Rab32 is important for autophagy and lipid storage in Drosophila

Lipids are essential components of all organisms. Within cells, lipids are mainly stored in a specific type of organelle, called the lipid droplet. The molecular mechanisms governing the dynamics of lipid droplets have been little explored. The protein composition of lipid droplets has been analyzed in numerous proteomic studies, and a large number of lipid droplet-associated proteins have been identified, including Rab small GTPases. Rab proteins are known to participate in many intracellular membranous events; however, their exact role in lipid droplets is largely unexplored. Here we systematically investigate the roles of Drosophila Rab family proteins in lipid storage in the larval adipose tissue, fat body. Rab32 and several other Rabs were found to affect the size of lipid droplets as well as lipid levels. Further studies showed that Rab32 and Rab32 GEF/Claret may be involved in autophagy, consequently affecting lipid storage. Loss-of-function mutants of several components in the autophagy pathway result in similar effects on lipid storage. These results highlight the potential functions of Rabs in regulating lipid metabolism.     

Tissue-autonomous function of Drosophila seipin in preventing ectopic lipid droplet formation

Obesity is characterized by accumulation of excess body fat, while lipodystrophy is characterized by loss or absence of body fat. Despite their opposite phenotypes, these two conditions both cause ectopic lipid storage in non-adipose tissues, leading to lipotoxicity, which has health-threatening consequences. The exact mechanisms underlying ectopic lipid storage remain elusive. Here we report the analysis of a Drosophila model of the most severe form of human lipodystrophy, Berardinelli-Seip Congenital Lipodystrophy 2, which is caused by mutations in the BSCL2/Seipin gene. In addition to reduced lipid storage in the fat body, dSeipin mutant flies accumulate ectopic lipid droplets in the salivary gland, a non-adipose tissue. This phenotype was suppressed by expressing dSeipin specifically within the salivary gland. dSeipin mutants display synergistic genetic interactions with lipogenic genes in the formation of ectopic lipid droplets. Our data suggest that dSeipin may participate in phosphatidic acid metabolism and subsequently down-regulate lipogenesis to prevent ectopic lipid droplet formation. In summary, we have demonstrated a tissue-autonomous role of dSeipin in ectopic lipid storage in lipodystrophy.     

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.     

Intracellular origin and secretion of milk fat globules

The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are surrounded by cellular membranes. In this review we discuss what is known about how these droplets are formed in and secreted by mammary epithelial cells during lactation. This secretion mechanism, which appears to be unique, is unlike the exocytotic mechanism used by other cell types to secrete lipids. Milk fat globules originate as small, triacylglycerol-rich, droplets that are formed on or in endoplasmic reticulum membranes. These droplets are released from endoplasmic reticulum into the cytosol as microlipid droplets coated by proteins and polar lipids. Microlipid droplets can fuse with each other to form larger cytoplasmic lipid droplets. Droplets of all sizes appear to be unidirectionally transported to apical cell regions by as yet unknown mechanisms that may involve cytoskeletal elements. These lipid droplets appear to be secreted from the cell in which they were formed by being progressively enveloped in differentiated regions of apical plasma membrane. While plasma membrane envelopment appears to be the primary mechanism by which lipid droplets are released from the cell, a mechanism involving exocytosis of lipid droplets from cytoplasmic vacuoles also has been described. As discussed herein, while we have a general overview of the steps leading to the fat globules of milk, virtually nothing is known about the molecular mechanisms involved in milk fat globule formation, intracellular transit, and secretion.     

Connecting lipid droplet biology and the metabolic syndrome

In the recent years, new advances in the biology of lipid droplets led these structures specialized for lipid storage to be considered as new universal intracellular organelles playing active roles in cell physiology. Concomitantly, studies on the pathogenesis of metabolic diseases such as type 2 diabetes or atherosclerosis, associated with ongoing epidemic obesity, have pointed out the importance of lipotoxic effects in metabolic dysfunction, generated by ectopic lipid storage in non-adipose tissues. The purpose of this paper is to establish connections between recent discoveries in lipid droplet biology and novel views in the pathology of the metabolic syndrome. Bringing together the new concepts produced in these two separated fields might show the way towards the definition of innovative strategies to treat metabolic diseases. Particular attention is given to the role of adipocyte-specific proteins that interact with lipid droplets and confer unique functions to adipocyte lipid storage by limiting the spill-over of fatty acids and their lipotoxic effects.     

Developmental changes in the protein composition of Manduca sexta lipid droplets

The lipid droplets (LDs) are intracellular organelles mainly dedicated to the storage and provision of fatty acids. To accomplish these functions the LDs interact with other organelles and cytosolic proteins. In order to explore possible correlations between the physiological states of cells and the protein composition of LDs we have determined and compared the proteomic profiles of lipid droplets isolated from the fat bodies of 5th-instar larvae and adult Manduca sexta insects and from ovaries. These LD-rich tissues represent three clearly distinct metabolic states in regard to lipid metabolism: 1) Larval fat body synthesizes fatty acids (FA) and accumulates large amounts as triglyceride (TG); 2) Fat body from adult insects provides FA to support reproduction and flight; 3) Ovaries do not synthesize FA, but accumulate considerable amounts of TG in LDs. Major qualitative and semi-quantitative variations in the protein compositions of the LDs isolated from these three tissues were observed by MS/MS and partially validated by immuno-blotting. The differences observed included changes in the abundance of lipid droplet specific proteins, cytosolic proteins, mitochondrial proteins and also proteins associated with the machinery of protein synthesis. These results suggest that changes in the interaction of LDs with other organelles and cytosolic proteins are tightly related to the physiological state of cells. Herein, we summarize and compare the protein compositions of three subtypes of LDs and also describe for the first time the proteomic profile of LDs from an insect ovary. The compositions and compositional differences found among the LDs are discussed to provide a platform for future studies on the role of LDs, and their associated proteins, in cellular metabolism.     

Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells

While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also 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.     

Tobacco pollen tubes – a fast and easy tool for studying lipid droplet association of plant proteins

In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site‐directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.     

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.     

Retinyl ester storage particles (retinosomes) from the retinal pigmented epithelium resemble lipid droplets in other tissues

Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored.     

Nuclear lipid droplets and nuclear damage in Caenorhabditis elegans

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.     

The control of lipid metabolism by mRNA splicing in Drosophila

The storage of lipids is an evolutionarily conserved process that is important for the survival of organisms during shifts in nutrient availability. Triglycerides are stored in lipid droplets, but the mechanisms of how lipids are stored in these structures are poorly understood. Previous in vitro RNAi screens have implicated several components of the spliceosome in controlling lipid droplet formation and storage, but the in vivo relevance of these phenotypes is unclear. In this study, we identify specific members of the splicing machinery that are necessary for normal triglyceride storage in the Drosophila fat body. Decreasing the expression of the splicing factors U1-70K, U2AF38, U2AF50 in the fat body resulted in decreased triglyceride levels. Interestingly, while decreasing the SR protein 9G8 in the larval fat body yielded a similar triglyceride phenotype, its knockdown in the adult fat body resulted in a substantial increase in lipid stores. This increase in fat storage is due in part to altered splicing of the gene for the β-oxidation enzyme CPT1, producing an isoform with less enzymatic activity. Together, these data indicate a role for mRNA splicing in regulating lipid storage in Drosophila and provide a link between the regulation of gene expression and lipid homeostasis.     

Interactomic study on interaction between lipid droplets and mitochondria

An increasing body of evidence shows that the lipid droplet, a neutral lipid storage organelle, plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria. However, the cellular functions and molecular mechanisms of the interaction remain ambiguous. Here we present data from transmission electron microscopy, fluorescence imaging, and reconstitution assays, demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro. Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae, we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes. The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75% of the interactions detected. Interestingly, interactions between 3 pairs of lipid metabolic enzymes were detected. Collectively, these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes, and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.     

Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.     

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.     

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.     

The lipid-droplet proteome reveals that droplets are a protein-storage depot

BACKGROUND: Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. RESULTS: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. CONCLUSIONS: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.     

Lipid oversupply,selective insulin resistance,and lipotoxicity: Molecular mechanisms

The accumulation of fat in tissues not suited for lipid storage has deleterious consequences on organ function, leading to cellular damage that underlies diabetes, heart disease, and hypertension. To combat these lipotoxic events, several therapeutics improve insulin sensitivity and/or ameliorate features of metabolic disease by limiting the inappropriate deposition of fat in peripheral tissues (i.e. thiazolidinediones, metformin, and statins). Recent advances in genomics and lipidomics have accelerated progress towards understanding the pathogenic events associated with the excessive production, underutilization, or inefficient storage of fat. Herein we review studies applying pharmacological or genetic strategies to manipulate the expression or activity of enzymes controlling lipid deposition, in order to gain a clearer understanding of the molecular mechanisms by which fatty acids contribute to metabolic disease.     

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.