Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism

Many cells store neutral lipids, as triacylglycerol and sterol esters, in droplets. PAT-domain proteins form a conserved family of proteins that are localized at the surface of neutral lipid droplets. Two mammalian members of this family, Perilipin and adipose differentiation-related protein, are involved in lipid storage and regulate lipolysis. Here, we describe the Drosophila PAT-family member Lsd2. We showed that Lsd2 is predominantly expressed in tissues engaged in high levels of lipid metabolism, the fat body and the germ line of females. Ultrastructural analysis in the germ line showed that Lsd2 localizes to the surface of lipid droplets. We have generated an Lsd2 mutant and described its phenotype. Mutant adults have a reduced level of neutral lipid content compared to wild type, showing that Lsd2 is required for normal lipid storage. In addition, ovaries from Lsd2 mutant females exhibit an abnormal pattern of accumulation of neutral lipids from mid-oogenesis, which results in reduced deposition of lipids in the egg. Consistent with its expression in the female germ line, we showed that Lsd2 is a maternal effect gene that is required for normal embryogenesis. This work demonstrates that Lsd2 has an evolutionarily conserved function in lipid metabolism and establishes Drosophila melanogaster as a new in vivo model for studies on the PAT-family of proteins.     

Characterization of the Drosophila lipid droplet subproteome

Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states.     

PERILIPIN-dependent control of lipid droplet structure and fat storage in Drosophila

Lipid droplets are intracellular organelles enriched in adipose tissue that govern the body fat stores of animals. In mammals, members of the evolutionarily conserved PERILIPIN protein family are associated with the lipid droplet surface and participate in lipid homeostasis. Here, we show that Drosophila mutants lacking the PERILIPIN PLIN1 are hyperphagic and suffer from adult-onset obesity. PLIN1 is a central and Janus-faced component of fat metabolism. It provides barrier function to storage lipid breakdown and acts as a key factor of stimulated lipolysis by modulating the access of proteins to the lipid droplet surface. It also shapes lipid droplet structure, transforming unilocular into multilocular fat cells. We generated flies devoid of all PERILIPIN family members and show that they exhibit impaired yet functional body fat regulation. Our data reveal the existence of a basal and possibly ancient lipid homeostasis system.     

S3-12, Adipophilin, and TIP47 package lipid in adipocytes

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.     

Purification and characterization of recombinant lipid storage protein-2 from Drosophila melanogaster

Lipid storage protein 2 (Lsd 2) is a conserved insect protein that belongs to the small PAT family of proteins. PAT proteins are found associated to the lipid droplets of adipocytes and play significant roles in the regulation of triacylglycerides metabolism. Here we describe the expression and purification of Lsd2, its reconstitution in lipoprotein particles, the location of putative lipid binding sites and its secondary structure. This study provides the starting point for future studies on the mechanism of function of Lsd2. The similarities and differences between Lsd1 and Lsd2, the only PAT proteins found in insects, are discussed.     

Lipid droplets as fat storage organelles in Caenorhabditis elegans: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

Lipid droplets are evolutionarily conserved organelles where cellular fat storage and mobilization are exquisitely regulated. Recent studies have defined lipid droplets in C. elegans and explored how they are regulated by genetic and dietary factors. C. elegans offers unique opportunities to visualize lipid droplets at single-cell resolution in live animals. The development of novel microscopy techniques and protein markers for lipid droplets will accelerate studies on how nutritional states and subcellular organization are linked in vivo. Together with powerful tools for genetic and biochemical analysis of metabolic pathways, alteration in lipid droplet abundance, size, and distribution in C. elegans can be readily connected to whole-animal energy homeostasis, behavior, and life span. Therefore, further studies on lipid droplets in C. elegans promise to yield valuable insights that complement our knowledge gained from yeast, Drosophila, and mammalian systems on cellular and organismal fat storage.     

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.     

The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid Metabolism, Appressorial Turgor Pressure, and Virulence

Cells store lipids in droplets. Studies addressing how mammals control lipid-based energy homeostasis have implicated proteins of the PAT domain family, such as perilipin that surrounds the lipid droplets. Perilipin knock-out mice are lean and resistant to obesity. Factors that mediate lipid storage in fungi are still unknown. Here we describe a gene (Mpl1) in the economically important insect fungal pathogen Metarhizium anisopliae that has structural similarities to mammalian perilipins. Consistent with a role in lipid storage, Mpl1 is predominantly expressed when M. anisopliae is engaged in accumulating lipids and ectopically expressed green fluorescent protein-tagged MPL1 (Metarhizium perilipin-like protein) localized to lipid droplets. Mutant M. anisopliae lacking MPL1 have thinner hyphae, fewer lipid droplets, particularly in appressoria (specialized infection structures at the end of germ tubes), and a decrease in total lipids. Mpl1 therefore acts in a perilipin-like manner suggesting an evolutionary conserved function in lipid metabolism. However, reflecting general differences between animal and fungal lineages, these proteins have also been selected to cope with different tasks. Thus, turgor generation by DeltaMpl1 appressoria is dramatically reduced indicating that lipid droplets are required for solute accumulation. This was linked with the reduced ability to breach insect cuticle so that Mpl1 is a pathogenicity determinant. Blast searches of fungal genomes revealed that perilipin homologs are found only in pezizomycotinal ascomycetes and occur as single copy genes. Expression of Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions.     

Functional conservation for lipid storage droplet association among Perilipin,ADRP, and TIP47 (PAT)-related proteins in mammals,Drosophila, and Dictyostelium

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.     

Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately α-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect.     

The contribution of the Drosophila model to lipid droplet research

Intracellular lipid droplets have long been misconceived as evolutionarily conserved but functionally frugal components of cellular metabolism. An ever-growing repertoire of functions has elevated lipid droplets to fully-fledged cellular organelles. Insights into the multifariousness of these organelles have been obtained from a range of model systems now employed for lipid droplet research including the fruit fly, Drosophila melanogaster. This review summarizes the progress in fly lipid droplet research along four main avenues: the role of lipid droplets in fat storage homeostasis, the control of lipid droplet structure, the lipid droplet surface as a dynamic protein-association platform, and lipid droplets as mobile organelles. Moreover, the research potential of the fruit fly model is discussed with respect to the prevailing general questions in lipid droplet biology.     

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. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that 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. We found that 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.     

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.     

Endocannabinoids and liver disease. IV. Endocannabinoid involvement in obesity and hepatic steatosis

Endocannabinoids are endogenous lipid mediators that interact with the same receptors as plant-derived cannabinoids to produce similar biological effects. The well-known appetitive effect of smoking marijuana has prompted inquiries into the possible role of endocannabinoids in the control of food intake and body weight. This brief review surveys recent evidence that endocannabinoids and their receptors are involved at multiple levels in the control of energy homeostasis. Endocannabinoids are orexigenic mediators and are part of the leptin-regulated central neural circuitry that controls energy intake. In addition, they act at multiple peripheral sites including adipose tissue, liver, and skeletal muscle to promote lipogenesis and limit fat elimination. Their complex actions could be viewed as anabolic, increasing energy intake and storage and decreasing energy expenditure, as components of an evolutionarily conserved system that has insured survival under conditions of starvation. In the era of plentiful food and limited physical activity, pharmacological inhibition of endocannabinoid activity offers benefits in the treatment of obesity and its hormonal/metabolic consequences.     

A Lipid Droplet-Associated GFP Reporter-Based Screen Identifies New Fat Storage Regulators in C.elegans

Fat storage disorders including obesity are pandemic human health problems. As a genetically amenable model organism, Caeno- rhabditis elegans has often been used to explore the molecular mechanisms of fat storage regulation. Dye staining of fixed animals and stimulated Raman scattering （SRS） microscopy methods have been used successfully to study fat storage, but a genetic screening system that takes full advantage of C. elegans transparency to perform live imaging of fluorescent protein reporters has not yet been reported. Here, we investigated the tissue-specific expression of the GFP fusion of Perilipin 1 （PLIN1）, a Drosophila lipid droplet-associated protein, in C. elegans. Our results indicate that PLINI：：GFP labels lipid droplets and can be used as a fat storage indicator in live worms. Through an RNAi screen, we further identified several previously uncharacterized new fat storage regulators.     

Seipin concentrates distinct neutral lipids via interactions with their acyl chain carboxyl esters

Lipid droplets (LDs) are essential for cellular lipid homeostasis by storing diverse neutral lipids (NLs), such as triacylglycerol (TAG), steryl esters (SE), and retinyl esters (RE). A proper assembly of TAG-containing LDs at the ER requires Seipin, a conserved protein often mutated in lipodystrophies. Here, we show that the yeast Seipin Sei1 and its partner Ldb16 also promote the storage of other NL in LDs. Importantly, this role of Sei1/Ldb16 is evolutionarily conserved as expression of human-Seipin restored normal SE-containing LDs in yeast Seipin mutants. As in the case of TAG, the formation of SE-containing LDs requires interactions between hydroxyl-residues in human Seipin or yeast Ldb16 with NL carboxyl esters. These findings provide a universal mechanism for Seipin-mediated LD formation and suggest a model for how Seipin distinguishes NLs from aliphatic phospholipid acyl chains in the center of the membrane bilayer.     

The central clock neurons regulate lipid storage in Drosophila

A proper balance of lipid breakdown and synthesis is essential for achieving energy homeostasis as alterations in either of these processes can lead to pathological states such as obesity. The regulation of lipid metabolism is quite complex with multiple signals integrated to control overall triglyceride levels in metabolic tissues. Based upon studies demonstrating effects of the circadian clock on metabolism, we sought to determine if the central clock cells in the Drosophila brain contribute to lipid levels in the fat body, the main nutrient storage organ of the fly. Here, we show that altering the function of the Drosophila central clock neurons leads to an increase in fat body triglycerides. We also show that although triglyceride levels are not affected by age, they are increased by expression of the amyloid-beta protein in central clock neurons. The effect on lipid storage seems to be independent of circadian clock output as changes in triglycerides are not always observed in genetic manipulations that result in altered locomotor rhythms. These data demonstrate that the activity of the central clock neurons is necessary for proper lipid storage.