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Lipid droplet (LD) coating proteins are essential for the formation and stability of intracellular LDs. Plin2 is an abundant LD coating protein in skeletal muscle, but its importance for muscle function is unclear. We show that myotubes established from Plin2−/− mice contain reduced content of LDs and accumulate less oleic acid (OA) in triacylglycerol (TAG) due to elevated LD hydrolysis in comparison with Plin2+/+ myotubes. The reduced ability to store TAG in LDs in Plin2−/− myotubes is accompanied by a shift in energy metabolism. Plin2−/− myotubes are characterized by increased oxidation of OA, lower glycogen synthesis, and reduced glucose oxidation in comparison with Plin2+/+ myotubes, perhaps reflecting competition between FAs and glucose as part of the Randle cycle. In accord with these metabolic changes, Plin2−/− myotubes have elevated expression of Ppara and Ppargc1a, transcription factors that stimulate expression of genes important for FA oxidation, whereas genes involved in glucose uptake and oxidation are suppressed. Loss of Plin2 had no impact on insulin-stimulated Akt phosphorylation. Our results suggest that Plin2 is essential for protecting the pool of skeletal muscle LDs to avoid an uncontrolled hydrolysis of stored TAG and to balance skeletal muscle energy metabolism. This article was originally published in the Journal of Lipid Research. © 2017 American Society for Biochemistry and Molecular Biology

The cytoplasmic lipid droplet (CLD) protein perilipin-2 (Plin2) is expressed in multiple nonadipose tissues, where it is thought to play a role in regulating their lipid storage properties. However, the extent to which Plin2 functions in nutrient utilization and metabolism, or how it influences the consequences of over-feeding, remains unclear. In this study, we demonstrate that the absence of Plin2 prevents high-fat diet(HFD)-induced obesity in male and female mice. This response is associated with increased formation of subcutaneous beige adipocyte cells with uncoupling protein 1 expression, and amelioration of inflammatory foci formation in white adipose tissue and steatosis in the liver. Experiments demonstrate that Plin2 loss results in reduced energy intake and increased physical activity in response to HFD feeding. Our study provides the first evidence that Plin2 contributes to HFD-induced obesity by modulating food intake, and that its absence prevents obesity-associated adipose tissue inflammatory foci and liver steatosis.

Regulation of hepatic very low density lipoprotein (VLDL) assembly and maturation is crucial in controlling lipid homeostasis and in the development of metabolic disorders, including obesity, hepatic steatosis, and insulin resistance. Cideb, a member of cell death-inducing DFF45-like effector (CIDE) protein family, has been previously shown to promote VLDL lipidation and maturation. However, the precise subcellular location of Cideb-mediated VLDL lipidation and the factors modulating its activity remain elusive. In addition to its localization to endoplasmic reticulum (ER) and lipid droplets (LD), we observed that Cideb was also localized to the Golgi apparatus. Mature and lipid-rich VLDL particles did not accumulate in the Golgi apparatus in Cideb(-/-) livers. Interestingly, we observed that hepatic perilipin 2/adipose differentiation-related protein (ADRP) levels were markedly increased in Cideb(-/-) mice. Liver-specific knockdown of perilipin 2 in Cideb(-/-) mice resulted in the reduced accumulation of hepatic triglycerides (TAG), increased VLDL-TAG secretion, and the accumulation of mature TAG-rich VLDL in the Golgi apparatus. These data reveal that Cideb and perilipin 2 play opposing roles in controlling VLDL lipidation and hepatic lipid homeostasis.

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

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.

Recent evidence suggests that fatty acids generated from intracellular triacylglycerol (TAG) hydrolysis may have important roles in intracellular signaling. This study was conducted to determine if fatty acids liberated from TAG hydrolysis regulate peroxisome proliferator-activated receptor alpha (PPARalpha). Primary rat hepatocyte cultures were treated with adenoviruses overexpressing adipose differentiation-related protein (ADRP) or adipose triacylglycerol lipase (ATGL) or treated with short interfering RNA (siRNA) targeted against ADRP. Subsequent effects on TAG metabolism and PPARalpha activity and target gene expression were determined. Overexpressing ADRP attenuated TAG hydrolysis, whereas siRNA-mediated knockdown of ADRP or ATGL overexpression resulted in enhanced TAG hydrolysis. Results from PPARalpha reporter activity assays demonstrated that decreasing TAG hydrolysis by ADRP overexpression resulted in a 35-60% reduction in reporter activity under basal conditions or in the presence of fatty acids. As expected, PPARalpha target genes were also decreased in response to ADRP overexpression. However, the PPARalpha ligand, WY-14643, was able to restore PPARalpha activity following ADRP overexpression. Despite its effects on PPARalpha, overexpressing ADRP did not affect PPARgamma activity. Enhancing TAG hydrolysis through ADRP knockdown or ATGL overexpression increased PPARalpha activity. These results indicate that TAG hydrolysis and the consequential release of fatty acids regulate PPARalpha activity.

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.

The adipose differentiation-related protein (ADFP)/adipophilin belongs to a family of PAT (for perilipin, ADFP, and TIP47) proteins that associate on the surface of lipid droplets (LDs). Except for LD association, a clear role for ADFP has not been found. We demonstrate that ADFP is transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) in mouse liver and rat and human hepatoma cells through a highly conserved direct repeat-1(DR-1) element. Although the ADFP mRNA is highly increased by a synthetic PPARalpha agonist, the ADFP protein is only substantially increased in cells containing LDs, such as hepatocytes incubated with fatty acids, and in livers of fasted mice. ADFP is induced by fasting even in the absence of a functional PPARalpha, in marked contrast to the PPARalpha target gene acyl-coenzyme A oxidase-1. Activation of LXRs, which stimulates LD formation through the activation of lipogenesis, does not affect ADFP mRNA levels. TIP47, another PAT member known to be expressed in liver, was unaffected by all treatments. This constitutively expressed PAT member seems to be less transcriptionally regulated than ADFP. These observations suggest that ADFP is primarily a fasting-induced protein in liver that coats the newly synthesized triacylglycerol-containing LDs formed during fasting.

The adipose differentiation-related protein (ADRP) was first characterized as a mRNA induced early during adipocyte differentiation (Jiang, H. P., and G. Serrero. 1992. Proc. Natl. Acad. Sci. USA. 89:7856-7860). The present study demonstrates that ADRP mRNA is expressed in a variety of tissues and cultured cell lines. Immunocytochemical examination revealed that ADRP localizes to neutral lipid storage droplets in cultured murine 3T3-L1 adipocytes, murine MA-10 Leydig cells, Chinese hamster ovary (CHO) fibroblasts, and human HepG2 hepatoma cells; the association of ADRP with lipid droplets was confirmed by subcellular fractionation of MA-10 Leydig cells. In addition to ADRP, steroidogenic cells and adipocytes express the perilipins, a family of lipid droplet-associated proteins that share a highly related sequence domain with ADRP. ADRP and perilipins co-localize on lipid droplets in MA-10 Leydig cells. While ADRP was found on small lipid droplets in 3T3-L1 preadipocytes and early differentiated adipocytes, it was absent in maturing adipocytes. In contrast, perilipins were absent early during differentiation, but were found on small and large lipid droplets at later stages. The transition in surface protein composition of adipocyte lipid droplets from ADRP to perilipins occurred 3 days after the initiation of differentiation when cells displayed co-localizatioin of both proteins on the same lipid droplets. The specific localization of adipose differentiation-related protein to lipid droplets in a wide variety of cells suggests that ADRP plays a role in management of neutral lipid stores.

PAT proteins (perilipin, adipophilin, and TIP47) are hypothesized to be critical regulators of lipid accumulation in eukaryotic cells. We investigated the developmental relationships between the expression of these proteins and cytoplasmic lipid droplet (CLD) accumulation in differentiating secretory epithelial cells in mouse mammary glands. Adipophilin (ADPH) specifically localized to CLD in differentiating and lactating mammary glands and was found exclusively in the secreted lipid droplet fraction of mouse milk. ADPH transcripts were selectively detected in secretory epithelial cells, and steady-state levels of both ADPH mRNA and protein increased during secretory differentiation in patterns consistent with functional linkage to CLD accumulation. TIP47 also was detected in secretory epithelial cells; however, it had a diffuse punctate appearance, and its mRNA and protein expression patterns did not correlate with CLD accumulation. Perilipin-positive adipose cells and steady-state levels of perilipin mRNA and protein decreased during mammary gland differentiation, suggesting a progressive loss of adipose lipid storage during this process. Collectively, these data demonstrate that increased ADPH expression is a specialized property of differentiated secretory epithelial cells and provide developmental evidence specifically linking increased ADPH expression to increased CLD accumulation. In addition, evidence is presented that the epithelial and adipose compartments of the mammary gland undergo concerted, developmentally regulated shifts in lipid metabolism that increase the availability of fatty acids necessary for lipid synthesis by milk-secreting cells.

The PAT family proteins, named after perilipin, adipophilin, and the tail-interacting protein of 47 kDa (TIP47), are implicated in intracellular lipid metabolism. They associate with lipid droplets, but how is completely unclear. From immunofluorescence studies, they are reported to be restricted to the outer membrane monolayer enveloping the lipid droplet and not to enter the core. Recently, we found another kind of lipid droplet-associated protein, caveolin-1, inside lipid droplets. Using freeze-fracture immunocytochemistry and electron microscopy, we now describe the distributions of perilipin and caveolin-1 and of adipophilin and TIP47 in lipid droplets of adipocytes and macrophages. All of these lipid droplet-associated proteins pervade the lipid droplet core and hence are not restricted to the droplet surface. Moreover, lipid droplets are surprisingly heterogeneous with respect to their complements and their distribution of lipid droplet-associated proteins. Whereas caveolin-1 is synthesized in the endoplasmic reticulum and is transferred to the lipid droplet core by inundating lipids during droplet budding, the PAT proteins, which are synthesized on free ribosomes in the cytoplasm, evidently target to the lipid droplet after it has formed. How the polar lipid droplet-associated proteins are accommodated among the essentially hydrophobic neutral lipids of the lipid droplet core remains to be determined.

Lipid droplets serve a vital function in many types of cells. In most cells of the body, cholesterol esters stored in lipid droplets are thought to serve as a source of cholesterol for membrane synthesis and repair when dietary cholesterol is scarce (1). In steroidogenic cells, cholesterol derived from the hydrolysis of stored cholesterol esters provides substrate for steroid hormone synthesis. In other cells, the cores of lipid droplets store primarily triacylglycerols that, following lipolysis, provide a source of fatty acids to drive ATP production; fatty acids and partially hydrolyzed neutral lipids also support the synthesis of phospholipids for membranes. In heart and skeletal muscle, fatty acids are a major energy substrate and triacylglycerols stored in lipid droplets are an important source of fatty acids during fasting. Stored neutral lipids are hydrolyzed by cytosolic neutral lipid lipases. Lipolysis has been best characterized in adipocytes, where the release of fatty acids from triacylglycerols is orchestrated by the coordinated activity of three lipases and several accessory factors (2). Adipose triglyceride lipase (ATGL) is the major triacylglycerol hydrolase; its activity is modulated by a coactivator, CGI-58, and an inhibitory protein, G0S2. The hydrolysis of triacylglycerol releases a fatty acid and diacylglycerol, which is further cleaved by hormone-sensitive lipase (HSL). The final fatty acid is released by monoglyceride lipase. The same trio of lipases can be detected in myocytes of heart and skeletal muscle; however, the identity of relevant lipases remains unclear for many tissues where neutral lipids are stored on a relatively small scale. The hydrolysis of stored neutral lipids is finely tuned by the interaction of lipid droplet-associated proteins with these cytosolic lipases. In chordates, some of the most abundant lipid droplet-associated proteins are perilipins. Perilipins are encoded by 5 genes, and are numbered according to the order of discovery (3). Lipid droplets in most tissues are coated by two or more members of the perilipin family of proteins. Perilipins 2 (formerly adipophilin or ADRP) and 3 (formerly TIP47) are ubiquitously expressed and hence, components of lipid droplets in most tissues (1). Perilipin 1 expression is restricted primarily to adipocytes of white and brown adipose tissue and to a lesser extent, steroidogenic cells of adrenal cortex, testes, and ovaries. Perilipin 4 (formerly S3-12) is expressed primarily in adipocytes of white adipose tissue. Perilipin 5 (formerly OXPAT, MLDP, or LSDP5) is expressed in myocytes of heart and skeletal muscle, and adipocytes of brown adipose tissue; each of these cells relies upon lipolysis to provide fatty acids to mitochondria for β-oxidation to drive the production of either ATP or heat. The perilipin composition of lipid droplets within a tissue is an important component of the regulation of lipolysis. In the current issue of the Journal of Lipid Research, two articles written by Wang et al. (4) and Pollak et al. (5) provide important insight into the control of lipolysis by perilipin 5 in cardiac myocytes. In each study, perilipin 5 was overexpressed in cardiac myocytes of mice, using the α-myosin heavy chain promoter. Increased perilipin 5 expression in these transgenic mice led to storage of excessive triacylglycerol in lipid droplets and cardiac steatosis (4, 5). Pollak et al. (5) noted that the observed steatosis resembled that of mice with a genetic deletion of ATGL (6), suggesting impaired hydrolysis of triacylglycerol; however, while ATGL-null mice succumb to early death due to heart failure, the hearts of perilipin 5 transgenic mice function relatively normally and the mice have normal life spans (4, 5). Investigation of the mechanisms behind the steatosis revealed that, while ATGL-null mice have reduced cytosolic lipase activity, lysates of cardiac tissue from perlipin 5 transgenic mice have elevated triacylglycerol hydrolase activity with corresponding elevated protein levels of ATGL and its coactivator CGI-58 (5). Further investigation revealed that lipid droplets isolated from Cos7 cells expressing perilipin 5 are less susceptible to the activity of either ATGL or HSL than lipid droplets from control cells (coated with perilipin 2), suggesting that perilipin 5 provides effective barrier function to reduce lipase access to lipid droplets (5). These studies are consistent with several previous studies characterizing reduced lipolysis in cultured cells in which perilipin 5 has been overexpressed (7–9). Conversely, the phenotype of perilipin 5-null mice reveals decreased triacylglycerol storage in cardiac myocytes accompanied by increased oxidation of fatty acids and increased oxidative stress (10), suggesting increased lipolysis. All of these studies support the idea that perilipin 5 functions similarly to perilipin 1 in reducing lipolysis. Thus, perilipin 5 may keep lipolysis in check to sequester potentially lipotoxic fatty acids and reduce β-oxidation. Stored triacylglycerols provide fuel for cardiac myocyte function, so a major remaining question is, “how does perilipin 5 permit or facilitate lipolysis in times of need?” One possible mechanism is through control of CGI-58 availability to activate ATGL. Previous studies have shown that perilipin 5 binds both ATGL and CGI-58 via carboxyl terminal binding sites (9, 11) while inhibiting lipolysis (9); however, both proteins cannot bind to the same molecule of perilipin 5 at the same time (11). This protein binding may serve to sequester CGI-58, preventing interaction with and activation of ATGL. Moreover, incubation of cells expressing ectopic perilipin 5 with forskolin to activate adenylyl cyclase and consequently protein kinase A increases lipolysis (9). Phosphorylation of perilipin 5 increases following the addition of forskolin to cells (9); however, the functional consequence of perilipin 5 phosphorylation is as yet unknown. It is tempting to speculate that phosphorylation of perilipin 5 may be part of the mechanism by which lipolysis is increased, perhaps by triggering the release of CGI-58 and ATGL from their binding sites on perilipin 5, in turn, facilitating interaction of the released proteins. The current work extends previous observations that perilipin 5 links lipid droplets to mitochondria. Histology of cardiac muscle from perilipin 5 transgenic mice reveals that perilipin 5-coated lipid droplets are closely surrounded by mitochondria (4, 5). Previous studies have revealed that a highly conserved carboxyl terminal sequence of perilipin 5 is required for recruitment of mitochondria to lipid droplets (12). The physical nature of the linking mechanism is as yet unknown, as are the consequences of the linkage. Importantly, Wang et al. (4) reveal that perilipin 5 overexpression impairs mitochondrial function, reducing the function of mitochondrial enzymes, and altering mitochondrial respiration, despite increases in mitochondrial size. The functional significance of the link between perilipin 5, lipid droplets, and mitochondria requires further study. Finally, both recent studies reveal that changes in gene expression accompany perilipin 5 overexpression in cardiac muscle (4, 5). The expression of peroxisome proliferator-activated receptor (PPAR)α, PPARγ coactivator 1α PGC1α, and PGC1β were decreased, as were mRNA levels for downstream target genes, including components of mitochondrial fatty acid import and oxidation. Prior studies have revealed that lipolysis of stored triacylglycerols provides ligands for PPARα, activating gene expression (13, 14). When lipolysis is decreased by reducing ATGL (13) or increasing perilipin 5 (4, 5), PPAR-mediated gene expression is impaired. Changes in the expression of genes encoding mitochondrial proteins are likely responsible for the observed diminished mitochondrial function in perilipin 5 transgenic mice (4); similar changes were observed in the hearts of ATGL-null mice and could be reversed by treatment of animals with a PPARα agonist (13). Interestingly, genes encoding proteins that mitigate oxidative stress responses were upregulated despite decreased β-oxidation (4). Protein levels of NF-E2-related factor 2, a transcription factor that induces antioxidant mechanisms in response to stress, were elevated in cardiac tissue of perilipin 5 transgenic mice, as were mRNA levels of downstream target genes. Thus, despite decreased lipolysis and flux of fatty acids to mitochondria, the cells show signs of increased oxidative stress. Further study is needed to elucidate these mechanisms. In summary, these studies have added to a growing body of evidence that perilipin 5 is a negative regulator of lipolysis in cardiac myocytes. By putting the brakes on lipolysis, perilipin 5 is an important component of the mechanisms controlling energy homeostasis.