InteractiveFly: GeneBrief

bummer : Biological Overview | References

BIOLOGICAL OVERVIEW

Energy homeostasis, a fundamental property of all organisms, depends on the ability to control the storage and mobilization of fat, mainly triacylglycerols (TAG), in special organs such as mammalian adipose tissue or the fat body of flies. Malregulation of energy homeostasis underlies the pathogenesis of obesity in mammals including human. A screen was performed to identify nutritionally regulated genes that control energy storage in the Drosophila. The brummer (bmm) gene encodes the lipid storage droplet-associated TAG lipase Brummer, a homolog of adipocyte triglyceride lipase (ATGL). Food deprivation or chronic bmm overexpression depletes organismal fat stores in vivo, whereas loss of bmm activity causes obesity in flies. These study identifies a key factor of insect energy homeostasis control. Their evolutionary conservation suggests Brummer/ATGL family members to be implicated in human obesity and establishes a basis for modeling mechanistic and therapeutic aspects of this disease in the fly (Grönke, 2005).

Providing constant energy supply despite variability in food access and metabolic energy demand is a fundamental property of animals. Key to an individual's survival during food deprivation is the ability to mobilize stored energy resources accumulated during periods of excessive energy supply. In organisms as different as humans and the fruit fly Drosophila, energy-rich diet components are converted into glycogen and, to a larger extent, triacylglycerols (TAG), the storage forms of carbohydrate and fat, respectively. Storage fat is deposited in intracellular lipid droplets of specialized organs called the adipose tissue in mammals or the fat body in Drosophila. In mammals, adipose tissue cooperates with the digestive tract and the central nervous system to hardwire an integrated molecular communication network ensuring the lifelong integrity of an organism's energy homeostasis under varying environmental conditions. In the peripheral fat storage tissue, a regulated balance between lipogenesis and lipolysis is believed to continuously match acute energy needs by TAG mobilization and readjust organismal storage fat content to a genetically determined setpoint during periods of excessive energy supply. Chronic imbalance of energy storage control by the lack or malfunction of regulatory genes results in excessive fat accumulation and is causative to the obesity pandemics in human populations as well as to related phenotypes in rodent models (Grönke, 2005).

A key regulator of storage fat lipolysis in mammalian adipocytes is the hormone-sensitive lipase (HSL) (Holm, 2003). Activated HSL interacts with perilipin at the lipid droplet membrane to eventually mobilize TAG. Acute HSL activation relies on posttranslational modification by protein kinase A (PKA) in response to hormonal β-adrenoceptor stimulation and subsequent activation of the cAMP second messenger signaling pathway. In addition, extensive fasting causes upregulation of mouse HSL mRNA and protein, supporting the enzyme's importance in acute and chronic TAG mobilization control. However, HSL knockout mice are viable and not obese, having substantial residual lipolytic activity. Accordingly, additional TAG lipases of the nutrin family (Villena, 2004), such as the most recently identified human adipose triglyceride lipase (ATGL) (Zimmermann, 2004), have been implicated in mammalian lipolysis. However, the in vivo relevance of this lipase family in fat storage control on the organismal level waits to be analyzed (Grönke, 2005 and references therein).

In insects, storage fat lipolysis is stimulated by adipokinetic hormone (AKH) in various species including the grasshopper Locusta migratoria, the tobacco hornworm Manduca sexta, and Drosophila, suggesting a general role in insect energy balance control. Like in mammalian TAG mobilization, AKH-stimulated lipolysis in the insect fat body relies on signaling via a G protein-coupled receptor (see Drosophila Gonadotropin-releasing hormone receptor/Adipokinetic hormone receptor), increase in intracellular cAMP, and activation of PKA. An insect TAG lipase, however, which makes the storage fat metabolically accessible for the energy-demanding target tissues, is currently unknown (Grönke, 2005).

Given the intriguing similarities in the regulatory mechanisms of TAG mobilization between mammals and insects, a genome-wide transcriptome profiling was performed in Drosophila to screen for nutritionally regulated and evolutionary conserved lipolysis effectors. This study presents the functional in vivo analysis of brummer, which encodes a TAG lipase of the nutrin family, whose lack causes obesity in the fly (Grönke, 2005).

To screen for nutritionally regulated genes, a genome-wide transcriptome analysis was performed, comparing gene expression of fed and food-deprived adult Drosophila flies. Sorting the total of 223 starvation-responsive genes according to their predicted function reveals that most of the starvation-induced genes are coding for metabolic enzymes (n = 44). In addition, genes coding for cytochromes (n = 10), metabolite transporters (n = 6), kinases (n = 5), and proteins involved in lipid metabolism (n = 7) are upregulated under starvation. Few metabolic enzymes (n = 7) are downregulated in response to starvation, whereas proteases and protease inhibitors form the largest group (n = 38). Nearly half of the starvation-induced metabolic enzymes are involved in carbohydrate catabolism, including key regulators like hexokinase (encoded by Hex-C), transketolase (CG8036), and phosphoglucomutase (Pgm) or enzymes involved in the breakdown of sugars like an α-Amylase (AmyD), a α-Glucosidase (CG11909), and six maltases (CG11669, CG8690, CG30359, CG30360, CG14934, CG14935). Protein degradation is reflected by the upregulation of genes involved in amino acid catabolism, including two aminotransferases encoded by got2 and spat, a phenylalanine-4-monooxygenase (henna), a 4-hydroxyphenylpyruvate-dioxygenase (CG11796), and a homogentisate-1,2-dioxygenase (hgo). The starvation-induced metabolic activation is further reflected by the transcriptional upregulation of five regulatory kinases or kinase subunits, which have all been implicated in energy homeostasis control. While the pyruvate dehydrogenase kinase encoded by pdk is critical for the regulation of oxidative glucose metabolism, the β subunit of the SNF1/AMP-activated protein kinase (AMPK) acts as a cellular energy sensor, and the cAMP-activated protein kinase A (PKA) promotes glycogen and TAG catabolism. The SNF4 γ subunit loechrig has been implicated in cholesterol homeostasis control. In addition, Lk6 kinase mutants have recently been described to have increased organismal TAG content, suggesting a function of the kinase in the control of organismal lipid storage (Grönke, 2005 and references therein).

Among the seven upregulated genes involved in lipid metabolism are genes encoding a putative TAG-lipase (CG5966), phospholipase A₂ (CG1583), low-density lipoprotein receptor (LpR2), long chain fatty acid CoA ligase (CG9009), and carnitine-O-palmitoyltransferase (CPTI). Anabolic reactions of the lipid metabolism are repressed under starvation, as indicated by the transcriptional downregulation of a lipogenic 1-acylglycerol-3-P-O-acyltransferase (CG4753) and a long chain fatty acid elongase (CG6261). Moreover, the PAT domain containing lipid storage droplet-associated protein Lsd-1 and three TAG lipases are among the nine genes involved in lipid metabolism that are downregulated in response to starvation (Grönke, 2005).

Taken together, genome-wide transcriptome profiling of fed versus food-deprived flies displays various regulatory aspects of the metabolic starvation response in Drosophila, including carbohydrate, amino acid, and lipid catabolism. However, no function has been assigned to 25% of the 223 starvation-responsive genes. Among those, the gene CG5295 was found in region 70F5 on chromosome 3L, termed brummer (bmm), is upregulated upon starvation. The single bmm transcript, which encodes a 507 amino-acid-long protein (BMM) closely related to TTS-2/ATGL of mouse and human, is expressed during all ontogenetic stages of the fly. It is highly enriched in the energy storage tissue as well as the food-absorbing parts of the digestive tract, i.e., the larval midgut and gastric caeca. Quantitative Northern blot analysis confirms sustained transcriptional upregulation in response to food-deprivation and downregulation upon refeeding. The nutritional regulation and the patterns of bmm expression suggest that bmm participates in the control of energy homeostasis (Grönke, 2005).

BMM contains a patatin-like domain (PLD) including a serine hydrolase motif, originally described in plant acyl-hydrolases, and a so-called Brummer box (BB) of unknown function. The BB motif is found in a number of PLD-containing proteins, which is referred to as the Brummer/Nutrin subfamily. It includes the Anopheles BMM ortholog, a Drosophila paralogue called doppelgänger von brummer (dob; CG5560), the human proteins Adiponutrin, GS2-like, TTS-2/ATGL and GS2, Caenorhabditis elegans C05D11.7 and D1054.1 as well as Arabidopsis NP_174597 (Grönke, 2005).

PLD proteins are phospholipases in plants, human (van Tienhoven, 2002), and Pseudomonas or TAG lipases, as recently shown for the human Brummer/Nutrin family members TTS-2.2/ATGL, GS2, and Adiponutrin (Jenkins, 2004; Zimmermann, 2004). Recombinant BMM exhibits esterase activity on an esterified fatty acid (6,8-difluoro-4-methylumbelliferyl octanoate) as substrate but fails to catalyze the release of fatty acid from either the A2 position of a phospholipid (PAP), the glycosylphosphatidylinositol (GPI) membrane glycolipid membrane anchor of GPI-modified proteins (5′-nucleotidase, Gce1), or monoacylglycerol (MAG). However, it cleaves TAG in vitro, whereas the BMM^S38A mutant, in which serine residue 38 of the catalytic center had been replaced by alanine, is enzymatically inactive. Thus, bmm as its mammalian homologs are candidates for nutritionally regulated in vivo effectors of TAG mobilization (Grönke, 2005).

To test whether bmm promotes fat mobilization in vivo, bmm loss-of-function mutant alleles (bmm¹ and bmm²) were generated by mobilization of a transposable P element located in the first exon of bmm. Precise excision of the P element, as obtained with bmm^rev, served as genetically matched control for phenotypic analysis. Embryos lacking both maternal and zygotic bmm activity are lethal, indicating that bmm carries an essential function. They develop pleiotropic degeneration phenotypes and have increased TAG levels in late embryogenesis. Embryonic lethality can be partially rescued by a paternally provided functional bmm gene and almost completely reverted by ubiquitous bmm expression from a cDNA-bearing transgene. Similar phenotypes and a reduced embryonic hatching rate have been reported for mutants of the perilipin-like fly gene Lsd-2. These results suggest that bmm fulfils a vital function in TAG mobilization during embryogenesis (Grönke, 2005).

Flies lacking only zygotic BMM lipase activity develop normally but show progressive obesity accumulating 17% (immature adults, <1 day old) to 101% (mature adults, 6 days old) more storage fat compared to control flies. Conversely, transgene-dependent bmm overexpression in fat body cells of fed flies, which mimics the effect of starvation-induced upregulation of bmm transcription, depletes the TAG content of immature and mature adults by 96% and 46%, respectively. These effects were not observed upon transgenic expression of the enzymatically inactive bmm^S38A mutant, indicating that the TAG mobilization is caused by the lipase activity of BMM. bmm-dependent differences of organismal TAG content are also reflected by the lipid storage phenotype of fat body cells showing variously sized storage droplets in bmm¹ mutant fat body cells and their reduction in size and number upon overexpression of the gene. The effect of BMM is specific for the fat-based aspect of energy storage, since the glycogen content is not affected in bmm mutant or bmm-overexpressing flies (Grönke, 2005).

Excessive fat storage in flies lacking bmm function reduces the median lifespan by only 10%. Acute TAG mobilization is impaired but not abolished in bmm mutants. While controls deplete their storage TAG during starvation, bmm mutants are able to consume 73% of their prestarvation fat content. Accordingly, food-deprived bmm mutants outlive controls by 56% on the expense of their increased prestarvation fat storage. The lipolytic activity present in bmm mutants allows fuelling their extended survival under food deprivation by metabolizing in total 65% more TAG than controls. Thus, as in mammals, mobilization of TAG storage in flies is controlled by more than one TAG lipase. Candidate effectors of bmm-independent TAG mobilization are the bmm paralogue dob and the genes CG5966 and CG11055, which code for a starvation-induced putative TAG lipase and a Drosophila HSL homolog, respectively (Grönke, 2005).

To possibly extend the functional similarity between mammalian and Drosophila TAG lipases, it was asked whether BMM localizes at the surface of lipid droplets. Transgenic flies expressing BMM:EGFP fusion protein variants in their fat body cells allow examination of BMM intracellular localization and lipolytic activity in vivo. Ubiquitous expression of BMM:EGFP or BMM reverts the obese phenotype of bmm mutant flies. Targeting of BMM:EGFP but not BMM^S38A:EGFP expression to the fat body of otherwise wild-type flies depletes the organismal TAG storage and reduces both the number and size of lipid droplets in fat body cells. BMM:EGFP localizes at islands on the droplet surface, often at interdroplet contact sites). In contrast, nonfunctional BMM^S38A:EGFP distributes homogenously over the droplet surfaces. The evolutionary conserved part of BMM including the Brummer box is sufficient to properly localize the protein on lipid droplets, likely to represent active sites of BMM-dependent TAG mobilization. Other BMM-related lipases, such as hamster desnutrin (Liu, 2004) and human TTS-2.1/ATGL (Umlauf, 2004), also localize on lipid droplets, but their localization sequences are presently unknown (Grönke, 2005).

The results indicate that the surface of lipid droplets is an evolutionary conserved intracellular compartment boundary for organismal TAG storage control, as has been suggested for mammalian adipocytes where perilipin modulates activity of HSL (Zhang, 2003) and possibly non-HSL lipases such as ATGL. Lack of perilipin results in lean mice with increased lipolysis and reverses the obese phenotype of leptin receptor-deficient mutants. The perilipin-like LSD-2 of fly localizes to lipid droplets of fat cells and adjusts organismal TAG content in a dosage-dependent manner, suggesting that it functions as an evolutionary conserved modulator of lipolysis. In fact, bmm⁻ Lsd-2⁻ double mutants have wild-type TAG levels, indicating that loss of Lsd-2 activity compensates for the lack of bmm. Conversely, combined overexpression of bmm and Lsd-2 in the fat body can partially revert the complementary phenotypes caused by the overexpression of each of the two genes. These data demonstrate that the lipid droplet-associated factors Brummer and LSD-2, which have opposite roles in organismal fat storage, act in an antagonistic manner (Grönke, 2005).

This first in vivo analysis of any insect lipase demonstrates a remarkable conservation of effectors controlling organismal fat storage in mammals and flies, emphasizing the value of Drosophila for research in energy homeostasis. On the basis of these results in the fly, it is speculated that mammalian members of the brummer/nutrin gene family like ATGL play an essential role in organismal fat mobilization and that malfunction of Brummer-homologous TAG lipases might contribute to mammalian obesity. Accordingly, stimulating Brummer-like lipase activity is a potential therapeutic approach to control TAG release from adipose tissue in obese patients, and lipase activators could be tested in the fly model (Grönke, 2005).

Dual lipolytic control of body fat storage and mobilization in Drosophila

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. A Drosophila mutant was generated lacking the receptor (AKHR; FlyBase name -- Gonadotropin-releasing hormone receptor or GRHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ss-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. These data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis (Grönke, 2007).

Expression studies in a heterologous tissue culture system and in Xenopus oocytes identified AKH-responsive G protein-coupled receptors in Drosophila, such as the one encoded by the AKHR (or CG11325) gene. AKHR is expressed during all ontogenetic stages of the fly. It consists of seven exons, which encode a predicted protein of 443 amino acids. In late embryonic and larval stages, AKHR is expressed in the fat body. This finding is consistent with its predicted role as transmitter of the lipolytic AKH signal in this organ (Grönke, 2007).

In order to examine the effect of AKHR signaling on fat storage and mobilization in vivo, two different P element-insertion mutants were used, CG11188^A1332 and AKHR^G6244, which are located close to and within the AKHR gene, respectively. CG11188^A1332 flies carrying the transposable element integration designated A1332 allow for the transcriptional activation of the adjacent AKHR gene. This ability was used for AKHR gain-of-function studies by overexpression of AKHR in the fat body of flies. Overexpression of AKHR in response to a fat body-specific Gal4 inducer causes dramatic reduction of organismal fat storage. This finding could be recapitulated by fat body-targeted AKHR expression from a cDNA-based upstream activation sequence (UAS)-driven AKHR transgene. These gain-of-function results suggest a critical in vivo role for AKHR in storage-lipid homeostasis of the adult fly (Grönke, 2007).

Flies of strain AKHR^G6244, which carry a P element integration in the AKHR untranslated leader region, were used to generate the AKHR deletion mutants AKHR¹ and AKHR², as well as the genetically matched control AKHR^rev, which possesses a functionally restored AKHR allele. As exemplified for embryonic and larval stages, AKHR¹ mutants lack AKHR transcript. Ad libitum-fed flies without AKHR function are viable, fertile, and have a normal lifespan. However, such flies accumulate lipid storage droplets in the fat body and have 65%-127% more body fat than the controls. These results indicate that AKHR¹ mutants develop an obese phenotype. The same result was obtained with AKHR² and AKHR¹/AKHR² transheterozygous mutant flies, as well as with flies lacking the AKH-producing cells of the neuroendocrine system due to targeted ablation by the cell-directed activity of the proapoptotic gene reaper. Conversely, chronic overexpression of AKH provided by a fat body-targeted AKH transgene of otherwise wild-type flies largely depletes lipid storage droplets and organismal fat stores. However, the obese phenotype of AKHR mutants is unresponsive to AKH, indicating that AKHR is the only receptor transmitting the lipolytic signal induced by AKH in vivo. Collectively, these data demonstrate that AKH-dependent AKHR signaling is critical for the chronic lipid-storage homeostasis in ad libitum-fed flies (Grönke, 2007).

Studies on various insect species helped elucidate several components and mediators of the lipolytic AKH/AKHR signal transduction pathway (for review, see [Van der Horst, 2001). However, the identity of the TAG lipase(s) executing the AKH-induced fat mobilization program remained elusive. Besides the Drosophila homolog of the TG lipase from the tobacco hornworm Manduca sexta (Arrese, 2006), the recently identified Brummer lipase, a homolog of the mammalian ATGL, is a candidate member of the AKH/AKHR pathway. This is based on the striking similarity between the phenotypes of AKHR and bmm mutants. Ad libitum-fed flies lacking either AKHR or bmm activity, store excessive fat. Both mutants show incomplete storage-fat mobilization (Grönke, 2005) and starvation resistance (Grönke, 2005) in response to food deprivation. Starvation resistance of these mutants might be caused by their increased metabolically accessible fat stores and/or changes in their energy expenditure due to locomotor activity reduction as described for flies with impaired AKH signaling. Despite the phenotypic similarities of their mutants, however, AKHR and bmm are members of two different fat-mobilization systems in vivo. Several lines of evidence support this conclusion. On one hand, AKH overexpression reduces the excessive TAG storage of bmm mutants, while on the other, bmm-induced fat mobilization can be executed in AKHR mutants. Thus, AKH/AKHR signaling is not a prerequisite for Brummer activity. Moreover, genetic epistasis experiments support this idea that AKHR and bmm belong to different control systems of lipocatabolism in vivo. Double-mutant analysis reveals that the obesity of AKHR and bmm single mutants is additive. Accordingly, AKHR bmm double-mutant flies store about four times as much body fat as control flies and accumulate excessive lipid droplets in their fat body cells (Grönke, 2007).

Thin layer chromatography (TLC) analysis was used to compare the storage-fat composition of AKHR and bmm single mutants with AKHR bmm double-mutant and control flies. Excessive body fat accumulation in AKHR bmm double mutants is on the one hand due to TAG, which is increased compared to AKHR and bmm single-mutant flies. Additionally, an uncharacterized class of TAG (TAGX) appears exclusively in AKHR bmm double mutants. In contrast to TAG, changes in diacylglycerol (DAG) content do not substantially contribute to the differences in body fat content in any of the analyzed genotypes. Taken together a quantitative increase and a qualitative change in the TAG composition account for the extreme obesity in AKHR bmm double-mutant flies (Grönke, 2007).

To address the in vivo response of AKHR bmm double mutants to induced energy-storage mobilization, flies were starved and their survival curve monitored. AKHR bmm double mutants die rapidly after food deprivation. In contrast to the starvation-resistant obese AKHR and bmm single mutants, the double mutants are not capable of mobilizing even part of their excessive fat stores. AKHR bmm double mutants do not, however, suffer from a general block of energy-storage mobilization because they can access and deplete their carbohydrate stores. These data demonstrate that energy homeostasis in AKHR bmm double-mutant flies is imbalanced by a severe and specific lipometabolism defect, which cannot be compensated in vivo (Grönke, 2007).

The nature of Brummer as a TAG lipase and AKHR as a transmitter of lipolytic AKH signaling suggests that the extreme storage-fat accumulation and starvation sensitivity of ad libitum-fed AKHR bmm double mutants is due to severe lipolysis dysfunction. To address this possibility in vitro, lipolysis rate measurements on fly fat body cell lysates and lysate fractions of control flies were performed. Results show that the cytosolic fraction of fat body cells contains the majority of basal and starvation-induced lipolytic activity against TAG, similar to the activity distribution in mammalian adipose tissue. Little basal and induced total TAG cleavage activity localizes to the lipid droplet fraction, whereas the pellet fraction including cellular membranes shows low basal, non-inducible TAG lipolysis. Lipolysis activity against DAG is similarly distributed between fat body cell fractions. However, in accordance with the function of DAG as major transport lipid in Drosophila, DAG lipolysis in fat body cells is not induced in response to starvation (Grönke, 2007).

Based on the lysate fraction analysis of control flies, cytosolic fat body cell extracts were used to assess the basal and starvation-induced lipolytic activity of mutant and control flies on TAG, DAG, and cholesterol oleate substrates. Whereas DAG and cholesterol oleate cleavage activity of fat body cells is comparable between all genotypes and physiological conditions tested, TAG lipolysis varies widely. Compared to control flies, basal TAG lipolysis of AKHR bmm double mutants is reduced by 80% and induced TAG cleavage is completely blocked, consistent with the flies' extreme obesity and their inability to mobilize storage fat. The impairment of basal lipolysis in the double mutants is largely due to the absence of bmm function, because it is also detectable in bmm single-mutant cells, whereas basal lipolysis in AKHR mutants is not reduced. Interestingly, bmm mutants mount a starvation-induced TAG lipolysis response after short-term (6 h), but not after extended (12 h), food deprivation. Conversely, AKHR mutant cells lack an early lipolysis response, but exhibit strong TAG cleavage activity after extended food deprivation. These data suggest that induced storage-fat mobilization in fly adipocytes relies on at least two lipolytic phases: an early, AKH/AKHR-dependent phase and a later, Brummer-dependent phase. Accordingly, it is speculated that the obesity of bmm and AKHR mutant flies is caused by different mechanisms: chronically low basal lipolysis in bmm mutants and, in AKHR mutants, lack of induced lipolysis during short-term starvation periods that is characteristic of organisms with discontinuous feeding behavior. It is acknowledged, however, that in vitro lipolysis assays on artificially emulsified substrates allow only a limited representation of the lipocatabolism in vivo, because lipid droplet-associated proteins modulate the lipolytic response in the insect fat body (Patel, 2005; Patel, 2006) and mammalian tissue. Moreover, excessive fat accumulation in AKHR mutants may be in part due to increased lipogenesis because AKH signaling has been demonstrated to repress this process in various insects (Grönke, 2007).

Fat body cells of control flies (AKHR^rev bmm^rev) exhibit basal TAG lipolysis, which is doubled by starvation-induced lipolysis after 6 h or 12 h of food deprivation. bmm mutant cells have reduced basal lipolysis and lack induced lipolysis after 12 h starvation. AKHR mutant cells lack early (6 h) induced lipolysis, but show strong starvation-induced lipolysis after 12 h food deprivation. AKHR bmm double mutants have reduced basal lipolysis and lack starvation-induced lipolysis altogether (Grönke, 2007).

The finding of the dual lipolytic control in the fly raises the question of whether the two systems involved act independently of each other or whether one system responds to the impairment of the other. Modulation of transcription is an evolutionarily conserved regulatory mechanism of lipases from the ATGL/Brummer family. ATGL is transcriptionally up-regulated in fasting mice, as is bmm transcription in starving flies. Moreover bmm overexpression depletes lipid stores in the fat body of transgenic flies. Accordingly, bmm transcription was analyzed in response to modulation of AKH/AKHR signaling to assess a potential regulatory interaction between the two lipolytic systems. Compared to the moderate starvation-induced up-regulation of bmm in control flies, the gene is hyperstimulated in flies with impaired AKH signaling. As early as 6 h after food deprivation, bmm transcription is up-regulated by a factor of 2.5-3 in flies lacking the AKH-producing neuroendocrine cells (AKH-ZD) or in AKHR mutant flies. Conversely, chronic expression of AKH in the fat body suppresses bmm transcription. Bmm hyperstimulation in AKHR mutants is consistent with a subsequent strong increase of starvation-induced TAG lipolysis observed 12 h after food deprivation. Taken together, these data demonstrate an AKH/AKHR-independent activation mechanism of bmm and suggest the existence of compensatory regulation between bmm and the AKH/AKHR lipolytic systems, the mechanism of which is currently unknown (Grönke, 2007).

The results presented in this study provide in vivo evidence that the fly contains two induced lipolytic systems. One system confers AKH/AKHR-dependent lipolysis, a signaling pathway, which assures rapid fat mobilization by cAMP signaling and PKA activity. Drosophila's second lipolytic system involves the Brummer lipase, which is responsible for most of the basal and part of the induced lipolysis in fly fat body cells, likely via transcriptional regulation. Currently, it is unknown whether Brummer activity is post-translationally modulated by an α/β hydrolase domain-containing protein like the regulation of its mammalian homolog ATGL by CGI-58. Homology searches between mammalian and Drosophila genomes identify the CGI-58-related fly gene CG1882 and the putative Hsl homolog CG11055, providing additional support for the evolutionary conservation of fat-mobilization systems. However, differences in lipid transport physiology (i.e., DAG transport in Drosophila, and FFA in mammals) suggest a different substrate specificity or tissue distribution of fly Hsl compared to its mammalian relative (Grönke, 2007).

Future studies will not only unravel the crosstalk between the two Drosophila lipocatabolic systems, but also disclose the identity of additional genes involved in this process, such as the upstream regulators of bmm. This study substantiates the emerging picture of the evolutionary conservation between insect and mammalian fat-storage regulation and emphasizes the value of Drosophila as a powerful model system for the study of human lipometabolic disorders (Grönke, 2007).

Opposite and redundant roles of the two Drosophila perilipins in lipid mobilization

Lipid droplets are the main lipid storage sites in cells. Lipid droplet homeostasis is regulated by the surface accessibility of lipases. Mammalian adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are two key lipases for basal and stimulated lipolysis, respectively. Perilipins, the best known lipid droplet surface proteins, can either recruit lipases or prevent the access of lipases to lipid droplets. Mammals have five perilipin proteins, which often exhibit redundant functions, precluding the analysis of the exact role of individual perilipins in vivo. Drosophila have only two perilipins, PLIN1/LSD-1 and PLIN2/LSD-2. Previous studies revealed that PLIN2 is important for protecting lipid droplets from lipolysis mediated by Brummer (BMM), the Drosophila homolog of ATGL. This study reports the functional analysis of (Lipid storage droplet-1) PLIN1 and Drosophila Hormone-sensitive lipase ortholog (HSL). Loss-of-function and overexpression studies reveal that unlike PLIN2, PLIN1 probably facilitates lipid mobilization. HSL is recruited from the cytosol to the surface of lipid droplets under starved conditions and PLIN1 is necessary for the starved induced lipid droplet localization of HSL. Moreover, phenotypic analysis of plin1;plin2 double mutants revealed that PLIN1 and PLIN2 might have redundant functions in protecting lipid droplets from lipolysis. Therefore, the two Drosophila perilipins have both opposite and redundant roles. Domain swapping and deletion analyses indicate that the C-terminal region of PLIN1 confers functional specificity to PLIN1. This study highlights the complex roles of Drosophila perilipin proteins and the evolutionarily conserved regulation of HSL translocation by perilipins (Bi, 2012).

The analysis of dHSL reveals several interesting points. Under fed conditions, both the TAG level and the size of lipid droplets are slightly increased in dHSL mutant larvae, indicating that dHSL may function under basal condition. In supporting that, dHSL mutation enhances the large lipid droplet phenotype of bmm mutants. The location of dHSL-EGFP to lipid droplets under starvation highlights that the mechanism by which HSL regulates stimulated lipolysis is likely conserved from Drosophila to mammals. This study took advantage of a dHSL-EGFP reporter to establish a strong connection between defective fat mobilization in plin1 mutants and the lipid droplet surface localization of dHSL (Bi, 2012).

The fact that plin1 mutant larvae have larger lipid droplets than bmm;dHSL double mutants can be explained by the proposed structural role of PLIN1 in lipid droplets (Beller, 2010). Since plin1 mutants have giant lipid droplets, it is possible that PLIN1 may be involved in lipid droplet fission or fusion. Several recent studies have revealed that phosphatidic acid (PA) is important for the formation of supersized lipid droplets in Seipin mutants. It remains to be determined whether PLIN1 affects the metabolism of fatty acids or phospholipids, such as PA. Moreover, these results also extend previous findings (Beller, 2010) by showing that PLIN1 has PLIN2-like function in protecting lipid droplets from lipolysis. Currently, it is not known how PLIN1 performs this protective role. It is possible that it acts by blocking the access of BMM. Previous finding (Beller, 2010) that more BMM localizes to lipid droplets in plin1 mutants is consistent with this possibility (Bi, 2012).

The dual role of Drosophila PLIN1 prompts comparison between Drosophila PLIN1 and mammalian Perilipin1. Both PLIN1 and Perilipin1 have two opposing functions in lipid droplets: preventing lipolysis and facilitating lipolysis. The two roles of Perilipin1 are regulated by phosphorylation. Unphosphorylated Perilipin1 protects lipid droplets from lipolysis by blocking the access of lipases, while phosphorylated Perilipin1 releases the ATGL activator CGI58, resulting in activation of ATGL, which promotes lipolysis (Zimmermann, 2004). Phosphorylated Perilipin1 can also elicit translocation of HSL from the cytosol to the lipid droplet surface. Similarly, studies using purified Drosophila PLIN1 implied that PKA phosphorylation of PLIN1 had a direct effect on lipase activity. Moreover, this study found that PLIN1 is important for dHSL lipid droplet location. Therefore, the regulation of HSL localization by Perilipins is likely highly conserved from Drosophila to mammals. It remains to be determined whether PLIN1 regulates the activity of BMM, the Drosophila ATGL. In contrast, plin1 differs from Perilipin1 in the following ways. First, plin1 mutants show different phenotypes under normal conditions to Perilipin1 mutants. Unlike Perilipin1 knockout mice, Drosophila plin1 mutants are not lean; indeed, a recent study showed that plin1 mutant animals develop adult-onset obesity (Beller, 2010). Second, overexpression of Perilipin1 results in aggregated lipid droplets (Marcinkiewicz, 2006), while overexpression of plin1 leads to small lipid droplets. Lastly, the partially redundant function of PLIN1 was revealed in the plin2 mutant background. It is not known whether Perilipin1 has other functions in the absence of other Perilipins in vivo (Bi, 2012).

The results suggest that PLIN2, together with PLIN1, may protect small lipid droplets at an early stage of lipid droplet biogenesis from BMM- and probably dHSL-mediated lipolysis, while PLIN1 facilitates dHSL-mediated lipolysis in large lipid droplets. Based on the phenotypic analysis, it is thought that the major function of PLIN1 is in facilitating fat mobilization. Because large lipid droplets have greater lipid content, lipolysis of large droplets may be an efficient way to support the cell’s energy needs and to balance lipid usage with lipid droplet biogenesis. Such fine regulation is important for maintaining lipid homeostasis. Moreover, the functional complexity of PLIN1 may reflect the evolution of ancient Perilipins from simple barriers that protect lipid droplets to more active regulators of lipid homeostasis. How are the dual functions of PLIN1 regulated? It is possible that PLIN1 may have different structures/states and binding partner(s) in lipid droplets of different sizes. Phosphorylated PLIN1 was found to affect the activity of lipase in in vitro assays (Arrese, 2008). Therefore, the phosphorylation state of PLIN1 may be different in small and large lipid droplets. Although the functional importance of PLIN1 phosphorylation remains to be determined in vivo, a recent study showed that the canonical PKA target sites are not important for PLIN1 function (Beller, 2010). Therefore, identification of the phosphorylation site of PLIN1 will lead to better understanding of the regulation of PLIN1 function. Since the C-terminal region of PLIN1 determines its functional specificity, regulation of the dual role may be a property of the C terminus. The N-terminal portion of PLIN1 may be sufficient for its function in protecting lipid droplets from lipolysis. The C-terminal region of PLIN1 is highly conserved among Drosophila species. Identifying protein partners of the C-terminal region could help to reveal the regulatory mechanisms involved. Similarly, compared to ADRP and TIP47, Perilipin1 has an extended C-terminal region. Phosphorylation of key residues in the C-terminal region of Perilipin1 is important for ATGL activation and lipid droplet dispersal. Frame-shift mutations at the C-terminal region of Perilipin1 result in dominant partial lipodystrophy in human, supporting the functional importance of the C-terminal region (Bi, 2012).

The study reveals the functions of the only two Perilipins in Drosophila. The fact that plin1;plin2 double mutants have small lipid droplets indicates that Perilipins are dispensable for the initial biogenesis of lipid droplets, but are required for the growth of lipid droplets. Together with a recent study on PLIN1 (Beller, 2010), these findings provide a better understanding of the exact function of Perilipins in vivo. plin1, plin2, and dHSL mutants can be used as models to further probe the homeostasis of lipid droplets. More functional studies of Drosophila lipid-related genes may facilitate a deeper understanding of diseases related to fat metabolism, such as obesity and diabetes (Bi, 2012).

Intergenerational inheritance of high fat diet-induced cardiac lipotoxicity in Drosophila

Obesity is strongly correlated with lipotoxic cardiomyopathy, heart failure and thus mortality. The incidence of obesity has reached alarming proportions worldwide, and increasing evidence suggests that the parents' nutritional status may predispose their offspring to lipotoxic cardiomyopathy. However, to date, mechanisms underlying intergenerational heart disease risks have yet to be elucidated. This study reports that cardiac dysfunction induced by high-fat-diet (HFD) persists for two subsequent generations in Drosophila and is associated with reduced expression of two key metabolic regulators, adipose triglyceride lipase (ATGL/bmm) and transcriptional cofactor PGC-1. Evidence is provided that targeted expression of ATGL/bmm in the offspring of HFD-fed parents protects them, and the subsequent generation, from cardio-lipotoxicity. Furthermore, it was found that intergenerational inheritance of lipotoxic cardiomyopathy correlates with elevated systemic H3K27 trimethylation. Lowering H3K27 trimethylation genetically or pharmacologically in the offspring of HFD-fed parents prevents cardiac pathology. This suggests that metabolic homeostasis is epigenetically regulated across generations (Guida, 2019).

Several studies have established a critical role for triacylglyceride (TAG) hydrolysis in cardiac metabolism and function, in both healthy and diseased hearts. This study shows that acute HFD (5 days of food supplemented with 30% coconut oil) induces lipotoxic cardiomyopathy that can be inherited by the next two generations, via the parental germlines, even when the offspring are raised on normal food diet (NFD). Similar to what has been described by epidemiological studies on the offspring of obese pregnant women, this study found in the fly model that parental HFD led to first generation progeny with increased adult body weight and increased fat content in late-stage embryos. This was no longer the case in second generation offspring, and adult progeny did not exhibit an increase in fat content relative to body weight in either generations. In contrast, metabolic reprogramming across generations was particularly evident in the systemic reduction in the transcript levels of ATGL/bmm lipase and its downstream target PGC-1/spargel, a key regulator of energy metabolism. The intergenerational lipotoxic cardiomyopathy model was further validated by genetically reducing PGC-1 expression in the parents (PGC-1xp heterozygotes), which causes similar cardiac lipotoxicity as HFD exposure. This was sufficient to alter the +/+ offspring's metabolic state, leading to lipotoxic cardiomyopathy later in life, even though these flies carry two wild-type copies of PGC-1. Of note, the partial reduction of PGC-1 induced by acute and parental HFD in control flies and PGC-1XP heterozygous mutant flies is likely having a profound effect on mitochondrial biogenesis that could be underlying the observed heart dysfunction (Guida, 2019).

The experiments presented in this study indicate that HFD and reduced PGC-1 expression levels have the ability to modify the offspring's metabolism leading to heart dysfunction. Thus, it is speculated that parental HFD-dependent metabolic reprogramming and associated lipotoxic cardiomyopathy in the progeny could be prevented by increasing ATGL/bmm levels. Indeed, targeted transgenic expression of bmm is able to reset the altered metabolic state induced by parental HFD, and thus protects the progeny from cardiac lipotoxicity. Remarkably, induction of bmm expression in the early embryo is sufficient to render the adult progeny (G1), as well as the following generation (G2), resistant to acute HFD (Guida, 2019).

The presented data support the hypothesis that the quality of nutrition during gestation leads to fetal programming that functions as a key determinant in establishing predisposition and/or susceptibility to metabolic and cardiovascular disorders later in life. While this hypothesis is sustained by several epidemiological studies, including the Dutch Hunger Winter studies, and many animal models with different environmental stressors, the underling mechanisms on the intergenerational inheritance of lipotoxic cardiomyopathy induced by parental HFD remain mostly unknown. The Drosophila model in this study indicates that the inheritance of altered histone modifications is a key mechanism in the propagation of lipotoxic cardiomyopathy across generations (Guida, 2019).

Histone modifications are essential in regulating chromatin packaging and gene expression for proper development and cell function. In turn, metabolites serve as co-factors for chromatin modifying enzymes, allowing protein activity and gene expression to match the specific energy requirements. It was found that HFD, and potentially its maladaptive fluctuation in metabolites, can lead to changes in chromatin structure and gene expression that are transmitted to the next generation. Indeed, a major finding presented in this study is the involvement of the PRC2 complex in regulating metabolism and heart function in response to a HFD, via H3K27me3 gene repression across generations. The increase in H3K27me3 global levels in the adult flies upon acute or parental HFD supports the hypothesis that HFD causes changes in the epigenome that have lifelong consequences. The results are in line with previous findings in Caenorhabditis elegans and mice, which provide evidence that PRC2-mediated epigenetic modifications in the germ cells can be transmitted to embryos by sperm and/or oocytes. In addition, this study found that reduction of H3K27me3, either by overexpression of UTX or inhibition of EzH2, can prevent the deleterious effects of a parental HFD on heart function and metabolism. Of note, at this point it cannot be ruled out that UTX overexpression and EzH2 inhibition might also have H3K27me3-independent effects (Guida, 2019).

Overall, using the genetic model of Drosophila, this study provides first evidence that early embryonic and tissue-specific modulation of lipolysis in myocardial progenitors, adipose tissue, and the germline leads to tissue-specific and/or systemic metabolic reprogramming or pre-programming that persists into adulthood. Importantly, the imposed metabolic state appears to be inherited by the next two generations as either a predisposition to metabolic imbalance and cardiac dysfunction, or a protection from HFD insult when bmm is overexpressed. Moreover, this study provides evidence that metabolic re-programming that leads to fat accumulation and cardiac lipotoxicity correlates with overall levels of H3K27me3 epigenetic marks, which can be reversed by genetic or pharmacological reduction of H3K27me3 levels. The findings shed light on possible causes of obesogenic heritability and early adult onset of cardiovascular disease and diabetes, which appear to have their roots in the diet and overall metabolic state of the parents. Importantly, this study demonstrates that targeted genetic or pharmacological interventions in the progeny are able to counteract the detrimental effects on cardiac function of parental dietary insults, a protection that persists even in the subsequent generation against acute HFD. These findings provide new perspectives for tackling metabolic syndrome effects across generations and preventing lipotoxic cardiomyopathies (Guida, 2019).

NF-kappaB shapes metabolic adaptation by attenuating Foxo-mediated lipolysis in Drosophila

Metabolic and innate immune signaling pathways have co-evolved to elicit coordinated responses. However, dissecting the integration of these ancient signaling mechanisms remains a challenge. Using Drosophila, this study uncovered a role for the innate immune transcription factor nuclear factor kappaB (NF-kappaB)/Relish in governing lipid metabolism during metabolic adaptation to fasting. Relish in fat bodies was found to be required to restrain fasting-induced lipolysis, and thus conserve cellular triglyceride levels during metabolic adaptation, through specific repression of ATGL/Brummer lipase gene expression in adipose tissue (fat body). Fasting-induced changes in Brummer expression and, consequently, triglyceride metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, a critical metabolic transcription factor. Relish limits Foxo function by influencing fasting-dependent histone deacetylation and subsequent chromatin modifications within the Bmm locus. These results highlight that the antagonism of Relish and Foxo functions are crucial in the regulation of lipid metabolism during metabolic adaptation, which may further influence the coordination of innate immune-metabolic responses (Molaei, 2019).

In order to explore mechanistic connections between NF-κB and various metabolic control networks, lipid homeostasis was assessed in Drosophila lacking functional Relish (utilizing the rel^E20 allele) independent of pathogenic infection. Relish is similar to mammalian p100/p105 NF-κB proteins and contains a Rel-homology domain, as well as ankyrin repeats (found in mammalian inhibitory IκBs). During ad libitum feeding, NF-κB/Rel mutant adult female flies (rel^E20/rel^E20) have significantly less organismal TAGs than genetically matched controls (either OreR or rel^E20 / + heterozygote flies, 7 days old post eclosion). However, these changes in TAG correlated with decreases in acute and chronic feeding and can be rescued by high-calorie (sugar) diets, suggesting that steady-state differences in lipid homeostasis are potentially driven by changes in feeding behavior. Assaying the major fat storage tissues, this study also found that TAG-level reduction in mutant animals correlates with strong, but variable, decreases in neutral lipid content in fat body and/or adipose but not in the intestine (Molaei, 2019).

Since ad libitum effects on lipid homeostasis appear to correspond with feeding deficits (i.e., they are potentially indirect), changes in fat metabolism were assayed in Relish mutant animals during metabolic adaptation to fasting. rel^E20/rel^E20 mutant flies were sensitive to starvation. Furthermore, Relish-deficient animals displayed accelerated decreases in organismal TAG levels during acute fasting (at time points before significant death occurred), while controls flies showed little to no change at the same time points (always comparing within sibling genotypes). These changes in TAG levels correlate with a strong reduction of stored neutral lipids and/or lipid droplets in carcass fat body, suggesting that there is enhanced or accelerated lipid breakdown during metabolic adaptation in these animals (Molaei, 2019).

The insect fat body acts as a key sensor to link nutrient status and energy expenditure and, as such, is the major lipid depository (mainly TAGs) that combines energy storage, de novo synthesis, and breakdown functions of vertebrate adipose and hepatic tissues. This tissue is also essential for Toll- and Relish-mediated innate immune responses to bacterial infection. Critically, the fat body is integral to properly balance lipid catabolism and anabolism in order to modulate organismal energy homeostasis (through lipid supply to other tissues) in response to metabolic or dietary adaptation. Expression of full-length Relish in the fat body (CGGal4>UAS-Rel) can rescue reduced starvation survival rates and the accelerated loss of lipid storage in rel^E20/rel^E20 mutant flies during fasting. These data suggest that Relish function in the fat body is required to acutely maintain lipid homeostasis throughout the course of metabolic adaptation (Molaei, 2019).

To further confirm an autonomous and potentially direct role for Relish in the regulation of fasting-mediated changes in lipid metabolism, Relish was specifically in the fat body (using multiple, independent RNAi lines: named UAS-RelRNAi KK and GD). Attenuating Relish in the fat body of female flies (CGGal4>UAS-RelRNAi) leads to starvation sensitivity, as well as accelerated loss of organismal TAG levels and fat body lipid storage in response to fasting. As expected, fasting-induced changes in fat body lipid storage occur before significant decreases in total TAG levels of whole animals are observed. Phenotypes were confirmed with an independent fat body driver (PplGal4; Figures S2A-S2C), and similar results were found utilizing males but not when utilizing another immune cell (hemocyte) driver (HmlGal4). Conversely, over-expressing full-length Relish (CGGal4>UAS-Rel) or a constitutively active N-terminal fragment (CGGal4>UAS-Rel.68) in the fat body significantly limits fasting-mediated decreases in lipids compared to controls (Molaei, 2019).

Furthermore, attenuation of upstream components of the Relish signaling pathway phenocopies these Relish loss-of-function effects on lipid metabolism during metabolic adaption. Relish is governed by conserved regulators TAK1 and the IKK (IκB Kinase) signalosome (which consists of homologs of both IKKβ [Drosophila Ird5] and IKKgamma [Drosophila Kenny (key)]), while the apical caspase DREDD is required for the proteolytic cleavage of the IkB domain, allowing for nuclear translocation. Inhibiting Kenny or DREDD in the fat body of female flies (CGGal4>UAS-DREDDRNAior KeyRNAi) leads to starvation sensitivity, as well as accelerated loss of organismal TAG levels and fat body lipid storage in response to fasting (compared to control flies (CGGal4>w1118). Similarly, attenuating upstream receptors usually required for NF-κB/Relish activation (PGRP family members PGRP-LC [trans-membrane] or PGRP-LE ) also leads to decreased lipid storage in the fat body after starvation, suggesting that at least part of the canonical innate immune pathway is required for these metabolic phenotypes (Molaei, 2019).

Taken together, these data show that Relish can autonomously regulate lipid metabolism in the fat body during metabolic adaptation and suggest that Relish may direct specific metabolic responses to control the breakdown of TAGs (Molaei, 2019).

Properly balancing energy homeostasis in response to metabolic adaptation depends on the ability to coordinate storage, breakdown, and mobilization of lipids, primarily TAG. This coordination requires precise control of metabolic response networks, including changes in metabolic gene expression. To determine potential mechanisms by which the Relish transcription factor could direct cellular TAG metabolism during fasting, transcriptional changes of various metabolic genes related to lipid catabolism or anabolism were measured in Relish-deficient animals. Specifically, the lipase Brummer (Bmm) was identified as being regulated by Relish. Bmm is the Drosophila homolog of mammalian adipose TAG lipase (ATGL), an enzyme that is critical for lipolysis. Bmm plays an essential and conserved role in TAG breakdown and, subsequently, fatty acid mobilization, from lipid droplets in fat storage tissues during metabolic adaptation. In control flies, bmm transcription is mildly induced during acute fasting, but in rel^E20/rel^E20 mutant flies, bmm expression is strongly up-regulated (from whole flies). These Relish-dependent changes in bmm transcription appear unique, as Relish deficiency does not impact fasting-induced changes in other lipases such as Drosophila hormone-sensitive lipase (dHSL), Drosophila lipase 4 (dlip4), or CG5966. Similar results were found in dissected fat body with specific attenuation of NF-κB/Relish in the same tissue (CGGal4>UAS-RelRNAi KK). These results suggest that Relish function is required to repress or limit Bmm expression in response to metabolic adaptation and subsequently restrain TAG breakdown (Molaei, 2019).

To correlate this difference in gene expression to differences in lipolysis, an assay was employed to measure dynamic changes in lipid content based on the incorporation of radiolabeled glucose (14C-glucose) into lipids during fatty acid synthesis in vivo. After acute feeding (1.5 h) of a diet containing 14C-glucose, Relish-mutant flies show drastic changes in glucose incorporation (synthesis) that is likely due to changes in feeding behavior. 16 h of feeding minimized these differences in synthesis, and subsequent analysis of newly synthesized 14C-labeled lipids during fasting showed an increased rate of breakdown in rel^E20/rel^E20mutant flies (47% in mutants compared to 20% in controls). This change in the rate of breakdown correlated with increases in free fatty acids. Finally, genetically attenuating Bmm lipase in the fat body (CGGal4>UAS-BmmRNAi) can rescue the accelerated loss of TAGs in rel^E20/rel^E20 mutant flies during fasting (Molaei, 2019).

These data collectively reveal that Relish function is required to limit fasting-induced Bmm gene expression and subsequently restrain TAG lipolysis during metabolic adaptation (Molaei, 2019).

Following these results, the mechanism by which the Relish transcription factor can context-dependently attenuate Bmm expression was explored. Utilizing Cis-element OVERrepresentation (Clover) software, this study identified conserved NF-κB DNA binding motifs (κB sequence sites identified as GGG R N YYYYY) throughout the first intron of the Bmm locus. To assess binding, a previously characterized Relish antibody was used to perform chromatin immunoprecipitation (ChIP)-qPCR experiments. Relish binding in fed or fasted wild-type flies is significantly enriched (compared to immunoprecipitations using serum controls) at binding motif(s) approximately 1 kb downstream from the transcriptional start site. This putative Bmm regulatory region was cloned upstream of RFP (red fluorescent protein) in order to generate in vivo expression reporters. While the unaltered region only slightly influenced RFP reporter activity in fed or fasted conditions, eliminating the Relish binding site leads to minimal enhanced reporter activity under fed conditions and strong increases in RFP activity during fasting (primarily in the fat body of the carcass and head). Thus, this Relish-binding site within the first Bmm intron acts as an important regulatory region to limit induced gene expression (Molaei, 2019).

Relish binding at this region is similar in fed and fasted states. No evidence was found of classical Relish transcriptional activation function during acute fasting. First, innate immune target gene expression (Drosomycin and Diptericin) and Relish DNA binding to innate immune gene promoters (Diptericin) were not changed during fasting. Second, metabolic adaptation did not significantly alter nuclear localization of Relish in the fat body. Thus, in order to explore how Relish limits or represses fasting-induced Bmm expression, despite its constitutive binding to DNA and distinct from its transcriptional activation function, histone and/or chromatin changes were assessed in Relish-deficient flies. Histone deacetylases (HDACs) have been shown to accumulate in the nucleus during metabolic adaptation, influencing gene expression in a fasting-dependent manner through chromatin regulation and transcription factor deacetylation. Furthermore, previous studies have linked interactions of NF-κB transcription factors and HDACs with NF-κB-dependent transcriptional repression. It was thus hypothesized that Relish might repress Bmm gene expression through influencing histone modifications during fasting, when histone modifiers (such as HDACs) in the nucleus are elevated. Using ChIP-qPCR, histone 3 lysine 9 acetylation (H3K9ac, a post-translational modification generally associated with transcriptional activation) was monitored at this Bmm regulatory region in Relish-deficient animals and controls. During feeding, there is no change in H3K9ac enrichment at this locus between genotypes. However, during fasting rel^E20/rel^E20 mutant flies display a significant enrichment (compared to controls) of H3K9ac at the site of Relish binding, indicative of promoter or enhancer activation. Analysis of modEncode ChIP sequencing databases associated with histone modifications (in adult female flies) also revealed that this site is generally enriched for other modifications linked to gene expression regulation (such as H3K27ac, H3K4me3, and H3K4me1), further indicating that this locus is an important regulatory region. Additionally, inhibiting a single HDAC in the fat body (Rpd3 [Drosophila HDAC1]; CGGal4>UAS-Rpd3RNAi) can drive small, but significant, increases in fasting-induced Bmm transcription (from whole flies; Figure 3D) and accelerate fat body lipid usage (Molaei, 2019).

Taken together, these data show that Relish can bind to a putative regulatory region within the Bmm locus during both feeding and fasting. In response to fasting, the presence of Relish can influence fasting-dependent histone acetylation and chromatin changes that are consistent with transcriptional repression (Molaei, 2019).

The unique ability of Relish to limit or repress fasting-induced Bmm transcription correlates with attenuation of H3K9ac at Bmm regulatory regions. It was thus hypothesized that Relish binding to the Bmm locus leads to fasting-dependent chromatin changes, which subsequently limit transcription activation function of other factors that are induced during metabolic adaptation. Various metabolic transcription factors were assessed and Foxo, a critical regulator of lipolysis and catabolism in general, was shown to be required for Relish-dependent changes in ATGL/Bmm expression during metabolic adaptation. Firstly, Foxo (of which there is a single ortholog in Drosophila) is activated during metabolic adaptation and required for fasting-induced ATGL/Bmm expression across taxa, including in the fly fat body. Full Relish/Foxo double mutant animals (using the foxo²⁴ allele) are synthetic lethal during metamorphosis (rel^E20, foxo24/rel^E20). However, simply reducing Foxo gene dose in NF-κB/Relish mutant flies (rel^E20, foxo²⁴/rel^E20) completely rescues fasting-dependent increases in Bmm expression, starvation survival rates, and increases in lipolysis (accelerated loss of lipid storage) in Relish-deficient flies during metabolic adaptation. Molecular analysis of Foxo transcription activation function also showed that Foxo binding to the Bmm promoter is slightly, but significantly, elevated in Relish-deficient flies only during fasting. Furthermore, attenuating Foxo specifically in the fat body (CGGal4>UAS-FoxoRNAi) rescues the enhanced depletion of lipid storage and starvation sensitivity associated with rel^E20/rel^E20 mutant flies during fasting. Foxo transcription activation function is thus required for Relish-dependent changes in lipid metabolism, highlighting that Relish/Foxo integration and antagonism is critical to maintain TAG metabolism throughout the course of metabolic adaptation (Molaei, 2019).

In summary, this study has uncovered a role for the innate immune transcription factor Relish in governing lipid metabolism during metabolic adaptation to fasting utilizing Drosophila. Relish is required to restrain fasting-induced lipolysis and thus conserve cellular TAG levels and promote survival during metabolic adaptation, through specific repression of Bmm lipase gene induction in the fat body/adipose. Fasting-induced changes in Bmm expression and TAG metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, likely through regulation of histone acetylation (Molaei, 2019).

These findings thus suggest that association of Relish with histone modifiers (and subsequent changes in chromatin accessibility at gene regulatory regions) functions to control or limit the induced level of certain metabolic genes. Indeed, a few previous studies have highlighted that mammalian p65/RelA or Drosophila Relish can negatively regulate gene expression, including innate immune targets, through changes in chromatin. This modification of chromatin and repression of gene expression likely occurs through recruitment of HDACs (HDAC 1/2) to promoter or enhancer regions of target genes, thus influencing histone acetylation, chromatin remodeling, and function of NF-κB itself or other transcriptional activators. This study shows that Relish can bind a metabolic target gene during both feeding and fasting but that uniquely during fasting, it can influence H3K9 acetylation levels at this target gene and subsequently limit transcriptional activation function of positive regulators (such as Foxo). This allows NF-κB transcription factors precise control of specific metabolic gene expression, potentially through metabolic changes in HDAC nuclear localization, in a context-dependent manner. To this end, this study has uncovered other metabolic target genes of Relish (in unique tissues with unique metabolic functions) regulated through similar mechanisms (Molaei, 2019).

It remains unclear whether Relish binding to metabolic target genes in fed states, independent of metabolic stress, can influence gene expression and physiology. However, this may represent a constitutive or steady-state function of NF-κB (described in mammals) and thus could act as a priming mechanism that promotes the maintenance of metabolic homeostasis during acute stress (such as fasting) (Molaei, 2019).

While these data show that NF-κB can direct lipid metabolism independent of infection, Foxo (as well as HDACs) is integrated with innate immune responses through a variety of mechanisms. Additional experimental evidence suggests that Relish and, perhaps, Relish-Foxo antagonism and Bmm gene regulation manipulate TAG metabolism after a chronic systemic bacterial infection. It is thus possible that NF-κB-dependent regulation of TAG catabolism, by limiting Foxo-mediated lipolysis, also plays a role in governing innate immune homeostasis in response to pathogenic infections (Molaei, 2019).

Specialized hepatocyte-like cells regulate Drosophila lipid metabolism

Lipid metabolism is essential for growth and generates much of the energy needed during periods of starvation. In Drosophila, fasting larvae release large quantities of lipid from the fat body but it is unclear how and where this is processed. This study identified the oenocyte as the principal cell type accumulating lipid droplets during starvation. Tissue-specific manipulations of the Slimfast amino-acid channel, the Lsd2 fat-storage regulator and the Brummer lipase indicate that oenocytes act downstream of the fat body. In turn, oenocytes are required for depleting stored lipid from the fat body during fasting. Hence, lipid-metabolic coupling between the fat body and oenocytes is bidirectional. When food is plentiful, oenocytes have critical roles in regulating growth, development and feeding behaviour. In addition, they specifically express many different lipid-metabolizing proteins, including Cyp4g1, an omega-hydroxylase regulating triacylglycerol composition. These findings provide evidence that some lipid-processing functions of the mammalian liver are performed in insects by oenocytes (Gutierrez, 2007).

In mammals, specialized cells of the adipose tissue and liver are critical for coordinating fat metabolism. This physiological axis regulates a complex set of lipid uptake, storage, synthesis, modification and degradation reactions essential for normal growth and development. Lipid metabolism also has a particularly critical role in providing energy during periods of starvation. Food deprivation (fasting) stimulates the lipolysis of triglycerides (also called triacylglycerol, TAG) stored in adipocyte fat droplets via increases in hormone-sensitive lipase activity and adipose triglyceride lipase (ATGL) expression (Zechner, 2005). A large proportion of the fatty acids and other lipids thereby released into the circulation are then captured, modified and broken down by the major cell type of the liver, the hepatocyte. An intriguing feature of the starvation response is that, in contrast to many other cell types, hepatocytes accumulate large numbers of fat droplets, resulting in hepatic steatosis. Fatty acids are released from hepatic lipid droplets during starvation and oxidized into shorter chain fatty acids and ultimately into soluble ketone bodies that can be discharged into the circulation for use as an energy source by many tissues. This fatty acid oxidation process involves chain shortening by α- and β-oxidation pathways active in peroxisomes and mitochondria. Although lipid catabolism predominates during starvation, in the postprandial state, hepatocytes are highly active in synthesizing fatty acids for incorporation into triglycerides. These can then be assembled into lipoprotein particles, delivered to adipocytes and stored in lipid droplets. One critical step for incorporating newly synthesized fatty acids into TAG is catalysed by stearoyl CoA-desaturase-1 (SCD-1), a hepatic enzyme converting palmitic (C_16:0) and stearic (C_18:0) acids into monounsaturated palmitoleic (C_16:1) and oleic (C_18:1) acids, respectively. The importance of maintaining an appropriate balance between hepatic fatty acid synthesis and oxidation is highlighted by human diseases arising from mutations in fatty acid oxidation enzymes, and also by widespread diet-influenced pathologies such as non-alcoholic fatty liver disease and metabolic syndrome (Gutierrez, 2007).

Invertebrate model organisms offer a powerful means to identify and functionally analyse lipid-metabolising genes. In Caenorhabditis elegans, fat is stored by intestinal epithelial cells and many regulators of this process have been identified using reverse genetic screens. In contrast, Drosophila and other insects store lipid in a specialized tissue that resembles the adipose tissue of mammals, the fat body. Diet-derived lipids, exported from the midgut as lipoproteins, are taken up from the haemolymph by the fat body via a mechanism involving Low-Density Lipoprotein (LDL) receptor-like molecules called Lipophorin receptors. These lipids accumulate in fat body cells in the form of intracellular droplets but, when larvae are food-deprived, there is a net efflux of lipid into the haemolymph. The mobilization process is regulated by TSC/TOR signalling and a nutrient sensor in the fat body that monitors amino-acid levels via the Slimfast (Slif) amino-acid channel. Fasting-induced fat release is accompanied by increased lipolysis, at least in part associated with upregulation of Brummer, an ATGL-related lipase localized to lipid droplets. Fat mobilization is also influenced by Lsd2, a lipid droplet protein related to a mammalian negative regulator of TAG hydrolysis called perilipin. In addition to its involvement in lipid storage and release, the fat body produces a humoral signal regulating larval tissue growth in response to food availability. Thus far, efforts to harness the power of Drosophila genetics to model human fat metabolism have been limited by the lack of information on how and where insect lipids are processed once they have been released from the fat body. For example, it is not known whether there is a specialized Drosophila tissue that synthesizes, modifies and oxidizes fatty acids in a similar way to the mammalian liver, nor is it clear to what degree the mammalian biochemical pathways metabolizing fatty acids are conserved in Drosophila. This study addresses both of these issues using a combination of bioinformatics, genetics and integrative physiology (Gutierrez, 2007).

How fat is redistributed throughout the larval body after food deprivation was studied. Using Oil Red O staining, three cell types in the third instar (L3) larva were found to contain numerous large (0.5-2.5 microm) lipid droplets under fed and/or fasting conditions: fat body cells, midgut epithelial cells and larval oenocytes. The fat body of L3 larvae has such a large capacity for lipid storage that, despite lipid loss over a 14-h period of fasting, intense Oil Red O staining persists. Lipid release from the L3 fat body during starvation correlates with lipid droplet aggregation. However, droplet aggregation is not a reliable indicator of fasting at some other larval ages and durations of fasting. In contrast to the fat body, regions of the gut (including the proventriculus and anterior midgut) staining strongly with Oil Red O under fed conditions have only limited storage capacity, losing most lipid droplets after 14 h of fasting. The third cell type, larval oenocytes (distinct from adult oenocytes but abbreviated hereafter as oenocytes), are large cells of unknown function that are attached to the basal surface of the lateral epidermis in clusters of ~6 cells per abdominal hemi-segment. L3 oenocytes do not stain strongly with Oil Red O under fed conditions but they do contain numerous large lipid droplets after a 14-h fast. This change in droplet abundance is consistent from oenocyte-to-oenocyte within one cluster and also from one cluster to another. Thus, oenocytes are highly atypical cells, in that they accumulate large numbers of lipid droplets specifically during fasting. As this is a hallmark of hepatocytes, the possibility is raised that insect oenocytes might process lipids in a similar way to the mammalian liver (Gutierrez, 2007).

Next, whether the accumulation of lipid droplets in oenocytes is regulated by the fat body nutrient sensor was tested. An antisense transgene directed against the amino-acid transporter Slimfast was expressed in the fat body (ppl-GAL4 driving UAS-slif^Anti; hereafter called ppl>slif^Anti). As reported previously (Colombani, 2003), it was observed that ppl>slif^Anti larvae raised to L3 on a standard diet resemble starved wild-type larvae in that lipid droplets aggregate in the fat body. Notably, it was also found that oenocytes contain numerous lipid droplets, regardless of whether ppl>slif^Anti larvae are fed on a standard diet or food-deprived for 14 h. This indicates that amino-acid monitoring via Slif in the fat body is required to ensure that lipid accumulation in oenocytes is kept low under standard nutritional conditions. TSC/TOR signalling, another component of the fat body nutrient sensor, is also involved; overexpressing TSC1 and TSC2 (ppl>Tsc1+2) leads to a marked accumulation of large lipid droplets in the oenocytes of 100% (n = 11) of fed larvae. Similarly, inhibiting the phosphatidylinositol-3 kinase pathway, which intersects with TOR signalling, by overexpressing the lipid phosphatase PTEN, also produces a build up of lipid droplets in the oenocytes of 89% of fed ppl>PTEN larvae. Hence, the fat body nutrient sensor regulates lipid accumulation in oenocytes but this could be directly via lipid release or indirectly, in response to a TSC/TOR-dependent signal (Gutierrez, 2007).

To assess directly the effect of lipid mobilization from the fat body, the balance between TAG storage and hydrolysis was altered in two ways. First, Brummer (Bmm) lipase, which is normally limiting for lipid release from the fat body, was overexpressed. This is sufficient to produce specific accumulation of lipid droplets in the oenocytes of 92% of fed ppl>bmm larvae. Second, TAG release from lipid droplets was decreased by overexpressing Lsd2. This reduces the accumulation of oenocyte lipid droplets in 78% of starved ppl>Lsd2 larvae, with ~4-fold fewer large droplets per oenocyte. A second driver, Lsp2-GAL4, was used that unlike ppl-GAL4 is activated in the fat body only at the mid-L3 stage. This temporally restricted driver nevertheless suffices to induce oenocyte lipid droplet accumulation in 100% of fed Lsp2>bmm larvae and also in fed Lsp2>slif^Anti animals. Together, the Slif, TSC, PTEN, Lsd2 and Bmm results demonstrate metabolic regulation from the fat body to the oenocytes, although they do not exclude the involvement of intermediate tissues such as the gut. Either way, these results strongly suggest that, when nutrition is poor, falling amino-acid levels stimulate lipid release from the fat body and subsequent lipid uptake from the haemolymph by oenocytes (Gutierrez, 2007).

To determine the in vivo roles of oenocytes during fasting and normal development, a targeted binary cell ablation system was developed. Larvae carrying sal[BO,7.6kb]GAL4, a purpose-built oenocyte driver, and also UAS-reaper, an inducible pro-apoptotic transgene, lack 100% of oenocytes from L1 onwards and die before reaching pupariation (hereafter called BO>rpr larvae). As a specificity control, BO>rpr animals were rescued to viable adults by expressing Gal80, an inhibitor of Gal4, under the regulation of an independent oenocyte enhancer from seven up (svp). As svp[3kb]GAL80 suppresses sal[BO,7.6kb]GAL4 activity in oenocytes but not in secondary larval sites, BO>rpr lethality results from the ablation of oenocytes and not some other cell type (Gutierrez, 2007).

BO>rpr larvae raised on a standard diet attain a similar mass to UAS-rpr controls during L1 but, after the L1-to-L2 transition, they grow at a much slower rate. Notably, reduced growth correlates with aberrant feeding behaviour, with most BO>rpr larvae dispersing away from the yeast food source during L2. This dispersal is distinct from premature wandering behaviour; BO>rpr larvae enter and exit the yeast source multiple times, retain food in the gut and do not pupariate precociously. Since BO>rpr larvae spend less time in the food source and grow more slowly than L2 controls, whether they show increased mouth-hook contractions, a behavioural response to hunger, was investigated. However, reduced mouth-hook contractions were observed that are not significantly increased by the motivation of a 2-h period of food deprivation. Thus, rather than stimulating hunger-driven feeding behaviour, oenocyte ablation seems to block it, although this effect could be very indirect. Either way, reduced feeding is likely to contribute to the slow growth rate of BO>rpr larvae during L2 (Gutierrez, 2007).

Since reduced growth resulting from inadequate nutrition before 70 h after egg laying (just before the L2/L3 moult) is associated with larval arrest rather than smaller-than-normal adult flies, morphological criteria were used to stage oenocyte-ablated animals. It was observed that BO>rpr larvae arrest at several different stages after the L1/L2 transition, thus displaying a polyphasic lethality profile. Although arrested development can result from reduced signalling by ecdysteroids, the BO>rpr polyphasic lethality profile is not significantly altered by adding 20-hydroxyecdysone or its precursor ecdysone. Therefore, a deficiency in these ecdysteroids is not the sole reason for BO>rpr arrest, but the possibility cannot be excluded that it, together with some other oenocyte deficit, contributes to the moulting phenotype (Gutierrez, 2007).

Unlike many larval tissues, oenocytes persist for much of pupal development. To address whether oenocytes are required for metamorphosis, a temperature-sensitive version of Gal80 (GAL80^ts ) was used to attenuate Gal4 activity, thus bypassing BO>rpr larval lethality. Combining tub-GAL80^ts with BO>rpr suppresses apoptosis in approximately 50% of oenocytes at 25 °C (from L1 onwards) and allows developmental progression until pupal stages. However, no animals complete pupal development, with many failing to separate from the puparial case during eclosion. Together, the 50% and 100% oenocyte ablation phenotypes demonstrate that oenocytes are required for growth and developmental progression during both larval and pupal stages (Gutierrez, 2007).

Whether lipid metabolism is altered in larvae lacking all oenocytes was examined. At early L2, when BO>rpr larvae are the same size as controls, no significant abnormalities in TAG content or in the relative amounts of the major long-chain fatty acids were detected. The fat storage capacity of larvae at early L2 is much less than at L3 such that a 12-h period of food withdrawal is sufficient to deplete ~60% of stored TAG in control animals. However, during this same fasting period, BO>rpr larvae only lose ~10% of total TAG. This deficit in TAG depletion correlates with a higher density of fat-body lipid droplets in 100% of starved BO>rpr larvae compared to controls after fasting. Since ~80% of larval fatty acids are stored as TAG, the proportions of individual fatty acids were examined in fasting BO>rpr larvae. In early L2 controls, lauric (C_12:0) and myristic (C_14:0) acids are depleted more efficiently than longer-chain (C₁₆-C₂₀) fatty acids such that their mass, relative to stearic acid (C_18:0), is reduced twofold after 12 h fasting. However, in fasted BO>rpr larvae, the C_12:0/C_18:0 and C_14:0/C_18:0 ratios remain close to those before starvation, corresponding to approximately twice the value of starved controls. Together, these results indicate that oenocytes are required for efficiently depleting fatty acids, stored largely in the fat body as TAG, during nutrient deprivation. With the previous Slif, TSC, PTEN, Bmm and Lsd2 results, it is proposed that lipid-metabolic coupling between the fat body and oenocytes is bidirectional (Gutierrez, 2007).

To identify the metabolic pathways processing lipids within oenocytes, 51 genes expressed selectively or exclusively in oenocytes were identified. About 40% of these encode orthologues of known human lipid-metabolizing/processing proteins. The high degree of conservation of most Drosophila proteins, together with some previous functional studies, suggests that oenocytes express lipid metabolic pathways strikingly similar to those of hepatocytes. By analogy, oenocytes would capture lipid from lipophorin in the haemolymph via LpR1 and LpR2, two Lipophorin receptors. Fatty acids released from lipid droplets by lipases such as the CG17292 product, could then be modified by a variety of enzymes, including the Desat1 and CG9743 acyl-CoA desaturases, the CG18609 and CG6921 fatty acid elongases and the microsomal lipid omega-hydroxylase, Cytochrome P450-4g1 (Cyp4g1). Fatty acids could also be chain shortened, at least partially, by the actions of peroxisomal β-oxidation components including those encoded by CG11151 (similar to Sterol carrier protein 2), CG12428 (Carnitine O-octanoyl transferase), CG9527 (Pristanoyl-CoA oxidase) and Catalase, the peroxisomal enzyme inactivating oxygen free radicals produced by pristanoyl-CoA oxidases. In addition, oenocytes strongly express Hnf4 and Svp, orthologues of the mammalian nuclear receptors Hnf4-α and COUP-TF, known regulators of hepatocyte differentiation and lipid-metabolic genes. Thus, the oenocyte/hepatocyte analogy includes a shared set of lipid-metabolizing genes and at least two of their transcriptional regulators (Gutierrez, 2007).

To explore the functions of fatty acid metabolism specifically within Drosophila oenocytes, two lethal protein-null alleles were generated for the predicted lipid omega-hydroxylase encoded by Cyp4g1. Cyp4g1 is known to be expressed in oenocytes, and it was found that this is the only site of detectable expression in embryos and larvae. Animals homozygous for either the Cyp4g1^Delta4 or Cyp4g1^Delta4-9 allele develop normally through larval and early pupal stages but arrest during mid-to-late pupal stages, with many failing during adult eclosion. This pupal phenotype is strikingly similar to the 50% oenocyte ablation phenotype. Moreover, although late-L3 Cyp4g1 mutant larvae appear morphologically indistinguishable from controls, they manifest a twofold increase in the oleic acid:stearic acid ratio (C_18:1/C_18:0). Notably, this imbalance in fatty acid desaturation is found in the TAG fraction but not in the phospholipid fraction. This selectivity strongly suggests that the Cyp4g1 defect is specific to fatty acids in metabolic storage form, most of which reside in the fat body, rather than fatty acids present in the structural lipids of all cell membranes. Taken together, the metabolic profiles of oenocyte-ablated and Cyp4g1 mutant larvae provide two independent lines of evidence that oenocytes regulate the lipid composition of the fat body (Gutierrez, 2007).

Functions of larval oenocytes, described in insects over 140 yr ago, have remained unclear, with largely descriptive studies implicating them in processes such as cuticle synthesis and the regulation of haemolymph composition. Using cell ablation to test their functions directly for the first time, clear requirements for larval growth and pupal development were found. Although the subset of oenocyte genes mediating the larval developmental functions remains to be identified, for pupal development it was shown that the lipid omega-hydroxylase Cyp4g1 is required. At least one important role of Cyp4g1 is to downregulate the ratio of oleic-to-stearic acid, widely used as a marker of SCD-1 activity in mammals. This prompts speculation that Cyp4g1 may repress the activity of stearoyl CoA-desaturases like Desat1, thereby inhibiting inappropriate monounsaturated fatty acid synthesis during long non-feeding periods such as late L3 and pupal stages (Gutierrez, 2007).

Four lines of evidence argue that at least some of the lipid-metabolizing roles of insect oenocytes are analogous to those of mammalian hepatocytes: (1) oenocytes express 22 orthologues of human fat-metabolizing genes expressed in hepatocytes; (2) like hepatocytes, they are atypical cells in that they accumulate fat droplets during starvation; (3) like the liver, oenocytes lie downstream of a nutrient sensor in a major fat depot; (4) Brummer lipase and Lsd2 in the fat body regulate oenocyte lipid content in a broadly similar way as ATGL and perilipin in adipose tissue regulate hepatic fat influx. However, whereas hepatocytes store large quantities of glycogen, this role in Drosophila is primarily carried out by the fat body. Thus, mammalian liver functions in glycogen storage and lipid processing seem to be divided in Drosophila between the fat body and oenocytes (Gutierrez, 2007).

This study suggests the existence of two-way metabolic coupling between the fat body and oenocytes. Analogous to the mammalian adipose-liver axis, lipid mobilization from the fat body during starvation produces lipid droplet accumulation in oenocytes, a metabolic change resembling hepatic steatosis. In Drosophila, a reciprocal regulation was also found, namely that oenocytes are required for efficiently depleting lipid from the fat body during fasting. This suggests a feedback mechanism for matching lipid supply to demand, whereby oenocytes keep haemolymph lipids low and also promote lipid mobilization from the fat body. Thus, in oenocyte-less larvae, excess circulating lipids might underlie the behavioural syndrome of larval dispersal and reduced feeding in a similar way as reported for elevated amino-acid levels (Zinke, 1999). Central to the proposed feedback model is the signal acting on the lipogenesis/lipolysis balance within the fat body. The data presented in this study are equally compatible with this signal corresponding to a haemolymph lipid/metabolite or to a separate signal generated by oenocytes. Regarding the latter possibility, it is interesting that recent work in mammals indicates that the liver secretes signalling factors (hepatokines) that promote lipolysis in adipose tissue (Oike, 2005). This suggests that Drosophila may prove useful, not only for modelling hepatic steatosis, but also some regulatory roles of the liver in metabolic homeostasis (Gutierrez, 2007).

COPI complex is a regulator of lipid homeostasis

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. This study identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system. These regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets (Beller, 2008).

Lipid homeostasis is critical in health and disease, but remains poorly understood. Non-esterified free fatty acid (NEFA) is used for energy generation in beta-oxidation, membrane phospholipid synthesis, signaling, and in regulation of transcription factors such as the peroxisome proliferator-activated receptors (PPARs). Essentially all cells take up excess NEFA and convert it to energy-rich neutral lipids in the form of triglycerides (TG). TG is packaged into specialized organelles called lipid droplets. NEFA is regenerated from lipid droplet stores to meet metabolic and energy needs, and lipid droplets protect cells against lipotoxicity by sequestering excess NEFA. Lipid droplets are the main energy storage organelles and are thus central to the understanding of energy homeostasis. Despite their importance, little is known about the ontogeny and regulation of these organelles (Beller, 2008).

Lipid droplets are believed to form in the ER membrane by incorporating a growing TG core between the leaflets of the bilayer, and ultimately are released surrounded by a phospholipid monolayer. Cytosolic lipid droplets possess a protein coat and grow by synthesis of TG at the lipid droplet surface and by fusion with other lipid droplets. Formation of nascent droplets and aggregation of existing droplets is likely to require a dynamic exchange of lipids and proteins from and to the droplet. Indeed, the range of proteins identified in lipid droplet proteomic studies suggests extensive trafficking between lipid droplets and other cellular compartments, including the endoplasmic reticulum (ER). Additionally, lipid droplet-associated proteins translocate between the cytosol and lipid droplets. For example, tail interacting protein of 47 kDa (TIP47) associates with small, putative nascent, lipid droplets, but is not found on larger droplets, which are coated by other members of the perilipin, adipocyte differentiation related protein (ADRP), TIP47 (PAT) protein family. Intriguingly, TIP47 mediates mannose 6-phosphate receptor trafficking between the lysosome and Golgi, raising the possibility that trafficking is involved in lipid droplet ontogeny or fate. However, unlike the well-studied Golgi trafficking system, the routes to and from the lipid droplet are unknown (Beller, 2008).

Once lipid droplets are formed, stored TG is mobilized in a regulated manner. Triglyceride, diglyceride (DG), and monoglyceride lipases convert TG back into NEFA. Most of the knowledge concerning lipolysis is based on extensively studied adipocytes in which at least two lipolytic enzymes have been identified: adipocyte triglyceride lipase (ATGL; Drosophila brummer) and hormone sensitive lipase (HSL). Due to the hydrophobic properties of the lipid droplet TG core, lipases are likely to act at the surface of lipid droplets, where members of the PAT protein family regulate lipase access to the TG core. Mammalian genomes encode at least five PAT-proteins. Whereas perilipin is the dominant PAT protein in adipocytes, ADRP is the dominant PAT protein in nonadipose tissues in which it is tightly associated with the lipid droplet surface. PAT members appear to have a hierarchical affinity for the lipid droplet surface. In nonmammalian genomes, there are fewer PAT proteins. For example, two PAT proteins termed lipid storage droplet 1 and 2 (LSD-1 and LSD-2) are found in Drosophila (Miura, 2002). The crucial role of PAT proteins is evolutionary conserved as the absence of perilipin in mice, or LSD-2 in flies results in lean animals. Overexpression of LSD-2 results in obese flies. These data indicate the conserved PAT proteins at the lipid droplet surface are important regulators of energy storage (Beller, 2008 and references therein).

It seems likely that PAT proteins protect lipid from lipolysis, but the role of PAT proteins may not be limited to passive steric hindrance of lipase access to the TG core, as illustrated by perilipin. Unphosphorylated perilipin protects the lipid droplet from lipase activity. Following stimulation by protein kinase A (PKA), however, phospho-perilipin acts as a docking site for HSL, which translocates from the cytosol to the droplet surface. Whereas phospho-perilipin promotes massive NEFA release from the droplet, this is not mediated exclusively by HSL, as mice lacking HSL function show a relatively mild phenotype marked by the accumulation of DG, thus demonstrating that HSL acts as a DG lipase in vivo. The TG lipase functioning in HSL null mice is ATGL. In the current view of adipocyte lipolysis, ATGL is responsible for the first step in TG hydrolysis, liberating DG and NEFA, whereas HSL acts as a DG lipase. Very little is known about how ATGL is targeted to the lipid droplet (Beller, 2008).

In contrast to the lean phenotype in animals lacking perilipin (mouse) or LSD-2 (fly), both mice and flies lacking ATGL are obese. In mice, the absence of ATGL results in excessive TG accumulation in liver and muscle. Similarly, human patients suffering from neutral lipid storage disease carry mutations resulting in truncated ATGL isoforms. ATGL function is evolutionary conserved, as flies lacking the Drosophila ATGL ortholog, Brummer, accumulate copious amounts of body fat. The lipid droplet-associated protein Comparative Gene Identification-58 (CGI-58) acts as an ATGL colipase (Lass, 2006). Mutations in the CGI-58 gene result in ectopic fat accumulation in patients suffering from Chanarin Dorfman Syndrome (CDS), supporting the idea that both ATGL and CGI-58 are required for mobilizing lipid stores in nonadipose tissue. Interestingly, CGI-58 physically interacts with perilipin as demonstrated by both coimmunoprecipitation and fluorescence resonance energy transfer (FRET) studies. In addition, there are other lipases and probably many more cofactors encoded in the genome. Understanding which ones act at the lipid droplet surface and how their localization is regulated will be important (Beller, 2008).

Drosophila is a powerful model for pathway discovery due to well-developed genetics. Additionally, greater than 60% of the genes associated with human disease have clear orthologs in Drosophila. Drosophila is highly relevant to lipid droplet study, as lipid droplets in Drosophila and mammals are associated with many of the same proteins. Finally, the emerging model of lipid storage and endocrine regulation are similar in humans and Drosophila, suggesting that Drosophila will be a good genetic model for lipid storage and lipid storage diseases in humans. This study therefore utilized genome-wide RNA interference (RNAi) screening in Drosophila tissue culture cells to identify and characterize novel regulators of lipid storage. The function of these regulators was tested in mouse lipid droplet regulation by directed RNAi studies. 318 Drosophila genes were identified that were required to limit lipid storage and 208 Drosophila genes were identified that were required to promote lipid storage. These genes encode known regulators of lipid storage as well as genes not previously associated with lipid storage regulation (Beller, 2008).

Positive regulation of lipolysis by the COPI retrograde-vesicle trafficking pathway was the most striking and unexpected result of the screen. Interference with COPI function, either by RNAi or compounds, in Drosophila Kc₁₆₇ or S3 cells, or in mouse 3T3-L1 or AML12 cells, results in increased lipid storage. Furthermore, recent and parallel studies in yeast and Drosophila S2 cells (Guo, 2008) also suggested a role of COPI function in lipid droplet regulation. Interestingly, only the epsilon-subunit of the COPI complex failed to result in a lipid droplet deposition phenotype on knockdown. Although limited RNAi efficacy or increased protein stability cannot be ruled out, epsilonCOP was the only canonical COP subunit not resulting in a lipid storage phenotype in a parallel study using different cells and reagents (Guo, 2008), and targeting of epsilonCOP transcripts by RNAi in AML12 cells had a weak effect on lipid storage at best. Finally, epsilonCOP is the only dispensable subunit in a recent study identifying COPI activity coupled with fatty acid biosynthesis as a host factor important for Drosophila C virus replication (Cherry, 2006). This is especially interesting, since certain enveloped viruses, including Hepatitis C virus, assemble on lipid droplets. Taken together, these results indicate that six out of the seven wild-type COPI subunits mediate lipid storage by positively regulating lipolysis (Beller, 2008).

COPI could have a direct or indirect effect on lipid storage. The indirect mechanism is poorly defined, but if the Golgi is a 'sink' for phospholipids derived from TG stores, then decreased Golgi function could simply decrease demand for TG substrate. If non-esterified free fatty acid (from the media in fed cells, and from biosynthesis in unfed cells) conversion to TG continues, then increased lipid droplet volume would occur. It is also possible that canonical COPI function transporting lipids and proteins from the Golgi to the ER is ultimately responsible for lipid droplet utilization and protein composition at the lipid droplet surface. For example, COPI might be required for the particular phospholipid composition in hemimembranes formed on nascent droplets, which secondarily alter TIP47 and ATGL localization in mature lipid droplets (Beller, 2008).

However, evidence that Golgi function per se is not linked to lipid storage phenotypes, as well as direct association of COPI members and regulators with the lipid droplet or PAT proteins supports a more direct model. The COPI and COPII pathways have established roles as constitutive vesicle transport systems that cycle proteins as well as lipid from the Golgi to the ER (COPI), or vice versa (COPII). Interference with either of the COP trafficking systems results in disturbed ER and Golgi function. The lipid overstorage phenotype was seen only in the case of interference with COPI trafficking. This indicates that the lipid overstorage phenotype is not a simple consequence of ER and Golgi function. Finally, in an indirect model in which COPI shuttles only between the Golgi and the ER, COPI should not be lipid droplet associated. However, COPI subunits are directly associated with the lipid droplet surface as shown by proteomics. Additionally, Arf1 binds to ADRP, which is exclusively associated with the lipid droplet surface. Arf79F, the Drosophila homolog of mammalian Arf1, also localizes to lipid droplets in Drosophila S2 cells (Beller, 2008 and references therein).

It is proposed that COPI is likely to function directly at the lipid droplet surface and not indirectly through the Golgi. Perhaps COPI is a destination-specific transporter returning lipid droplet surface hemimembrane and Golgi membrane to the ER. The transport system that brings nascent lipid droplets from the ER to the lipid droplet has not been elucidated, but it is intriguing that the transport/PAT protein TIP47 is found preferentially on small lipid droplets. Small lipid droplets derived from the ER are thought to help build larger droplets by fusion. TIP47-coated droplets might form in the ER, and then COPI could return TIP47 to the ER after the lipid cargo is deposited. In this model, TIP47 becomes trapped at the lipid droplet surface in the absence of COPI (Beller, 2008).

Although increased TIP47 was observed on ADRP-positive droplets by both western blot and cell staining, the cell staining result was more dramatic. The current model might also explain why. The punctate staining of TIP47 in untreated cells could be due to TIP47 on nascent droplets that might also cofractionate with the larger ADRP-positive droplets in the western blots, leading to a less dramatic enrichment for TIP47 relative to ADRP in that experiment. However, other explanations cannot be ruled out, such as nonlinear detection of antigen concentration or epitope masking in the cell staining experiments (Beller, 2008).

COPI perturbation increases stored TG by decreasing the lipolysis rate indicating that the wild-type COPI complex promotes lipolysis. COPI directly or indirectly removes TIP47 from the lipid droplet surface and promotes ATGL localization to the droplet surface, where lipolysis occurs. ATGL has a key role in lipid droplet utilization, and ATGL association with the droplet is reduced by ADRP and Tip47 (Bell, 2008). The epistasis experiments combining siRNA-mediated ATGL knockdown and BFA or Exo1 compound treatment demonstrated that the decrease in lipolysis rate is due to loss of ATGL activity. COPI activity specifically alters lipid droplet surface composition by increasing the amount of TIP47 and reducing the amount of ATGL at ADRP-coated lipid droplets. It is suggested that COPI negatively regulates localization of TIP47. TIP47 in turn prevents ATGL localization. The rescue of the double-knockdown phenotype of TIP47 and ADRP by BFA or Exo1 suggests that COPI has an independent feed-forward effect on ATGL levels at the lipid droplet surface (Beller, 2008).

Although this study focused attention here on COPI, systematic and genome-wide exploration of gene functions required for lipid storage in Drosophila significantly increases experimental access to the complex molecular processes regulating lipid storage and utilization. Further, the use of multiple screens using different cell types and different organisms greatly increases confidence in the genes in the intersection. Given widespread concerns about RNAi screening efficacy and off-target effects, as well as the time and effort required for downstream analysis, systematic use of multiple species and libraries to address a single biological question might be cost effective in addition to resulting in more durable datasets. Primary screens in Drosophila cells followed by secondary screens in mouse cells are much less expensive than a similar genome-wide screen in mammalian cells. Additionally, the availability of mutants in most Drosophila genes, along with demonstrated translation to mammalian systems, provides a valuable entry point for in-depth analyses in both fly and mouse; and eventually for the selection of therapeutic targets for emerging problems associated with obesity and other metabolic disorders (Beller, 2008).

The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila

Triacylglycerol (TAG) homeostasis is an integral part of normal physiology and essential for proper energy metabolism. This study shows that the single Drosophila ortholog of the PXR and CAR nuclear receptors, DHR96, plays an essential role in TAG homeostasis. DHR96 mutants are sensitive to starvation, have reduced levels of TAG in the fat body and midgut, and are resistant to diet-induced obesity, while DHR96 overexpression leads to starvation resistance and increased TAG levels. DHR96 function is required in the midgut for the breakdown of dietary fat, and it exerts this effect through the CG5932 gastric lipase, which is essential for TAG homeostasis. This study provides insights into the regulation of dietary fat metabolism in Drosophila and demonstrates that the regulation of lipid metabolism is an ancestral function of the PXR/CAR/DHR96 nuclear receptor subfamily (Sieber, 2009).

Fat metabolism is central to the process of energy homeostasis. When nutrients are abundant, dietary fat in the form of triacylglycerol (TAG) is broken down by gastric TAG lipases to release fatty acids. These fatty acids are absorbed by the intestine and used to resynthesize TAG in peripheral tissues. These TAG reserves can be accessed upon nutrient deprivation through the action of specific lipid droplet-associated lipases that release the fatty acids for energy production through mitochondrial fatty acid β-oxidation. Defects in these processes can lead to dramatic changes in TAG levels and a range of physiological disorders, including obesity, diabetes, and cardiovascular disease. The alarming rise in the prevalence of these disorders in human populations has focused attention on understanding the molecular mechanisms that coordinate dietary nutrient uptake with TAG homeostasis. As a result, many regulators of TAG metabolism have been identified, including SREBP, PPAR, and adiponectin. In spite of these advances, however, the molecular mechanisms that coordinate dietary fat uptake, synthesis, storage, and utilization remain poorly understood (Sieber, 2009).

Nuclear receptors (NRs) are ligand-regulated transcription factors that play a central role in metabolic control. They are defined by a conserved zinc-finger DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD) that can impart multiple functions, including hormone binding, receptor dimerization, and transactivation. Many NRs are regulated by small lipophilic compounds that include dietary signals and metabolic intermediates, and exert their effects by directing global changes in gene expression that act to maintain metabolic homeostasis. This is exemplified by members of the mammalian PPAR, LXR, and FXR subfamilies, which play critical roles in adipogenesis, lipid metabolism, and cholesterol and bile acid homeostasis, respectively (Sieber, 2009 and references therein).

This study used Drosophila as a model system to characterize the NR subfamily represented by the Pregnane X Receptor (PXR, NR1I2), Constitutive Androstane Receptor (CAR, NR1I3), and Vitamin D Receptor (VDR, NR1I1) in mammals. Previous studies have defined central roles for these receptors in sensing xenobiotic compounds and directly regulating genes involved in detoxification. Initial studies showed that the single ancestral Drosophila ortholog of this NR subclass, DHR96, has similar functions. A DHR96 null mutant displays increased sensitivity to the sedative effects of phenobarbital and the pesticide DDT as well as defects in the expression of phenobarbital-regulated genes. These studies, however, revealed other potential roles for the receptor -- in particular, unexpected effects on the expression of genes that are predicted to regulate lipid and carbohydrate metabolism. This observation is in line with recent studies that have implicated roles for the mammalian PXR and CAR NRs in metabolic control. The molecular mechanism by which they exert this effect, however, remains undefined (Sieber, 2009).

This study shows that DHR96 null mutants are sensitive to starvation and have reduced levels of TAG, while DHR96 overexpression leads to starvation resistance and elevated TAG levels. A series of studies using metabolic assays, diets, and the drug Orlistat revealed that DHR96 mutants are defective in their ability to break down dietary lipid. This model was supported by microarray studies, which showed that many genes expressed in the midgut are misregulated in DHR96 mutants, including highly reduced expression of the gastric lipase gene CG5932. CG5932 is required for proper whole-animal TAG levels, and selective expression of CG5932 in the midgut of DHR96 mutants is sufficient to rescue their lean phenotype. Taken together, these data support a role for the PXR/CAR/DHR96 NR subclass in lipid metabolism and define DHR96 as a key regulator of dietary TAG breakdown in the Drosophila midgut (Sieber, 2009).

Although DHR96 null mutants are viable and fertile, with no morphological defects, they die significantly more rapidly than genetically matched control flies under starvation conditions, while DHR96 overexpression leads to starvation resistance. The effects of these genotypes on the major forms of stored energy in the animal, glycogen and TAG, are consistent with their effects on the starvation response. DHR96 mutants have reduced levels of TAG under both fed and starved conditions, while DHR96 overexpression leads to increased TAG levels. Although no effects are seen on whole-animal glycogen levels in fed animals that either lack or overexpress DHR96, the mutants consume significantly more glycogen upon starvation than do controls. This rapid utilization of glycogen stores is most likely due to the decreased energy contribution from TAG. Taken together, these observations suggest that the starvation sensitivity of DHR96 mutants can be attributed to their lean phenotype, while the starvation resistance of the DHR96^2X strain is due to their excess energy in the form of TAG. This proposal is supported by the observation that genetically elevating the levels of TAG in DHR96 mutants by introducing mutations in brummer or AKHR, which control distinct aspects of fat body TAG lipolysis, effectively rescues their starvation sensitivity. In addition, the opposite effects of DHR96 loss of function and gain of function on both the starvation response and TAG levels argue that this receptor plays a central role in maintaining whole-animal TAG homeostasis (Sieber, 2009)

Several lines of evidence support the conclusion that DHR96 exerts its primary metabolic functions through the midgut. These include the initial observation that DHR96 mutants are resistant to treatment with the gastric lipase inhibitor Orlistat and display reduced levels of midgut lipolytic activity. In addition, dietary supplementation with free fatty acids, but not TAG, is sufficient to rescue the lean phenotype of DHR96 mutants, as is midgut-specific expression of wild-type DHR96 in a DHR96 mutant background. A dramatic effect is seen on lipid levels in the midgut, where almost no neutral lipids are detectable in DHR96 mutants and enlarged lipid droplets are evident in DHR96^2X flies maintained on a normal diet. Interestingly, while the lumen of the midgut is not evident in control animals, material was clearly seen in the lumen of DHR96 mutant midguts or in control flies that are treated with Orlistat. In some cases, this material is stained by oil red O, suggesting that it may represent an increased level of undigested fat in these animals. This phenotype would be similar to that seen in humans who have defects in intestinal lipase activity. Likewise, an increase in the passage of undigested dietary fat is a complication associated with Orlistat treatment in patients. Taken together, these observations support the conclusion that DHR96 acts in the midgut to regulate the breakdown of dietary fat (Sieber, 2009).

An essential role for DHR96 in the midgut is further supported by the microarray study, which revealed that many DHR96-regulated genes are primarily expressed in this tissue. Interestingly, many of these genes have predicted roles related to the breakdown of dietary nutrients. These include downregulation of multiple genes with predicted α-mannosidase activity, which is involved in the breakdown of the complex sugars found on glycoproteins. Many genes that encode trypsins and endopeptidases are also expressed at reduced levels in DHR96 mutants as well as a few genes that encode predicted α-glucosidases, which are involved in the breakdown of dietary carbohydrates. In addition, a number of genes involved in the formation of the peritrophic matrix are more abundantly expressed in DHR96 mutants. This matrix is comprised of chitin and peritrophic proteins and acts as a protective layer for the epithelial surface of the midgut. The peritrophic matrix also has critical roles in facilitating digestion. Only smaller molecules that arise from the initial digestion of complex nutrients, including peptides, sugars, and lipids, can move through the peritrophic matrix for final digestion and absorption by the midgut epithelium. These events are controlled by the selective partitioning of digestive enzymes to different sides of the peritrophic matrix as well as within the matrix itself. Thus, while midgut morphology appears normal in DHR96 mutants, the effect of the mutation on the peritrophic matrix could impact nutrient digestion and absorption (Sieber, 2009).

In addition to genes that regulate different aspects of lipid metabolism, microarray study of DHR96 mutants identified widespread effects on the expression of Drosophila homologs of NPC disease genes. The npc1b gene, which encodes an essential cholesterol transporter and the ortholog of mammalian NPC1L1, is the tenth most highly upregulated gene in DHR96 mutants. In addition, five of the eight Drosophila NPC2 genes are misregulated in DHR96 mutants: npc2c, npc2d, npc2e, npc2g, and npc2h. These genes encode homologs of mammalian NPC2, which is involved in intracellular cholesterol trafficking. Remarkably, two of these genes are the most highly up- and downregulated genes identified in the mutant (npc2e and npc2d, respectively). Moreover, many of these npc genes are located in clusters, suggesting that they are coregulated by the receptor. Although the function of these npc2 genes is unknown, their disproportionate representation within the list of DHR96-regulated genes implies a critical role for the receptor in regulating cholesterol trafficking. Indeed, dietary cholesterol triggers a widespread transcriptional response in Drosophila that is dependent on DHR96 function (Horner, 2010). Moreover, this study showed that DHR96 mutants display defects in their ability to maintain cholesterol homeostasis when grown on a high-cholesterol diet. Taken together with the results presented here, these studies suggest that DHR96 plays an essential role in the midgut to coordinate the processes of TAG and cholesterol breakdown, absorption, and trafficking (Sieber, 2009)

Two genes with predicted TAG lipase activity are expressed at lower levels in DHR96 mutants. One of these genes, CG5932, is abundantly expressed in the larval and adult midgut, encodes a protein that is highly related to human gastric lipase (LIPF, 37% identity over 358 amino acids), and is essential for maintaining whole-animal TAG levels. In addition, restoring CG5932 expression in the midguts of DHR96 mutants is sufficient to rescue their lean phenotype, defining this gene as a critical functional target of the receptor. Interestingly, CG5932 expression is also regulated by starvation, with reduced expression in the absence of food and increased expression upon refeeding. This regulation is consistent with an essential role for CG5932 in the breakdown of dietary fat, where its expression is upregulated when food is present. This response, however, is unaffected in DHR96 mutants, indicating that it is under independent control, possibly by known regulators of the starvation response such as dFOXO (Sieber, 2009).

The identification of CG5932 as a key functional target of DHR96 raises the question of how that regulation is achieved. A range of dietary parameters and candidate ligands were tested using the GAL4-DHR96 ligand sensor but conditions have not been identified that activate the DHR96 LBD. In addition, the DHR96-binding site remains undefined. DHR96 has a unique P box sequence within its DBD, which determines its DNA-binding specificity. This sequence is only shared by three C. elegans NRs, DAF-12, NHR-48, and NHR-8. Consistent with this observation, it was found that DHR96 protein fails to bind to most canonical NR-binding sites, except for weak binding to a palindromic EcR response element. One study has shown that DAF-12 displays preferential binding to a direct repeat of two distinct hexanucleotide sequences (AGGACA and AGTGCA), separated by five nucleotides (DR5). Whether DAF-12 contacts these sequences as a homodimer or a heterodimer with another NR, however, remains to be determined. The observation that many DHR96-regulated genes are arranged in clusters and DHR96 binds directly to a region upstream from the CG5932 start site provides an ideal context for defining its DNA-binding specificity as well as determining the molecular mechanisms by which DHR96 coordinates target gene transcription (Sieber, 2009).

These studies of DHR96 raise the interesting possibility that the defects in xenobiotic detoxification seen in DHR96 mutants may arise, at least in part, from its role in regulating midgut metabolic activity. As noted in the original study, most of the genes that are regulated by phenobarbital in wild-type flies do so independently of DHR96. These genes include representatives of the major classes associated with xenobiotic detoxification: cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), carboxylesterases, and UDP-glucuronosyl transferases (UGTs). Moreover, some phenobarbital-regulated genes that are misregulated in DHR96 mutants still show a transcriptional response to the drug, although that response is muted. These observations indicate that one or more other factors contribute to the transcriptional response to xenobiotic challenge in Drosophila. Interestingly, similar results have been observed in vertebrates, where less then half of the genes that are regulated by xenobiotics are affected in PXR and CAR mutant mice. It remains unclear, however, whether this lack of regulation might be due to functional redundancy between these mammalian receptors (Sieber, 2009).

There are several possible mechanisms by which the metabolic functions of DHR96 could impact xenobiotic responses. First, a recent study of the expression patterns of Drosophila P450 genes showed that 34 of 60 genes that could be detected in third-instar larvae are expressed in the midgut or hindgut. A similar overrepresentation of P450 genes is evident in a microarray study of midgut expressed genes. These observations indicate that, contrary to previous assumptions, the gut, and not the fat body, may be a critical site for xenobiotic detoxification. If this is true, then the sensitivity of DHR96 mutants to phenobarbital or DDT treatment may be affected by the effects of this mutation on midgut physiology. This could occur through defects in the peritrophic matrix or through changes in the ability of the midgut to absorb lipophilic xenobiotic compounds such as DDT. An alternative possibility is that the sensitivity of DHR96 mutants to xenobiotics might be due to their decreased energy stores. Detoxification requires energy expenditure. For example, P450s consume NADPH or NADH for their oxidation of xenobiotics, UGTs consume glucose, and GSTs consume glutathione. Thus, the reduced levels of stored energy in DHR96 mutants might compromise their ability to properly inactivate toxic compounds. In addition, the reduced lipid stores in DHR96 mutants might exert an indirect effect on xenobiotic responses by lowering the ability of the animal to sequester toxins in the fat reserves of the animal. Thus, there are multiple pathways by which the midgut-specific metabolic defects associated with the DHR96 mutation might indirectly affect xenobiotic responses in these animals. Further studies are required to test this possibility and clarify the functional overlaps between the roles of DHR96 in lipid metabolism and xenobiotic detoxification (Sieber, 2009).

Several recent studies have demonstrated roles for both PXR and CAR in lipid metabolism. CAR can repress the transcription of genes encoding carnitine palmitoyltransferase and enoyl-CoA isomerase, key steps in lipid β-oxidation. CAR mutant mice are also sensitive to starvation, much as was observed for DHR96 mutants, and lose weight more rapidly than wild-type mice when maintained on a low-calorie diet. Transgenic expression of a constitutively active form of PXR in the mouse liver leads to hepatic steatosis along with reduced expression of lipid catabolic genes and increased expression of genes involved in lipid synthesis. Importantly, similar effects were observed upon pharmacological activation of PXR using a specific agonist, indicating that the endogenous receptor can contribute to lipid homeostasis. Most recently, a mutation in CAR has been shown to normalize the elevated serum TAG levels seen in leptin-deficient mice or in wild-type mice maintained on a high-fat diet. Conversely, treatment of wild-type mice with a selective CAR agonist leads to increased serum TAG levels, and this response fails to occur in a CAR mutant background. Taken together, these studies indicate that activation of PXR/CAR receptors leads to lipid accumulation while a loss of PXR/CAR activity leads to reduced lipid levels, defining a central role for these NRs in lipid homeostasis. Their role in normal lipid metabolism, however, remains unknown, although it may be masked by functional redundancy between the two receptors. Similarly, the molecular mechanisms by which PXR and CAR can modulate lipid levels remain to be defined (Sieber, 2009)

These genetic studies of DHR96 have demonstrated that metabolic activities of PXR and CAR have been conserved through evolution, and represent an essential ancestral function for this NR subfamily. Moreover, the observation that DHR96 overexpression leads to lipid accumulation and DHR96 mutants are lean suggests that the molecular mechanisms that underlie these effects are also conserved across species. This conclusion is supported by genetic studies of the C. elegans member of this subfamily, DAF-12, which indicate that this receptor is also required for proper levels of stored fat. This study provides further evidence of a role for this NR subfamily in normal lipid homeostasis and defines the control of dietary fat breakdown as a key step at which this regulation is achieved. In addition, this work provides a foundation for understanding how dietary lipid uptake can impact normal lipid metabolism in Drosophila and provides a genetic model for characterizing how dietary factors can lead to lipid metabolic disorders such as obesity (Sieber, 2009)

Adipose Snail1 regulates lipolysis and lipid partitioning by suppressing Adipose Triacylglycerol Lipase expression

Lipolysis provides metabolic fuel; however, aberrant adipose lipolysis results in ectopic lipid accumulation and lipotoxicity. While adipose triacylglycerol lipase (ATGL) (see Drosophila Brummer) catalyzes the first step of lipolysis, its regulation is not fully understood. This study demonstrated that adipocyte Snail1 (see Drosophila Snail) suppresses both ATGL expression and lipolysis. Adipose Snail1 levels are higher in fed mice than in fasted mice and higher in obese mice as opposed to lean mice. Insulin increases Snail1 levels in both murine and human adipocytes, wherein Snail1 binds to the ATGL promoter to repress its expression. Importantly, adipocyte-specific deletion of Snail1 increases adipose ATGL expression and lipolysis, resulting in decreased fat mass and increased liver fat content in mice fed either a normal chow diet or a high-fat diet. Thus, this study has identified a Snail1-ATGL axis that regulates adipose lipolysis and fatty acid release, thereby governing lipid partitioning between adipose and non-adipose tissues (Sun, 2016).

Search PubMed for articles about Drosophila Brummer

Arrese, E. L., Patel, R. T., Soulages, J. L. (2006). The main triglyceride-lipase from the insect fat body is an active phospholipase A1: Identification and characterization. J. Lipid Res. 47: 2656-2667. PubMed ID: 17005997

Arrese, E. L., Rivera, L., Hamada, M., Mirza, S., Hartson, S. D., Weintraub, S. and Soulages, J. L. (2008). Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes. Arch Biochem Biophys 473: 42-47. PubMed ID: 18342616

Bell, M., et al. (2008). Consequences of lipid droplet coat protein downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance. Diabetes 57: 2037-2045. PubMed ID: 18487449

Beller, M., Sztalryd, C., Southall, N., Bell, M., Jäckle, H., Auld, D. S. and Oliver, B. (2008). COPI complex is a regulator of lipid homeostasis. PLoS Biol. 6(11): e292. PubMed ID: 19067489

Beller, M., Bulankina, A. V., Hsiao, H. H., Urlaub, H., Jackle, H. and Kuhnlein, R. P. (2010). PERILIPIN-dependent control of lipid droplet structure and fat storage in Drosophila. Cell Metab 12: 521-532. PubMed ID: 21035762

Bi, J., Xiang, Y., Chen, H., Liu, Z., Gronke, S., Kuhnlein, R. P. and Huang, X. (2012). Opposite and redundant roles of the two Drosophila perilipins in lipid mobilization. J Cell Sci 125: 3568-3577. PubMed ID: 22505614

Cherry, S., Kunte, A., Wang, H., Coyne, C., Rawson, R. B., et al. (2006). COPI activity coupled with fatty acid biosynthesis is required for viral replication. PLoS Pathog 2: e102. PubMed ID: 17040126

Colombani, J., et al. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed ID: 14505573

Grönke, S., et al. (2005). Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. Cell Metab. 1(5): 323-30. PubMed ID: 16054079

Grönke, S., Müller, G., Hirsch, J., Fellert, S., Andreou, A., Haase, T., Jäckle, H. and Kühnlein, R. P. (2007). Dual lipolytic control of body fat storage and mobilization in Drosophila. PLoS Biol. 5(6): e137. PubMed ID: 17488184

Guida, M. C., Birse, R. T., Dall'Agnese, A., Toto, P. C., Diop, S. B., Mai, A., Adams, P. D., Puri, P. L. and Bodmer, R. (2019). Intergenerational inheritance of high fat diet-induced cardiac lipotoxicity in Drosophila. Nat Commun 10(1): 193. PubMed ID: 30643137

Guo, Y., Walther, T. C., Rao, M., Stuurman, N., Goshima, G., et al. (2008). Functional genomic screen reveals genes involved in lipid-droplet formation and utilization. Nature 453: 657-661. PubMed ID: 18408709

Gutierrez, E., Wiggins, D., Fielding, B. and Gould, A. P. (2007). Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 445(7125): 275-80. PubMed ID: 17136098

Holm, C. (2003). Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem. Soc. Trans. 31: 1120-1124. PubMed ID: 14641008

Jenkins, C. M., et al. (2004). Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J. Biol. Chem. 279: 48968-48975. PubMed ID: 15364929

Lass, A., et al. (2006). Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome. Cell Metab 3: 309-319. PubMed ID: 16679289

Liu, P., et al. (2004). Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 279: 3787-3792. PubMed ID: 14597625

Marcinkiewicz, A., Gauthier, D., Garcia, A. and Brasaemle, D. L. (2006). The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion. J Biol Chem 281: 11901-11909. PubMed ID: 16488886

Molaei, M., Vandehoef, C. and Karpac, J. (2019). NF-kappaB shapes metabolic adaptation by attenuating Foxo-mediated lipolysis in Drosophila. Dev Cell 49(5):802-810. PubMed ID: 31080057

Miura, S., Gan, J. W., Brzostowski, J., Parisi, M. J., Schultz, C. J., et al. (2002). Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium. J. Biol. Chem. 277: 32253-32257. PubMed ID: 12077142

Oike, Y., Akao, M., Kubota, Y. and Suda, T. (2005). Angiopoietin-like proteins: potential new targets for metabolic syndrome therapy. Trends Mol. Med. 11: 473-479. PubMed ID: 16154386

Patel, R. T., Soulages, J. L., Hariharasundaram, B. and Arrese, E. L. (2005). Activation of the lipid droplet controls the rate of lipolysis of triglycerides in the insect fat body. J. Biol. Chem. 280: 22624-22631. PubMed ID: 15829485

Patel, R. T., Soulages, J. L. and Arrese, E. L. (2006). Adipokinetic hormone-induced mobilization of fat body triglyceride stores in Manduca sexta: Role of TG-lipase and lipid droplets. Arch Insect Biochem Physiol 63: 73-81. PubMed ID: 16983668

Sieber, M. H. and Thummel, C. S. (2009). The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila. Cell Metab. 10(6): 481-90. PubMed ID: 19945405

Sun C, Jiang L, Liu Y, Shen H, Weiss SJ, Zhou Y, Rui L. (2016). Adipose Snail1 regulates lipolysis and lipid partitioning by suppressing Adipose Triacylglycerol Lipase expression. Cell Rep 17(8):2015-2027. PubMed ID: 27851965

Umlauf, E., et al. (2004). Association of stomatin with lipid bodies. J. Biol. Chem. 279: 23699-23709. PubMed ID: 15024010

Van der Horst, D. J., Van Marrewijk, W. J. and Diederen, J. H. (2001). Adipokinetic hormones of insect: release, signal transduction, and responses. Int. Rev. Cytol. 211: 179-240. PubMed ID: 11597004

van Tienhoven, M., Atkins, J., Li, Y. and Glynn, P. (2002). Human neuropathy target esterase catalyzes hydrolysis of membrane lipids. J. Biol. Chem. 277: 20942-20948. PubMed ID: 11927584

Villena, J. A., et al. (2004). Desnutrin, an adipocyte gene encoding a novel patatin-domain containing protein, is induced by fasting and glucocorticoids. Ectopic expression of desnutrin increases triglyceride hydrolysis. J. Biol. Chem. 279: 47066-47075. PubMed ID: 15337759

Zechner, R., et al. (2005). Lipolysis: pathway under construction. Curr. Opin. Lipidol. 16: 333-340. PubMed ID: 15891395

Zhang, H. H., et al. (2003). Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis. J. Biol. Chem. 278: 51535-51542. PubMed ID: 14527948

Zimmermann, R., Strauss, J. G., Haemmerle, G., Schoiswohl, G., Birner-Gruenberger, R., Riederer, M., Lass, A., Neuberger, G., Eisenhaber, F., Hermetter, A. and Zechner, R. (2004). Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306: 1383-1386. PubMed ID: 15550674

Zinke, I., Kirchner, C., Chao, L. C., Tetzlaff, M. T. and Pankratz, M. J. (1999). Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126: 5275-5284. PubMed ID: 10556053

date revised: 22 September 2023

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