Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.

orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control.

BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.

The α/ß-hydrolase domain-containing 4- and 5-related phospholipase Pummelig controls energy storage in Drosophila.

Triglycerides (TGs) are the main energy storage form that accommodates changing organismal energy demands. In Drosophila melanogaster, the TG lipase Brummer is centrally important for body fat mobilization. Its gene brummer (bmm) encodes the ortholog of mammalian adipose TG lipase, which becomes activated by α/ß-hydrolase domain-containing 5 (ABHD5/CGI-58), one member of the paralogous gene pair, α/ß-hydrolase domain-containing 4 (ABHD4) and ABHD5 In Drosophila, the pummelig (puml) gene encodes the single sequence-related protein to mammalian ABHD4/ABHD5 with unknown function. We generated puml deletion mutant flies, that were short-lived as a result of lipid metabolism changes, stored excess body fat at the expense of glycogen, and exhibited ectopic fat storage with altered TG FA profile in the fly kidneys, called Malpighian tubules. TG accumulation in puml mutants was not associated with increased food intake but with elevated lipogenesis; starvation-induced lipid mobilization remained functional. Despite its structural similarity to mammalian ABHD5, Puml did not stimulate TG lipase activity of Bmm in vitro. Rather, Puml acted as a phospholipase that localized on lipid droplets, mitochondria, and peroxisomes. Together, these results show that the ABHD4/5 family member Puml is a versatile phospholipase that regulates Drosophila body fat storage and energy metabolism.

Drosophila as a model to study obesity and metabolic disease.

Excess adipose fat accumulation, or obesity, is a growing problem worldwide in terms of both the rate of incidence and the severity of obesity-associated metabolic disease. Adipose tissue evolved in animals as a specialized dynamic lipid storage depot: adipose cells synthesize fat (a process called lipogenesis) when energy is plentiful and mobilize stored fat (a process called lipolysis) when energy is needed. When a disruption of lipid homeostasis favors increased fat synthesis and storage with little turnover owing to genetic predisposition, overnutrition or sedentary living, complications such as diabetes and cardiovascular disease are more likely to arise. The vinegar fly Drosophila melanogaster (Diptera: Drosophilidae) is used as a model to better understand the mechanisms governing fat metabolism and distribution. Flies offer a wealth of paradigms with which to study the regulation and physiological effects of fat accumulation. Obese flies accumulate triacylglycerols in the fat body, an organ similar to mammalian adipose tissue, which specializes in lipid storage and catabolism. Discoveries in Drosophila have ranged from endocrine hormones that control obesity to subcellular mechanisms that regulate lipogenesis and lipolysis, many of which are evolutionarily conserved. Furthermore, obese flies exhibit pathophysiological complications, including hyperglycemia, reduced longevity and cardiovascular function - similar to those observed in obese humans. Here, we review some of the salient features of the fly that enable researchers to study the contributions of feeding, absorption, distribution and the metabolism of lipids to systemic physiology.

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. In this study, we report the functional analysis of PLIN1 and Drosophila 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. Our study highlights the complex roles of Drosophila perilipin proteins and the evolutionarily conserved regulation of HSL translocation by perilipins.

Thematic review series: Lipid droplet synthesis and metabolism: from yeast to man. Lipid droplet-based storage fat metabolism in Drosophila.

The fruit fly Drosophila melanogaster is an emerging model system in lipid metabolism research. Lipid droplets are omnipresent and dynamically regulated organelles found in various cell types throughout the complex life cycle of this insect. The vital importance of lipid droplets as energy resources and storage compartments for lipoanabolic components has recently attracted research attention to the basic enzymatic machinery, which controls the delicate balance between triacylglycerol deposition and mobilization in flies. This review aims to present current insights in experimentally supported and inferred biological functions of lipogenic and lipolytic enzymes as well as regulatory proteins, which control the lipid droplet-based storage fat turnover in Drosophila.

Drosophila Lipase 3 Mediates the Metabolic Response to Starvation and Aging.

The human LIPA gene encodes for the enzyme lysosomal acid lipase, which hydrolyzes cholesteryl ester and triacylglycerol. Lysosomal acid lipase deficiency results in Wolman disease and cholesteryl ester storage disease. The Drosophila genome encodes for two LIPA orthologs, Magro and Lipase 3. Magro is a gut lipase that hydrolyzes triacylglycerides, while Lipase 3 lacks characterization based on mutant phenotypes. We found previously that Lipase 3 transcription is highly induced in mutants with defects in peroxisome biogenesis, but the conditions that allow a similar induction in wildtypic flies are not known. Here we show that Lipase 3 is drastically upregulated in starved larvae and starved female flies, as well as in aged male flies. We generated a lipase 3 mutant that shows sex-specific starvation resistance and a trend to lifespan extension. Using lipidomics, we demonstrate that Lipase 3 mutants accumulate phosphatidylinositol, but neither triacylglycerol nor diacylglycerol. Our study suggests that, in contrast to its mammalian homolog LIPA, Lipase 3 is a putative phospholipase that is upregulated under extreme conditions like prolonged nutrient deprivation and aging.

Drosophila as a lipotoxicity model organism--more than a promise?

Looking back over the century long research career of the fruit fly, Drosophila melanogaster has frequently been in the scientific spotlight with respect to fundamental discoveries in biology. The last decade witnessed the increasing importance of the fly as a human disease model but studies on energy homeostasis and lipometabolism remain in their infancy. This perspective, addressing readers largely unfamiliar with the Drosophila model system, aims to highlight the starting points for which the fly could be employed to gain a deeper understanding of lipotoxicity and possibly contribute to strategies for the identification of novel drug targets relevant to type 2 diabetes mellitus and the metabolic syndrome.

Lipidome remodeling in aging normal and genetically obese Drosophila males.

Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. We here used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. Our comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, we present first evidence for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life.

Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success.

Triacylglycerol (TG) and steryl ester (SE) lipid storage is a universal strategy to maintain organismal energy and membrane homeostasis. Cycles of building and mobilizing storage fat are fundamental in (re)distributing lipid substrates between tissues or to progress ontogenetic transitions. In this study, we show that Hormone-sensitive lipase (Hsl) specifically controls SE mobilization to initiate intergenerational sterol transfer in Drosophila melanogaster. Tissue-autonomous Hsl functions in the maternal fat body and germline coordinately prevent adult SE overstorage and maximize sterol allocation to embryos. While Hsl-deficiency is largely dispensable for normal development on sterol-rich diets, animals depend on adipocyte Hsl for optimal fecundity when dietary sterol becomes limiting. Notably, accumulation of SE but not of TG is a characteristic of Hsl-deficient cells across phyla including murine white adipocytes. In summary, we identified Hsl as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

Endocrine signals fine-tune daily activity patterns in Drosophila.

Animals need to balance competitive behaviors to maintain internal homeostasis. The underlying mechanisms are complex but typically involve neuroendocrine signaling. Using Drosophila, we systematically manipulated signaling between energy-mobilizing endocrine cells producing adipokinetic hormone (AKH), octopaminergic neurons, and the energy-storing fat body to assess whether this neuroendocrine axis involved in starvation-induced hyperactivity also balances activity levels under ad libitum access to food. Our results suggest that AKH signals via two divergent pathways that are mutually competitive in terms of activity and rest. AKH increases activity via the octopaminergic system during the day, while it prevents high activity levels during the night by signaling to the fat body. This regulation involves feedback signaling from octopaminergic neurons to AKH-producing cells (APCs). APCs are known to integrate a multitude of metabolic and endocrine signals. Our results add a new facet to the versatile regulatory functions of APCs by showing that their output contributes to shape the daily activity pattern under ad libitum access to food.

Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila.

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. We performed a screen to identify nutritionally regulated genes that control energy storage in the model organism Drosophila. The brummer (bmm) gene encodes the lipid storage droplet-associated TAG lipase Brummer, a homolog of human 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. Our 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.

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. We generated a Drosophila mutant lacking the receptor (AKHR) 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. Our 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.

Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila.

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. Here we introduce RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. We show that organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, our study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases.

Triacylglycerol Metabolism in Drosophila melanogaster.

Triacylglycerol (TAG) is the most important caloric source with respect to energy homeostasis in animals. In addition to its evolutionarily conserved importance as an energy source, TAG turnover is crucial to the metabolism of structural and signaling lipids. These neutral lipids are also key players in development and disease. Here, we review the metabolism of TAG in the Drosophila model system. Recently, the fruit fly has attracted renewed attention in research due to the unique experimental approaches it affords in studying the tissue-autonomous and interorgan regulation of lipid metabolism in vivo Following an overview of the systemic control of fly body fat stores, we will cover lipid anabolic, enzymatic, and regulatory processes, which begin with the dietary lipid breakdown and de novo lipogenesis that results in lipid droplet storage. Next, we focus on lipolytic processes, which mobilize storage TAG to make it metabolically accessible as either an energy source or as a building block for biosynthesis of other lipid classes. Since the buildup and breakdown of fat involves various organs, we highlight avenues of lipid transport, which are at the heart of functional integration of organismic lipid metabolism. Finally, we draw attention to some "missing links" in basic neutral lipid metabolism and conclude with a perspective on how fly research can be exploited to study functional metabolic roles of diverse lipids.

Chronic low-dose pro-oxidant treatment stimulates transcriptional activity of telomeric retroelements and increases telomere length in Drosophila.

It has been proposed that oxidative stress, elicited by high levels of reactive oxygen species, accelerates telomere shortening by erosion of telomeric DNA repeats. While most eukaryotes counteract telomere shortening by telomerase-driven addition of these repeats, telomeric loss in Drosophila is compensated by retrotransposition of the telomeric retroelements HeT-A, TART and TAHRE to chromosome ends. In this study we tested the effect of chronic exposure of flies to non-/sub-lethal doses of paraquat, which is a redox cycling compound widely used to induce oxidative stress in various experimental paradigms including telomere length analyses. Indeed, chronic paraquat exposure for five generations resulted in elevated transcriptional activity of both telomeric and non-telomeric transposable elements, and extended telomeric length in the tested fly lines. We propose that low oxidative stress leads to increased telomere length within Drosophila populations. For a mechanistic understanding of the observed phenomenon we discuss two scenarios: adaption, acting through a direct stimulation of telomere extension, or positive selection favoring individuals with longer telomeres within the population.

Diacylglycerol triggers Rim101 pathway-dependent necrosis in yeast: a model for lipotoxicity.

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.

Control of fat storage by a Drosophila PAT domain protein.

In Drosophila, the masses and sheets of adipose tissue that are distributed throughout the fly are collectively called the fat body. Like mammalian adipocytes, insect fat body cells provide the major energy reserve of the animal organism. Both cell types accumulate triacylglycerols (TAG) in intracellular lipid droplets; this finding suggests that the strategy of energy storage as well as the machinery and the control to achieve fat storage might be evolutionarily conserved. Studies addressing the control of lipid-based energy homeostasis of mammals identified proteins of the PAT domain family, such as Perilipin, which reside on lipid droplets. Perilipin knockout mice are lean and resistant to diet-induced obesity. Conversely, Perilipin expression in preadipocyte tissue culture increases lipid storage by reducing the rate of TAG hydrolysis. Factors that mediate corresponding processes in invertebrates are still unknown. We examined the function of Lsd2, one of only two PAT domain-encoding genes in the Drosophila genome. Lsd2 acts in a Perilipin-like manner, suggesting that components regulating homeostasis of lipid-based energy storage at the lipid droplet membrane are evolutionarily conserved.

Spastic paraplegia-linked phospholipase PAPLA1 is necessary for development, reproduction, and energy metabolism in Drosophila.

The human PAPLA1 phospholipase family is associated with hereditary spastic paraplegia (HSP), a neurodegenerative syndrome characterized by progressive spasticity and weakness of the lower limbs. Taking advantage of a new Drosophila PAPLA1 mutant, we describe here novel functions of this phospholipase family in fly development, reproduction, and energy metabolism. Loss of Drosophila PAPLA1 reduces egg hatchability, pre-adult viability, developmental speed, and impairs reproductive functions of both males and females. In addition, our work describes novel metabolic roles of PAPLA1, manifested as decreased food intake, lower energy expenditure, and reduced ATP levels of the mutants. Moreover, PAPLA1 has an important role in the glycogen metabolism, being required for expression of several regulators of carbohydrate metabolism and for glycogen storage. In contrast, global loss of PAPLA1 does not affect fat reserves in adult flies. Interestingly, several of the PAPLA1 phenotypes in fly are reminiscent of symptoms described in some HSP patients, suggesting evolutionary conserved functions of PAPLA1 family in the affected processes. Altogether, this work reveals novel physiological functions of PAPLA1, which are likely evolutionary conserved from flies to humans.

Thermal stress depletes energy reserves in Drosophila.

Understanding how environmental temperature affects metabolic and physiological functions is of crucial importance to assess the impacts of climate change on organisms. Here, we used different laboratory strains and a wild-caught population of the fruit fly Drosophila melanogaster to examine the effect of temperature on the body energy reserves of an ectothermic organism. We found that permanent ambient temperature elevation or transient thermal stress causes significant depletion of body fat stores. Surprisingly, transient thermal stress induces a lasting "memory effect" on body fat storage, which also reduces survivorship of the flies upon food deprivation later after stress exposure. Functional analyses revealed that an intact heat-shock response is essential to protect flies from temperature-dependent body fat decline. Moreover, we found that the temperature-dependent body fat reduction is caused at least in part by apoptosis of fat body cells, which might irreversibly compromise the fat storage capacity of the flies. Altogether, our results provide evidence that thermal stress has a significant negative impact on organismal energy reserves, which in turn might affect individual fitness.