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Drosophila gains traction as a repurposed tool to investigate metabolism Divya Padmanabha and Keith D. Baker Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, 1220 East Broad Street Room 2052, Richmond, VA 23298, USA

The use of fruit flies has recently emerged as a powerful experimental paradigm to study the core aspects of energy metabolism. The fundamental need for lipid and carbohydrate processing and storage across species dictates that the central regulators that control metabolism are highly conserved through evolution. Accordingly, the Drosophila system is being used to identify human disease genes and has the potential to model successfully human disorders that center on excessive caloric intake and metabolic dysfunction, including dietinduced lipotoxicity and type 2 diabetes. We review here recent progress on this front and contend that increasing such efforts will yield unexpectedly high rates of experimental return, thereby leading to novel approaches in the treatment of obesity and its comorbidities. Introduction Healthcare providers are increasingly confronted with patient populations suffering from obesity-associated conditions that were once rare but are now commonplace. One major concern is that containment of the problem has been lost; according to the World Health Organization, every region of the world has seen a doubling in obesity rates since 1980. The stark reality of an aging and prevalently obese population has prompted the need for new research paths. The past decade has seen a paradigm shift in how metabolic research is performed, with innovative technologies allowing unprecedented interrogation into etiologies associated with chronic caloric surplus (obesity, insulin resistance, hyperglycemia, and metabolic syndrome). Emblematic of the change is the emergence of studies that rely on simple model organisms for discovery and analysis. Inasmuch as these novel approaches are revealing new aspects about model organisms such as the fly, they also underline the shared mechanisms between species and the similar phenotypes that result from their dysfunction. This review highlights recent progress in Drosophila metabolic research and argues that expanding such efforts

Corresponding author: Baker, K.D. ([email protected]). Keywords: Drosophila; metabolism; obesity; insulin signaling; endocrine. 1043-2760/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.03.011

will continue to provide viable pathways that lead to clinical solutions for obesity-related disorders. The Drosophila metabolic system at a glance Flies have only recently been used as tools to investigate the core aspects of metabolism but, in this short history, findings from approaches utilizing the Drosophila model have provided key insights into conserved metabolic processes [1,2]. Considering the similarities in how flies and vertebrates uptake, regulate, and traffic their nutrients, these successes are not surprising; what is surprising is that it took so long to appreciate the utility of the similarities. Nutrient intake As in mammals, nutrients in the fly are taken in by a segmented digestive tract that is analogous to the stomach/ intestines and which is highly specialized in organization and function (Figure 1). Recent studies demonstrate the Drosophila adult midgut has as many as 14 functional zones along its anterior–posterior axis and is directly innervated by the central nervous system (CNS) [3–5]. Each zone has distinct RNA expression profiles that help to facilitate sequential processing, modification, and absorption of ingested food as it traverses through luminal regions that are locally controlled by such cells as acidsecreting copper cells [6]. Zone-specific gut cells are replenished by populations of stem cells that are functionally and spatially distinct, and these differentiate into multiple cell lineages including enterocytes or enteroendocrine cells in a manner that greatly resembles the self-renewing abilities of mammalian gut epithelial cells [3,4,7]. Because the larval midgut is programmatically destroyed by autophagy upon pupariation [8], the extent of local specialization that it has and its putative role in patterning of the adult midgut remain clear. Nevertheless, it too is complex, having an unmistakable morphology (Figure 1), including diverticula (gastric caeca), and regions of specialized functional capacity [9,10]. Hormone and nutrient dissemination Absorbed nutrients are released by enterocytes and dispersed through an open circulatory system by hemolymph, a blood-like fluid that bathes internal tissues. Efficient metabolic exchange is enhanced by the pumping action Trends in Endocrinology and Metabolism xx (2014) 1–10

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IPCs Lipid droplets Oenocytes

Trachea Aorta

Spiracle

Proventriculus

Mouth hooks Mob iliz e

AKH

n

AKHR

TAG

TAG

Trehalose

S ta r v

a ti

o

InR

Heart CC

InR

Esophagus

InR

Midgut

Foregut

DAG

DAG

Dilps

Ring gland

DAG

Systemic growth

Monosaccharides

Fat body

Brain Storage

InR Glycogen

TAG

Trehalose VLCFAs

Gastric caecum TRENDS in Endocrinology & Metabolism

Figure 1. Schematic of the Drosophila metabolic network. The metabolism of Drosophila is carried out by a well-balanced network of organs and tissues that perform many of the same basic functions as those found in mammals. Shown is a dorsolateral view of an anterior third-instar larva packed with fat. The fly insulin receptor (InR) mediates functions similar to those of insulin and insulin-like growth factor receptors. The neurosecretory cells located in the brain and the ring gland [a collective of the corpus cardiacum (CC), corpus allatum, and prothoracic gland] secrete insulin and adipokinetic hormone (AKH, Drosophila glucagon) into the open hemolymph. Efficient metabolic exchange is enhanced by the pumping action of a tubular heart. Excess sugar is converted to glycogen and accumulates in the muscles and fat body (akin to the vertebrate liver and white adipose tissues). Absorbed dietary lipids are trafficked from the gut by lipophorins in the hemolymph as diacylglycerides (DAGs) and are stored in lipid droplets as triacylglycerides (TAGs). Upon starvation, AKH stimulates the release of trehalose into the hemolymph. Fat is released from the fat body into the hemolymph and is then taken in by the oenocytes. Additional to its role as a storage depot, the fat body coordinates systemic larval growth with nutrient availability by integrating insulin and target of rapamycin (TOR) signaling.

of a tubular heart. The aorta extends from the heart into the head and maintains neuronal connections with the brain, where small clusters of median neurosecretory cells (known as insulin-producing cells, IPCs) are located and produce Drosophila insulin-like peptides (Dilp2, -3, -5) [11]. Adjacent to the IPCs is a group of glandular tissues that are directly connected to the aorta and are known collectively as the ring gland [corpus cardiacum (CC), corpus allatum, and prothoracic gland]. The specialized tissues of the ring gland produce lipophilic hormones such as ecdysone and juvenile hormone, as well as the glucagonlike peptide, adipokinetic hormone (AKH) [12]. This architecture (shown for the larva in Figure 1) allows rapid dissemination of metabolism-altering hormones that are released from these tissues upon changing nutrient conditions [13,14] (Table 1). It should be noted, however, that during metamorphosis the brain and ring gland undergo extensive remodeling. Changes include the posterior migration of the ring gland toward the proventriculus and the complete degeneration of the ecdysone-producing prothoracic gland cells [14]. Nutrient breakdown, trafficking, and storage Dietary sugars are broken down by glucosidases in the gut lumen, usually into monosaccharides, allowing complex sugars to be absorbed and then released into hemolymph by enterocytes [15]. The closely situated fat body takes these in and either stores them in reserve as glycogen or 2

converts them into the glucose–glucose disaccharide trehalose which is subsequently released into the hemolymph. The strategy of circulating carbohydrate primarily in the form of trehalose allows the fly to circulate sugars at high levels while still allowing passive influx of dietary sugars [16]. Absorbed dietary lipids are modified and trafficked from the gut as lipoprotein particles by the ApoB (apolipoprotein B)-family lipoprotein lipophorin (Lpp), which is crucial for fat storage and peripheral tissue membrane homeostasis [17]. Additionally, lipids are transiently stored as triacylglycerides (TAG) in midgut-localized lipid droplets (LDs) that are mobilized upon starvation [18,19]. Lpp prefers to carry lipid cargo in the form of diacylglycerides (DAG) over TAG by a factor of 15:1 (mole:mole), and additionally has a high affinity for sterols [20]. Apo-Lpp particles are made and originally lipidated with sterols and long-chain phosphatidylethanolamine (PE) in the fat body, but need another fat body-derived protein, lipid transfer particle (LTP), also an ApoB-family member, in the gut for loading diet-derived and gut-synthesized lipid cargoes [17]. Although the fat body is the primary depot for stored fat in the animal, LDs can be found throughout the animal. The primary regulator of energy metabolism in Drosophila is the fat body. This loosely organized and dispersed tissue serves as the functional equivalents of the white adipose tissue and the liver, acting both as a source and a sink for sugars and fats throughout the life of the

Class of molecule

Mammalian ortholog/ functional equivalent

Dilp1 Dilp2 Dilp3 Dilp4 Dilp5 Dilp6 Dilp7 Dilp8

Insulin-like peptides

Insulin/IGFs

Effect on insulin pathway

Function

Site of expression/ production

Refs

 Systemic regulators of growth in fed-state  Altered expression of DILP2, 3, 5, and 6 results in modulated IIS, increases lifespan, and confers several metabolic consequences. Partial ablation of median neurosecretory cells (IPCs) extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism, and increases oxidative stress resistance  Knockdown of Dilp2 is compensated for by increases in Dilp3 and Dilp5  Starvation reduces expression of Dilp3 and Dilp5, but not Dilp2  Knockdown of Dilp5 along with the DTKR and InR result in increased survival following nutritional or oxidative stress, suggesting that stress regulates the hormonal release of tachykinin that then regulates Dilp5. The transcription factor Dachshund physically interacts with Drosophila Eyeless to promote Dilp5 expression  Dilp6 is produced during pupal development via ecdysone pathway, also is induced by FoxO in starvation  Dilp8 delays metamorphosis by inhibiting ecdysone biosynthesis, mutants are asymmetric

Dilp1 – pupal CNS

[11,82–86]

 Coordinates cellular metabolism with nutritional conditions, mutants are small

Ubiquitous

[45,47]

Dilp2 – IPCs Dilp3 – IPCs Dilp4 Dilp5 – IPCs, Malpighian tubules Dilp6 – fat body Dilp7 – thoracic abdominal ganglion Dilp8 – imaginal disks

Insulin-like receptor

Insulin/IGF receptor

ImpL2

Secreted ligand

IGF-BP7

Inhibits Dilp2

 Inhibits growth non-autonomously through complex formation with of Dilp2 and dALS  Loss-of-function mutants have overgrowth, overexpression results in small animals  Dampens insulin signal in starvation

Ubiquitous

[48]

dALS/ convoluted

Secreted ligand

IGF-binding protein acid-labile subunit (ALS)

Inhibits Dilp2

 Forms a circulating trimeric complex with Dilp and ImpL2  Antagonizes Dilp function to control animal growth as well as carbohydrate and fat metabolism  Essential gene; mutants have tracheal defects

Crop, hindgut, trachea

[49]

Sdr (secreted decoy of InR)

Secreted receptor

Insulin receptor ectodomain

Inhibits several Dilps

 Negative regulator of insulin signaling; structurally similar to the extracellular domain of InR, interacts with several Dilps in vitro independent of ImpL2  Larvae lacking SDR grow at a faster rate, thereby increasing adult body size  SDR is constantly secreted into the hemolymph independent of nutritional status and is essential for adjusting insulin signaling under adverse food conditions

Brain, hindgut

[50]

AKH (Adipokinetic hormone) AKHR (Adipokinetic hormone receptor)

Secreted peptide and GPCR

Glucagon and receptor

Antagonizes insulin

 Similar to glucagon, AKH acts antagonistically to insulin by activating glycogen phosphorylase and mobilizing carbohydrates  AKH induces both hypertrehalosemia and hyperlipidemia  AKH-ablated flies show increased starvation resistance  Ectopic expression of AKH in the fat body resulted in both increased circulating trehalose and a decrease in stored lipids  AKHR mutants display an obese phenotype and increased levels of stored carbohydrates

AKH – corpora cardiaca, thoracic abdominal ganglion

[12,46,87]

NLaz (Neural Lazarillo)

Secreted peptide

Apolipoprotein D (ApoD), retinol binding protein 4 (RBP4), lipocalin 2

Involved in Dilp resistance

 NLaz expression is strongly increased upon high sugar diet and NLaz null mutants are fully protected from high-sugar diet-induced Dilp resistance  NLaz is a target for JNK signaling in mammals  Isolated in a screen for molecules involved in Dilp resistance

Gut, Malpighian tubules, fat body, salivary glands

[73]

sNPF (short neuropeptide F precursor) sNPF-R (short neuropeptide F receptor) NPF (neuropeptide F) NPFR (neuropeptide F receptor)

Secreted peptides and GPCRs

Neuropeptide Y (NPY) and neuropeptide Y receptor (NPY1R)

Regulates Dilp expression

 sNPF and its receptor sNPF-R regulate expression of Dilps; body size is increased by overexpression of sNPF or sNPFR1  sNPF mutants showed downregulated Akt, nuclear localized FOXO, upregulated 4E-BP, and reduced cell size in the fat body  Circulating levels of glucose were elevated and lifespan extended in sNPF mutants  Insulin interacts with the sNPF pathway by acting as a satiety signal to decrease sNPFR expression in the odorant receptor neurons, and in turn this decreases motivated feeding  Impairment of Stim leads to hyperphagia via control of sNPF expression in brain  sNPF promotes sleep, independently of feeding, through clock neuron modulation  NPF and NPFR regulate circadian locomotor rhythm as well as integrate internal state of hunger and appetitive memory; stimulation of NPF-expressing neurons mimics starvation response

sNPF – brain, thoracic abdominal ganglion

[39,88–92]

AKHR – fat body, heart, head, hindgut

sNPF-R – brain, thoracic-abdominal ganglion, hindgut, fat body, heart NPF – brain, midgut NPFR – brain, gut, thoracic-abdominal ganglion, Malpighian tubules

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dInR

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Review

Table 1. Secreted endocrine regulators, their receptors, and their impacts on metabolism.

Class of molecule

Mammalian ortholog/ functional equivalent

Effect on insulin pathway

Function

Site of expression/ production

Refs

DTK (tachykinin) DTKR (tachykinin-like receptor at 99D)

Secreted peptide and GPCR

Tachykinin-related peptides and receptor

Regulates Dilp5 expression; inhibits Dilp2/3 expression

 DTKR knockdown in IPCs in fed flies resulted in increases in Dilp2 and Dilp3 transcripts, whereas starvation resulted in marked reduction in the Dilp3 transcript with concomitant increase in the Dilp2 transcript; this shows that circulating DTK affects the IPCs  Targeted knockdown of DTKR, DILP5, or the insulin receptor dInR in principal cells, or Dilp5 mutation, resulted in increased survival following stress, whereas overexpression of these produced the opposite phenotype; thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to positively regulate DILP5 signaling

DTK – brain, midgut, hindgut

[93,94]

Crz (corazonin) CrzR (corazonin receptor)

Secreted peptide and GPCR

S

 In the adult fly, the sNPF-expressing dorsolateral peptidergic neurons (DLPNs) coexpress corazonin  Corazonin from the DLPNs stimulates the IPCs and thereby affects carbohydrate and lipid levels, but it doesn’t affect Dilp transcription  Corazonin appears to be released into the hemolymph to act on the fat body  Genetic ablation of the DLPNs results in flies with reduced trehalose levels, indicating that corazonin may modulate the AKH producing cells  AKH-ablated flies do not show starvation-induced hyperlocomotion

Crz – DLPNs

Asta (Allatostatin A) AstA-R1 (Allatostatin A receptor 1) Ast-R2 (Allatostatin A receptor 2)

Secreted peptide and GPCRs

S

 AstA inhibits starvation-induced changes in the feeding behavior in Drosophila, including increased food intake and enhanced behavioral responsiveness to sugar  AstA promotes aversion to food  AstA-R1 -?  AstA-R2 -?

AstA – brain, thoracic-abdominal ganglion, midgut, hindgut

[97]

Hugin

Secreted peptides

S

 Hugin gene encodes a neuropeptide precursor containing two putative neuropeptides: hugin-g and hugin-PK  Upregulated in the feeding mutants pumpless and klumpfuss  Linked to the inhibition of feeding because ectopic expression in larvae resulted in reduced food intake  Ectopic expression results in decreased growth and the larvae don’t survive until adulthood  Downregulated in starved or amino acid-deprived larvae

Brain

[98–100]

Lk (leucokinin) Lkr (leucokinin receptor)

Secreted peptide and GPCR

Tachykinin and receptor

 Mutants show increased meal size and a compensatory reduction in meal frequency  Increased meal size in mutants could be due to a meal termination defect, perhaps arising from impaired communication of gut distension signals to the brain  Acts in non-neuronal tissues to maintain water homeostasis; hence, it regulates food intake in starvation and water in fed state

Lk – brain, thoracic-abdominal ganglion Lkr – brain, thoracic-abdominal ganglion hindgut, Malpighian tubules

[5,101]

SP (sex peptide)

Secreted peptide

Upd2 (Unpaired2)

Type I cytokine-like peptide and type I cytokine receptor

Leptin and receptor

dAdipoR (adiponectin receptor)

Hemolysin-IIIrelated integral membrane protein

Adiponectin receptor 1

Drosulfakinin (Dsk)

Secreted peptide

Cholecystokinin-like peptide

[95,96]

CrzR – brain, fat body, heart, salivary gland

 The primary trigger of post-mating responses in Drosophila. SP is produced in the male accessory gland and transferred to the female in the seminal fluid  The post-mating response of enhanced food intake is induced by SP and female flies mated to males lacking SP failed to increase food intake  Mated sterile females did not display enhanced food intake and virgin females with an experimentally increased egg production showed increased feeding

[102–105]

 Upd2 secreted from fat body in fed-state to control systemic growth  Fat body-specific knockdown of Upd2, but not of Upd1 or Upd3, results in small flies  Mutation produces small flies with accumulated lipid in oenocytes, mimicking the starvation phenotype  Severely downregulated when starved on 1% sucrose  Overexpression is able to suppress TAG mobilization of fat stores  Signals through Jak/Stat pathway in GABAergic neurons to relieve inhibition of IPCs, resulting in Dilp release  Stat92E knockdown in dome-expressing neurons recapitulates Upd2 phenotype  Human leptin functionally substitutes for Upd2 in vivo

Upd2 – fat body dome – ubiquitous

[54]

Promotes DILP protein accumulation in IPCs

 IPC-specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed increased sugar levels in the hemolymph and elevated triglyceride level in whole body  However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein accumulated in IPCs, the level of circulating Dilp2 decreased, and insulin signaling reduced in fat body

Ubiquitous

[106]

Positive feedback regulation between Dsk and Dilp

 IPC-specific reduction of dsk increases food consumption in fed Drosophila  Dilp transcript levels also reduced in dsk knockdown flies, suggesting that dsk and Dilps mediate satiety signals in flies

IPCs

[107]

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Domeless (dome)

DTKR – brain, midgut, hindgut, thoracic ganglion, Malpighian tubules, heart

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Table 1 (Continued )

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Review animal. In larvae, when rapid growth is of primary concern, the fat body stockpiles vast reserves of carbohydrates and lipids in the forms of glycogen and TAG. However, the fat body is not simply a storage depot of fuel reserves. Instead, it remotely coordinates systemic larval growth with nutrient availability by integrating insulin and target of rapamycin (TOR) signaling (reviewed in [1]). The growth-regulatory role of the larval fat body is replaced by an adult fat body that serves a primary role of homeostatic balance, with the ability to release necessary energy stores in nutrient-deprived states [21–23]. Complementing the activities of the fat body are specialized secretory cells called oenocytes. In larvae, they are bilaterally dispersed in small packs of 6 cells, whereas adult oenocytes are found in the abdomen in dorsally localized ribbons and in groups along the ventral midline. Upon starvation, oenocytes accumulate mobilized lipid from the fat body in a manner that resembles the actions of the mammalian liver, with oenocyte ablation in larvae resulting in lethality [18]. These findings, and others that demonstrate oenocytes are crucial for very long chain fatty acid processing and cuticular lipid production [24,25], are consistent with oenocytes having a central role in lipid metabolism, although much remains to be learned about the integrated physiologic role they likely provide in systemic adiposity. This is particularly true in the adult where they are relatively unstudied [26]. Oxygen delivery is linked to nutrition and lifespan Analogous to the mammalian vasculature, the fly tracheal system comprises an extensive open tubular network throughout the body and can undergo dynamic remodeling in response to hypoxia [27]. It was recently demonstrated that terminal tracheal branching is also directly responsive to nutritional status – the poorer the food quality the less extensive the branching, specifically in the gut [28]. The larval and adult branch remodeling requires neurons producing Dilp7 and Pdf (pigment dispersing factor) peptide hormones. In poor nutrient conditions, the remodeling does not delay developmental timing but it does extend lifespan in the adult. The results suggest that the Drosophila tracheal system can be used to model vascular plasticity that is subject to local and systemic metabolic cues. Applying Drosophila genetics to questions on human obesity Genetic tools The best reason to use the Drosophila model is for the genetics. The repertoire of genetic tools continues to expand and many, if not all, of the components necessary to perform the studies are available through publicly available repositories and stock centers [e.g., the Drosophila Genomics Resource center (DGRC, https://dgrc.cgb.indiana.edu/Home) and the Bloomington Drosophila Stock Center (BDSC; http://flystocks.bio.indiana.edu/)]. Among the experimental tools of choice are those that utilize the Gal4/ UAS (galactose gene transcription factor 4/upstream activation sequence) binary expression system [29], where a wide array of Gal4 lines can drive ubiquitous, conditional, or spatiotemporal expression of UAS-sensitive transgenes to knockdown, rescue, visualize, or otherwise alter a

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system in the context of an intact organism. Furthermore, new site-specific nuclease technologies are being introduced into the Drosophila model for targeted genome editing, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the CRISPR (clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR-associated nuclease) system [30– 32]. Despite their limited use in the fly thus far, these new approaches show great transformative promise for fly genetics and are already redefining the efficiency of germline targeting, and the speed with which it can take place (under 1 month). Genetic screens to identify genes affecting adiposity A major difficulty confronting human obesity researchers has been the limited success in identification of genes that impart susceptibility; only a small fraction of obesity cases can be explained based upon the genes identified [33]. These difficulties have been made even more frustrating given that the heritability of obesity is thought to be 40– 70% [34]. It might be argued, however, that because nearly 80% of human disease genes have recognizable Drosophila orthologs [35], finding human obesity susceptibility genes may be more easily accomplished in the fly. Several recent genetic screens suggest this may be the case. In one approach, cultured S2 cells were pre-loaded with TAG and changes to LD morphology were screened using an RNA interference (RNAi) library [36]. Their results indicated that a surprising number of genes are involved in LD formation/maintenance (1.5% of all genes). The Arf1 (ADP-ribosylation factor 1)–COP1 (coat protein complex 1) vesicular transport proteins were shown to be involved in TAG mobilization, a previously unappreciated role for these proteins in LD biology that was later confirmed in mammalian cells. In another excellent study, Pospisilik et al. performed a genome-wide RNAi screen in adults and identified 500 genes that are candidate obesity genes, 1/3 of which when specifically knocked down only in neurons change total TAG levels by >25% [37]. They also identified hedgehog signaling components as major regulators of fat body TAG storage, and confirmed that white adipose-specific activation of the hedgehog pathway in the mouse results in a near-complete loss of white fat due to a block in adipocyte differentiation. Notably, Palm et al. recently showed that Drosophila S2 cells and cultured human HeLa cells secrete hedgehog independently of lipoprotein, or in combination with it, demonstrating that lipidated and non-lipidated forms of hedgehog have distinct functions in hedgehog signaling [38]. Finally, Baumbach et al. screened for changes in adult adiposity by expressing fat body-specific RNAi constructs that targeted nearly 6800 individual genes [39]. Of the 77 positive hits, 75% had not previously been shown to impact adiposity. They found that increases or reductions in intracellular Ca2+ concentration, via impairment of storeoperated calcium entry (SOCE), result in a lean or obese phenotype, respectively. Through knockdown of the SOCE core component, Stromal interaction molecule (Stim), they demonstrated that intracellular (i) Ca2+ levels in the fat body remotely induce expression of the orexigenic short 5

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Review neuropeptide F in the brain, resulting in increased fat deposition through hyperphagia. It will be interesting to see if similar iCa2+-dependent effects are at play in mammalian systems. Genome-wide association studies (GWAS) GWAS have been extensively used to identify loci associated with variations in metabolism and obesity [40]. Pendse et al. recently used a Drosophila model for T2D to survey orthologs of human GWAS-identified candidate genes, and found that a homeobox (HOX) class transcription factor (dHHEX; Drosophila hematopoietically expressed homeobox) is an important mediator of insulin insensitivity [41]. However, with the introduction of the Drosophila Genetic Reference Panel (DGRP; http:// dgrp.gnets.ncsu.edu/), a collection of 192 naturally isolated and fully sequenced inbred strains [42], a more direct route for identification of susceptibility loci in flies is now available. He et al. used this resource to identify variations in eye degeneration brought about by expression of a misfolded protein, human mutant preproinsulin [43]. Using mean eye area as a quantitative trait, they found an association within the sulfateless gene, later confirmed through RNAi, that heparin sulfate biosynthetic pathway members are important in proteostasis. A growing list of endocrine regulators of metabolism The most influential of the pioneering reports on Drosophila endocrine regulators of metabolism established the existence of a pancreatic-like axis in flies, with insulinand glucagon-like peptides [11,12,44–46]. Dilps are produced in the b cell-like IPCs of the brain, although not exclusively. The Dilps have profound impact on growth through systemic phosphoinositide 3-kinase (PI3K) activation in a manner that integrates TOR signaling from the fat body [21,47]. Furthermore, the insulin pathway is counterbalanced by AKH, which is released from the a cell-like CC cells in response to low circulating sugars levels in a calcium-sensitive manner to initiate glycogenolysis and lipolysis in the fat body [12]. Given the surge of interest in Drosophila metabolic research, a host of newly found conserved endocrine regulators of metabolism has recently been reported, many of which impinge directly on the activities of the insulin pathway. We highlight only some of those findings here, but direct readers to Table 1 for brief synopses from these genetic analyses, along with the impact of each factor on the insulin signaling pathway when known. Only factors with confirmed roles in metabolism are included, and only secreted ligands and their receptors are listed. Circulating antagonists of insulin Two elegant studies demonstrated that ImpL2 and dALS, the Drosophila functional equivalents to insulin-like growth factor-binding protein 7 (IGFBP7) and acid-labile subunit (ALS) in vertebrates, form a trimeric complex with circulating Dilps (2 and 5) to antagonize insulin signaling upstream of the insulin receptor (InR) [48,49]. Another factor, secreted decoy of InR (SDR), was also found to bind to Dilps in circulation, thereby dampening their systemic effects [50]. SDR is released primarily from glia, irrespective of 6

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nutritional status, and acts independently of ImpL2/ALS; however, like ImpL2, SDR is crucial for survival upon starvation. As would be anticipated for negative regulators of insulin, mutation of Sdr, ImpL2, or dALS results in nonautonomous overgrowth phenotypes. Nutritional control of stem cell proliferation Neuroblast exit from quiescence in development is dependent upon a nutritional checkpoint. A still unidentified factor emanates from the fat body, from where it is released in response to dietary amino acids that signal through the Slimfast transporter and TOR [21,51]. Recent reports indicate that this fat body mitogen directs CNS-specific production of Dilps in glia, which in turn activate insulin/TOR pathways in neuroblasts to drive proliferation [52,53]. Interestingly, inactivation of the upstream fat body signal can be overridden through CNS-specific activation of PI3K or TOR. Leptin pathway Because the Drosophila genome does not encode a protein that bears recognizable sequence similarity to leptin, this pathway was thought to be nonexistent in flies. However, Rajan and Perrimon showed that the cytokine-related Upd2 (unpaired 2) protein, which is released from the fat body in late larvae, mediates a fed-state response that is strikingly similar to the leptin pathway [54]. They demonstrated that Upd2 acts upon the Jak/Stat (Janus kinase/signal transducer and activator of transcription) receptor dome (domeless) in g-amino butyric acid (GABA)-ergic neurons to disinhibit IPCs, thereby initiating Dilp secretion. Furthermore, with perhaps the most convincing evidence yet of the relevance of the Drosophila system, they demonstrated that human leptin can functionally substitute for Upd2 in vivo. These collective findings bode well for the potential applicability that Drosophila metabolism genes have in human disease, even in the absence of strong sequence identity. At a bare minimum, they suggest that we should be systematically looking at uncharacterized genes that are already known to impact adiposity, such as those identified in the above-mentioned screens [37]. Roles of nuclear hormone receptors (NRs) in metabolism Drosophila NRs have long been studied for their roles in steroid-induced transcriptional cascades. More recently, however, their metabolic and growth regulatory activities have come under scrutiny. The peroxisome proliferator activated receptors (PPARs) are not amongst the 18 highly conserved NRs that are present in the Drosophila genome [55], despite their profound affects on glucose and lipid metabolism in mammals [56]. Nevertheless, the metabolic roles of other superfamily members can still be modeled in the fly system. As might be anticipated, the ecdysone receptor (EcR) plays a crucial role that coordinates growth with developmental maturation through actions in the fat body (reviewed in [57]). It achieves this immediately before the onset of metamorphosis by antagonizing the pro-growth activities of dMyc and insulin in a ligand-dependent fashion [58,59]. The

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Review insulin signaling inhibition by EcR also promotes FOXO (forkhead box O) nuclear translocation and upregulation of the EcR coactivator dDOR (from diabetes- and obesity regulated), which further potentiates the effect [60]. Sieber and Thummel recently showed that the DHR96 (Drosophila hormone receptor 96) orphan nuclear receptor plays a key role in the gut by regulating the expression of magro, a dual-function esterase and LipA (lipase A) homolog [61]. Knockdown of magro or mutation of DHR96 lead to a cholesterol efflux deficiency in the midgut and to depleted levels of stored TAG, similar to the reported phenotypes of LipA mutant mice and patients with LIPA mutations suffering from cholesteryl ester storage disease (CESD) and Wolman’s disease [62,63]. Thus, DHR96 actions in the gut are consistent with those of its mammalian homologs, the LXRs, in maintaining appropriate lipid balance [64]. Another orphan receptor in the fly, the Drosophila estrogen-related receptor (dERR), regulates nearly all aspects of carbohydrate catabolism. In the embryo, dERR activates an essential transcriptional program that promotes rapid larval growth via aerobic glycolysis [65], reflecting the same type of metabolic activity often seen in cancers. Further, dERR is an essential participant in adaptive hypoxic responses. Although the mechanisms for these actions remain unclear, dERR facilitates hypoxiainducible factor (HIF)-dependent and -independent transcriptional responses in hypoxia, including upregulation of glycolytic transcripts [66]. Not surprisingly, mammalian ERRs also have heavy-handed influences regulating carbohydrate catabolic enzymes and are necessary for appropriate hypoxic responses in the presence and absence of HIF-1 [67–69]. Modeling diet-induced lipotoxicity in flies The storage capacity of healthy adipose tissue in mammals is limiting. In the face of a consistent lipogenic diet, the conversion of dietary glucose into stored fat overwhelms the adipose tissue, and thus ectopic lipid deposition ensues, causing lipid-intolerant tissues to sustain lipotoxic damage. This lipid spillover, combined with age-dependent leptin resistance, is what Unger and Scherer argue to be the real culprit behind the comorbidities associated with the metabolic syndrome [70]. From this perspective, fat accumulation by adipocytes leading to obesity is not problematic; rather, obesity is a protective physiological action that holds off disease progression for as long as possible. Interestingly, flies seem to also use the conversion of glucose-derived carbons into fat body-stored TAG as a protective measure when confronted with a high-calorie carbohydrate-rich diet [71]. Although effective, this metabolic strategy can be overwhelmed, as it is in mammals, when the caloric excess is unrelenting. Intriguing new studies in Drosophila demonstrate that lipogenic diets, fed to otherwise normal flies, can induce the same suite of metabolic phenotypes seen in patients with the metabolic syndrome (Figure 2). Although the effects of diet on metabolic phenotypes is in its infancy in Drosophila, there is good reason to believe that recent advances in metabolite identification via mass spectrometric analysis will be of great aid in decoding the

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Drosophila fed lipogenic diet

Cardiomyopathyy

Hyperglycemia

LLoss of insulin sensivity

Obesity

Drosophila expanded adipose ssue TRENDS in Endocrinology & Metabolism

Figure 2. Lipotoxic spillover results in similar phenotypes in flies and man. As in mammals, consistent lipogenic diets in Drosophila result in ectopic lipid deposition and incidence of classic metabolic syndrome phenotypes, including diet-induced obesity, cardiomyopathy, and hyperglycemia.

meaning of complex metabolic signatures. For example, Carvalho et al. demonstrated that the diet exerts a direct effect on the phospholipid constituency of cell membranes in flies [20]. Similarly, fatty acid chain lengths tend to shorten whereas unsaturation increases when flies are fed a high-sugar diet (HSD) [71]. This same lipid signature is also apparent in insulin-resistance in humans [72]. HSD-induced phenotypes Musselman et al. showed that wild type larvae fed a HSD consisting of 86% carbohydrate calories from 1 M sucrose specifically developed an array of phenotypes that are consistent with T2D [73]. This includes hyperglycemia, insulin-resistance (small-size phenotype and developmental delay), molecular obesity (high TAG levels, large fatbody LDs), elevated free fatty acids, and activation of metabolic genes regulated by the forkhead transcription factor FOXO. Pasco and Leopold subsequently demonstrated that the HSD-induced peripheral insulin-resistance depends entirely on a secreted adipokine, Neural Lazarillo (NLaz), which is highly upregulated in the fat body upon HSD treatment [74]. Interestingly, the expression of the homolog of NLaz, retinol-binding protein 4 (RBP4), is also elevated in humans and mice that are obese and precedes frank diabetes [75,76], suggesting that lipocalins share a functionally similar role in the etiological progression of insulin resistance. When adult flies are subjected to a HSD regimen, Na et al. showed that fibrotic lesions begin to appear in the heart, accompanied by cardiac arrhythmias [77]. Importantly, this study demonstrated that dysfunctions induced by a HSD are due, at least in part, to increased flux of substrates through enzymes of the hexosamine biosynthetic pathway. Although their findings indicate that these proteins may be candidates for therapeutic targeting in cardiac dysfunction, it remains unclear whether or not hexosamine pathway inhibition would provide similar relief in mammals [78]. 7

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Review High-fat diets (HFDs) and fatty acid taste perception An array of diabetic cardiomyopathy-like phenotypes (reduced insulin signaling, elevated TAGs and glucose, defects in cardiac function, cardiac steatosis) result when flies are fed a HFD comprising 30% coconut oil [79]. Remarkably, systemic inhibition of insulin/TOR signaling alleviated cardiac fat accumulation, as did increased lipase expression in the myocardium. As it is in humans, the appetite for fats is perceived in a phospholipase C (PLC)dependent manner, which is specifically mediated through gustatory neurons that additionally sense sugars [80]. Interestingly, the responsiveness to sugars in unaffected in norpA (no receptor potential A)/PLC mutants, but extinguishes attraction to fatty acids. In addition, when flies are reared on a HFD they have decreased tolerance to stress, and this phenotype is positively altered by exposure to intermittent hypoxia but negatively altered in constant hypoxia [81]. Concluding remarks and future perspectives The studies mentioned here clearly demonstrate that Drosophila is a system that offers distinct advantages over other model systems and approaches. Despite its relative simplicity, it provides a pathway to translational applicability for human metabolic disorders. In the future, reverse genetic approaches that utilize Drosophila as a means to establish new models of human metabolic diseases, particularly those that have been difficult to model in other systems, are warranted. A good candidate for such an approach may be mature-onset diabetes of the young (MODY), an autosomal dominant form of diabetes that has not been successfully recapitulated in murine models, but which the factors known to be causal are highly conserved in Drosophila. Similarly, the pursuit of open-ended forward genetic screens that use random mutagenesis will be important to interrogate specific metabolic processes and phenotypes. Although difficult and more expensive to perform, these types of screens are more likely to uncover novel, unexpected phenotypes that may otherwise remain unseen in RNAi or enhancer-trap approaches. Furthermore, with the advent of next-generation sequencing, random mutational approaches should become an attractive option because this technology has made what once was the most arduous part of the screen (allele identification) increasingly affordable and much faster to complete. Despite the fact that Drosophila research is now in its second century, much still remains to learned about how this simple organism can be exploited to reveal novel aspects about our own biology. However, if the rapid advance of metabolic research in the fly in recent years is any indication of their utility, flies should expect to do some heavy lifting for the long-term. Acknowledgments Research in our lab is currently supported by Virginia Commonwealth University School of Medicine. We thank Y. Li for critical comments and help in preparing this manuscript.

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