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Drosophila Insulin-Like Peptides DILP2 and DILP5 Differentially Stimulate Cell Signaling and Glycogen Phosphorylase to Regulate Longevity

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Drosophila Insulin-Like Peptides DILP2 and DILP5 Differentially Stimulate Cell Signaling and Glycogen Phosphorylase to Regulate Longevity ORIGINAL RESEARCH published: 28 May 2018 doi: 10.3389/fendo.2018.00245 Drosophila Insulin-Like Peptides DILP2 and DILP5 Differentially Stimulate Cell Signaling and Glycogen Phosphorylase to Regulate Longevity Stephanie Post1,2*, Galina Karashchuk 2, John D. Wade 3,4, Waseem Sajid 5, Pierre De Meyts6,7 and Marc Tatar 2* 1 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, United States, 2 Department of Ecology and Evolutionary Biology, Brown University, Providence, RI, United States, 3 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia, 4 School of Chemistry, University of Melbourne, Melbourne, VIC, Australia, 5 LEO Pharma A/S, Ballerup, Denmark, 6 Department of Cell Signalling, de Duve Institute, Brussels, Belgium, 7 Department of Stem Cell Research Novo Nordisk A/S, Måløv, Denmark Edited by: Ian Orchard, Insulin and IGF signaling (IIS) is a complex system that controls diverse processes University of Toronto including growth, development, metabolism, stress responses, and aging. Drosophila Mississauga, Canada melanogaster IIS is propagated by eight Drosophila insulin-like peptides (DILPs), Reviewed by: Gert Jansen, homologs of both mammalian insulin and IGFs, with various spatiotemporal expres- Erasmus University sion patterns and functions. DILPs 1–7 are thought to act through a single Drosophila Rotterdam, Netherlands Leslie Pick, insulin/IGF receptor, InR, but it is unclear how the DILPs thereby mediate a range of University of Maryland, physiological phenotypes. We determined the distinct cell signaling effects of DILP2 College Park, United States and DILP5 stimulation upon Drosophila S2 cells. DILP2 and DILP5 induced similar *Correspondence: transcriptional patterns but differed in signal transduction kinetics. DILP5 induced Stephanie Post [email protected]; sustained phosphorylation of Akt, while DILP2 produced acute, transient Akt phos- Marc Tatar phorylation. Accordingly, we used phosphoproteomic analysis to identify distinct [email protected] patterns of non-genomic signaling induced by DILP2 and DILP5. Across all treatments Specialty section: and replicates, 5,250 unique phosphopeptides were identified, representing 1,575 This article was submitted to proteins. Among these peptides, DILP2, but not DILP5, dephosphorylated Ser15 on Neuroendocrine Science, glycogen phosphorylase (GlyP), and DILP2, but not DILP5, was subsequently shown a section of the journal Frontiers in Endocrinology to repress enzymatic GlyP activity in S2 cells. The functional consequences of this dif- Received: 19 March 2018 ference were evaluated in adult Drosophila dilp mutants: dilp2 null adults have elevated Accepted: 02 May 2018 GlyP enzymatic activity relative to wild type, while dilp5 mutants have reduced GlyP Published: 28 May 2018 activity. In flies with intact insulin genes, GlyP overexpression extended lifespan in a Citation: Ser15 phosphorylation-dependent manner. In dilp2 mutants, that are otherwise long- Post S, Karashchuk G, Wade JD, Sajid W, De Meyts P and Tatar M lived, longevity was repressed by expression of phosphonull GlyP that is enzymatically (2018) Drosophila Insulin- inactive. Overall, DILP2, unlike DILP5, signals to affect longevity in part through its Like Peptides DILP2 and DILP5 Differentially Stimulate Cell Signaling control of phosphorylation to deactivate glycogen phosphorylase, a central modulator and Glycogen Phosphorylase of glycogen storage and gluconeogenesis. to Regulate Longevity. Front. Endocrinol. 9:245. Keywords: insulin, IGF, Drosophila insulin-like peptides, glycogen phosphorylase, glycogen, metabolism, aging, doi: 10.3389/fendo.2018.00245 signaling bias Frontiers in Endocrinology | www.frontiersin.org 1 May 2018 | Volume 9 | Article 245 Post et al. DILP2, Not DILP5, Regulates GlyP INTRODUCTION sustained Akt phosphorylation than insulin, which generates transient Akt phosphorylation. Analogs such as [B-Asp10] insulin Insulin and insulin-like growth factor signaling (IIS) is an and [A-His8, B-His4, B-Glu10, B-His27] insulin, which have strong extensive network crucial for development, growth, nutrient receptor-binding affinity, induced sustained receptor phospho- sensing, aging, and stress responses (1–3). Dysfunction in rylation and increased mitogenicity (26), and insulin analogs IIS contributes to metabolic syndrome, diabetes, and cancer with weaker receptor-binding affinity produced less mitogenic (4, 5), yet genetic modification of IIS can extend lifespan in potential (27, 28). Recently, decreased mitogenicity was reported many animals (6–8). Mammalian insulin and IGF ligands each for the insulin analog dicarba where the A6-A11 disulfide bond have respective receptors, although each ligand can activate is replaced with a rigid bridge to reduce flexibility in its receptor either receptor, receptors can form hybrid dimers, and the interaction (29). Collectively, these data suggest that insulin, IGF, receptors themselves activate similar kinase cascades with mul- and insulin analogs can differentially bind to receptors to produce tiple redundant components (9). It is currently unknown how distinct signals and generate different downstream phenotypes. IIS ligands use common receptors and pathways to produce Drosophila insulin-like peptide 2 and DILP5 are two princi- different cellular and organism phenotypes such as glucose pal hormones made in the insulin-producing cells of the adult homeostasis for insulin and development and differentiation for Drosophila brain (30). They are thought to regulate sugar and pro- IGF. Here, we use Drosophila melanogaster as a model system tein metabolism respectively (31), and to uniquely respond to die- to understand how various insulin-like peptides [Drosophila tary sugar and protein (32). We compared cells stimulated in vitro insulin-like peptides (DILPs)] function through the fly’s sole with DILP2 or DILP5 to determine their cell signaling outputs insulin/IGF tyrosine kinase receptor (InR) to mediate specific measured as Akt and InR phosphorylation, transcript profiles and physiological traits. global patterns of protein phosphorylation. In S2 cells, DILP2 and In Drosophila, ligand-activated InR phosphorylates a single DILP5 regulate highly similar sets of mRNA transcripts and are insulin receptor substrate (IRS) chico, the homolog of mamma- equally potent in their ability to stimulate Akt phosphorylation. lian IRS1–4, to induce the phosphorylation cascade of phosphoi- On the other hand, at a given dose DILP2 and DILP5 differen- nositide-3-kinase (PI3K), phosphoinositide-dependent-kinase-1, tially modulate the kinetics of Akt phosphorylation, with DILP2 and Akt (protein kinase B) (10). This signal transduction cul- producing acute stimulation and DILP5 generating prolonged minates to repress the forkhead transcription factor dFOXO, signal. Expanding this non-genomic view of biased signaling, we the homolog of mammalian FOXO1, 3a, and 4 (11). Fly IIS and used global phosphoproteomic analysis and found substantial FOXO mutants affect larval growth, adult size, lipid metabolism, qualitative differences between DILP2 and DILP5. In particular, stress responses and aging (8, 12, 13). Data suggest that DILPs DILP2 uniquely regulates dephosphorylation of glycogen phos- have distinct spatial and temporal expression patterns and regu- phorylase (GlyP) at Ser15, the residue that represses enzymatic late shared and specific functions (13, 14). For instance, embryos activity in mammals and Drosophila (33–35). In concordance, express dilp4, larvae express dilp2, dilp3, and dilp5, and pupae we find that dilp2 mutant flies have elevated GlyP enzymatic upregulate dilp1 and dilp6 expression (1, 13, 14). In adults, dilp2 activity, and that overexpression of GlyP extended longevity in modulates adult lifespan and blood sugar (8, 15), dilp5 mediates adults with normal insulin in a GlyP phosphorylation-dependent protein metabolism (16), and dilp3 is suggested to regulate lipid manner that corresponded with GlyP enzymatic activity. In a metabolism (17). Given the diversity of these functions, it is complementary way, we infer that the phosphorylation status and poorly understood how specificity can be produced by similar enzymatic activity of GlyP contribute to the lifespan extension of ligands signaling through a common InR receptor. dilp2 mutants because expression of phosphonull, inactive GlyP The unique spatiotemporal expression patterns of DILPs may partially rescues extended longevity of dilp2 mutants. Together, be sufficient to confer their specific phenotypes. An alternative, these data demonstrate that non-genomic signaling bias at the but not mutually exclusive model proposes that DILPs differen- cellular level plays a role in differential physiological and life his- tially activate InR to induce distinct cell signaling patterns that tory functions of two insulin-like peptides. communicate specific downstream phenotypes. Studies from mammalian systems support such a signaling bias model: cells engineered to express only the insulin receptor (IR), IGF-1R, or MATERIALS AND METHODS IGF-2R produce distinct signaling and gene expression patterns in response to insulin, IGF-1, or IGF-2 (18, 19). At a biochemical Cell Culture and DILP Stimulation level, Cieniewicz et al. (20) found that the IR was differentially S2 cells were cultured in Schneider’s media (Gibco) with 10% phosphorylated and dephosphorylated on several individual FBS (Gibco) and maintained at 28°C with ambient
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