The perilipin family of lipid droplet proteins: Gatekeepers of intracellular lipolysis.

Lipid droplets in chordates are decorated by two or more members of the perilipin family of lipid droplet surface proteins. The perilipins sequester lipids by protecting lipid droplets from lipase action. Their relative expression and protective nature is adapted to the balance of lipid storage and utilization in specific cells. Most cells of the body have tiny lipid droplets with perilipins 2 and 3 at the surfaces, whereas specialized fat-storing cells with larger lipid droplets also express perilipins 1, 4, and/or 5. Perilipins 1, 2, and 5 modulate lipolysis by controlling the access of lipases and co-factors of lipases to substrate lipids stored within lipid droplets. Although perilipin 2 is relatively permissive to lipolysis, perilipins 1 and 5 have distinct control mechanisms that are altered by phosphorylation. Here we evaluate recent progress toward understanding functions of the perilipins with a focus on their role in regulating lipolysis and autophagy. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization.

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS(239)S(240), we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno-blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.

Comparative gene identification 58/α/ß hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity.

Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/ß hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity.

Packaging of fat: an evolving model of lipid droplet assembly and expansion.

Lipid droplets (LDs) are organelles found in most types of cells in the tissues of vertebrates, invertebrates, and plants, as well as in bacteria and yeast. They differ from other organelles in binding a unique complement of proteins and lacking an aqueous core but share aspects of protein trafficking with secretory membrane compartments. In this minireview, we focus on recent evidence supporting an endoplasmic reticulum origin for LD formation and discuss recent findings regarding LD maturation and fusion.

Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein.

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.

Adipose-selective overexpression of ABHD5/CGI-58 does not increase lipolysis or protect against diet-induced obesity.

Adipose triglyceride lipase (ATGL) catalyzes the first step of triacylglycerol hydrolysis in adipocytes. Abhydrolase domain 5 (ABHD5) increases ATGL activity by an unknown mechanism. Prior studies have suggested that the expression of ABHD5 is limiting for lipolysis in adipocytes, as addition of recombinant ABHD5 increases in vitro TAG hydrolase activity of adipocyte lysates. To test this hypothesis in vivo, we generated transgenic mice that express 6-fold higher ABHD5 in adipose tissue relative to wild-type (WT) mice. In vivo lipolysis increased to a similar extent in ABHD5 transgenic and WT mice following an overnight fast or injection of either a ß-adrenergic receptor agonist or lipopolysaccharide. Similarly, basal and ß-adrenergic-stimulated lipolysis was comparable in adipocytes isolated from ABHD5 transgenic and WT mice. Although ABHD5 expression was elevated in thioglycolate-elicited macrophages from ABHD5 transgenic mice, Toll-like receptor 4 (TLR4) signaling was comparable in macrophages isolated from ABHD5 transgenic and WT mice. Overexpression of ABHD5 did not prevent the development of obesity in mice fed a high-fat diet, as shown by comparison of body weight, body fat percentage, and adipocyte hypertrophy of ABHD5 transgenic to WT mice. The expression of ABHD5 in mouse adipose tissue is not limiting for either basal or stimulated lipolysis.

Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins.

The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the Human Genome Organization Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each gene member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase.

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an alpha/beta-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.

Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.

Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.

ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells.

Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [(14)C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.

Developmental biology: holding pattern for histones.

New research on lipid droplets in Drosophila embryos has led to the surprising conclusion that these poorly understood organelles have a novel role as a regulated storage depot of maternally supplied proteins, particularly histones.

Perilipin A and the control of triacylglycerol metabolism.

Perilipin A is the most abundant protein associated with the lipid droplets of adipocytes and functions to control both basal and stimulated lipolysis. Under basal or fed conditions, perilipin A shields stored triacylglycerols from cytosolic lipases, thus promoting triacylglycerol storage. When catecholamines bind to cell surface receptors to initiate signals that activate cAMP-dependent protein kinase (PKA), phosphorylated perilipin A facilitates maximal lipolysis. Mutagenesis studies have revealed that central sequences of moderately hydrophobic amino acids are required to target nascent perilipin A to lipid droplets and provide an anchor into the hydrophobic environment of lipid droplets. Sequences of amino acids in the unique carboxyl terminus of perilipin A and those in amino terminal sequences flanking the first hydrophobic stretch are required for the barrier function of perilipin A in promoting triacylglycerol storage. Site-directed mutagenesis studies of serine residues within six PKA consensus sites of perilipin A reveal functions for phosphorylation of at least three of the sites. Phosphorylation of one or more of the serines within three amino terminal PKA sites is required to facilitate hormone-sensitive lipase access to lipid substrates. Phosphorylation of serines within two carboxyl terminal sites is also required for maximal lipolysis. Phosphorylation of serine 492 (site 5) triggers a massive remodeling of lipid droplets, whereby large peri-nuclear lipid droplets fragment into myriad lipid micro-droplets that scatter throughout the cytoplasm. We hypothesize that perilipin A binds accessory proteins to provide assistance in carrying out these functions.

Caveolin-1 expression is essential for proper nonshivering thermogenesis in brown adipose tissue.

Recently, we have shown that loss of caveolin-1 leads to marked alterations in insulin signaling and lipolysis in white adipose tissue. However, little is known about the role of caveolin-1 in brown adipose tissue (BAT), a tissue responsible for nonshivering thermogenesis. Here, we show that caveolin-1 null mice have a mildly, yet significantly, decreased resting core body temperature. To investigate this in detail, we next subjected the mice to fasting (for 24 h) or cold treatment (4 degrees C for 24 h), individually or in combination. Interestingly, caveolin-1 null mice showed markedly decreased body temperatures in response to fasting or fasting/cold treatment; however, cold treatment alone had no effect. In addition, under these conditions caveolin-1 null mice failed to show the normal increase in serum nonesterified fatty acids induced by fasting or fasting/cold treatment, suggesting that these mice are unable to liberate triglyceride stores for heat production. In accordance with these results, the triglyceride content of BAT was reduced nearly 10-fold in wild-type mice after fasting/cold treatment, but it was reduced only 3-fold in caveolin-1 null mice. Finally, electron microscopy of adipose tissue revealed dramatic perturbations in the mitochondria of caveolin-1 null interscapular brown adipocytes. Taken together, our data provide the first molecular genetic evidence that caveolin-1 plays a critical functional and structural role in the modulation of thermogenesis via an effect on lipid mobilization.

A proposed model of fat packaging by exchangeable lipid droplet proteins.

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3-12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.

Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation.

Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. © 2016 by John Wiley & Sons, Inc.

Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation.

Recently, it was shown that caveolin-1 can be redirected from the cell surface to intracellular lipid droplets in a variety of cell types. Here, we directly address the role of caveolin-1 in lipid droplet formation and breakdown, showing that caveolin-1 null mice exhibit markedly attenuated lipolytic activity. Mechanistically, although the activity of protein kinase A (PKA) was greatly increased in caveolin-1 null adipocytes, the phosphorylation of perilipin was dramatically reduced, indicating that caveolin-1 may facilitate the PKA-mediated phosphorylation of perilipin. In support of this hypothesis, coimmunoprecipitation experiments revealed that treatment with a beta(3)-adrenergic receptor agonist resulted in ligand-induced complex formation between perilipin, caveolin-1, and the catalytic subunit of PKA in wild-type but not in caveolin-1 null fat pads. We also show that caveolin-1 expression is important for efficient lipid droplet formation because caveolin-1 null embryonic fibroblasts stably transfected with perilipin accumulated approximately 4.5-fold less lipid than perilipin-transfected wild-type cells. Finally, high-pressure freeze-substitution electron microscopy of adipose tissue revealed dramatic perturbations in the architecture of the "lipid droplet cortex" (the interface between the lipid droplet surface and the cytoplasm) in caveolin-1 null perigonadal adipocytes. Taken together, our data provide the first molecular genetic evidence that caveolin-1 plays a critical functional and structural role in the modulation of both lipid droplet biogenesis and metabolism in vivo.

Fixation methods for the study of lipid droplets by immunofluorescence microscopy.

The study of proteins associated with lipid droplets in adipocytes and many other cells is a rapidly developing area of inquiry. Although lipid droplets are easily visible by light microscopy, few standardized microscopy methods have been developed. Several methods of chemical fixation have recently been used to preserve cell structure before visualization of lipid droplets by light microscopy. We tested the most commonly used methods to compare the effects of the fixatives on cellular lipid content and lipid droplet structure. Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. We found this method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are discovered and studied, it is critical to use appropriate methods to avoid studying artifacts.

Inhibitory effects of grape seed extract on lipases.

OBJECTIVE: The aim of the present study was to assess the effects of grape seed extract (GSE) on the fat-metabolizing enzymes pancreatic lipase, lipoprotein lipase, and hormone-sensitive lipase in vitro and evaluate its potential application as a treatment for obesity. METHODS: Crushed grape seeds were extracted in ethanol, and the extract was assayed for the measurement of inhibitory effects on pancreatic lipase and lipoprotein lipase activities and on lipolysis of 3T3-L1 adipocytes. RESULTS: The GSE rich in bioactive phytochemicals showed inhibitory activity on the fat-metabolizing enzymes pancreatic lipase and lipoprotein lipase, thus suggesting that GSE might be useful as a treatment to limit dietary fat absorption and the accumulation of fat in adipose tissue. The observed reduction in intracellular lipolytic activity of cultured 3T3-L1 adipocytes may reduce the levels of circulating free fatty acids that have been linked to insulin resistance in obese patients. CONCLUSION: The GSE rich in compounds that inhibit lipases may provide a safe, natural, and cost-effective weight control treatment.

CGI-58/ABHD5-derived signaling lipids regulate systemic inflammation and insulin action.

Mutations of comparative gene identification 58 (CGI-58) in humans cause Chanarin-Dorfman syndrome, a rare autosomal recessive disease in which excess triacylglycerol (TAG) accumulates in multiple tissues. CGI-58 recently has been ascribed two distinct biochemical activities, including coactivation of adipose triglyceride lipase and acylation of lysophosphatidic acid (LPA). It is noteworthy that both the substrate (LPA) and the product (phosphatidic acid) of the LPA acyltransferase reaction are well-known signaling lipids. Therefore, we hypothesized that CGI-58 is involved in generating lipid mediators that regulate TAG metabolism and insulin sensitivity. Here, we show that CGI-58 is required for the generation of signaling lipids in response to inflammatory stimuli and that lipid second messengers generated by CGI-58 play a critical role in maintaining the balance between inflammation and insulin action. Furthermore, we show that CGI-58 is necessary for maximal TH1 cytokine signaling in the liver. This novel role for CGI-58 in cytokine signaling may explain why diminished CGI-58 expression causes severe hepatic lipid accumulation yet paradoxically improves hepatic insulin action. Collectively, these findings establish that CGI-58 provides a novel source of signaling lipids. These findings contribute insight into the basic mechanisms linking TH1 cytokine signaling to nutrient metabolism.