Molecular mechanisms of metabolic regulation by insulin in ... · Molecular mechanisms of metabolic regulation by insulin in Drosophila Aurelio A. TELEMAN1 German Cancer Research

Biochem. J. (2010) 425, 13–26 (Printed in Great Britain) doi:10.1042/BJ20091181 13

REVIEW ARTICLEMolecular mechanisms of metabolic regulation by insulin in DrosophilaAurelio A. TELEMAN1

German Cancer Research Center (Deutsches Krebsforschungszentrum), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany

The insulin signalling pathway is highly conserved from mam-mals to Drosophila. Insulin signalling in the fly, as in mammals,regulates a number of physiological functions, including carbo-hydrate and lipid metabolism, tissue growth and longevity. Inthe present review, I discuss the molecular mechanisms by whichinsulin signalling regulates metabolism in Drosophila, comparing

and contrasting with the mammalian system. I discuss both theintracellular signalling network, as well as the communicationbetween organs in the fly.

Key words: Akt, Drosophila metabolism, insulin signalling, targetof rapamycin (TOR), total body glucose, total body lipid.

INTRODUCTION

In the past decade, Drosophila has been one of the importantmodel systems for studying the insulin and IGF (insulin-likegrowth factor)/TOR (target of rapamycin) signalling pathway(Figure 1). For instance, some components of the pathway suchas Rheb, Tsc1 (tuberous sclerosis complex 1) and Tsc2 (tuberoussclerosis complex 2) were first placed into the TOR pathway in thefly [1–5]. Such work has been supported by the powerful genetictools available in Drosophila, allowing researchers to quicklygenerate loss-of-function animals to query gene function, andepistasis experiments to query gene relationships, both of whichare fundamental for elucidating signalling networks.

More recently, Drosophila has also become a model systemfor studying how insulin signalling regulates organismal andcellular metabolism (Table 1). Although the metabolic regulationof Drosophila is interesting in itself, work in this field has alsobeen sparked by an increased interest in organismal metabolismdue to the emerging world-wide epidemic of Type 2 diabetesand obesity. At first, one might be sceptical that the metabolicregulation of flies and humans have anything in common, yet asurprising amount of conservation and parallelism between thetwo systems has emerged. As discussed below, the conservationoccurs not only at the level of the molecular components of theinsulin signalling pathway, but also at the level of the physiologicaloutputs of the pathway. This is probably because multicellularorganisms needed to regulate their response to varying nutrientconditions early in evolutionary history. Indeed Drosophila, likemammals, regulate their circulating sugar levels and store excessenergy in the form of glycogen and lipids [6]. These energy storesare then mobilized when energy is needed [7]. In the light ofthis, in this review I will highlight both the similarities and thedifferences between the fly and mammalian systems.

INSULIN MEDIATES MUCH OF THE NUTRIENT-DEPENDENTSIGNALLING IN DROSOPHILA

Two sets of observations support the conclusion that most of thenutrient sensing in Drosophila takes place via the insulin pathway.First, mutations in components of the insulin pathway phenocopythe effects of nutrient deprivation. For instance, mutation ofthe kinase TOR phenocopies amino acid deprivation, leadingto a block in tissue growth, reduced nucleolar size and cellcycle arrest despite the presence of nutrients [8]. As anotherexample, reduction in S6K [RPS6 (ribosomal protein S6)-p70-protein kinase] activity in the Drosophila brain leads to similarchanges in feeding behaviour to those observed upon fasting,including an increased rate of food intake and reduced aversion tounappealing food [9]. Secondly, artificially elevating signallingthrough the insulin pathway is sufficient to bypass the needfor nutrients for many cell-biological processes. This meansthat signalling through the pathway is ‘epistatic’ to nutrientavailability. For instance, activation of PI3K (phosphoinositide3-kinase) signalling bypasses the usual nutrient requirement ofmany larval cell types for growth and DNA replication [10].Thus PI3K activation can lead to cell growth and proliferationdespite a lack of nutrient availability, causing death of the animal[10].

It is worth mentioning that in Drosophila, organismal growth isrestricted to the larval stages of development, which can thereforebe considered similar to the childhood and teenage years inhumans. In contrast, after metamorphosis, the adult fly no longerincreases in size. This means that genetic manipulations that alterinsulin signalling in the fly during larval stages result in bothgrowth and metabolic phenotypes, whereas genetic manipulationsthat alter insulin signalling in the adult only result in metabolicconsequences.

Abbreviations used: 4E-BP, 4E-binding protein; AKH, adipokinetic hormone, AMPK, AMP-activated protein kinase; Atg1, autophagy-specific gene 1; CC,corpus cardiacum; CREB, cAMP-response-element-binding protein; dALS, Drosophila acid-labile subunit; dLip4, Drosophila lipase 4; eIF4B, eukaryoticinitiation factor 4B; eIF4E, eukaryotic initiation factor 4E; ERK, extracellular-signal-regulated kinase; FOXO, forkhead box O; GAP, GTPase-activating pro-tein; GEF, guanine-nucleotide-exchange factor; GFP, green florescent protein; GLUT4, glucose transport protein 4; GRB2, growth-factor-receptor-boundprotein 2; GSK-3β, glycogen synthase kinase 3β; IGF, insulin-like growth factor; ILP, insulin-like peptide; Imp-L2, imaginal morphogenesis protein-late 2;InR, insulin-like receptor; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; MAP4K3, MAPK kinase kinase kinase-3; MAPK, mitogen-activatedprotein kinase; Melt, Melted; NLaz, neural lazarillo; NS3, nucleostemin 3; NSC, neurosecretory cell; Path, pathetic; PDK1, phosphoinositide-dependentkinase 1; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PP2A, protein phosphatase 2A; PRAS40, proline-rich Aktsubstrate of 40 kDa; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RNAi, RNA interference; S6K, RPS6 (ribosomal proteinS6)-p70-protein kinase; Sgg, shaggy; SIK2, salt-inducible kinase 2; Slif, slimfast; sNPF, short neuropeptide F; NPFR1, sNPF receptor; SREBP, sterol-regulatory-element-binding protein; Step, steppke; TIF-IA, transcriptional intermediary factor-IA; Tobi, target of brain insulin; TOP, terminal oligopyrimidinetract TOR, target of rapamycin; TOR-C1, TOR complex 1; TOR-C2, TOR complex 2; Tsc1, tuberous sclerosis complex 1; Tsc2, tuberous sclerosis com-plex 2; Wdb, widerborst.

1 email [email protected]

c© The Authors Journal compilation c© 2010 Biochemical Society

14 A. A. Teleman

Figure 1 The ‘core’ intracellular insulin signalling pathway in Drosophila

Functional relationships between components are indicated. Arrows indicate activation, but notnecessarily indicate direct physical interactions, whereas bar-ended lines indicate inhibitoryinteractions. Broken lines indicate indirect interactions or interactions requiring further study.Red arrows indicate transcriptional regulation. Details of each interaction are described in themain text, and outlined in Table 1.

Metabolic regulation is inherently a complex system, designedto maintain homoeostasis in a robust way, yet to be responsive tovarying inputs. As a consequence, it is rich in feedback loopsand interconnections, both within a single cell and betweenorgans. In the following sections I will first describe theintracellular insulin/IGF signalling network in flies, togetherwith the metabolic phenotypes that have been reported forcomponents of the pathway. I will then address the roles andinterconnections of various organs involved in insulin signallingin the fly. Finally, I will point out some differences between theDrosophila and mammalian insulin systems.

COMPONENTS OF THE INSULIN PATHWAY AND METABOLICPHENOTYPES

ILPs (Insulin-like peptides)

Signalling through the insulin/IGF pathway commences uponbinding of ligand to the insulin receptor, in DrosophilaInR (insulin-like receptor) (Figure 1). Drosophila has seven ILPs,termed ILP1–7, which are homologues of human insulin andIGFs. Mammalian insulin and Drosophila ILPs are homologues

at the protein level [11], as well as being functionally equivalent;mammalian insulin can activate the Drosophila InR [12,13] andDrosophila extract has insulin bioactivity in mice [14]. Althoughit was initially surprising that Drosophila has seven ILPs, as thecomplexity of Drosophila insulin signalling tends to be lowerthan that of its mammalian counterpart, it is now apparent thatmammals also have a large number of ILPs as at least nine havebeen identified in rodents {insulin, Igf1, Igf2, relaxin, Insl3, Ins4,Ins5 and Ins6 (for a review see [15])}. The seven DrosophilaILPs are similar at the amino acid level with the exception ofILP6, which has some structural differences that might distinguishit functionally from the others [16]. However, each ILP has adifferent expression pattern and unique regulation, suggesting thatthe functions of the various ILPs are not overlapping. ILPs 1, 2, 3and 5 are expressed in seven median NSCs (neurosecretory cells)of the Drosophila brain [11,17,18], ILPs 4, 5 and 6 are expressed inthe midgut, ILP2 is expressed in the imaginal discs and the salivarygland [11,17] and ILP7 is expressed in the ventral nerve cord ofthe brain [11]. In the larva, expression of ILPs 2, 3 and 5 reduceupon fasting [17,19] in a manner analogous to that of humaninsulin, whereas, surprisingly, expression of ILPs 6 and 7 increase[19].

Overexpression of any of the seven ILPs during larvaldevelopment leads to increased body size [17] indicating thatall seven ILPs can activate the insulin receptor. Larval ablationof the NSCs that produce ILPs 2, 3 and 5 yields adultsthat are developmentally delayed, reduced in body size andhave elevated circulating sugars, total body glycogen and lipidlevels [18,20,21]. Ablation of the same cells in adults leads toincreased trehalosaemia without the growth phenotypes [21].Selective knockdown of ILP2 expression in the NSCs byRNAi (RNA interference) yields adults with elevated totalbody trehalose levels, but none of the other defects, possiblydue to compensatory changes in ILP 3 and 5 expression[22].

These physiological effects of ablating the NSCs cells, reducinginsulin levels systemically and hence causing elevated circulatingsugars and lipid accumulation, are analogous to the effects seenin diabetic patients or in mice when there is generalized insulinresistance, such as in insulin receptor knockouts (for a review see[15]). Furthermore, the attenuated growth observed in animalswith reduced insulin signalling (either via ablation of the NSCsor other manipulations as described below) parallel the reductionin body size seen in mice mutant for insulin and IGF receptors[23].

Although it is probable that the various ILPs have distinctfunctions, this has not yet been carefully studied and the roleof each individual ILP in fly physiology is only starting to beelucidated. For example, overexpression of ILPs 2 and 4 in adultNSCs induce behavioural changes that are not induced by ILP3.When larvae are starved of nutrients, they undertake a set ofhunger-induced behaviours, including an enhanced feeding rate.This is caused by reduced insulin production from the NSCs.Expression of ILPs 2 and 4 are able to suppress this hunger-driven feeding activity, whereas expression of ILP3 does not[9]. The above example also illustrates that in Drosophila, ILPsform part of a homoeostatic feedback loop. Nutrient conditionsregulate expression of several ILPs, and expression of ILPs inturn regulate feeding behaviour. The generation of knockout fliesfor each of the individual ILPs should shed light on the functionof each ILP.

Three secreted inhibitors of ILP function have recently beendescribed, Imp-L2 (imaginal morphogenesis protein-late 2, alsoknown as Ecdysone-inducible gene L2) [24], dALS (Drosophilaacid-labile subunit, also known as convoluted) [25] and NLaz


Metabolic regulation by insulin in Drosophila 15

Table 1 Summary of insulin genes with demonstrated metabolic phenotypes in Drosophila

The metabolic phenotypes are given for the loss-of-funtion condition unless otherwise stated.

Protein CG number(s) Function Inputs Metabolic phenotypes Experimental model Source(s)

Positive factorsILP1–7 CG14173; CG8167;

CG14167; CG6736;CG33273; CG14049;CG13317

activates insulin receptor nutrient availability; stress viap53, JNK and FOXO; ERKsignalling

elevated circulating sugars,glycogen and lipids;overexpression in adultNCSs leads to alteredfeeding behaviour

larvae and adults [9,18,20,21,22]

InR CG18402 transduces insulin signalintracellularly; activatesPI3K

transcriptionally regulated byFOXO

elevated total body sugars andlipids

adult [30]

Chico CG5686 recruits downstream signallingcomponents to InR

inhibited by S6Kphosphorylation

elevated lipids adult [31]

Lnk CG17367 recruits downstream signallingcomponents to InR

not known elevated lipids adults [32]

Melt CG8624 recruits Akt targets (FOXO andTsc1/2) to Akt

none known lean adults [66]

TOR-C1 CG5092 promotes cellular translationand growth and nutrientuptake; inhibits autophagy

activated by insulin signalingvia Akt; activated by aminoacid availability

decreased lipid stores inadipose tissue; reducedcirculating sugar levels

larvae [67,94, 98,100,102,110,119]

Tobi CG11909 glucosidase induced transcriptionally byinsulin

reduced glycogen uponoverexpression

adult [147]

sNPF CG13968 neuropeptide; activates ERKsignaling and ILP secretionin NSCs

not known reduced circulatingcarbohydrates uponoverexpression

larvae [143]

Negative factorsImp-L2 CG15009 binds and antagonizes ILP2 transcriptionally induced upon

adverse nutrient conditionsincreased mortality in adverse

nutrient conditionslarvae [24]

dALS CG8561 binds and modulates ILP2activity

transcriptionally down-regulated upon starvation

decreased haemolymphtrehalose; increasedhaemolymph lipids uponfat-body-specificknockdown

larvae [25]

NLaz CG33126 lipocalin activated transcriptionallyby JNK

reduced trehalose, glycogenand triacylglycerol levels

adults [26]

PTEN CG5671 phosphatase counteractingPI3K action

not known reduced total body glycogenand lipids

adults [39]

Wdb CG5643 phosphatasedephosphorylating Akt

not known enlarged lipid dropletsin oocytes

oocyte nurse cells [54]

FOXO CG3143 transcription factor activatingstress-response andcatabolic genes

inhibited by Aktphosphorylation

required for wasting inresponse to infection; onoverexpression arrest infeeding and growthand accumulationof lipid aggregates

adults; lavae [57,58,62,64]

4E-BP CG8846 translation inhibitor viabinding of eIF4E

inhibited by TOR-C2phosphorylation

elevated rate of lipid useupon starvation

adults [68]

TORC CG6064 CREB transcriptionalco-activator

inhibited by insulin signallingand activated uponstarvation

reduced glycogen andlipid stores

adults [93]

(neural lazarillo) [26]. These will be described in detail in thesection on insulin signalling in tissues.

Insulin receptor and IRS (insulin receptor substrate)

Drosophila has one insulin receptor, InR, which is similar insequence to the mammalian insulin receptor, except that itcontains 400 additional amino acids at the C-terminus. Thisextension contains three YXXM motifs similar to those foundin mammalian IRS1, allowing Drosophila InR to bind PI3K inthe absence of an IRS [27,28]. Whereas complete removal ofInR in Drosophila throughout development causes early larvallethality [12,29], a more mild reduction in InR levels, specificallyin the adult, leads to live adults with increased total body freesugar and lipid levels [30].

Upon ligand binding, InR autophosphorylates [28], recruitingthe IRSs Chico and Lnk [31,32]. Although a large number ofadapter proteins have been described as binding to the mammalianinsulin/IGF receptors {IRS1, IRS2, IRS3, IRS4, SHC, CBL,APS, SH2B, GAB1, GAB2, DOCK1 (dedicator of cytokinesis ordownstream of Crk-180 homologue 1), DOCK2 and CEACAM1(carcinoembryonic antigen-related cell-adhesion molecule 1); fora review see [33]}, only Chico (the fly homologue of IRS1),dreadlocks (which is involved in axon guidance [34]) andLnk [the fly homologue of SH2B (SH2B adaptor protein 1)]have been described as binding to the Drosophila InR. AsDrosophila has homologues for some of these other adapterproteins, future work might shed light on whether they are alsoinvolved in transducing signals downstream of the fly insulinreceptor. Both Chico and Lnk mutant adults are strongly reducedin size and viability, and have elevated total body lipid levels


16 A. A. Teleman

[31,32], whereas simultaneously mutation of Chico and Lnkis lethal, suggesting these two proteins transduce the majorityof the signal downstream of InR. The metabolic phenotypesobserved in InR, Chico and Lnk mutants, which are essentiallyinsulin-resistant in the whole body, are consistent with themetabolic phenotypes observed upon ablation of the NSCsas described above, which also causes a reduction in insulinsignalling.

In mammals, a number of connections have been describedbetween insulin and Ras signalling. In mammals, activation of theinsulin receptor induces interaction of the GRB2 (growth-factor-receptor-bound protein 2)–SOS (Son of sevenless) complex withIRS1 thus activating Ras and subsequently ERK (extracellular-signal-regulated kinase) [35,36]. Furthermore, Ras-GTP bindsto and activates the catalytic subunit of PI3K [37]. Theseinterconnections are starting to be studied in the fly, yieldinginsights into the physiological relevance of these interactions.Although insulin treatment of Drosophila cells leads to activationof ERK [38], mutating the consensus binding site for the Raspathway adaptor Drk (downstream of receptor kinase)/GRB2 inthe Chico protein does not interfere with growth [39]. As Rasis required for viability, this suggests that either Ras is activatedin a Chico-independent manner in Drosophila, perhaps via theC-terminal extension of the Drosophila insulin receptor, or thatthe effect of insulin signalling on Ras is modulatory and notrequired for insulin-mediated growth. A study has shown thatmutating the Ras-binding domain in the PI3K Drosophila p110protein (also known as PI3K92E), the catalytic subunit of PI3K,in vivo is dispensable for viability but is required for maximalPI3K signalling, leading to a phenotype of small flies withdramatically lowered egg production [40]. This lowered eggproduction might reflect a defect in lipid metabolism given lipidsare required in large amounts in eggs. As PI3K is requiredfor viability, these results suggest the impact of Ras on insulinsignalling is modulatory in function.

PI3K, PTEN (phosphatase and tensin homologue deleted onchromosome 10), PDK1 (phosphoinositide-dependent kinase 1)and Akt/PKB (protein kinase B)

Upon phosphorylation of InR and Chico, PI3K (p110) [41,42],together with its adapter subunit p60 (also known as PI3K21B)[43], is recruited to the cell membrane and activated. Thisleads to the generation and accumulation of PtdIns(3,4,5)P3

at the cell membrane. The kinase activity of PI3K is opposedby the phosphatase activity of the tumour suppressor PTEN. Step(Steppke), a member of the cytohesin GEF (guanine-nucleotide-exchange factor) family, has now been found to act upstream ofPI3K to regulate growth and metabolism [44,45]. Step mutantflies are small, and blocking the function of the Step homologuesin mice causes elevated gene transcription of insulin-repressedgluconeogenic genes, as well as inhibition of glycogen and fattyacid synthesis [44,45]. The exact mechanism by which Stepaffects PI3K function remains to be elucidated.

Accumulation of PtdIns(3,4,5)P3 recruits the two kinasesPDK1 [also known as PK61C (protein kinase 61C)] and Aktto the plasma membrane, via their lipid-binding PH (pleckstrinhomology) domains, and leads to their phosphorylation andactivation (for a review see [46]). These proteins and interactionsare highly conserved between flies and mammals. The mostdramatic phenotypes observed in mutants for these componentsare growth-related. Overexpression of PI3K, Akt or PDK1 leadsto tissue overgrowth, whereas loss-of-function leads to tissueundergrowth and lethality [41,42,47,48]. Because of this, it

is difficult to reveal any ‘metabolic’ phenotypes. In contrast,mutation of the counteracting phosphatase PTEN has the oppositesize effects and inactivating mutations in PTEN are not lethal[49–52]. In PTEN mutants, which can be considered insulin‘hyperactive’ in the entire body, it would be expected that wewould see the opposite phenotype from that in diabetic patients.Indeed both total body glycogen and lipid levels are reducedin PTEN mutant adult flies [39] (although, as will be discussedbelow, in one specific cell type in Drosophila, the nurse cells,PTEN mutation leads to the opposite effect, namely formation ofenlarged lipid droplets [53]).

Akt activity is regulated by two additional inputs. First, recentwork has shown that Akt is dephosphorylated and inactivated bythe phosphatase PP2A (protein phosphatase 2A) via its PP2A-B′

subunit, Wdb (widerborst) [54]. Mutation of Wdb leads toactivation of Akt and to the same lipid-droplet phenotype in nursecells as seen in PTEN mutants [53,54]. It would be interesting toknow whether, at the whole organism level, Wdb mutants havethe same phenotypes of reduced body glycogen and lipid levelsas are observed in PTEN mutants. Secondly, Akt is phosphory-lated and activated by TOR-C2 (TOR complex 2) both inDrosophila and in mammals [55]. This regulation appears to bemodulatory, as removal of TOR-C2 activity by mutation of theessential component Rictor (rapamycin-insensitive companionof Tor) only resulted in mild growth impairment and noobservable metabolic effects ([55]; V. Hietakangas, personalcommunication).

Akt targets and FOXO (forkhead box O) proteins

In mammals, Akt phosphorylates a large number of proteinsinvolved in metabolic control, including GSK-3β [glycogensynthase kinase 3β; the Drosophila orthologue is known asSgg (shaggy)], TBC1D4 (TBC1 domain family, member 4), theFOXO transcription factors, Tsc2, PRAS40 (proline-rich Aktsubstrate of 40 kDa; the Drosophila orthologue is known as Lobe),6-phosphofructo-2-kinase and ATP-citrate lyase [46,56]. Anumber of these interactions have also been studied inDrosophila.

One important target of Akt in Drosophila is the singlehomologue of the FOXO transcription factors, dFOXO [57,58].In Drosophila, as in mammals, phosphorylation of FOXOby Akt leads to its retention in the cytoplasm, inhibiting itsnuclear transcriptional activity [57,59]. As one of the principletranscription factors in the insulin signalling pathway, FOXO hasa profound impact on animal metabolism and a large numberof functional studies have been performed in the fly [57–66].These studies all converge at the concept that part of the anaboliceffect of insulin results from blocking FOXO activity, whichotherwise promotes the conservation of energy or, in extremecases, even catabolism. Overexpression of dFOXO in larvaephenocopies starvation, leading to growth arrest and causinglarvae to wander away from food [58]. In contrast targetedoverexpression of dFOXO in fly tissues reduces their size byreducing cell number [57]. Analogously, growth suppression canbe seen when endogenous FOXO is activated by reducing Aktactivation [60,61]. Along these lines, one study showed thatflies infected with Mycobacterium marinum undergo a processsimilar to wasting; they progressively lose metabolic stores, inthe form of fat and glycogen, and become hyperglycaemic. Thisprocess is mediated in part via dFOXO, as dFOXO mutantswere found to exhibit less wasting [62]. Finally, a recent reportshowed that dFOXO is activated upon amino acid starvation ofthe animal and that this activation is required for the animals



to survive the adverse conditions [63]. Surprisingly, dFOXOfunctions both autonomously and non-autonomously in cells;expression of dFOXO in adult head fat body causes reducesILP2 secretion from the NSCs via an unknown mechanism [64]and causes the same metabolic consequences as in flies withreduced insulin production, including increased longevity [65].This will be discussed in more detail in the section on tissuespecificity below. A further Drosophila scaffolding protein, calledMelt (melted), has been found to modulate the ability of Aktto inhibit FOXO in vivo, possibly by encouraging the physicalinteraction between Akt and FOXO [66]. Melt mutants displayelevated FOXO activity and reduced total body lipid levels, aphenotype which could be rescued by removal of FOXO [66].

FOXO exerts its effects by modulating transcription of avery large number of target genes. One canonical target ofdFOXO is the translational repressor 4E-BP (4E-binding protein,in Drosophila also known as Thor). Upon activation, 4E-BP binds eIF4E (eukaryotic initiation factor 4E) and blocksrecruitment of the ribosome to the 5′ end of mRNAs. As aresult, cellular translation rates are dampened [67]. Althoughthis has been suggested to regulate tissue growth, no growthdefects are observed in 4E-BP-null flies [68], nor in 4E-BP1-or 4E-BP2-null mice [69, 70]. Instead, both 4E-BP-null flies and4E-BP1-null mice have elevated metabolic rates [68,69], which isconsistent with the idea that protein synthesis is an energeticallyexpensive process and that its regulation impacts overall organis-mal energy balance. Studies have shown that FOXO regulatesclose to 2000 genes, half of which are regulated in a tissue-specificmanner [19,71]. Among these are many components of the transla-tional apparatus, as well as mitochondrial components, leading toan overall reduction in translation and in mitochondrial biogenesisupon dFOXO activation. Furthermore, dFOXO was found toregulate expression of Myc (the Drosophila orthologue is alsoknown as diminutive), which in turn will also affect mitochondrialbiogenesis [19,72]. Finally, dFOXO also regulates expression ofan acid lipase (dLip4; Drosophila lipase 4) [73], and so is involvedin lipid homoeostasis. In summary, FOXO regulates expressionof a large number of genes that have an impact upon cellularmetabolism. Furthermore, a large number of metabolism-relatedtargets for FOXO1 have been described in mammals (for a reviewsee [74]), which have not yet been studied in depth in the fly.

A second important phosphorylation target of Akt is the tumoursuppressor Tsc2. This phosphorylation presents a conundrum.Tsc2 inhibits TOR-C1 (TOR complex 1) activity both in flies andin mammals via the small GTPase Rheb. Tsc2 acts as a GAP(GTPase-activating protein) for Rheb, thereby inhibiting it and,in turn, inhibiting TOR (Figure 1). In both systems, inhibition ofTsc2 therefore leads to TOR hyperactivation and consequentlystrong effects on tissue growth and metabolism [5,75–77].Phosphorylation of Tsc2 by Akt can inhibit Tsc2 function, atleast when Tsc2 is overexpressed in mammalian cell culture, inDrosophila cell culture or in vivo in the fly [78–81]. However, thein vivo functional significance of this phosphorylation event is notclear, as flies lacking Akt phosphorylation sites on Tsc2, or fliessimultaneously lacking Akt phosphorylation sites on Tsc1 andTsc2, are viable and normal in size, with only very mild metabolicdefects [82,83]. Therefore, although this phosphorylation doesoccur in vivo, the functional consequences of this regulationare not yet clear. Further work, perhaps with ‘knockin’ mice,should shed light on whether this reflects a difference betweenmammals and flies, or whether phenotypes are being masked byredundant regulatory mechanisms such as the possible mechanismmediated via PRAS40/Lobe. In an analogous manner to Tsc2,mammalian PRAS40 was identified as a TOR-C1 inhibitor thatis phosphorylated and inhibited by Akt [84,85]. However, the

functional role of PRAS40 is not completely clear, as it hasalso been reported that PRAS40 is actually a TOR-C1 target,required for signalling downstream of the complex [86–88].PRAS40 and Tsc2 therefore could represent two redundant path-ways by which Akt activates TOR. Knockdown of the Drosophilaorthologue Lobe in tissue culture does activate phosphorylationof TOR-C1 targets, suggesting that Lobe/PRAS40 function mightbe conserved in flies [85]. However, Lobe mutants displayphenotypes suggestive of Notch pathways function, rather thanTOR-C1 function [89], so further work will be required to shedlight on the possible role of Lobe in TOR-C1 signalling.

In mammals, Akt phosphorylates and inactivates GSK-3β,leading to the dephosphorylation and activation of glycogensynthase and hence to an acceleration of glycogen synthesis [90].The Drosophila orthologue of GSK-3β, Sgg, was also shownto be phosphorylated downstream of PI3K signalling in vivo[91]. Future work will shed light on whether this regulation hasmetabolic consequences.

Another phosphorylation target of mammalian Akt is thekinase SIK2 (salt-inducible kinase 2; the Drosophila orthologueis CG4290) [92]. Upon feeding, Akt phosphorylates SIK2,leading to phosphorylation and degradation of the transcriptionalco-activator TORC (in mammals also known as CRTC2[CREB (cAMP-response-element-binding protein)-regulatedtranscription co-activator 2]. As a consequence, TORC no longerfunctions as a CREB co-activator and inducer of genes involved incatabolism and gluconeogenesis. Recently, this signalling cascadewas found to be conserved in Drosophila. The Drosophila TORChomologue is phosphorylated by Drosophila SIK2 in an Akt-dependent manner [93]. TORC mutant adult flies are sensitive tostarvation and oxidative stress [93]. Surprisingly, TORC mutantflies have reduced glycogen and lipid stores [93], rather than theelevated levels of nutrient stores that might be expected frommutation of a catabolic gene.

The Tsc1/2, Rheb and TOR signalling cassette

A central regulator of cellular metabolism is the anabolickinase TOR. TOR exists in one of two complexes, termed TOR-C1and TOR-C2, with some components that are shared and somethat are distinct between the two complexes (for a review see[94]). TOR-C1 is traditionally thought of as a regulator of cellsize and cell growth, as manipulation of TOR-C1 activity affectscell size in eukaryotes from yeast to humans. However, TOR alsoregulates processes that at first seem quite disparate, includingcarbohydrate metabolism, lipid metabolism and autophagy. Oneway to unify these functions is to consider TOR-C1 principallyas a regulator of cellular metabolism, controlling a cell’s decisionon whether to use energy and nutrients or whether to conservethem. Growth can then be considered a metabolic process. Togrow, a cell needs biochemical building blocks, including aminoacids, proteins, lipids and carbohydrates, which in turn needto by synthesized via metabolic pathways. Furthermore, proteinsynthesis is a highly energetically expensive process, using 35–80% of a cell’s energy [95,96]. Therefore cell size and growthare readouts or consequences of a cell-metabolic decision. WhenTOR-C1 activity is high, this leads to accumulation of glycogen,lipids and cell mass, whereas low TOR-C1 activity in a cell leadsto the catabolism of carbohydrate and lipid stores, turnover ofexcess mass and, in extreme cases, autophagy. For this reason, Ialso mention growth phenotypes in the present review, as a readoutof cellular metabolism.

In Drosophila, TOR-C1 function is conserved relative tomammals. Genetic manipulation of TOR activity results in


18 A. A. Teleman

significant changes in tissue and animal size. Loss-of-functionreduces tissue size, by reducing cell size and cell number, whereasgain-of-function leads to the opposite effects [8,97]. Loss of TORalso leads to lipid vesicle aggregation in the larval fat body [8]and mild impairment of TOR-C1 activity in the whole larva alsoresults in decreased lipid stores in adipose tissue and reducedcirculating sugar levels [98]. This is consistent with the phenotypeof mice lacking one of the TOR-C1 targets, S6K1, which arehypersensitive to insulin and are protected against obesity [99].Strong reduction in TOR-C1 activity in the Drosophila adiposetissue induces autophagy [100] (for a review see [101]) andTOR-C1 activation is sufficient to suppress starvation-inducedautophagy [102].

Mechanistically, one of the main cellular processes that TOR-C1 regulates is translation. This occurs at a number of levels. TOR-C1 regulates ribosome biogenesis by controlling the productionof both components of the ribosome, ribosomal proteins andrRNA. In mammals and in the fly, TOR-C1 activity promotesthe function of the transcription factor TIF-IA (transcriptionalintermediary factor-IA), which is required for RNA polymerase I-mediated expression of rRNA [103,104]. Although the regulationis conserved, the mechanistic details might be different inthe two systems, as phosphorylation of TIF-IA by mTORinfluences TIF-IA subcellular localization in mammalian cells[104], whereas this does not seem to be the case inDrosophila (A. A. Teleman, unpublished work). Furthermore,in mammals, TOR-C1 also regulates activity of another rRNAtranscription factor, UBF (upstream binding transcription factor)[105]; however, Drosophila contains no obvious homologue.In Drosophila, TOR-C1 also regulates transcription of genesinvolved in ribosome synthesis and assembly [106]. This wasrecently shown to occur via regulation of the transcription factorMyc [19]. Inhibition of TOR-C1 leads to a rapid reductionin Myc protein levels, and consequently in expression of genesinvolved in ribosome biogenesis. Consistent with this result, Mycactivity is also required for TOR-C1 to drive tissue growth [19,72].Close connections between TOR-C1 and Myc appear to also existin mammals [107]. In addition to regulating ribosome biogenesis,in mammals TOR-C1 also regulates expression of tRNAs byRNA polymerase III. Mammalian TOR-C1 phosphorylates thetranscription factor Maf1, causing it to relocalize to the nucleoluswhere it activates expression of tRNA genes [108]. Whether thisis also the case in Drosophila has not yet been investigated.Finally, TOR-C1 regulates translation initiation and elongation.Both in mammals and in flies TOR-C1 phosphorylates theinhibitor of translation initiation 4E-BP [109] (for reviews see[67,94,110]). This leads to the dissociation of 4E-BP fromthe initiation factor eIF4E, allowing recruitment of the ribosometo the cap complex at the 5′ end of cellular mRNAs, andan increase in overall cellular translation rates. TOR-C1 alsophosphorylates and activates S6K, which phosphorylates the40S ribosomal protein S6 [109] (for reviews see [67,94,110]).S6K requires phosphorylation at two sites for full activationand the second site is phosphorylated by PDK1. Activation ofS6K was initially thought to regulate translation of mRNAscontaining 5′ TOPs (terminal oligopyrimidine tracts), whichinclude many mRNAs encoding components of the translationapparatus, although this has now been shown not to be the case,as mice lacking S6K have normal translation of 5′ TOP mRNAs[111]. Therefore it is likely that TOR-C1 regulates translation of5′ TOP mRNAs in an S6K-independent manner, which remainsto be explored. Nonetheless, mammalian S6K phosphorylatestwo other regulators of translation initiation and elongation,eIF4B (eukaryotic initiation factor 4B) and eEF2K (eukaryoticelongation factor-2 kinase) [112,113].

Regulation of translation by these TOR-C1 effectors has astrong impact on organismal size and metabolism. TIF-IA mutantsarrest as first instar larvae and fail to grow [103]. Drosophiladeficient in S6K exhibit a strong delay in development and a severereduction in body size [114]. Although the S6K mutant phenotypeis less severe than that of dTOR mutants, S6K appears to be a keymediator of TOR-C1 activity, as overexpression of S6K in vivocan rescue dTOR the viability of mutant animals [8] and removalof one copy of S6K can rescue the lethality of Tsc1/2 loss-of-function [115]. As mentioned above, mutants for the other TORtarget, 4E-BP, do not display growth abnormalities, but ratherhave elevated metabolic rates, leading to reduced organismal lipidlevels, consistent with translation being an energetically expensiveprocess [68].

In addition to translation, TOR-C1 regulates a number of othercellular processes. TOR-C1 inhibits autophagy in the Drosophilafat body, and perhaps in other tissues as well [100,102]. Althoughthe molecular mechanism by which this occurs is not completelyclear, it is known to be S6K-independent [102] and that Atg1(autophagy-specific gene 1), an important regulator of autophagy,is involved. TOR-C1 physically interacts with Atg1 and promotesits phosphorylation, although more work will be required tounderstand the implications of this regulation [116]. Thereare probably other molecular mechanisms by which TOR-C1regulates autophagy and these remain to be explored. In addition,Atg1 was shown to inhibit S6K phosphorylation [117] suggestinga regulatory feedback loop between TOR activity and autophagy.Autophagy is important for lipid mobilization as inhibition ofautophagy leads to increased lipid storage, indicating this isprobably one means by which TOR-C1 regulates metabolism[118].

TOR signalling also stimulates bulk endocytic uptake andtargeted uptake of nutrients, although again the mechanism isnot yet completely elucidated [119]. This may occur in part viainhibition of the endocytic degradation of nutrient transporterssuch as Slif (slimfast) [119]. Additionally, calderon [also knownas Orct2 (organic cation transporter 2)], an organic cationtransporter similar to transporters believed to function in theuptake of sugars and other organic metabolites, was shown tobe transcriptionally induced by S6K activity [120]. Calderon isrequired cell autonomously and downstream of S6K for normalgrowth and proliferation of larval tissues [120].

Finally, a recent study in mammalian cells showed that TOR-C1 activity is required for Akt-induced activation of de novo lipidsynthesis [121]. Inhibition of mTOR-C1 by rapamycin preventedAkt-dependent accumulation of unsaturated and saturated fattyacids as well as phosphatidylcholine and phosphatidylglycerol[121]. This probably occurs via regulation of SREBP [sterol-regulatory-element-binding protein, also known in Drosophliaas HLH106 (helix-loop-helix 106)] by TOR-C1. SREBP is atranscription factor that regulates expression of genes involved insterol biosynthesis. The nuclear accumulation of mouse SREBP1in response to Akt activation was prevented by rapamycin [121].This interaction is probably conserved in flies as the study showedthat silencing of Drosophila SREBP blocked the induction of cellgrowth caused by PI3K [121].

It is clear that the regulation of TOR-C1 is complex andnot completely understood (for reviews see [67,110,122,123]).Briefly, TOR-C1 is activated in response to growth factors,especially insulin, and nutrient availability. Activation of TOR-C1 in response to insulin signalling occurs via Akt, but the exactmechanism is still unclear and, as described above, this activationmight occur via two redundant pathways: by phosphoryla-tion and inhibition of the Tsc1/2 complex or by phosphorylationand inhibition of PRAS40/Lobe. The Tsc1/2 complex acts as a



GAP, and inhibitor, for the small GTPase Rheb, which in turnactivates TOR. Rheb was reported to be activated by the GEFTCTP (translationally controlled tumour protein) [124]; however,a later study called this into question [125]. In mammalian cellsTOR-C1 is also regulated by cellular energy status via AMPK(AMP-activated protein kinase). High intracellular AMP levelsactivate AMPK, which directly phosphorylates Tsc2, leading toTsc2 activation and TOR-C1 inhibition. Whether activation ofAMPK also leads to TOR-C1 inhibition in the fly remains tobe directly tested. TOR-C1 is also regulated by the availabilityof amino acids in mammals and in Drosophila. Three factorshave been reported to mediate the amino acid availability signalto TOR-C1, Vps34 (also known in Drosophila as PI3K59F),the Rag GTPases and MAP4K3 [MAPK (mitogen-activatedprotein kinase) kinase kinase kinase-3; the Drosophila orthologueis CG7097]. In mammals, activity of the class III PI3K Vps34 isregulated by amino acid availability via a calcium-dependentmechanism [126]. This leads to accumulation of PtdIns3P in cells,which is thought to cause the recruitment of proteins recognizingPtdIns3P to early endosomes, forming an intracellular signallingplatform that leads to TOR-C1 activation [127,128]. This featureof the pathway may be specific for vertebrates, as flies mutant forVps34 have been reported not to show TOR-C1 signalling defects[129]. Recently, two groups discovered that Rag GTPases mediateamino acid signalling to TOR-C1, both in mammals and in flies[130,131]. The emerging picture is that amino acids change theGDP/GTP loading of the Rag GTPases, thereby stimulatingthe binding of Rag heterodimeric complexes to TOR-C1. Thisin turn causes TOR-C1 to change its intracellular localization,perhaps relocalizing it to vesicles containing the activator Rheb.Finally, Lamb and co-workers identified human MAP4K3 in cellculture as a kinase that is activated by amino acid sufficiency, andin turn it is necessary for S6K activation in response to aminoacids [132]. This mechanism also appears to be conserved fromflies to humans; flies mutant for MAP4K3 have reduced TOR-C1activity levels and display TOR-C1 hypomophorphic phenotypes(B. Bryk, K. Hahn, S. M. Cohen and A. A. Teleman, unpublishedwork). The relationships between Vps34, the Rag GTPases andMAP4K3 remain to be investigated. In addition, novel regulatorsof TOR-C1 have been identified, whose mechanisms of action arenot yet clear. TOR-C1 activity was found to be down-regulatedupon exposure of cells to hypoxic conditions [133]. This down-regulation was mediated by Ptp61F (protein tyrosine phosphatase61F), although the mechanism remains to be elucidated [133].TOR-C1 activity is also modulated by the amino acid transporterPath (pathetic). Path mutants are reduced in size and yield geneticinteractions consistent with reduced TOR-C1 activity [134].When expressed in Xenopus oocytes, Path allows extracellularamino acids to activate TOR-C1. Surprisingly, however, Path hasvery low transport capacity for amino acids, suggesting Path maybe acting as an amino acid sensor which regulates TOR-C1 via anunknown intracellular mechanism [134].

Outputs of the insulin signalling pathway

As described above, the ‘upstream’ signalling events in the insulinsignalling pathway are now fairly well understood. In brief, theylead to activation of a number of kinases such as Akt and TORvia a relay of phosphorylation events. In comparison, however,at least in Drosophila, the outputs of the pathway are not asclearly defined. Activation of the kinases in the insulin cascade,in one way or another, has an impact on cellular metabolism byregulating the enzymes that act as effectors, controlling lipid andcarbohydrate biosynthesis and breakdown. One well-understoodexample is the regulation of GSK-3β by Akt. Activation of Akt

leads to phosphorylation and inactivation of GSK-3β, leadingto the dephosphorylation and activation of glycogen synthase[90]. However, there are probably a large number of linksbetween the insulin pathway and metabolic enzymes that arenot yet known. Work in the future on this ‘downstream’ partof the insulin signalling pathway will be fundamental for us tounderstand how metabolic pathways are regulated by insulin.This will require the coming together of multiple fields ofbiological research, including cell signalling, biochemistry andmetabolomics. Furthermore, such research will probably requirethe study of phenotypes that are more subtle and quantitative innature, compared with the phenotypes the field has studied sofar. This is because the few ‘master regulator’ kinases of theinsulin pathway, such as Akt and TOR, control both directlyand indirectly a large number of effector enzymes. Thereforemutation of these master regulatory kinases leads to obvious andeasily studied phenotypes. In contrast, single mutations of eacheffector enzymes will only lead to a subset of effects. Nonetheless,an understanding of the molecular connections between insulinsignalling components and cellular metabolic enzymes will benecessary for us to have a complete picture of how insulinregulates metabolism.

Intracellular feedback loops

The molecular mechanisms regulating animal metabolism need tosimultaneously meet two constraints. First, the system needsto be exquisitely sensitive and reactive to nutrient conditions ofthe animal, in order to respond quickly to environmental changes.This is best illustrated by insulin levels and insulin intracellularsignalling in humans; the reaction to changing blood glucoselevels occurs within minutes to maintain the glucose concentrationin human blood between 80 and 110 mg/dl [135]. Secondly, thesystem needs to be robust and to function properly in a wide rangeof individuals with differing genetic makeups. This ‘engineering’problem of making a machine that is both flexible and inflexible isprobably partly solved by utilizing the large number of feedbackloops present in the system. In the case of insulin signalling, thereare at least three feedback loops worth mentioning.

First, activation of the insulin signalling pathway leads to activ-ation of PDK1 and TOR-C1, both of which phosphorylate and ac-tivate S6K. S6K, in mammalian cells, then phosphorylates IRS1,inhibiting its function and down-regulating signalling throughthe pathway [110]. This mechanism is probably conserved inDrosophila, as removal of S6K in Drosophila cell culture leadsto increased Akt phosphorylation [136]. Thus this is one negativefeedback loop which attenuates signalling through the pathway.Furthermore, reducing S6K phosphorylation specifically shouldlead to increased signalling through Akt and TOR, and thereforeincreased phosphorylation of the remaining TOR targets.

A second feedback loop occurs when activation of the insulinsignalling pathway leads to activation of Akt and inhibitionof the FOXO transcription factor. As the insulin receptoris a transcriptional target of FOXO, signalling through theinsulin receptor leads to its own transcriptional down-regulation,attenuating signalling. Conversely, low insulin signalling leadsto increased transcription of insulin receptor levels, sensitizingthe system to renewed insulin signalling. This occurs both inmammalian cells and in Drosophila [137].

One further mechanism is the antagonistic relationship betweenactivation of TOR-C1 and activation of TOR-C2. This can beseen in Drosophila cells by monitoring phosphorylation of S6Kat Thr398 (a readout for TOR-C1 activity) and phosphorylation ofAkt at Ser505 (a readout for TOR-C2 activity) when Rheb, Tsc1


20 A. A. Teleman

or Tsc2 are knocked-down [136]. For instance, knockdown ofRheb in Drosophila S2 cells causes decreased phosphorylationof S6K and increased phosphorylation of Akt, indicating thatTOR-C1 activity is reduced, whereas TOR-C2 activity isincreased [136]. In mammalian cells, this also appears to be thecase, as mouse embryonic fibroblasts lacking Tsc1 or Tsc2 haveelevated TOR-C1 activity and severely blunted TOR-C2 activity[138]. As a consequence, a negative feedback loop is createdbetween Akt and TOR (Figure 1). Activation of Akt leads toactivation of TOR-C1 and inactivation of TOR-C2. This causes areduction in the ability of TOR-C2 to enhance Akt activation viaphosphorylation of its hydrophobic motif (Ser505 in Drosophila)[55].

Although the metabolic and growth consequences of these threefeedback loops have not been carefully explored, they constitutean integral part of the insulin signalling network and thereforewarrant attention.

INSULIN SIGNALLING IN TISSUE CONTEXT

The insulin signalling pathway described above can be consideredthe core pathway, which functions in almost all cells andtissues of the animal. In addition to this core pathway, thereare tissue-specific upstream regulators and downstream effectorsof the pathway. Furthermore, the metabolic consequences ofmanipulating insulin signalling depend strongly on the identityof the tissue in which the manipulation takes place. Thereforeit is essential to consider insulin signalling in a tissue-specificcontext. Work in the mouse has been at the forefront of ourunderstanding of how various tissues contribute to metabolicregulation. Nonetheless, recent studies in the fly have also takentissue specificity into account. Future work will probably taketissue specificity increasingly into account, as tissue-specificmanipulations are relatively easy in Drosophila.

Tissue-specific effects of insulin signalling

A wealth of mouse knockout studies have shown thatthe physiological effects of reduced insulin signalling differenormously depending on the tissue in which the manipulationtakes place (for a review see [139]). For instance, mice completelylacking the insulin receptor in the whole body, or lacking oneof the two insulin genes, die soon after birth due to highlyelevated circulating glucose levels. Along the same lines, micelacking insulin receptor specifically in muscle develop a metabolicsyndrome with increased fat stores and hypertriglyceridemia,confirming the central role of this tissue in glucose disposal. Instark contrast, however, mice lacking insulin receptor in whiteand brown adipose tissue have a 30% decrease in whole bodytriacylglycerol (triglyceride) content and have normal levels ofcirculating lipids, non-esterified fatty acids (free fatty acids) andglycerol. This is similar to leanness observed [upon adipose-specific removal of TOR-C1 function [via knockout of the Raptor(regulatory associated protein of mTOR) protein] in mice [140],indicating that insulin and TOR-C1 signalling help promotefat accumulation in this tissue. These differing effects reflectthe function of each individual organ in organismal metaboliccontrol. Furthermore, they highlight an antagonistic relationship,in which insulin signalling in muscle promotes the reduction oftotal body nutrient stores, whereas insulin signalling in adiposetissue promotes accumulation of nutrient stores.

Although it has not been tested directly, various studies inDrosophila suggest that the antagonistic relationship describedabove also holds true for the fly. Reduction of insulin signalling

in the entire animal, for instance via ablation of the NSCs thatproduce ILPs 2, 3 and 5, yields animals that have elevated levels ofcirculating sugars and total body glycogen and lipids [18,20,21].As another example, flies lacking the IRS Chico similarly haveelevated whole body lipid levels [31]. In contrast, reducing insulinsignalling specifically in adipose tissue, as observed both inMelt and miR-278 (microRNA-278) mutant animals, has theopposite effect, leading to reduced adiposity [66,141]. The sameis observed with conditions that up-regulate insulin signalling.Flies mutant for PTEN, an inhibitor of the pathway, have reducedtotal body glycogen and lipid levels [39]. In contrast, adipose-specific activation of TOR activity leads to increased total bodylipid levels [66].

In addition to these results, which parallel those in the mouse,other studies have highlighted that other tissues respond in aspecific manner to insulin signalling in the fly. For example,although PTEN mutants have reduced total body glycogen andlipid levels, the nurse cells in the developing oocytes of PTENmutants actually have enlarged lipid droplets [53], suggesting thatnurse cells respond differently to the PTEN mutation comparedwith other tissues of the animal. Another recent study looked at thetranscriptional response of adipose tissue and muscle to alteredinsulin signalling, and found that insulin signalling modulates theexpression of approx. 2000 genes in each tissue [19]. However,only half of these genes were affected in both tissues, and theother half were specific for each tissue, reflecting the differentrole of each tissue in organismal metabolism.

Communication between tissues

Metabolic regulation by insulin takes place within the contextof an intricate network of communication between varioustissues (Figure 2). For instance, inhibition of insulin signallingspecifically in larval muscles autonomously reduces muscle sizeand non-autonomously reduces the size of the entire body,most likely by regulating feeding behaviour [72]. Although thisis not seen in mice [142], this highlights the importance ofcommunication between tissues, which will be discussed below.

Brain

One important organ for insulin signalling in Drosophila is thebrain. The brain contains seven NSCs in each hemisphere, whichexpress ILPs 1, 2, 3 and 5 [11,17,18]. These NSCs have axonterminals in the larval ring gland and on the aorta, where theILPs are secreted into the haemolymph. Reduced ILP2 expressionin NSCs correlates with reduced insulin signalling in peripheraltissues, observed by reduced phosphorylation of Akt or increasednuclear localization of FOXO [64,143–146], which is consistentwith a model in which ILPs secreted from NSCs activate insulinsignalling in most tissues of the animal. Secretion of ILPsfrom these cells has profound effects on other tissues of theanimal, affecting tissue growth, lipid metabolism, carbohydratemetabolism and animal longevity. Knockdown of ILP2 expressionin the NSCs by RNAi yields animals with elevated total bodytrehalose levels [22]. Ablation of the NSCs causes developmentaldelay, growth retardation, elevated carbohydrate levels in larvalhaemolymph and an extension of lifespan [18,20]. Recently, an α-1,4-glucosidase named Tobi (target of brain insulin) was identifiedas a peripheral transcriptional target of ILP signalling emanatingfrom the brain [147]. Ablation of NSCs leads to a 17-fold down-regulation of Tobi expression in the gut. Consistent with itsglucosidase function, Tobi overexpression causes a reduction intotal body glycogen levels in adult flies [147].



Figure 2 Communication between Drosophila tissues in the insulin signalling pathway

NSCs are one tissue that produces ILPs, which are secreted to influence insulin signalling in all other tissues of the animal. ILP transcription is a central node of regulation, modulated by a number ofinputs including FOXO and S6K activity, as well as sNPF signalling from nearby neurons. Furthermore, FOXO activation in the fat body of the head regulates ILP expression in NSCs by an unknownmechanism and serotonin (5-hydroxytryptamine) signalling from nearby neurons regulates secretion of ILP2. ILP function is inhibited by a number of secreted factors including dALS, Imp-L2 andNLaz. Several feedback loops exist in the animal. For instance, insulin signalling in the prothoracic gland promotes ecdysone production, which in turn inhibits insulin signalling in other tissuessuch as the peripheral fat body. Insulin signalling in gut promotes expression of the α-1,4-glucosidase Tobi. Further details are described in the main text. a.a., amino acids; EcR, ecdysone receptor.

A large number of factors have been reported to regulate ILPexpression by NSCs, suggesting this is a central node of metabolicregulation in Drosophila. Neurons adjacent to the NSCs expresssNPF (short neuropeptide F), the fly orthologue of mammalianneuropeptide Y [143]. In an elegant study, Lee et al. [143]showed that activation of sNPF signalling, via NPFR1 (sNPFreceptor), in NSCs leads to activation of ERK signalling, inducingexpression of ILPs 1, 2 and 3. This connection was confirmedin cell culture using CNS (central nervous system)-derivedneural BG2-c6 cells, and is therefore direct. Thus increasedsNPF production by sNPFnergic neurons resulted in increasedinsulin signalling in peripheral tissues like fat body, leading toa reduction in larval haemolymph carbohydrates. In contrast,reduced sNPF signalling caused an elevation in circulating sugarsand increased median lifespan by 16–21 %. Another set ofneurons adjacent to the NSCs, serotonergic neurons, were alsoshown to regulate ILP activity [145]. This study focused onthe function of NS3 (nucleostemin 3), a nucleostemin familyGTPase. NS3 mutants have elevated serotonin signalling inserotonergic neurons, leading to inhibition of ILP2 secretion,but not expression, from NSCs by an unknown mechanism.This causes NS3 mutant animals to have low insulin si-gnalling in peripheral tissues, to grow slowly and to reach anadult weight of only ∼60% that of control animals [145,148].Adjacent to the adult brain in the Drosophila head is fat tissue.Activity of the transcription factor FOXO in this adult head fat

body somehow regulates ILP2 expression in NSCs [64]. Increasedactivity of FOXO in head fat leads to reduced ILP2 expression inNSCs, leading to reduced insulin signalling in peripheral tissues,such as peripheral fat, leading to increased animal lifespan. Themechanism by which FOXO activity in head fat causes reducedexpression of ILP2 in NSCs remains to be investigated.

In addition to these inputs from nearby cells, several factorswithin the NSCs themselves have been found to regulate ILPexpression. NSCs have high JNK (c-Jun N-terminal kinase)activity levels [146]. As in other tissues, this JNK activity wasshown to activate FOXO in a cell autonomous manner in NSCs,causing a reduction in ILP2 expression and consequently areduction in growth and an extension of lifespan [146]. Likewise,increased S6K activity in NSCs has been proposed as a mechanismto increase ILP expression [9], although further work will benecessary to test this directly. In both cases, the mechanism bywhich S6K and FOXO activity in NSCs regulates ILP expressionremains unclear. A recent study showed that PKA (protein kinaseA) activity in NSCs also affects insulin signalling in peripheraltissues, and regulates production of ecdysone in the prothoracicgland [149]. This presumably occurs via modified ILP expressionin the NSCs caused by the altered PKA activity, although this wasnot tested directly. Finally, overexpression of Drosophila p53 inNSCs causes nuclear accumulation of FOXO in NSCs. Consistentwith the studies mentioned above, this leads to reduced ILP2expression, reduced insulin signalling in peripheral tissues and


22 A. A. Teleman

increased lifespan [144]. Future work will shed light on whetherp53 loss-of-function in NSCs has the opposite effect to that seenwith overexpression.

Ring gland

The ring gland in Drosophila is a central endocrine organ whichproduces ecdysteroids and juvenile hormones. It is composed ofthree parts, the prothoracic gland, the corpus allatum and the CC(corpus cardiacum). A number of connections involving insulinsignalling have been described between the ring gland and otherorgans of the fly.

The CC secretes several factors that counteract insulinsignalling. A recent study showed that CC cells produce Imp-L2, which binds ILP2 inhibiting its function and reducing growthnon-autonomously [24]. This function is particularly necessarywhen animals are in adverse nutritional conditions. Under theseconditions, Imp-L2 mutant animals are not able to reduce theirinsulin signalling in peripheral tissues such as fat body, leadingto increased mortality [24]. In addition, CC cells, togetherwith fat body cells, produce the Drosophila homologue of thevertebrate IGF-binding protein acid-labile subunit, dALS. dALSbinds dILPs in a tertiary complex with IMP-L2, antagonizing ILPfunction, hence controlling animal growth as well as carbohydrateand fat metabolism [25]. Finally, CC cells produce AKH(adipokinetic hormone), a functional homologue of glucagon[150]. CC cells express ATP-sensitive potassium channels, thetargets of sulfonylureas, such as glyburide and tolbutamide, usedto stimulate glucagon secretion in diabetic patients. Exposure ofDrosophila to tolbutamide led to a 40% increase in haemolymphglucose in a manner dependent on CC cells. This indicates thatCC cells regulate glucose homoeostasis using glucose-sensingand response mechanisms similar to islet cells [150].

The second component of the ring gland, the prothoracicgland, is the site of synthesis of ecdysone. Ecdysone counteractsinsulin signalling in a number of tissues including the fat body.Increased ecdysone levels lead to reduced PI3K activity in thefat body, assayed via the PtdIns(3,4,5)P3 level, as well as byincreased nuclear FOXO [151,152]. The mechanistic details ofhow ecdysone signalling counteracts insulin signalling are notknown. Nonetheless, the physiological results of this inhibitionare clear. During metamorphosis, increased ecdysone titreslead to the induction of autophagy in the fat body, which isnormally suppressed by TOR signalling [100,102]. Experimentalmanipulations that lead to increased ecdysone during larvallife inhibit the growth-promoting function of insulin, leadingto reduced growth of the entire animal [151,152]. Surprisingly,ecdysone is part of a feedback loop involving insulin signalling.Synthesis of ecdysone is positively regulated by PI3K andTOR activity in the prothoracic gland [151–153]. This hasseveral physiological consequences. First, as TOR can sensenutrients directly, independently of insulin signalling, this couplesthe availability of nutrients to developmental timing, which isregulated by ecdysone. When TOR activity is reduced in theprothoracic gland, the ecdysone peak that marks the end of larvaldevelopment and the beginning of metamorphosis is abrogated,extending the duration of growth. Thus the developmental delayinduced by food deprivation works in part via TOR signalling inthe prothoracic gland [153]. A second consequence is that ILP2produced by the NSCs is less ‘powerful’ than ILP2 produced inother tissues, due to two counteracting functions; ILP2 producedby NSCs acts directly on peripheral tissues to regulate theirgrowth and metabolism but also increases insulin signalling inthe prothoracic gland, which the NSCs innervate and this leads

to increased ecdysone signalling which counteracts ILP function[149].

Fat body

In mammals, adipose tissue not only acts as a fat reservoirbut also functions as an endocrine organ. The same holds truein Drosophila. The fat body appears to act as a sensor ofnutrients, in particular amino acids, and secretes factors that non-autonomously regulate insulin signalling in other tissues. Thiswas discovered by studying mutants for the cationic amino acidtransporter called Slif [154]. When Slif is knocked-down in fatbody, this causes a non-autonomous reduction in insulin signallingin other tissues, such as salivary gland cells, and a reduction inthe total body size of the animal. These effects are partiallyrescued by co-expressing S6K in adipose tissue, implicating theTOR pathway in this process. Indeed, down-regulation of TORfunction in adipose tissue also non-autonomously reduces insulinsignalling and growth in other tissues [154]. This suggests thatremoval of Slif in fat body causes a reduction in intracellularamino acids, which is sensed by TOR. The authors of that studygo on to show that knockdown of Slif in adipose tissue stronglysuppresses expression of dALS in adipose tissue, suggesting thatdALS might function as the secreted second messenger [154].However, subsequent work has shown that dALS functions asan ILP antagonist, and not as an agonist, as would be requiredby this model [25]. Therefore further work will be required toidentify the secreted messenger by which fat body signals to othertissues.

A third secreted factor, in addition to dALS and IMP-L2,was recently identified as an inhibitor of insulin signallingin Drosophila. Stress-responsive JNK signalling, as well asstarvation were found to induce expression of the secretedlipocalin, NLaz, in fat body [26]. NLaz mutant larvae havereduced insulin signalling levels, as measured via localizationof a reporter for PI3K activity, GFP–PH [GFP (green florescentprotein) fused to the PH domain of GRP1 (general receptor ofphosphoinositides-1)]. Conversely, overexpression of NLaz in fatbody caused a decrease in the GFP–PH signal. Overexpression ofNLaz in fat body also led to a decrease in GFP–PH signal in nursecells of the Drosophila oocyte, demonstrating non-autonomy ofNLaz action [26]. NLaz mutant animals display all the canonicalphenotypes of animals with increased systemic insulin signalling,such as larger size and reduced stores of glucose, trehalose,glycogen and triacylglycerols [26]. This is a second mechanism,in addition to its effects on ILP2 secretion from NSCs, by whichJNK signalling regulates insulin signalling and hence metabolismin Drosophila.

SOME DIFFERENCES BETWEEN MAMMALS AND FLIES

Although the insulin signalling pathway is strikingly conservedbetween flies and mammals, there are also differences between thetwo systems, some of which are mentioned above. Some furtherdifferences are also described in this section.

Insulin and Ras

In mammalian cells, Ras activation has been implicated asan important mediator of the mitogenic effects of insulinstimulation [36,155]. Results so far in Drosophila have seenonly a modulatory effect of Ras on the physiological effectsof insulin (discussed above) and this might reflect a differencebetween Drosophila and mammals. Alternatively, the Drosophila



results agree with those presented in a study performedin CHO (Chinese-hamster ovary) cells, where inhibition ofRas and MAPK activation downstream of insulin signallingdoes not have an impact on proliferation (assayed via DNAsynthesis), activation of S6K or glycogen synthase [156],suggesting that in this system the effect of Ras is alsomodulatory.

GLUT4 (glucose transport protein 4) and glucose

In mammals, one of the principal effects of insulin signalling isto promote cellular glucose update via the GLUT4 transporter[157]. In response to insulin signalling, GLUT4 translocatesto the cell surface resulting in an increased glucose uptake. InDrosophila, this appears not to be the case. Insulin stimulationdoes not alter [3H]-labelled 2-deoxyglucose uptake in DrosophilaKc cells [158], nor does manipulation of PI3K or Akt activity inS2 cells [159]. However, one difference between Drosophila andmammals is that the major circulating sugar in Drosophila istrehalose, not glucose. Knocking-down ILP2 expression in theNSCs by RNAi yields animals with elevated total body trehaloselevels [22], indicating that insulin signalling is indeed involvedin homoeostasis of circulating trehalose. Therefore it would beinteresting to test whether cellular uptake of trehalose is inducedupon insulin stimulation.

Glucocorticoids

In mammals, signalling through the glucocorticoid receptor isknown to have strong effects on the insulin signalling pathway[160]. Drosophila has no obvious orthologue of the glucocorticoidreceptor [161], suggesting that one major layer of regulation mightbe missing in the fly.

Reduced complexity of the pathway

Although Drosophila contains a homologue for virtually allcomponents of the insulin signalling pathway, the overallcomplexity of the pathway is somewhat reduced in flies comparedwith mammals. This is because flies often have a singlehomologue that corresponds to multiple mammalian ones. Forinstance, humans have two S6K proteins, three 4E-BP proteinsand multiple FOXOs, whereas flies only have a single S6K,4E-BP and FOXO protein. On the one hand, this means thatremoval of gene function in the fly is more likely to give strongand clear effects, as there is less redundancy compared withthe mammalian system. On the other hand, direct comparisonbetween the fly protein functions and the mammalian ones maynot be easy, as the proteins in the mammalian system may havespecialized functions.

CONCLUSIONS

Some aspects of mammalian metabolic control, such as the leptinsignalling pathway, do not exist in the fly. These mechanisms canbe considered additional layers of metabolic regulation that wereadded during mammalian evolutionary on top of the more basicones found in all animals. Clearly these mechanisms cannot bestudied in the fly. Insulin signalling, however, appears to be bothpresent and highly conserved, both at the molecular level and thephysiological level, probably due to the basic cellular functions itregulates. Therefore it is fruitful to exploit the powerful genetictools available in the Drosophila model organism in order to

effectively study the molecular mechanisms by which insulinregulates metabolism.

FUNDING

The work in my laboratory was supported by a HelmholtzYoung Investigator grant and an European Union FP7 grant underthe MITIN (integration of the system models of mitochondrialfunction and insulin signalling and its application in the study ofcomplex diseases) project.

REFERENCES

1 Stocker, H., Radimerski, T., Schindelholz, B., Wittwer, F., Belawat, P., Daram, P.,Breuer, S., Thomas, G. and Hafen, E. (2003) Rheb is an essential regulator of S6Kin controlling cell growth in Drosophila. Nat. Cell Biol. 5, 559–565

2 Saucedo, L. J., Gao, X., Chiarelli, D. A., Li, L., Pan, D. and Edgar, B. A. (2003) Rhebpromotes cell growth as a component of the insulin/TOR signalling network.Nat. Cell Biol. 5, 566–571

3 Zhang, Y., Gao, X., Saucedo, L. J., Ru, B., Edgar, B. A. and Pan, D. (2003) Rheb is a directtarget of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5, 578–581

4 Potter, C. J., Huang, H. and Xu, T. (2001) Drosophila Tsc1 functions with Tsc2 toantagonize insulin signaling in regulating cell growth, cell proliferation, and organ size.Cell 105, 357–368

5 Gao, X. and Pan, D. (2001) TSC1 and TSC2 tumor suppressors antagonize insulinsignaling in cell growth. Genes Dev. 15, 1383–1392

6 Palanker, L., Tennessen, J. M., Lam, G. and Thummel, C. S. (2009) Drosophila HNF4regulates lipid mobilization and β-oxidation. Cell Metab. 9, 228–239

7 Baker, K. D. and Thummel, C. S. (2007) Diabetic larvae and obese flies: emergingstudies of metabolism in Drosophila. Cell Metab. 6, 257–266

8 Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C. and Neufeld, T. P. (2000) Regulation ofcellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14, 2712–2724

9 Wu, Q., Zhang, Y., Xu, J. and Shen, P. (2005) Regulation of hunger-driven behaviors byneural ribosomal S6 kinase in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 102,13289–13294

10 Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. and Edgar, B. A. (2002) Drosophila ’sinsulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions.Dev. Cell 2, 239–249

11 Brogiolo, W., Stocker, H., Ikeya, T., Rintelen, F., Fernandez, R. and Hafen, E. (2001) Anevolutionarily conserved function of the Drosophila insulin receptor and insulin-likepeptides in growth control. Curr. Biol. 11, 213–221

12 Fernandez, R., Tabarini, D., Azpiazu, N., Frasch, M. and Schlessinger, J. (1995) TheDrosophila insulin receptor homolog: a gene essential for embryonic developmentencodes two receptor isoforms with different signaling potential. EMBO J. 14,3373–3384

13 Yamaguchi, T., Fernandez, R. and Roth, R. A. (1995) Comparison of the signalingabilities of the Drosophila and human insulin receptors in mammalian cells.Biochemistry 34, 4962–4968

14 Meneses, P. and De Los Angeles Ortiz, M. (1975) A protein extract from Drosophilamelanogaster with insulin-like activity. Comp. Biochem. Physiol. A Comp. Physiol. 51,483–485

15 Nakae, J., Kido, Y. and Accili, D. (2001) Distinct and overlapping functions of insulinand IGF-I receptors. Endocr. Rev. 22, 818–835

16 Wu, Q. and Brown, M. R. (2006) Signaling and function of insulin-like peptides ininsects. Annu. Rev. Entomol. 51, 1–24

17 Ikeya, T., Galic, M., Belawat, P., Nairz, K. and Hafen, E. (2002) Nutrient-dependentexpression of insulin-like peptides from neuroendocrine cells in the CNS contributes togrowth regulation in Drosophila. Curr. Biol. 12, 1293–1300

18 Broughton, S. J., Piper, M. D., Ikeya, T., Bass, T. M., Jacobson, J., Driege, Y.,Martinez, P., Hafen, E., Withers, D. J., Leevers, S. J. and Partridge, L. (2005) Longerlifespan, altered metabolism, and stress resistance in Drosophila from ablation of cellsmaking insulin-like ligands. Proc. Natl. Acad. Sci. U.S.A. 102, 3105–3110

19 Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008) Nutritionalcontrol of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab. 7,21–32

20 Rulifson, E. J., Kim, S. K. and Nusse, R. (2002) Ablation of insulin-producing neurons inflies: growth and diabetic phenotypes. Science 296, 1118–1120

21 Belgacem, Y. H. and Martin, J. R. (2006) Disruption of insulin pathways alters trehaloselevel and abolishes sexual dimorphism in locomotor activity in Drosophila. J.Neurobiol. 66, 19–32


24 A. A. Teleman

22 Broughton, S., Alic, N., Slack, C., Bass, T., Ikeya, T., Vinti, G., Tommasi, A. M.,Driege, Y., Hafen, E. and Partridge, L. (2008) Reduction of DILP2 in Drosophila triages ametabolic phenotype from lifespan revealing redundancy and compensation amongDILPs. PLoS ONE 3, e3721

23 Baserga, R. (2007) Is cell size important? Cell Cycle 6, 814–81624 Honegger, B., Galic, M., Kohler, K., Wittwer, F., Brogiolo, W., Hafen, E. and Stocker, H.

(2008) Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteractsinsulin signaling in Drosophila and is essential for starvation resistance. J. Biol. 7, 10

25 Arquier, N., Geminard, C., Bourouis, M., Jarretou, G., Honegger, B., Paix, A. andLeopold, P. (2008) Drosophila ALS regulates growth and metabolism through functionalinteraction with insulin-like peptides. Cell Metab. 7, 333–338

26 Hull-Thompson, J., Muffat, J., Sanchez, D., Walker, D. W., Benzer, S., Ganfornina, M. D.and Jasper, H. (2009) Control of metabolic homeostasis by stress signaling is mediatedby the lipocalin NLaz. PLoS Genet. 5, e1000460

27 Yenush, L., Fernandez, R., Myers, Jr, M. G., Grammer, T. C., Sun, X. J., Blenis, J.,Pierce, J. H., Schlessinger, J. and White, M. F. (1996) The Drosophila insulin receptoractivates multiple signaling pathways but requires insulin receptor substrate proteinsfor DNA synthesis. Mol. Cell. Biol. 16, 2509–2517

28 Ruan, Y., Chen, C., Cao, Y. and Garofalo, R. S. (1995) The Drosophila insulin receptorcontains a novel carboxyl-terminal extension likely to play an important role in signaltransduction. J. Biol. Chem. 270, 4236–4243

29 Chen, C., Jack, J. and Garofalo, R. S. (1996) The Drosophila insulin receptor is requiredfor normal growth. Endocrinology 137, 846–856

30 Shingleton, A. W., Das, J., Vinicius, L. and Stern, D. L. (2005) The temporal requirementsfor insulin signaling during development in Drosophila. PLoS Biol. 3, e289

31 Bohni, R., Riesgo-Escovar, J., Oldham, S., Brogiolo, W., Stocker, H., Andruss, B. F.,Beckingham, K. and Hafen, E. (1999) Autonomous control of cell and organ size byCHICO, a Drosophila homolog of vertebrate IRS1–4. Cell 97, 865–875

32 Werz, C., Kohler, K., Hafen, E. and Stocker, H. (2009) The Drosophila SH2B familyadaptor Lnk acts in parallel to chico in the insulin signaling pathway. PLoS Genet. 5,e1000596

33 Taguchi, A. and White, M. F. (2008) Insulin-like signaling, nutrient homeostasis, and lifespan. Annu. Rev. Physiol. 70, 191–212

34 Song, J., Wu, L., Chen, Z., Kohanski, R. A. and Pick, L. (2003) Axons guided by insulinreceptor in Drosophila visual system. Science 300, 502–505

35 Skolnik, E. Y., Batzer, A., Li, N., Lee, C. H., Lowenstein, E., Mohammadi, M., Margolis, B.and Schlessinger, J. (1993) The function of GRB2 in linking the insulin receptor to Rassignaling pathways. Science 260, 1953–1955

36 Ogawa, W., Matozaki, T. and Kasuga, M. (1998) Role of binding proteins to IRS-1 ininsulin signalling. Mol. Cell. Biochem. 182, 13–22

37 Rodriguez-Viciana, P., Warne, P. H., Vanhaesebroeck, B., Waterfield, M. D. andDownward, J. (1996) Activation of phosphoinositide 3-kinase by interaction with Rasand by point mutation. EMBO J. 15, 2442–2451

38 Kim, S. E., Cho, J. Y., Kim, K. S., Lee, S. J., Lee, K. H. and Choi, K. Y. (2004) DrosophilaPI3 kinase and Akt involved in insulin-stimulated proliferation and ERK pathwayactivation in Schneider cells. Cell. Signalling 16, 1309–1317

39 Oldham, S., Stocker, H., Laffargue, M., Wittwer, F., Wymann, M. and Hafen, E. (2002)The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP3

levels. Development 129, 4103–410940 Orme, M. H., Alrubaie, S., Bradley, G. L., Walker, C. D. and Leevers, S. J. (2006) Input

from Ras is required for maximal PI3K signalling in Drosophila. Nat. Cell Biol. 8,1298–1302

41 Leevers, S. J., Weinkove, D., MacDougall, L. K., Hafen, E. and Waterfield, M. D. (1996)The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 15,6584–6594

42 Weinkove, D., Neufeld, T. P., Twardzik, T., Waterfield, M. D. and Leevers, S. J. (1999)Regulation of imaginal disc cell size, cell number and organ size by Drosophila class IAphosphoinositide 3-kinase and its adaptor. Curr. Biol. 9, 1019–1029

43 Weinkove, D., Leevers, S. J., MacDougall, L. K. and Waterfield, M. D. (1997) p60 is anadaptor for the Drosophila phosphoinositide 3-kinase, Dp110. J. Biol. Chem. 272,14606–14610

44 Fuss, B., Becker, T., Zinke, I. and Hoch, M. (2006) The cytohesin Steppke is essential forinsulin signalling in Drosophila. Nature 444, 945–948

45 Hafner, M., Schmitz, A., Grune, I., Srivatsan, S. G., Paul, B., Kolanus, W., Quast, T.,Kremmer, E., Bauer, I. and Famulok, M. (2006) Inhibition of cytohesins by SecinH3 leadsto hepatic insulin resistance. Nature 444, 941–944

46 Franke, T. F. (2008) PI3K/Akt: getting it right matters. Oncogene 27, 6473–6488

47 Verdu, J., Buratovich, M. A., Wilder, E. L. and Birnbaum, M. J. (1999) Cell-autonomousregulation of cell and organ growth in Drosophila by Akt/PKB. Nat. Cell Biol. 1,500–506

48 Rintelen, F., Stocker, H., Thomas, G. and Hafen, E. (2001) PDK1 regulates growththrough Akt and S6K in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 98, 15020–15025

49 Huang, H., Potter, C. J., Tao, W., Li, D. M., Brogiolo, W., Hafen, E., Sun, H. and Xu, T.(1999) PTEN affects cell size, cell proliferation and apoptosis during Drosophila eyedevelopment. Development 126, 5365–5372

50 Goberdhan, D. C., Paricio, N., Goodman, E. C., Mlodzik, M. and Wilson, C. (1999)Drosophila tumor suppressor PTEN controls cell size and number by antagonizing theChico/PI3-kinase signaling pathway. Genes Dev. 13, 3244–3258

51 Gao, X., Neufeld, T. P. and Pan, D. (2000) Drosophila PTEN regulates cell growth andproliferation through PI3K-dependent and -independent pathways. Dev. Biol. 221,404–418

52 Scanga, S. E., Ruel, L., Binari, R. C., Snow, B., Stambolic, V., Bouchard, D., Peters, M.,Calvieri, B., Mak, T. W., Woodgett, J. R. and Manoukian, A. S. (2000) The conservedPI3K/PTEN/Akt signaling pathway regulates both cell size and survival in Drosophila.Oncogene 19, 3971–3977

53 Vereshchagina, N. and Wilson, C. (2006) Cytoplasmic activated protein kinase Aktregulates lipid-droplet accumulation in Drosophila nurse cells. Development 133,4731–4735

54 Vereshchagina, N., Ramel, M. C., Bitoun, E. and Wilson, C. (2008) The proteinphosphatase PP2A-B′ subunit Widerborst is a negative regulator of cytoplasmicactivated Akt and lipid metabolism in Drosophila. J. Cell Sci. 121, 3383–3392

55 Hietakangas, V. and Cohen, S. M. (2007) Re-evaluating AKT regulation: role of TORcomplex 2 in tissue growth. Genes Dev. 21, 632–637

56 Nascimento, E. B. and Ouwens, D. M. (2009) PRAS40: target or modulator of mTORC1signalling and insulin action? Arch. Physiol. Biochem. 115, 163–175

57 Puig, O., Marr, M. T., Ruhf, M. L. and Tjian, R. (2003) Control of cell number byDrosophila FOXO: downstream and feedback regulation of the insulin receptor pathway.Genes Dev. 17, 2006–2020

58 Kramer, J. M., Davidge, J. T., Lockyer, J. M. and Staveley, B. E. (2003) Expression ofDrosophila FOXO regulates growth and can phenocopy starvation. BMC Dev. Biol.3, 5

59 Van Der Heide, L. P., Hoekman, M. F. and Smidt, M. P. (2004) The ins and outs of FOXOshuttling: mechanisms of FOXO translocation and transcriptional regulation. Biochem.J. 380, 297–309

60 Junger, M. A., Rintelen, F., Stocker, H., Wasserman, J. D., Vegh, M., Radimerski, T.,Greenberg, M. E. and Hafen, E. (2003) The Drosophila forkhead transcription factorFOXO mediates the reduction in cell number associated with reduced insulin signaling.J. Biol. 2, 20

61 Harvey, K. F., Mattila, J., Sofer, A., Bennett, F. C., Ramsey, M. R., Ellisen, L. W., Puig, O.and Hariharan, I. K. (2008) FOXO-regulated transcription restricts overgrowth of Tscmutant organs. J. Cell Biol. 180, 691–696

62 Dionne, M. S., Pham, L. N., Shirasu-Hiza, M. and Schneider, D. S. (2006) Akt and FOXOdysregulation contribute to infection-induced wasting in Drosophila. Curr. Biol. 16,1977–1985

63 Kramer, J. M., Slade, J. D. and Staveley, B. E. (2008) FOXO is required for resistance toamino acid starvation in Drosophila. Genome 51, 668–672

64 Hwangbo, D. S., Gershman, B., Tu, M. P., Palmer, M. and Tatar, M. (2004) DrosophiladFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature429, 562–566

65 Giannakou, M. E., Goss, M., Junger, M. A., Hafen, E., Leevers, S. J. and Partridge, L.(2004) Long-lived Drosophila with overexpressed dFOXO in adult fat body. Science305, 361

66 Teleman, A. A., Chen, Y. W. and Cohen, S. M. (2005) Drosophila Melted modulatesFOXO and TOR activity. Dev. Cell 9, 271–281

67 Hay, N. and Sonenberg, N. (2004) Upstream and downstream of mTOR. Genes Dev. 18,1926–1945

68 Teleman, A. A., Chen, Y. W. and Cohen, S. M. (2005) 4E-BP functions as a metabolicbrake used under stress conditions but not during normal growth. Genes Dev. 19,1844–1848

69 Tsukiyama-Kohara, K., Poulin, F., Kohara, M., DeMaria, C. T., Cheng, A., Wu, Z.,Gingras, A. C., Katsume, A., Elchebly, M., Spiegelman, M. et al. (2001) Adipose tissuereduction in mice lacking the translational inhibitor 4E-BP1. Nat. Med. 7, 1128–1132

70 Banko, J. L., Poulin, F., Hou, L., DeMaria, C. T., Sonenberg, N. and Klann, E. (2005) Thetranslation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity,and memory in the hippocampus. J. Neurosci. 25, 9581–9590

71 Gershman, B., Puig, O., Hang, L., Peitzsch, R. M., Tatar, M. and Garofalo, R. S. (2007)High-resolution dynamics of the transcriptional response to nutrition in Drosophila:a key role for dFOXO. Physiol. Genomics 29, 24–34



72 Demontis, F. and Perrimon, N. (2009) Integration of insulin receptor/FOXO signalingand dMyc activity during muscle growth regulates body size in Drosophila.Development 136, 983–993

73 Vihervaara, T. and Puig, O. (2008) dFOXO regulates transcription of a Drosophila acidlipase. J. Mol. Biol. 376, 1215–1223

74 Gross, D. N., van den Heuvel, A. P. and Birnbaum, M. J. (2008) The role of FOXO in theregulation of metabolism. Oncogene 27, 2320–2336

75 Ito, N. and Rubin, G. M. (1999) gigas, a Drosophila homolog of tuberous sclerosis geneproduct-2, regulates the cell cycle. Cell 96, 529–539

76 Tapon, N., Ito, N., Dickson, B. J., Treisman, J. E. and Hariharan, I. K. (2001) TheDrosophila tuberous sclerosis complex gene homologs restrict cell growth and cellproliferation. Cell 105, 345–355

77 Inoki, K. and Guan, K. L. (2009) Tuberous sclerosis complex, implication from a raregenetic disease to common cancer treatment. Hum. Mol. Genet. 18, R94–R100

78 Potter, C. J., Pedraza, L. G. and Xu, T. (2002) Akt regulates growth by directlyphosphorylating Tsc2. Nat. Cell Biol. 4, 658–665

79 Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J. and Cantley, L. C. (2002)Identification of the tuberous sclerosis complex-2 tumor suppressor gene producttuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell 10, 151–162

80 Inoki, K., Li, Y., Zhu, T., Wu, J. and Guan, K. L. (2002) TSC2 is phosphorylated andinhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657

81 Cai, S. L., Tee, A. R., Short, J. D., Bergeron, J. M., Kim, J., Shen, J., Guo, R.,Johnson, C. L., Kiguchi, K. and Walker, C. L. (2006) Activity of TSC2 is inhibited byAKT-mediated phosphorylation and membrane partitioning. J. Cell Biol. 173, 279–289

82 Dong, J. and Pan, D. (2004) Tsc2 is not a critical target of Akt during normal Drosophiladevelopment. Genes Dev. 18, 2479–2484

83 Schleich, S. and Teleman, A. A. (2009) Akt phosphorylates both Tsc1 and Tsc2 inDrosophila, but neither phosphorylation is required for normal animal growth. PLoSONE. 4, e6305

84 Vander Haar, E., Lee, S. I., Bandhakavi, S., Griffin, T. J. and Kim, D. H. (2007) Insulinsignalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9,316–323

85 Sancak, Y., Thoreen, C. C., Peterson, T. R., Lindquist, R. A., Kang, S. A., Spooner, E.,Carr, S. A. and Sabatini, D. M. (2007) PRAS40 is an insulin-regulated inhibitor of themTORC1 protein kinase. Mol. Cell 25, 903–915

86 Wang, L., Harris, T. E., Roth, R. A. and Lawrence, Jr, J. C. (2007) PRAS40 regulatesmTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.J. Biol. Chem. 282, 20036–20044

87 Oshiro, N., Takahashi, R., Yoshino, K., Tanimura, K., Nakashima, A., Eguchi, S.,Miyamoto, T., Hara, K., Takehana, K., Avruch, J. et al. (2007) The proline-rich Aktsubstrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target ofrapamycin complex 1. J. Biol. Chem. 282, 20329–20339

88 Fonseca, B. D., Smith, E. M., Lee, V. H., MacKintosh, C. and Proud, C. G. (2007)PRAS40 is a target for mammalian target of rapamycin complex 1 and is required forsignaling downstream of this complex. J. Biol. Chem. 282, 24514–24524

89 Chern, J. J. and Choi, K. W. (2002) Lobe mediates Notch signaling to controldomain-specific growth in the Drosophila eye disc. Development 129, 4005–4013

90 Cohen, P., Alessi, D. R. and Cross, D. A. (1997) PDK1, one of the missing links ininsulin signal transduction? FEBS Lett. 410, 3–10

91 Papadopoulou, D., Bianchi, M. W. and Bourouis, M. (2004) Functional studies ofshaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster.Mol. Cell. Biol. 24, 4909–4919

92 Dentin, R., Liu, Y., Koo, S. H., Hedrick, S., Vargas, T., Heredia, J., Yates, III, J., andMontminy, M. (2007) Insulin modulates gluconeogenesis by inhibition of the coactivatorTORC2. Nature 449, 366–369

93 Wang, B., Goode, J., Best, J., Meltzer, J., Schilman, P. E., Chen, J., Garza, D.,Thomas, J. B. and Montminy, M. (2008) The insulin-regulated CREB coactivatorTORC promotes stress resistance in Drosophila. Cell Metab. 7, 434–444

94 Wullschleger, S., Loewith, R. and Hall, M. N. (2006) TOR signaling in growth andmetabolism. Cell 124, 471–484

95 Pannevis, M. C. and Houlihan, D. F. (1992) The energetic cost of protein synthesis inisolated hepatocytes of rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B 162,393–400

96 Siems, W. G., Schmidt, H., Gruner, S. and Jakstadt, M. (1992) Balancing ofenergy-consuming processes of K 562 cells. Cell. Biochem. Funct. 10, 61–66

97 Oldham, S., Montagne, J., Radimerski, T., Thomas, G. and Hafen, E. (2000) Genetic andbiochemical characterization of dTOR, the Drosophila homolog of the target ofrapamycin. Genes Dev. 14, 2689–2694

98 Luong, N., Davies, C. R., Wessells, R. J., Graham, S. M., King, M. T., Veech, R.,Bodmer, R. and Oldham, S. M. (2006) Activated FOXO-mediated insulin resistance isblocked by reduction of TOR activity. Cell Metab. 4, 133–142

99 Um, S. H., Frigerio, F., Watanabe, M., Picard, F., Joaquin, M., Sticker, M., Fumagalli, S.,Allegrini, P. R., Kozma, S. C., Auwerx, J. and Thomas, G. (2004) Absence of S6K1protects against age- and diet-induced obesity while enhancing insulin sensitivity.Nature 431, 200–205

100 Rusten, T. E., Lindmo, K., Juhasz, G., Sass, M., Seglen, P. O., Brech, A. and Stenmark, H.(2004) Programmed autophagy in the Drosophila fat body is induced by ecdysonethrough regulation of the PI3K pathway. Dev. Cell 7, 179–192

101 Chang, Y. Y., Juhasz, G., Goraksha-Hicks, P., Arsham, A. M., Mallin, D. R., Muller, L. K.and Neufeld, T. P. (2009) Nutrient-dependent regulation of autophagy through the targetof rapamycin pathway. Biochem. Soc. Trans. 37, 232–236

102 Scott, R. C., Schuldiner, O. and Neufeld, T. P. (2004) Role and regulation ofstarvation-induced autophagy in the Drosophila fat body. Dev. Cell 7, 167–178

103 Grewal, S. S., Evans, J. R. and Edgar, B. A. (2007) Drosophila TIF-IA is required forribosome synthesis and cell growth and is regulated by the TOR pathway. J. Cell Biol.179, 1105–1113

104 Mayer, C., Zhao, J., Yuan, X. and Grummt, I. (2004) mTOR-dependent activation of thetranscription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev. 18,423–434

105 Hannan, K. M., Brandenburger, Y., Jenkins, A., Sharkey, K., Cavanaugh, A.,Rothblum, L., Moss, T., Poortinga, G., McArthur, G. A., Pearson, R. B. and Hannan, R. D.(2003) mTOR-dependent regulation of ribosomal gene transcription requires S6K1 andis mediated by phosphorylation of the carboxy-terminal activation domain of thenucleolar transcription factor UBF. Mol. Cell. Biol. 23, 8862–8877

106 Guertin, D. A., Guntur, K. V., Bell, G. W., Thoreen, C. C. and Sabatini, D. M. (2006)Functional genomics identifies TOR-regulated genes that control growth and division.Curr. Biol. 16, 958–970

107 Schmidt, E. V., Ravitz, M. J., Chen, L. and Lynch, M. (2009) Growth controls connect:interactions between c-myc and the tuberous sclerosis complex-mTOR pathway. CellCycle 8, 1344–1351

108 Wei, Y., Tsang, C. K. and Zheng, X. F. (2009) Mechanisms of regulation of RNApolymerase III-dependent transcription by TORC1. EMBO J. 28, 2220–2230

109 Miron, M., Lasko, P. and Sonenberg, N. (2003) Signaling from Akt to FRAP/TORtargets both 4E-BP and S6K in Drosophila melanogaster. Mol. Cell. Biol. 23,9117–9126

110 Bhaskar, P. T. and Hay, N. (2007) The two TORCs and Akt. Dev. Cell 12, 487–502111 Pende, M., Um, S. H., Mieulet, V., Sticker, M., Goss, V. L., Mestan, J., Mueller, M.,

Fumagalli, S., Kozma, S. C. and Thomas, G. (2004) S6K1−/−/S6K2−/− mice exhibitperinatal lethality and rapamycin-sensitive 5′-terminal oligopyrimidine mRNAtranslation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway.Mol. Cell. Biol. 24, 3112–3124

112 Raught, B., Peiretti, F., Gingras, A. C., Livingstone, M., Shahbazian, D., Mayeur, G. L.,Polakiewicz, R. D., Sonenberg, N. and Hershey, J. W. (2004) Phosphorylation ofeucaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases. EMBO J.23, 1761–1769

113 Wang, X., Li, W., Williams, M., Terada, N., Alessi, D. R. and Proud, C. G. (2001)Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J. 20,4370–4379

114 Montagne, J., Stewart, M. J., Stocker, H., Hafen, E., Kozma, S. C. and Thomas, G. (1999)Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129

115 Radimerski, T., Montagne, J., Hemmings-Mieszczak, M. and Thomas, G. (2002)Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducingdS6K signaling. Genes Dev. 16, 2627–2632

116 Chang, Y. Y. and Neufeld, T. P. (2009) An Atg1/Atg13 complex with multiple roles inTOR-mediated auTOPhagy regulation. Mol. Biol. Cell 20, 2004–2014

117 Lee, S. B., Kim, S., Lee, J., Park, J., Lee, G., Kim, Y., Kim, J. M. and Chung, J. (2007)ATG1, an auTOPhagy regulator, inhibits cell growth by negatively regulating S6 kinase.EMBO Rep. 8, 360–365

118 Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., Tanaka, K.,Cuervo, A. M. and Czaja, M. J. (2009) Autophagy regulates lipid metabolism.Nature 458, 1131–1135

119 Hennig, K. M., Colombani, J. and Neufeld, T. P. (2006) TOR coordinates bulk andtargeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth.J. Cell Biol. 173, 963–974

120 Herranz, H., Morata, G. and Milan, M. (2006) calderon encodes an organic cationtransporter of the major facilitator superfamily required for cell growth and proliferationof Drosophila tissues. Development 133, 2617–2625

121 Porstmann, T., Santos, C. R., Griffiths, B., Cully, M., Wu, M., Leevers, S., Griffiths, J. R.,Chung, Y. L. and Schulze, A. (2008) SREBP activity is regulated by mTORC1 andcontributes to Akt-dependent cell growth. Cell Metab. 8, 224–236

122 Bjornsti, M. A. and Houghton, P. J. (2004) The TOR pathway: a target for cancer therapy.Nat. Rev. Cancer 4, 335–348


26 A. A. Teleman

123 Avruch, J., Hara, K., Lin, Y., Liu, M., Long, X., Ortiz-Vega, S. and Yonezawa, K. (2006)Insulin and amino-acid regulation of mTOR signaling and kinase activity through theRheb GTPase. Oncogene 25, 6361–6372

124 Hsu, Y. C., Chern, J. J., Cai, Y., Liu, M. and Choi, K. W. (2007) Drosophila TCTP isessential for growth and proliferation through regulation of dRheb GTPase. Nature 445,785–788

125 Wang, X., Fonseca, B. D., Tang, H., Liu, R., Elia, A., Clemens, M. J., Bommer, U. A. andProud, C. G. (2008) Re-evaluating the roles of proposed modulators of mammaliantarget of rapamycin complex 1 (mTORC1) signaling. J. Biol. Chem. 283,30482–30492

126 Gulati, P., Gaspers, L. D., Dann, S. G., Joaquin, M., Nobukuni, T., Natt, F., Kozma, S. C.,Thomas, A. P. and Thomas, G. (2008) Amino acids activate mTOR complex 1 viaCa2+/CaM signaling to hVps34. Cell Metab. 7, 456–465

127 Byfield, M. P., Murray, J. T. and Backer, J. M. (2005) hVps34 is a nutrient-regulated lipidkinase required for activation of p70 S6 kinase. J. Biol. Chem. 280, 33076–33082

128 Nobukuni, T., Joaquin, M., Roccio, M., Dann, S. G., Kim, S. Y., Gulati, P., Byfield, M. P.,Backer, J. M., Natt, F., Bos, J. L. et al. (2005) Amino acids mediate mTOR/raptorsignaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc. Natl.Acad. Sci. U.S.A. 102, 14238–14243

129 Juhasz, G., Hill, J. H., Yan, Y., Sass, M., Baehrecke, E. H., Backer, J. M. and Neufeld, T. P.(2008) The class III PI3K Vps34 promotes auTOPhagy and endocytosis but not TORsignaling in Drosophila. J. Cell Biol. 181, 655–666

130 Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. and Guan, K. L. (2008) Regulation ofTORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945

131 Sancak, Y., Peterson, T. R., Shaul, Y. D., Lindquist, R. A., Thoreen, C. C., Bar-Peled, L.and Sabatini, D. M. (2008) The Rag GTPases bind raptor and mediate amino acidsignaling to mTORC1. Science 320, 1496–1501

132 Findlay, G. M., Yan, L., Procter, J., Mieulet, V. and Lamb, R. F. (2007) A MAP4 kinaserelated to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem. J. 403,13–20

133 Lee, S. J., Feldman, R. and O’Farrell, P. H. (2008) An RNA interference screen identifies anovel regulator of target of rapamycin that mediates hypoxia suppression of translationin Drosophila S2 cells. Mol. Biol. Cell 19, 4051–4061

134 Goberdhan, D. C., Meredith, D., Boyd, C. A. and Wilson, C. (2005) PAT-related aminoacid transporters regulate growth via a novel mechanism that does not require bulktransport of amino acids. Development 132, 2365–2375

135 Rahaghi, F. N. and Gough, D. A. (2008) Blood glucose dynamics. Diabetes Technol.Ther. 10, 81–94

136 Yang, Q., Inoki, K., Kim, E. and Guan, K. L. (2006) TSC1/TSC2 and Rheb havedifferent effects on TORC1 and TORC2 activity. Proc. Natl. Acad. Sci. U.S.A. 103,6811–6816

137 Puig, O. and Tjian, R. (2005) Transcriptional feedback control of insulin receptor bydFOXO/FOXO1. Genes Dev. 19, 2435–2446

138 Huang, J., Dibble, C. C., Matsuzaki, M. and Manning, B. D. (2008) The TSC1-TSC2complex is required for proper activation of mTOR complex 2. Mol. Cell. Biol. 28,4104–4115

139 Kitamura, T., Kahn, C. R. and Accili, D. (2003) Insulin receptor knockout mice.Annu. Rev. Physiol. 65, 313–332

140 Polak, P., Cybulski, N., Feige, J. N., Auwerx, J., Ruegg, M. A. and Hall, M. N. (2008)Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrialrespiration. Cell Metab. 8, 399–410

141 Teleman, A. A., Maitra, S. and Cohen, S. M. (2006) Drosophila lacking microRNAmiR-278 are defective in energy homeostasis. Genes Dev. 20, 417–422

142 Bruning, J. C., Michael, M. D., Winnay, J. N., Hayashi, T., Horsch, D., Accili, D.,Goodyear, L. J. and Kahn, C. R. (1998) A muscle-specific insulin receptor knockoutexhibits features of the metabolic syndrome of NIDDM without altering glucosetolerance. Mol. Cell 2, 559–569

143 Lee, K. S., Kwon, O. Y., Lee, J. H., Kwon, K., Min, K. J., Jung, S. A., Kim, A. K.,You, K. H., Tatar, M. and Yu, K. (2008) Drosophila short neuropeptide F signallingregulates growth by ERK-mediated insulin signalling. Nat. Cell Biol. 10, 468–475

144 Bauer, J. H., Chang, C., Morris, S. N., Hozier, S., Andersen, S., Waitzman, J. S. andHelfand, S. L. (2007) Expression of dominant-negative Dmp53 in the adult fly braininhibits insulin signaling. Proc. Natl. Acad. Sci. U.S.A. 104, 13355–13360

145 Kaplan, D. D., Zimmermann, G., Suyama, K., Meyer, T. and Scott, M. P. (2008) Anucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulinsignaling and control body size. Genes Dev. 22, 1877–1893

146 Wang, M. C., Bohmann, D. and Jasper, H. (2005) JNK extends life span and limitsgrowth by antagonizing cellular and organism-wide responses to insulin signaling. Cell121, 115–125

147 Buch, S., Melcher, C., Bauer, M., Katzenberger, J. and Pankratz, M. J. (2008) Opposingeffects of dietary protein and sugar regulate a transcriptional target of Drosophilainsulin-like peptide signaling. Cell Metab. 7, 321–332

148 Ruaud, A. F. and Thummel, C. S. (2008) Serotonin and insulin signaling team up tocontrol growth in Drosophila. Genes Dev. 22, 1851–1855

149 Walkiewicz, M. A. and Stern, M. (2009) Increased insulin/insulin growth factor signalingadvances the onset of metamorphosis in Drosophila. PLoS ONE 4, e5072

150 Kim, S. K. and Rulifson, E. J. (2004) Conserved mechanisms of glucose sensing andregulation by Drosophila corpora cardiaca cells. Nature 431, 316–320

151 Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C.,Antoniewski, C., Carre, C., Noselli, S. and Leopold, P. (2005) Antagonistic actions ofecdysone and insulins determine final size in Drosophila. Science 310, 667–670

152 Mirth, C., Truman, J. W. and Riddiford, L. M. (2005) The role of the prothoracic gland indetermining critical weight for metamorphosis in Drosophila melanogaster. Curr. Biol.15, 1796–1807

153 Layalle, S., Arquier, N. and Leopold, P. (2008) The TOR pathway couples nutrition anddevelopmental timing in Drosophila. Dev. Cell 15, 568–577

154 Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J. and Leopold, P.(2003) A nutrient sensor mechanism controls Drosophila growth. Cell 114, 739–749

155 Margolis, B. and Skolnik, E. Y. (1994) Activation of Ras by receptor tyrosine kinases.J. Am. Soc. Nephrol. 5, 1288–1299

156 Sakaue, M., Bowtell, D. and Kasuga, M. (1995) A dominant-negative mutant of mSOS1inhibits insulin-induced Ras activation and reveals Ras-dependent and -independentinsulin signaling pathways. Mol. Cell. Biol. 15, 379–388

157 Hou, J. C. and Pessin, J. E. (2007) Ins (endocytosis) and outs (exocytosis) of GLUT4trafficking. Curr. Opin. Cell Biol. 19, 466–473

158 Ceddia, R. B., Bikopoulos, G. J., Hilliker, A. J. and Sweeney, G. (2003) Insulin stimulatesglucose metabolism via the pentose phosphate pathway in Drosophila Kc cells. FEBSLett. 555, 307–310

159 Hall, D. J., Grewal, S. S., de la Cruz, A. F. and Edgar, B. A. (2007) Rheb–TOR signalingpromotes protein synthesis, but not glucose or amino acid import, in Drosophila. BMCBiol. 5, 10

160 van Raalte, D. H., Ouwens, D. M. and Diamant, M. (2009) Novel insights intoglucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options?Eur. J. Clin. Invest. 39, 81–93

161 King-Jones, K. and Thummel, C. S. (2005) Nuclear receptors: a perspective fromDrosophila. Nat. Rev. Genet. 6, 311–323

Received 4 August 2009/4 September 2009; accepted 7 September 2009Published on the Internet 14 December 2009, doi:10.1042/BJ20091181


Documents

Molecular mechanisms of metabolic regulation by insulin in ... · Molecular mechanisms of metabolic regulation by insulin in Drosophila Aurelio A. TELEMAN1 German Cancer Research