is increased during nonfeeding developmental stages (Y. Liu et al., 2016), may be required for starvation survival by inducing catabolism of nutrients such as glycogen, perhaps through the action of AKH. Dilp3 mRNA is increased in dilp2 mutants and similarly in dilp1-dilp2 double mutants, but not significantly increased in dilp1 mutants (Fig 3E). Dilp5 mRNA is increased to a greater extent in dilp1-dilp2 double mutants relative to its increase in either single mutant, representing synergistic genetic interaction between dilp1 and dilp2 (Fig 3F). We observed no induction or repression of mRNA for dilp6, dilp7 and dilp8 in single and double mutants of dilp1 and dilp2 (Fig S2A-C, Supporting Information). Fecundity is not significantly different among wildtype, single and double dilp1 and dilp2 mutants for adult females one or three weeks old, although at two weeks old, dilp2 mutants lay slightly more eggs per day than the other genotypes (Fig S2E, Supporting Information). Finally, survival measured from egg through pupal stage was 50% less in dilp1-dilp2 double mutants relative to wildtype and to single mutants, suggesting that these insulin loci have redundant functions in egg-to-pupal viability (Fig S2F, Supporting Information). In sum, lifespan, body size and AKH are mediated by classic genetic epistasis between dilp1 and dilp2, where dilp1 functions downstream of dilp2. Other measured phenotypes are jointly regulated by dilp1 and dilp2, in some cases by redundant functions of the ligands and in other through synergistic functional interactions.

Epistatic analysis of insulin/IGF and juvenile hormone signaling

To understand how dilp1 is required to extend longevity, we evaluated insulin/IGF signal (IIS) transduction and juvenile hormone (JH) signaling in single and double dilp1 and dilp2 mutants. Insulin ligands in Drosophila are described to induce phosphorylation of Akt and ERK, which in turn regulate activity of transcription factors including FOXO. Here, we measured Akt and ERK phosphorylation from thorax tissue, which primarily consists of flight muscle (Fig 4A-C). While loss of dilp1 had no impact on Akt phosphorylation, loss of dilp2 increased Akt phosphorylation in single and double mutants, suggesting that compensatory expression of other dilps (dilp3 and dilp5) are sufficient to maintain this branch of IIS in the absence of dilp2. In contrast, ERK phosphorylation in thorax is reduced in dilp2 mutants, is unaffected in dilp1 mutants, and is restored to wildtype levels in the dilp1-dilp2 double mutant. Dilp1 and dilp2 interact epistatically to control ERK phosphorylation. This pattern correlates with the epistatic interaction we observe for dilp1 and dilp2 in the control of longevity, and we note that ERK has been implicated in how IIS controls aging downstream of the insulin receptor substrate chico (Slack et al., 2015).

Figure 4.
Figure 4. Components of insulin/IGF and JH signaling regulated by dilp1 and dilp2.

(A) Dissected thorax pAkt is increased in dilp2 and double mutants, and pERK is decreased in dilp2 mutants in a dilp1-dependent manner, representative blot shown. (B) Quantification of thorax pERK/ERK phosphowesterns, two-way ANOVA dilp1 x dilp2 p=0.003, n=6 per genotype. (C) Quantification of thorax pAkt/Akt phosphowesterns, two-way ANOVA dilp2 factor p<0.001, dilp1 x dilp2 N.S, n=6 per genotype. (D) 4eBP expression is not elevated in dilp2 mutants, two-way ANOVA dilp1 x dilp2 p=0.01, n=7-9 per genotype. (E) InR expression is not elevated in dilp2 mutants, but differentially expressed between single and double mutants, two-way ANOVA dilp1 x dilp2 p=0.054, n=7-9 per genotype. (F) Kr-h1 expression is decreased in dilp2 mutants, two-way ANOVA dilp1 p=0.02, dilp2 p=0.013, dilp1 x dilp2 N.S., n=8-9 per genotype.

Reduced IIS extends Drosophila lifespan in part through activating the FOXO transcription factor (Bai, Kang, Hernandez, & Tatar, 2013; Hwangbo et al., 2004; Min, Yamamoto, Buch, Pankratz, & Tatar, 2008). Accordingly, we measured FOXO activation in dilp1 and dilp2 single and double mutants by measuring expression of FOXO transcriptional targets 4eBP and InR from whole animals (Fig 4D-E). Neither FOXO target was elevated in the long-lived dilp2 single mutant. Thus, with our current mutants we find no association between longevity and elevated 4eBP or InR expression, suggesting that FOXO activation is not responsible for how reduced dilp2 slows aging.

Juvenile hormone (JH) is an insect terpenoid hormone produced by the corpora allata and documented to modulate how IIS impacts aging, where eliminating adult JH production is sufficient to extend lifespan (Tatar et al., 2001; Yamamoto, Bai, Dolezal, Amdam, & Tatar, 2013). JH controls transcriptional programs by regulating expression of the transcription factor Kruppel-homolog 1 (Kr-h1) (S. Liu et al., 2018; Minakuchi, Zhou, & Riddiford, 2008). Unlike FOXO, the expression of Kr-h1 mRNA (and thus JH activity) follows a pattern consistent with the epistatic interaction between dilp1 and dilp2 as seen for longevity: Kr-h1 is reduced in dilp2 mutant and restored to wildtype levels in the dilp1-dilp2 double mutant, while dilp1 single mutants tend to have greater Kr-h1 mRNA than wildtype (Fig 4F). Dilp1 thus appears to normally repress JH activity, and we conjecture that this may be sufficient for dilp1 to extend longevity when induced in dilp2 mutants.

Overexpression of dilp1 to rescue phenotypes in dilp1-dilp2 double mutant

We sought to confirm our key inferences from the epistasis analyses by rescuing phenotypes in the double mutant by exogenously expressed dilp1. We generated and validated a new UAS-dilp1 stock to express this insulin protein using GAL4-drivers (Fig S4A, Supporting Information). To test whether dilp1 is sufficient to slow aging and increase Akh expression, we induced UAS-dilp1 in the line containing dilp1-dilp2 null mutations. Driving UAS-dilp1 in IPCs with dilp2-GAL4 (Fig 5A-B) or in all neurons with the RU486 inducible GeneSwitch elav-GSGal4 (Fig 5C-D) significantly extended lifespan by consistently decreasing age-specific mortality. Likewise, Akh mRNA was elevated by dilp1 transgene expression when driven by dilp2-GAL4 in the double mutant background (Fig 5E).

Figure 5.
Figure 5. Dilp1 expression in double mutants rescues longevity and AKH.

(A) Lifespan is extended by dilp2>dilp1 overexpression rescue in the dilp1-dilp2 double mutant background compared to dilp2-GAL4/+ controls, Cox hazard analysis p<0.0001, chi2=25.8, n=289-364 per genotype. (B) Mortality is decreased in dilp2>dilp1 overexpression rescue in the dilp1-dilp2 double mutants compared to dilp2-GAL4/+ controls. (C) Lifespan is extended by elav-GS>dilp1 overexpression in the dilp1-dilp2 double mutant background treated with RU486 in adulthood compared to elav-GS/+ controls, Cox hazard analysis p<0.0001, chi2=89.7, n=361-377 per genotype. (D) Mortality is decreased by elav-GS>dilp1 overexpression in the dilp1-dilp2 double mutant background treated with RU486 in adulthood compared to elav-GS/+ controls. (E) Dilp2>dilp1 rescue in the dilp1-dilp2 double mutant background increases Akh expression compared to dilp2-Gal4/+ controls, t test p<0.001, n=5-6 per genotype. (F) Model for epistasis and interactions between dilp1 and dilp2 in regulating lifespan. IPCs, insulin producing cells; CC, corpora cardiaca; CA, corpora allata; JH, Juvenile Hormone.

DILP1 functions as a pro-longevity factor – it is necessary and sufficient for mutants of dilp2 to extend longevity. This result could be readily explained if DILP1 peptide acts to inhibit the insulin receptor and its tyrosine kinase activity. DILP1 might then slow aging by decreasing insulin/IGF signaling, although our observed patterns of FOXO target gene expression are not consistent with this hypothesis. To further evaluate the potential for DILP1 to act as an IIS inhibitor, we measured body mass of adults when dilp1 was induced as a transgene in the double mutant background. When driven by dilp2-GAL4, flies were the same mass as control flies, contrary to the expectation that an insulin/IGF signaling inhibitor would decrease body mass (Fig S5A, Supporting Information). In addition, when UAS-dilp1 is driven by dilp2-GAL4, there was no decrease in Akt or ERK phosphorylation, again inconsistent with DILP1 acting as an IIS inhibitor (Fig S5B-D, Supporting Information). These data make it unlikely that DILP1 extends longevity by functioning as an insulin/IGF receptor inhibitor, and opens the possibility that DILP1 acts as an insulin signaling agonist that somehow induces unique signaling to assure longevity.

Discussion

Based on mutational analyses of the insulin receptor (daf-2, InR) and its associated adaptor proteins and signaling elements, numerous studies in C. elegans and Drosophila established that decreased insulin/IGF signaling (IIS) extends lifespan (Clancy et al., 2001; Kenyon, Chang, Gensch, Rudner, & Tabtiang, 1993; Tatar et al., 2001). Studies on how reduced IIS in Drosophila systemically slows aging also reveal systems of feed-back where repressed IIS in peripheral tissue decreases DILP2 production in brain insulin producing cells (IPC), which may then reinforce a stable state of longevity assurance (Bai et al., 2012; Hwangbo et al., 2004; Wang et al., 2005). Here we find that expression of dilp1 from the IPC is required for loss of dilp2 to extend longevity (Fig 5C). This novel observation contrasts with conventional interpretations where reduced IIS is required to slow aging: elevated dilp1 mRNA is associated with longevity in dilp2 mutants, and transgene expression of dilp1 increases longevity.

Dilp1 and dilp2 are encoded in tandem, likely having arisen from a duplication event (Tatar et al., 2003). Perhaps as a result, some aspects of dilp1 and dilp2 are regulated in common: both are expressed in IPCs (Y. Liu et al., 2016; Rulifson et al., 2002), are regulated by sNPF (Lee et al., 2008) and have strongly correlated responses to dietary composition (Post & Tatar, 2016). Nonetheless, the paralogs are differentially expressed throughout development (Brogiolo et al., 2001). While dilp2 is expressed in larvae, dilp1 expression is elevated in the pupal stage when dilp2 expression is minimal (Slaidina et al., 2009). In adults, dilp1 expression decreases substantially after eclosion and dilp2 expression increases (Slaidina et al., 2009).

Furthermore, as noted, DILP1 production is associated with adult reproductive diapause, (Y. Liu et al., 2016). IIS regulates adult reproductive diapause in Drosophila, where a somatic state is induced that prolongs survival during inclement seasons (Tatar & Yin, 2001). DILP1 may stimulate these diapause pro-longevity pathways, while expression in non-diapause adults is sufficient to extend survival even in optimal environments.

Our data suggest that dilp1 extends longevity in part through induction of adipokinetic hormone (AKH), which is also increased during reproductive diapause (Kucerova et al., 2016) and is the functional homolog of mammalian glucagon (Bednarova, Kodrik, & Krishnan, 2013). Critically, manipulation to stimulate AKH secretion has been shown to increase Drosophila lifespan, potentially from catabolism of triacylglycerides and free fatty acids (Waterson et al., 2014). Here we also note that dilp1 mutants were more sensitive to starvation than wildtype and dilp2 mutants, further corroborating evidence that DILP1 may help mobilize nutrients during fasting (Y. Liu et al., 2016). Mammalian insulin and glucagon have inverse functions in regulating glucose storage and glycogen breakdown, and insulin decreases glucagon mRNA expression (Petersen, Vatner, & Shulman, 2017). We propose that DILP2 in Drosophila indirectly regulates AKH by repressing dilp1 expression, where DILP1 otherwise induces AKH.

A further connection between dilp1 and diapause involves juvenile hormone (JH). In many insects, adult reproductive diapause and its accompanied longevity are maintained by the absence of JH (Tatar & Yin, 2001). Furthermore, ablation of JH producing cells in adult Drosophila is sufficient to extend lifespan, and JH is greatly reduced in long-lived Drosophila insulin receptor mutants (Tatar et al., 2001; Yamamoto et al., 2013). In each case, exogenous treatment of long-lived flies with a JH analog (methoprene) restores survival to the level of wildtype or non-diapause controls. JH is a terpenoid hormone that interacts with a transcriptional complex consisting of Met (methoprene tolerant), Taimen and Kruppel homolog 1 (Kr-h1) (Jindra, Bellés, & Shinoda, 2015). As well, JH induces expression of kr-h1 mRNA, which serves as a reliable proxy for functionally active JH. Here we find that dilp2 mutants have reduced kr-h1 mRNA, while the titer of this message is similar to that of wildtype in dilp1-dilp2 double mutants. DILP1 appears to normally repress JH activity, as would occur in diapause when DILP1 is highly expressed. Such JH repression may contribute to longevity assurance during diapause as well as in dilp2 mutant flies maintained in laboratory conditions.

Does DILP1 act as an insulin receptor agonist or inhibitor? As an inhibitor, DILP1 could interact with the insulin receptor to suppress IIS, potentially even in the presence of other insulin peptides. Such action could systemically regulate programs for longevity assurance that are stimulated by low IIS. Alternatively, DILP1 may act as an insulin receptor agonist, binding to the receptor and inducing autophosphorylation. In this case, DILP1 could stimulate signal transduction and cellular responses to confer longevity assurance that are distinct from responses associated with insulin peptides such as DILP2 or DILP5 (Post et al., 2018). To date, our limited observations on this question are ambiguous. Genetic epistasis analysis between dilp1 and dilp2 in terms of ERK phosphorylation suggests that DILP1 might act as an inhibitor: solo mutation of dilp2 represses ERK phosphorylation (while increasing dilp1 mRNA) but the double dilp1-dilp2 mutant has wildtype ERK phosphorylation. Through a third potential mechanism, DILP1 may interact with circulating binding proteins such as IMPL2 or dALS to indirectly inhibit IIS output (Alic et al., 2011; Okamoto et al., 2013). We expect to resolve this question in a future study using synthetic DILP1 applied to cells in culture.

A precedent exists with work in C. elegans to suggest some insulin-like peptides might function as antagonists (Matsunaga et al., 2018; Pierce et al., 2001). Ins-23 and ins-18 positively regulate larval diapause and longevity (Matsunaga et al., 2018) while ins-1 promotes dauer formation during development and longevity in adulthood (Pierce et al., 2001). Both studies suggested that the peptide chains’ altered amino acid lengths may contribute to their distinct functions. Similarly, DILP1 has an extension at the N-terminus of the B chain compared to the other DILP sequences (Brogiolo et al., 2001). Moreover, C. elegans ins-7 suppresses ins-6 to have contrasting and epistatic functions in particular neurons regulating olfactory learning, demonstrating a daf-2 antagonistic role for ins-7 (Chen et al., 2013).

While FOXO (dFOXO or DAF-16) is intimately associated with how reduced IIS regulates aging in Drosophila and C. elegans (Martins, Lithgow, & Link, 2016), we did not find evidence for this role in our epistatic analysis between dilp1 and dilp2 despite the robust epistatic signals for lifespan and AKH. Mutation of dilp2 appeared to not impact FOXO activity, as measured by expression of target genes InR and 4eBP, and interactions with dilp1 did not modify this negative result. Notably, some precedence suggests only a limited role for dfoxo as the mediator of reduced IIS in aging, as dfoxo only partially rescues longevity benefits of chico, revealing that IIS extends lifespan through some FOXO-independent pathways (Yamamoto & Tatar, 2011). On the other hand, our whole animal analysis of dFOXO targets may obscure its role in IIS signaling if IIS and FOXO regulate aging only through actions in a few specific tissues, as previously documented (Tain et al., 2017; Wolkow, Kimura, Lee, & Ruvkun, 2000). In this vein, we find that dilp2 controls thorax ERK signaling but not AKT, suggesting that dilp2 mutants may activate muscle-specific ERK/MAPK anti-aging programs, although their potential action through dFOXO remains to be determined.

Dilp1 and dilp2 interact in a redundant fashion to regulate glycogen levels and blood sugar. These loci interact synergistically to regulate dilp5 mRNA compensation and starvation sensitivity. In contrast, dilp1 and dilp2 interact in a classic epistatic fashion to modulate longevity and AKH. The distinctions and associations delineated by the classes of interactions may reflect unique ways DILP1 and DILP2 stimulate different outcomes from their common tyrosine kinase insulin-like receptor. Understanding how and what is stimulated by DILP1 in the absence of dilp2 will likely reveal critical outputs that specific longevity assurance.

Experimental Procedures

Fly husbandry

Flies were reared and maintained at 25°C, 40% relative humidity and 12-hour light/dark. Adults were maintained upon agar-based diet with cornmeal (0.8%), sugar (10%), and yeast (2.5%). Fly stocks from Bloomington Stock Center include w1118,dilp1 (#30880) and w1118,dilp2 (#30881) mutants. dilp2-Gal4 stock was originally obtained from Ernst Hafen and elav-GSGal4 stock was obtained from Steven Helfand (Brown University). All stocks were backcrossed to w1118 for at least five generations.

Homologous Recombination

Homologous recombination (HR) of dilp1 and dilp2 in tandem was conducted as previously performed (Grönke et al., 2010; Staber, Gell, Jepson, & Reenan, 2011). See Supporting Information for detailed procedures.

Production of UAS-dilp1

See Supporting Information for detailed cloning procedures. Embryos were injected with UAS-dilp1 by BestGene Inc. (thebestgene.com) yielding 5 independent transformants for dilp1. For this study we selected one transformant for dilp1 that produced the strongest DILP1 immunolabeling when testing various Gal4 lines in larval and adult flies (see Fig S3).

Lifespan assays

Two to three-day-old female adult flies, reared in density-controlled bottles and mated after eclosion, were collected with light CO2 anesthesia and pooled in 1 L demography cages at a density of 100-125 flies per cage. Three independent cages were used per genotype. Food vials were changed every day for the first three weeks, then every two days for the remainder of each experiment. Dead flies were removed and recorded every other day. Cox Proportional Hazard analysis was conducted in R using the “surv” package and “survdiff” function.

RNA purification and quantitative RT-PCR

Total RNA was extracted from 20 whole mated female flies (8-10 days old) in Trizol (Invitrogen, Grand Island, NY, USA) and treated with Turbo DNase (Invitrogen). RNA was quantified with a NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and reverse-transcribed with iScript cDNA synthesis (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Quantitative RT-PCR was conducted with SYBR Green PCR master mix (Applied Biosystems, Carlsbad, CA, USA) and measured on an ABI prism 7300 Sequence Detection System (Applied Biosystems). mRNA abundance was calculated by comparative CT relative to ribosomal protein 49 (RP49). Primer sequences are listed in Supplemental Table S1.

Glycogen quantification

Mated female flies aged 8-10 days were flash frozen and homogenized in PBS. Samples were heat-treated at 70°C for 10 min and spun down at 14K rpm for 3 min at 4°C. Diluted amyloglucosidase enzyme (Sigma, #A1602) was added to the glycogen standard dilutions and half of the sample wells in a 96-well clear plate. PBS was added to the glucose standards and the other half of the sample wells. After 1 hour at 37°C, Glucose Hexokinase Reagent (Thermo Scientific) was added and the plate was incubated at room temperature for 15 min. The absorbance was read at 340nm on a SpectraMax M5 platereader using Softmax Pro software. The glycogen absorbance was quantified by subtracting the glucose absorbance from the total glycogen + glucose absorbance, and normalized to protein content by BCA assay.

Hemolymph sugar quantification

Mated female flies aged 8-10 days were decapitated to collect hemolymph in a microcentrifuge tube containing mesh. Flies were centrifuged for 4.5 min at 3K rpm and 4°C, and about 1ul hemolymph was transferred to 9ul PBS. Diluted hemolymph was heat-treated at 70°C for 5 min and transferred to tubes containing 200ul of Glucose Hexokinase Reagent (Thermo Scientific) with or without Porcine Kidney Trehalase (1:1000; Sigma-Aldrich). Reactions were incubated at 37°C for 16 hours and then transferred to wells of a clear microplate. The absorbance was read at 340nm using a SpectraMax M5 platereader and Softmax Pro software.

Body Mass

Two females and two males in each vial were allowed to lay eggs for 24-36 hours or until proper density was attained (about 60-80 eggs). Eclosed flies were mated for two days and females were sorted to separate vials. Food was changed every other day and at 8-10 days old, flies were counted, briefly anesthetized on CO2 and collected in a pre-weighed microcentrifuge tube. Tubes were weighed and mass per fly was calculated.

Western Blots

Mated female flies age 8-10 days were harvested in NP40 lysis buffer (Invitrogen) supplemented with 1mM PMSF, PhosSTOP phosphatase inhibitor cocktail (Roche #04906837001) and Protease Inhibitor Cocktail (Invitrogen). Samples were run on SDS-PAGE (Invitrogen NuPAGE). Gels were transferred to nitrocellulose membrane (Whatman) for one hour at 30V and washed in TBS-T for 10 min. Membranes were blocked with 5% BSA in TBS-T for one hour, and incubated with antibody 1:1000 in 5% BSA overnight at 4°C. Antibodies used were from Cell Signaling Technology: Drosophila phospho-Akt Ser505 (#4054S), Pan-Akt (#4691S), Pan-phospho-ERK (#4370S), Pan-ERK (#9102S). Blots were washed 3 times for 5 min and incubated in HRP-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch) 1:5000 in 1% BSA for one hour at room temperature. Blots were washed and incubated with ECL reagent (Perkin Elmer #NEL121001EA) for 5 minutes. Blots were imaged and analyzed by volume densitometry in ImageLab (BioRad).

Antisera and immunocytochemistry

Tissues from larvae or 7-day-old female adults were dissected in 0.1 M PBS, then fixed for four hours in ice-cold 4% paraformaldehyde (PFA), and rinsed in PBS three times for one hour. Incubation with primary antiserum was performed for 48 hours at 4°C. After rinsing in PBS with 0.25% Triton-X 100 (PBS-Tx) four times, tissues were incubated with secondary antibody for 48 hours at 4°C. After washing in PBS-Tx, tissues were mounted in 80% glycerol with 0.1 M PBS. Primary antisera used were: Rabbit antisera to DILP1 C-peptide (Y. Liu et al., 2016) at a dilution of 1:10,000, rabbit antisera to DILP2 and DILP3 A-chains (Veenstra, Agricola, & Sellami, 2008) at a dilution of 1:2000, rabbit antisera to AKH was kindly donated by M. Brown (Athens, GA) used at 1:1000, rabbit anti-GFP at 1:000 (Invitrogen, Carlsbad, CA). Secondary antisera used were: goat anti-rabbit Alexa 546 antiserum and goat anti-rabbit Alexa 488 antiserum (Invitrogen, Carlsbad, CA). at 1:1000.

Image analysis

Confocal images were captured with a Zeiss LSM 780 confocal microscope (Jena, Germany) using a 40× oil immersion objective. The projection of z-stacks was processed using Fiji (https://imagej.nih.gov/ij/). The cell body outlines were extracted manually and the staining intensity was determined using Fiji. The background intensity for all samples was recorded by randomly selecting three small regions near the cell body of interest. The final intensity value of the cell bodies was determined by subtracting the background intensity.

Starvation resistance assays

Flies were mated for two days and females were separated and maintained for 6-7 days on normal food. For fasting, the flies were kept in vials containing 5 ml of 0.5% agarose (Sigma-Aldrich). Dead flies were counted at least every 12 hours. No less than 118 flies from 3 replicates were used for the analysis.

Author Contributions

SP and MT designed experiments and interpreted results. SP executed experiments. SF and RY executed experiments and contributed to experimental design. DN contributed to experimental design and interpretation of results. JV contributed to developing reagents. SP and MT wrote the manuscript and all authors edited the manuscript.

Acknowledgements

We thank Barry Pfeiffer for sharing the vector used to initiate production of the UAS-dilp1 transgenic fly. Funding for SP, RY and MT was supported by NIH R37 AG024360. SP was additionally supported by NIH T32 AG 41688-3 and by AFAR GR5290420. SL and DRN were supported by The Swedish Research Council (Vetenskapsrådet 2015-04626). JAV was supported by institutional funds from the CNRS.

Footnotes

  • sifang.liao{at}zoologi.su.se, rochele_yamamoto{at}brown.edu, jan-adrianus.veenstra{at}u-bordeaux.fr, dnassel{at}zoologi.su.se

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Supplementary Material

Email
Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2
Stephanie Post, Sifang Liao, Rochele Yamamoto, Jan A. Veenstra, Dick R. Nässel, Marc Tatar
bioRxiv 380410; doi: https://doi.org/10.1101/380410
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