Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes have distinct Plin gene members and additional protein forms derived from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to different types of lipid droplets that are composed of either triacylcerides or cholesterol esters. Furthermore, Plins with strong association preferences to triacylceride (or cholesterol ester) droplets can re-direct the relative intracellular triacylceride–cholesterol ester balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.
PLIN; ADRP; TIP47; LSDP5; S3-12; Triacylglyceride; Cholesterol; Fatty acids; Lipolysis
Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.
The PAT proteins, named after the three PLIN/ADRP/TIP47 (PAT) proteins, PLIN for perilipin, ADRP for adipose differentiation-related protein and TIP47 for tail-interacting protein of 47 kDa, now officially named M6PRBP1 for mannose-6-phosphate receptor binding protein 1, is a set of intracellular lipid droplet binding proteins. They are localized in the outer membrane monolayer enveloping lipid droplets and are involved in the metabolism of intracellular lipid. This work describes the cloning and sequencing of porcine PLIN and M6PRBP1 cDNAs, the chromosome mapping of these two genes, as well as the expression pattern of porcine PAT genes. Sequence analysis shows that the porcine PLIN cDNA contains an open reading frame of 1551 bp encoding 516 amino acids and that the porcine M6PRBP1 cDNA contains a coding region of 1320 bp encoding 439 amino acids. Comparison of PLIN and M6PRBP1 amino-acid sequences among various species reveals that porcine and bovine proteins are the most conserved. Porcine PLIN and M6PRBP1 genes have been mapped to pig chromosomes 7 and 2, respectively, by radiation hybrid analysis using the IMpRH panel. Expression analyses in pig showed a high expression of PLIN mRNA in adipose tissue, M6PRBP1 mRNA in small intestine, kidney and spleen and ADRP mRNA in adipose tissue, lung and spleen.
pig; PLIN; M6PRBP1; cDNA cloning; chromosome mapping; tissue expression pattern
Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, 14C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.
PLIN5; OXPAT; Perilipin; Lipid droplet; Fatty acid oxidation; Mitochondria
Perilipin (PLIN) is the major protein surrounding lipid droplets in adipocytes and regulates adipocyte metabolism by modulating the interaction between lipases and triacylglycerol stores. Associations between PLIN gene polymorphisms and obesity risk have been described, but interactions with dietary macronutrients require further attention. We examined whether dietary macronutrients (e.g. carbohydrates and fats) modulated the associations of the common PLIN 11482G > A (rs894160) single nucleotide polymorphism with obesity. We studied a population-based sample of Caribbean-origin Hispanics (n = 920, aged 45-74 y) living in the Boston area. Obesity measures (waist and hip circumference, BMI) did not differ between GG subjects and carriers of the A allele (GA and AA). In multivariate linear regression models, we found a significant interaction between complex carbohydrate intake as a continuous variable and PLIN 11482 G > A genotype for waist circumference (P = 0.002). By dichotomizing complex carbohydrate intake, we found significantly different effects across PLIN 11482G > A genotypes. When complex carbohydrate intake was <144 g/d, waist circumference was larger in PLIN 11482G > A carriers (P = 0.024). Conversely, when complex carbohydrate intake was ≥144 g/d, waist and hip circumferences were less in PLIN 11482G > A carriers (P < 0.05). These interactions were not found for simple sugars or total carbohydrates. We identified a significant gene-diet interaction associated with obesity at the PLIN locus. In subjects with higher complex carbohydrate intake, the minor allele was protective against obesity, whereas in subjects with lower carbohydrate intake, the minor allele was associated with increased obesity. These interactions may be relevant to dietary management of obesity.
Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.
Professor Steve O’Rahilly is one of the UK’s most renowned clinical researchers. He made his reputation by combining clinical practice with scientific and clinical studies focused on understanding the causes and consequences of obesity and insulin resistance. Here, he talks about his research philosophy, and his wider role as a spokesman for obesity research.
Perilipin1, a lipid droplet associated protein has an important role in the regulation of lipolysis and lipid storage in adipocytes. Perilipin1 is also expressed in foam cells of atheroma plaques and could therefore play a role in the accumulation of lipids in arterial wall and in the development of atherosclerosis. The aim of the study was to investigate this possible role of perilipin1 in atherogenesis.
Mice deficient in perilipin1 (Plin1-/-) were crossed with Ldlr-/- mice. Ldlr-/- and Plin1-/- Ldlr-/- mice received an atherogenic diet during 10 or 20 weeks. Blood pressure and plasma lipids concentrations were measured. Aortas were collected at the end of the atherogenic diet periods for quantification of atheroma lesions (en face method), histological and immunohistological studies
Ldlr-/- and Plin1-/- Ldlr-/- mice had comparable blood pressure and plasma lipids levels. Plin1-/- Ldlr-/- mice had a lower body weight and decreased adiposity. The atherosclerotic lesion area in Plin1-/-Ldlr-/- mice was moderately increased after 10 weeks of atherogenic diet (ns) and significantly higher after 20 weeks (p < 0.01). Histology of atheroma plaques was comparable with no sign of increased inflammation in Plin1-/- Ldlr-/- mice.
Perilipin1 ablation in mice results in increased atherosclerosis independently of modifications of risk factors such as raised blood pressure or plasma lipids levels. These data strongly support an atheroprotective role for perilipin1.
perilipin1; atherosclerosis; lipids
Human aging is associated with a progressive loss of muscle mass and strength and a concomitant fat accumulation in form of inter-muscular adipose tissue, causing skeletal muscle function decline and immobilization. Fat accumulation can also occur as intra-muscular triglycerides (IMTG) deposition in lipid droplets, which are associated with perilipin proteins, such as Perilipin2 (Plin2). It is not known whether Plin2 expression changes with age and if this has consequences on muscle mass and strength. We studied the expression of Plin2 in the vastus lateralis (VL) muscle of both healthy subjects and patients affected by lower limb mobility limitation of different age. We found that Plin2 expression increases with age, this phenomenon being particularly evident in patients. Moreover, Plin2 expression is inversely correlated with quadriceps strength and VL thickness. To investigate the molecular mechanisms underpinning this phenomenon, we focused on IGF-1/p53 network/signalling pathway, involved in muscle physiology. We found that Plin2 expression strongly correlates with increased p53 activation and reduced IGF-1 expression. To confirm these observations made on humans, we studied mice overexpressing muscle-specific IGF-1, which are protected from sarcopenia. These mice resulted almost negative for the expression of Plin2 and p53 at two years of age. We conclude that fat deposition within skeletal muscle in form of Plin2-coated lipid droplets increases with age and is associated with decreased muscle strength and thickness, likely through an IGF-1- and p53-dependent mechanism. The data also suggest that excessive intramuscular fat accumulation could be the initial trigger for p53 activation and consequent loss of muscle mass and strength.
Mature white adipocytes contain a characteristic unilocular lipid droplet. However, the molecular mechanisms underlying unilocular lipid droplet formation are poorly understood. We previously showed that Fsp27, an adipocyte-specific lipid droplet-associated protein, promotes lipid droplet growth by initiating lipid exchange and transfer. Here, we identify Perilipin1 (Plin1), another adipocyte-specific lipid droplet-associated protein, as an Fsp27 activator. Plin1 interacts with the CIDE-N domain of Fsp27 and markedly increases Fsp27-mediated lipid exchange, lipid transfer and lipid droplet growth. Functional cooperation between Plin1 and Fsp27 is required for efficient lipid droplet growth in adipocytes, as depletion of either protein impairs lipid droplet growth. The CIDE-N domain of Fsp27 forms homodimers and disruption of CIDE-N homodimerization abolishes Fsp27-mediated lipid exchange and transfer. Interestingly, Plin1 can restore the activity of CIDE-N homodimerization-defective mutants of Fsp27. We thus uncover a novel mechanism underlying lipid droplet growth and unilocular lipid droplet formation that involves the cooperative action of Fsp27 and Plin1 in adipocytes.
Adipocytes store lipid in spherical droplets whose size is determined by lipid exchange and transfer proteins. Sun et al. show that Perilipin1 promotes the growth of lipid droplets by activating the lipid transfer protein Fsp27.
In adipose tissue, access of adipose triglyceride and hormone-sensitive lipases (ATGL and HSL) to the lipid droplet depends on PLIN1 phosphorylation, however, PLIN1 is not expressed in skeletal muscle and the phosphorylation of the expressed PLINs has yet to be investigated. Further, direct interactions between skeletal muscle PLINs and HSL are unknown. We investigated the isolated and combined effects of epinephrine and contraction on PLIN-to-lipase interactions as well as phosphorylation. Isolated rat solei were assigned to one of four 30 min in vitro conditions (25°C): (1) rest; (2) intermittent tetanic stimulation (60 Hz for 150 msec; train rate 20/min); (3) 5 nmol/L epinephrine; (4) intermittent tetanic stimulation and 5 nmol/L epinephrine. Immunoprecipitation of serine phosphorylated proteins followed by Western blotting for PLIN2, PLIN3, PLIN5, revealed that only PLIN2 is not phosphorylated under any of the experimental conditions. This is the first study to show that in whole rat skeletal muscle PLIN3 and PLIN5 are serine phosphorylated. The degree of serine phosphorylation remained unchanged following adrenergic and/or contractile stimulation. Oil red O staining of muscle sections for lipid content shows a significant decrease following each condition, confirming lipolysis occurred (P < 0.05). PLIN2, 3, and 5 all interact with HSL and ATGL, but these interactions were unchanged following treatments. Our results show that in skeletal muscle, PLIN2 is not serine phosphorylated at rest or with lipolytic stimulation and that while PLIN3, PLIN5 are serine phosphorylated at rest, the degree of phosphorylation does not change with lipolytic stimulation.
ADRP; exercise; lipolysis; OXPAT; TIP47
Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin−/− mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin−/− mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin−/− WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.
Cytosolic lipid droplets (LDs), which are now recognized as multifunctional organelles, accumulate in leukocytes under various inflammatory conditions. However, little is known about the characteristic features of LDs in neutrophils. In this study, we show that perilipin-3 (PLIN3; formerly called TIP47) is involved in LD formation and the inflammatory response in HL-60-derived neutrophils. HL-60, a promyelocytic cell line, was differentiated into neutrophils via treatment with all-trans retinoic acid. After differentiation, cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g-LPS), a major pathogen in adult periodontitis. When HL-60-derived neutrophils were stimulated with P.g-LPS, LDs increased in both number and size. In the differentiated cells, PLIN3 was induced while PLIN1, PLIN2 and PLIN5 were not detected. PGE2 production and the PLIN3 protein level were increased by the P.g-LPS treatment of the cells in a dose-dependent manner. When PLIN3 was down-regulated with siRNA treatment, LDs essentially disappeared and the level of PGE2 secreted in the cell culture medium decreased by 65%. In addition, the suppression of PLIN3 repressed the PGE2 producing enzymes; i.e., microsomal PGE synthase-1, -2 and cyclooxygenase-2. These findings indicate that PLIN3 has a pivotal role in LD-biogenesis in HL-60-derived neutrophils, and that PLIN3 is associated with the synthesis and secretion of PGE2.
Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.
Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.
Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5). Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron– in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.
There is increasing interest in identifying new pathways and candidate genes that confer susceptibility to osteoporosis. There is evidence that adipogenesis and osteogenesis may be related, including a common bone marrow progenitor cell for both adipocytes and osteoblasts. Perilipin 1 (PLIN1) and Perilipin 4 (PLIN4) are members of the PATS family of genes and are involved in lipolysis of intracellular lipid deposits. A previous study reported gender-specific associations between one polymorphism of PLIN1 and bone mineral density (BMD) in a Japanese population. We hypothesized that polymorphisms in PLIN1 and PLIN4 would be associated with bone measures in adult Caucasian participants of the Framingham Osteoporosis Study (FOS). We genotyped 1,206 male and 1,445 female participants of the FOS for four single-nucleotide polymorphism (SNPs) in PLIN1 and seven SNPs in PLIN4 and tested for associations with measures of BMD, bone ultrasound, hip geometry, and height. We found several gender-specific significant associations with the measured traits. The association of PLIN4 SNP rs8887, G>A with height in females trended toward significance after simulation testing (adjusted P = 0.07) and remained significant after simulation testing in the combined-sex model (adjusted P = 0.033). In a large study sample of men and women, we found a significant association between one SNP in PLIN4 and height but not with bone traits, suggesting that PATS family genes are not important in the regulation of bone. Identification of genes that influence human height may lead to a better understanding of the processes involved in growth and development.
Perilipin 1; Perilipin 4; Bone mineral density; Bone geometry; Framingham Osteoporosis Study
Interventions on macrophages/foam cells to redirect intracellular cholesterol towards efflux pathways could become a very valuable addition to our therapeutic arsenal against atherosclerosis. However, certain manipulations of the cholesteryl ester cycle, such as the inhibition of ACAT1, an ER-resident enzyme that re-esterifies cholesterol, are not well tolerated. Previously we showed that targeting perilipin-2 (PLIN2), a major lipid droplet (LD)-associated protein in macrophages, prevents foam cell formation and protects against atherosclerosis. Here we have assessed the tolerance of PLIN2-deficient bone marrow derived macrophages (BMM) to several lipid loading conditions similar to the found during atherosclerosis development, including exposure to modified low-density lipoprotein (mLDL) and 7-ketocholesterol (7-KC), a free cholesterol (FC) metabolite, in media with or without cholesterol acceptors. BMM isolated from mice that do or do not express PLIN2 were tested for apoptosis (TUNEL and cleaved caspase-3), ER stress (CHOP induction and XBP-1 splicing), and inflammation (TNF-α and IL-6 mRNA levels). Like in other cell types, PLIN2 deficiency impairs LD buildup in BMM. However, while most stress parameters were elevated in macrophages under ACAT inhibition and 7-KC loading, PLIN2 inactivation was well tolerated. The data support the safety of targeting PLIN2 to prevent foam cell formation and atherosclerosis.
Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the β-adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A–phosphorylated HSL and perilipin A.
lipolysis; adipocytes; ADRP/adipophilin; HSL; lipid storage droplets
Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3′,5′-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.
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.
perilipin; adipocyte differentiation-related protein; adipophilin; tail-interacting protein of 47 kDa
In many species, the lactating mammary gland is one of the most lipogenic organs of the body. The majority of the lipid produced during lactation is secreted into milk by a novel process of membrane envelopment of cytoplasmic lipid droplets (CLDs). Adipophilin (ADRP/ADPH/PLIN2), a member of the perilipin (PAT) family of lipid droplet proteins, is hypothesized to play a pivotal role in both formation and secretion of milk lipids. Production of milk lipids is the only known example of CLD secretion, and the only process in which PAT family members undergo secretion. This review discusses emerging data about the structural and functional properties of adipophilin that determine its physiological actions and mediate its effects on milk lipid formation and secretion.
The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.
lipid droplet; lipolysis; lipogenesis; perilipin; PAT proteins; adipocyte