Juvenile diet restriction and the aging and reproduction of adult Drosophila melanogaster

Mutations of the insulin signal pathway in Drosophila melanogaster produce long-lived adults with many correlated phenotypes. Homozygotes of insulin-like receptor ( InR ) and insulin-like receptor substrate ( chico ) delay time to eclosion, reduce body size, decrease reproduction and increase life span. Because these mutations are expressed through all life stages it is unclear when insulin signals must be reduced to increase life span. As a first analysis of this problem in D. melanogaster we have manipulated the larval diet to determine if changes in metabolic regulation at this stage are sufficient to slow aging. We controlled the dietary yeast fed to third instar larvae and studied the size, mortality, fecundity and hormones of the resulting adults, which were fed a normal, yeast-replete diet. Adults from yeast-deprived larvae phenocopied many traits of InR and chico mutants: small body size, delayed eclosion, reduced ovariole number and reduced age-specific fecundity. But unlike constitutive mutants of the insulin/IGF system, adults from yeast-deprived larvae had normal patterns of demographic senescence, and this was accompanied by normal insulin-like peptide and juvenile hormone syntheses. Surprisingly, the normal aging in these adults was also associated with greatly reduced fecundity. Although nutritional conditions of the larvae can affect the subsequent body size and fecundity of adults, these are not sufficient to slow aging.     

The functions of insulin signaling: size isn't everything,even in Drosophila

Mammalian insulin and insulin-like growth factors (IGFs) signal through several receptors with different ligand specificities to regulate metabolism and growth. This regulation is defective in diabetes and in a wide variety of human tumors. Recent analysis in Drosophila melanogaster has revealed that insulin-like molecules (known as DILPs in flies) also control growth and metabolism, but probably do so by signaling through a single insulin receptor (InR). The intracellular signaling molecules regulated by this receptor are highly evolutionarily conserved. Work in flies has helped to dissect the network of InR-regulated intracellular signaling pathways and identify some of the critical players in these pathways and in interacting signaling cascades. Surprisingly, these studies have shown that DILPs control tissue and body growth primarily by regulating cell growth and cell size. Changes in cell growth produced by these molecules may subsequently modulate the rate of cell proliferation in a cell type-specific fashion. At least part of this growth effect is mediated by two small groups of neurons in the Drosophila brain, which secrete DILPs into the circulatory system at levels that are modulated by nutrition. This signaling center is also involved in DILP-dependent control of the fly's rate of development, fertility, and life span. These surprisingly diverse functions of InR signaling, which appear to be conserved in all higher animals, reflect a central role for this pathway in coordinating development, physiology, and properly proportioned growth of the organism in response to its nutritional state. Studies in flies are providing important new insights into the biology of this system, and the identification of novel components in the InR-regulated signaling cascade is already beginning to inform the development of new therapeutic strategies for insulin-linked diseases in the clinic.     

Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila

Insulin signaling pathways are implicated in several physiological processes in invertebrates, including the control of growth and life span; the latter of these has also been correlated with juvenile hormone (JH) deficiency. In turn, JH levels have been correlated with sex-specific differences in locomotor activity. Here, the involvement of the insulin signaling pathway in sex-specific differences in locomotor activity was investigated in Drosophila. Ablation of insulin-producing neurons in the adult pars-intercerebralis was found to increase trehalosemia and to abolish sexual dimorphism relevant to locomotion. Conversely, hyper-insulinemia induced by insulin injection or by over-expression of an insulin-like peptide decreases trehalosemia but does not affect locomotive behavior. Moreover, we also show that in the head of adult flies, the insulin receptor (InR) is expressed only in the fat body surrounding the brain. While both male and female InR mutants are hyper-trehalosemic, they exhibit similar patterns of locomotor activity. Our results indicate that first, insulin controls trehalosemia in adults, and second, like JH, it controls sex-specific differences in the locomotor activity of adult Drosophila in a manner independent of its effect on trehalose metabolism.     

Temporal control of differentiation by the insulin receptor/tor pathway in Drosophila

Multicellular organisms must integrate growth and differentiation precisely to pattern complex tissues. Despite great progress in understanding how different cell fates are induced, it is poorly understood how differentiation decisions are temporally regulated. In a screen for patterning mutants, we isolated alleles of tsc1, a component of the insulin receptor (InR) growth control pathway. We find that loss of tsc1 disrupts patterning due to a loss of temporal control of differentiation. tsc1 controls the timing of differentiation downstream or in parallel to the RAS/MAPK pathway. Examination of InR, PI3K, PTEN, Tor, Rheb, and S6 kinase mutants demonstrates that increased InR signaling leads to precocious differentiation while decreased signaling leads to delays in differentiation. Importantly, cell fates are unchanged, but tissue organization is lost upon loss of developmental timing controls. These data suggest that intricate developmental decisions are coordinated with nutritional status and tissue growth by the InR signaling pathway.     

A triangular connection between Cyclin G,PP2A and Akt1 in the regulation of growth and metabolism in Drosophila

Size and weight control is a tightly regulated process, involving the highly conserved Insulin receptor/target of rapamycin (InR/TOR) signaling cascade. We recently identified Cyclin G (CycG) as an important modulator of InR/TOR signaling activity in Drosophila. cycG mutant flies are underweight and show a disturbed fat metabolism resembling TOR mutants. In fact, InR/TOR signaling activity is disturbed in cycG mutants at the level of Akt1, the central kinase linking InR and TORC1. Akt1 is negatively regulated by protein phosphatase PP2A. Notably the binding of the PP2A B′-regulatory subunit Widerborst (Wdb) to Akt1 is differentially regulated in cycG mutants, presumably by a direct interaction of CycG and Wdb. Since the metabolic defects of cycG mutant animals are abrogated by a concomitant loss of Wdb, CycG presumably influences Akt1 activity at the PP2A nexus. Here we show that Well rounded (Wrd), another B' subunit of PP2A in Drosophila, binds CycG similar to Wdb, and that its loss ameliorates some, but not all, of the metabolic defects of cycG mutants. We propose a model, whereby the binding of CycG to a particular B′-regulatory subunit influences the tissue specific activity of PP2A, required for the fine tuning of the InR/TOR signaling cascade in Drosophila.     

Hmgcr in the corpus allatum controls sexual dimorphism of locomotor activity and body size via the insulin pathway in Drosophila

The insulin signaling pathway has been implicated in several physiological and developmental processes. In mammals, it controls expression of 3-Hydroxy-3-Methylglutaryl CoA Reductase (HMGCR), a key enzyme in cholesterol biosynthesis. In insects, which can not synthesize cholesterol de novo, the HMGCR is implicated in the biosynthesis of juvenile hormone (JH). However, the link between the insulin pathway and JH has not been established. In Drosophila, mutations in the insulin receptor (InR) decrease the rate of JH synthesis. It is also known that both the insulin pathway and JH play a role in the control of sexual dimorphism in locomotor activity. In studies here, to demonstrate that the insulin pathway and HMGCR are functionally linked in Drosophila, we first show that hmgcr mutation also disrupts the sexual dimorphism. Similarly to the InR, HMGCR is expressed in the corpus allatum (ca), which is the gland where JH biosynthesis occurs. Two p[hmgcr-GAL4] lines were therefore generated where RNAi was targeted specifically against the HMGCR or the InR in the ca. We found that RNAi-HMGCR blocked HMGCR expression, while the RNAi-InR blocked both InR and HMGCR expression. Each RNAi caused disruption of sexual dimorphism and produced dwarf flies at specific rearing temperatures. These results provide evidence: (i) that HMGCR expression is controlled by the InR and (ii) that InR and HMGCR specifically in the ca, are involved in the control of body size and sexual dimorphism of locomotor activity.     

PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids

Growth in normal and tumour cells is regulated by evolutionarily conserved extracellular inputs from the endocrine insulin receptor (InR) signalling pathway and by local nutrients. Both signals modulate activity of the intracellular TOR kinase, with nutrients at least partly acting through changes in intracellular amino acid levels mediated by amino acid transporters. We show that in Drosophila, two molecules related to mammalian proton-assisted SLC36 amino acid transporters (PATs), CG3424 and CG1139, are potent mediators of growth. These transporters genetically interact with TOR and other InR signalling components, indicating that they control growth by directly or indirectly modulating the effects of TOR signalling. A mutation in the CG3424 gene, which we have named pathetic (path), reduces growth in the fly. In a heterologous Xenopus oocyte system, PATH also activates the TOR target S6 kinase in an amino acid-dependent way. However, functional analysis reveals that PATH has an extremely low capacity and an exceptionally high affinity compared with characterised human PATs and the CG1139 transporter. PATH and potentially other PAT-related transporters must therefore control growth via a mechanism that does not require bulk transport of amino acids into the cell. As PATH is likely to be saturated in vivo, we propose that one specialised function of high-affinity PAT-related molecules is to maintain growth as local nutrient levels fluctuate during development.     

Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.     

Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila

Phagocytosis is a complex and apparently evolutionarily conserved process that plays a central role in the immune response to infection. By ultrastructural and functional criteria, Drosophila hemocyte (macrophage) phagocytosis resembles mammalian phagocytosis. Using a non-saturated forward genetic screen for larval hemocyte phagocytosis mutants, D-SCAR and profilin were identified as important regulators of phagocytosis in Drosophila. In both hemocytes ex vivo and the macrophage-like S2 cell line, lack of D-SCAR significantly decreased phagocytosis of Escherichia coli and Staphylococcus aureus. In contrast, profilin mutant hemocytes exhibited increased phagocytic activity. Analysis of double mutants suggests that D-SCAR and profilin interact during phagocytosis. Finally, RNA interference studies in S2 cells indicated that the D-SCAR homolog D-WASp also participates in phagocytosis. This study demonstrates that Drosophila provides a viable model system in which to dissect the complex interactions that regulate phagocytosis.     

Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early foetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis we find that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis we find that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, we have identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and have shown that the molecular circuitry required is lineage specific.     

Cytoplasmic activated protein kinase Akt regulates lipid-droplet accumulation in Drosophila nurse cells

The insulin/insulin-like growth factor signalling (IIS) cascade performs a broad range of evolutionarily conserved functions, including the regulation of growth, developmental timing and lifespan, and the control of sugar, protein and lipid metabolism. Recently, these functions have been genetically dissected in the fruit fly Drosophila melanogaster, revealing a crucial role for cell-surface activation of the downstream effector kinase Akt in many of these processes. However, the mechanisms regulating lipid metabolism and the storage of lipid during development are less well characterized. Here, we use the nutrient-storing nurse cells of the fly ovary to study the cellular effects of intracellular IIS components on lipid accumulation. These cells normally store lipid in a perinuclear pool of small neutral triglyceride-containing droplets. We find that loss of the IIS signalling antagonist PTEN, which stimulates cell growth in most developing tissues, produces a very different phenotype in nurse cells, inducing formation of highly enlarged lipid droplets. Furthermore, we show that the accumulation of activated Akt in the cytoplasm is responsible for this phenotype and leads to a much higher expression of LSD2, the fly homologue of the vertebrate lipid-storage protein perilipin. Our work therefore reveals a signalling mechanism by which the effect of insulin on lipid metabolism could be regulated independently of some of its other functions during development and adulthood. We speculate that this mechanism could be important in explaining the well-established link between obesity and insulin resistance that is observed in Type 2 diabetes.     

The IRS-signalling system during insulin and cytokine action

The discovery of the first intracellular substrate for insulin, IRS-1, redirected the field of diabetes research and has led to many important advances in our understanding of insulin action. Detailed analysis of IRS-1 demonstrates structure/function relationships for this modular docking molecule, including mechanisms of substrate recognition and signal propagation. Recent work has also identified other structurally similar molecules, including IRS-2, the Drosophila protein, DOS, and the Grb2-binding protein, Gab1, suggesting that this intracellular signalling strategy is conserved evolutionarily and is utilized by an expanding number of receptor systems. In fact, IRS-1 itself has been shown to be important in other growth factor and cytokine signalling systems, including growth hormone and several interleukins. Analysis of mice lacking IRS-1 confirms an important physiological role for this protein in glucose metabolism and general cell growth in the intact animal. Disregulation of the signalling pathways integrated by the IRS proteins may contribute to the pathophysiology of non-insulin-dependent diabetes mellitus or other diseases.     

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells.     

Rheb promotes cell growth as a component of the insulin/TOR signalling network

Insulin signalling is a potent stimulator of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin) [corrected]. Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity [corrected]. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, we identified Rheb (Ras homologue enriched in brain), a member of the Ras superfamily of GTP-binding proteins. Increased levels of Rheb in Drosophila melanogaster promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth.     

A Cullin1-Based SCF E3 Ubiquitin Ligase Targets the InR/PI3K/TOR Pathway to Regulate Neuronal Pruning

Pruning that selectively eliminates unnecessary axons/dendrites is crucial for sculpting the nervous system during development. During Drosophila metamorphosis, dendrite arborization neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone, whereas mushroom body γ neurons specifically eliminate their axon branches within dorsal and medial lobes. However, it is unknown which E3 ligase directs these two modes of pruning. Here, we identified a conserved SCF E3 ubiquitin ligase that plays a critical role in pruning of both ddaC dendrites and mushroom body γ axons. The SCF E3 ligase consists of four core components Cullin1/Roc1a/SkpA/Slimb and promotes ddaC dendrite pruning downstream of EcR-B1 and Sox14, but independently of Mical. Moreover, we demonstrate that the Cullin1-based E3 ligase facilitates ddaC dendrite pruning primarily through inactivation of the InR/PI3K/TOR pathway. We show that the F-box protein Slimb forms a complex with Akt, an activator of the InR/PI3K/TOR pathway, and promotes Akt ubiquitination. Activation of the InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning. Thus, our findings provide a novel link between the E3 ligase and the InR/PI3K/TOR pathway during dendrite pruning.     

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B''-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.