Sestrins are conserved proteins that accumulate in cells exposed to stress and potentiate adenosine monophosphate-activated protein kinase (AMPK) and inhibit activation of target of rapamycin (TOR). We show that abundance of Drosophila Sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species (ROS) that cause activation of c-Jun N-terminal kinase (JNK) and transcription factor FoxO (Forkhead box O). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration and cardiac malfunction, which were prevented by pharmacological activation of AMPK or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation, that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids.TOR (target of rapamycin) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis (1-3). Insulin (Ins) and insulin-like growth factors (IGF) are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT (2). Conversely, adenosine monophosphate activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation (2,4). TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) (1-3,5) and by decreasing autophagic † To whom correspondence should be addressed. karinoffice@ucsd.edu. Prolonged TOR signaling induces dSesnPersistent TOR activation in wing discs by a constitutively active form of insulin receptor (InR CA ) resulted in prominent dSesn protein accumulation, not seen in a dSesn-null larvae (Fig. 1, A to C). InR CA also induced accumulation of dSesn RNA (Fig. 1, D to F), indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. As dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb (Fig. 1G), or clonal loss of PTEN (phosphatase and tensin homolog) or TSC1 (tuberous sclerosis complex 1) (Fig. 1, H TOR signaling generates ROS to induce dSesnIn mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress (9,11) and we observed ROS accumulation, detected by oxidation of dihydroethidium (DHE), in the same region of the imaginal discs in which InR CA or Rheb were expressed (Fig. 2, A (Fig. 2F).FoxO and p53 are ROS-activated transcription factors that control mammalian Sesn genes (9-12,14). The dSesn locus contains 8 perfect FoxO-response elemen...

High Fat Diet (HFD)-induced obesity is a major contributor to diabetes and cardiovascular disease, but the underlying genetic mechanisms are poorly understood. Here, we use Drosophila to test the hypothesis that HFD-induced obesity and associated cardiac complications have early evolutionary origins involving nutrient-sensing signal transduction pathways. We find that HFD-fed flies exhibit increased triglyceride (TG) fat and alterations in insulin/glucose homeostasis, similar to mammalian responses. A HFD also causes cardiac lipid accumulation, reduced cardiac contractility, conduction blocks and severe structural pathologies, reminiscent of diabetic cardiomyopathies. Remarkably, these metabolic and cardiotoxic phenotypes elicited by HFD are blocked by inhibiting insulin-TOR signaling. Remarkably, reducing insulin-TOR activity by TSC1-2, 4EBP, FOXO) or increasing lipase expression in the myocardium suffices to efficiently alleviate cardiac fat accumulation and dysfunction induced by HFD. We conclude that deregulation of insulin-TOR signaling due to a HFD is responsible for mediating the detrimental effects on metabolism and heart function.

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antannal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). We show here that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using twophoton microscopy, we found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. We detect opposite behavioral phenotypes after reduction and over expression of DTKR in ORNs. Our findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe.olfactory behavior ͉ presynaptic inhibition ͉ tachykinin ͉ two-photon imaging I n Drosophila, odor detection begins when odor molecules activate olfactory receptor neurons (ORNs) in the antennae and maxillary palps. Each of the ORNs expresses only 1 or a few members of a large family of odorant receptor genes (1-4). These ORNs propagate activity to neurons with dendrites in the glomerular compartments of the antennal lobe; each glomerulus receives inputs from ORNs that express the same odorant receptor (1, 3, 5, 6). In the glomeruli, the activity is read by second-order neurons, designated projection neurons (PNs), which relay information to higher olfactory centers in the brain (7).Inhibitory circuits in the glomeruli, mediated by local interneurons (LNs), play a key role in modulating glomerular signal activity. Presynaptic GABAergic inhibition of the ORNs has been shown in both Drosophila (8, 9) and in mammals (10-12). Conversely, cholinergic LNs in the Drosophila antennal lobe have been suggested to increase and redistribute odor-evoked activity at low odor concentrations (13,14).In addition to GABA and acetylcholine it is likely that certain neuropeptides are used as neuromodulators in the antennal lobe circuitry of insects (15, 16), as also suggested in the olfactory bulb in mammals (17,18). One neuropeptide gene that has been implicated in olfactory processing is dtk (19), a gene encoding 5 tachykinin-related peptides, DTKs (20). The DTKs are expressed in Ϸ150 neurons in the Drosophila brain, and in the antennal lobe glomeruli, there are extensive DTK-immunoreactive arborizations derived from a subset of antennal lobe LNs (21). Two DTK receptors, DTKR and NKD, have been identified in Drosophila (22, 23) and 1 of these, DTKR, is strongly expressed in antennal lobe glomeruli (24). Behavioral evidence for a role of DTKs in olfaction was obtained from analysis of flies where dtk expression was knocked down globally using RNA interference (RNAi); these flies displayed diminished odor sensitivity (19).To gain insight into the neuromodulation provided by the DTK signaling system in the antennal lob...

SUMMARY Obesity and metabolic syndrome are associated with an increased risk for lipotoxic cardiomyopathy, which is strongly correlated with excessive accumulation of lipids in the heart. Obesity- and type 2 diabetes-related disorders have been linked to altered expression of the transcriptional cofactor PGC-1α, which regulates the expression of genes involved in energy metabolism. Using Drosophila, we identify PGC-1/spargel (PGC-1/srl) as a key antagonist of high-fat diet (HFD)-induced lipotoxic cardiomyopathy. We find that HFD-induced lipid accumulation and cardiac dysfunction are mimicked by reduced PGC-1/srl function and reversed by PGC-1/srl overexpression. Moreover, HFD feeding lowers PGC-1/srl expression by elevating TOR signaling and inhibiting expression of the Drosophila adipocyte triglyceride lipase (ATGL, Brummer), both of which function as upstream modulators of PGC-1/srl. The lipogenic transcription factor SREBP also contributes to HFD-induced cardiac lipotoxicity, likely in parallel with PGC-1/srl. These results suggest a regulatory network of key metabolic genes that modulates lipotoxic heart dysfunction.

Activation of G protein-coupled receptors (GPCR)leads to the recruitment of ␤-arrestins. By tagging the ␤-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTP␥S binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the ␤-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles.

SUMMARYDrosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophila the regulation of DILP production and release in adult fruit flies is poorly understood. Here we investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR and NKD, in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, we show DTK-immunoreactive axon terminations close to the presumed dendrites of the IPCs, and DTKR immunolabeling in these cells. Second, we utilized targeted RNA interference to knock down expression of the DTK receptor, DTKR, in IPCs and monitored the effects on Dilp transcript levels in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. We also measured trehalose and lipid levels as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, we targeted the IPCs with RNAi or ectopic expression of the other DTK receptor, NKD, but found no effect on survival at starvation. Our results suggest that DTK signaling, via DTKR, regulates the brain IPCs. Supplementary material available online at

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