The role of apolipoprotein A5 in obesity and the metabolic syndrome

Apolipoprotein A5 (apoA5) has an important role in lipid metabolism, specifically for triglyceride‐rich lipoproteins. Recently, evidence has emerged for an association between genetic variability at the APOA5 locus and increased risk of obesity and metabolic syndrome. However, its mechanism of action remains to be fully elucidated. Importantly, an intracellular role of apoA5 has been indicated since apoA5 is associated with cytoplasmic lipid droplets and affects intrahepatic triglyceride accumulation, as well as affecting intravascular triglyceride metabolism. Given that adipocytes provide the largest storage depot for energy in the form of triglyceride within the lipid droplets, and play a crucial role in the development of obesity, we highlight recent findings discussing the interaction of apoA5 with adipocytes or adipose tissue, indicating that apoA5 may act as a novel regulator to modulate triglyceride storage in adipocytes. We review the association of APOA5 gene polymorphisms with obesity and metabolic syndrome, and propose potential mechanisms by which apoA5 may increase susceptibility to these conditions. This review provides new insights into the physiological role of apoA5 and identifies a potential therapeutic target for obesity and associated disorders.     

Possible role for intracellular cholesteryl ester transfer protein in adipocyte lipid metabolism and storage

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.     

Establishment of a clonal cell line that differentiates into adipose cells in vitro

Summary From the fibroblastic cells of murine mammary tumor tissue we isolated a clonal cell line that could be induced to differentiate into fat-accumulating cells in vitro. Differentiation began after the cultures had reached confluence and was accompanied by (a) an increase in the incorporation of sodium acetate into the triglyceride (TG) fraction of cellular lipids, (b) a more than 50-fold increase in cellular TG content per milligram cell-layer protein basis and (c) the cessation of cellular DNA synthesis. The addition of insulin to the culture medium enhanced lipid accumulation and increased the fraction of cells that differentiated into adipocytes. When insulin (5 to 10 μg/ml) was added exogenously, 80% or more of the cells were induced to differentiate into mature adipocytes within 2 weeks. This cell line can be used as a model for mammalian cell differentiation and also as a convenient material for the study of lipid metabolism in adipocytes. This work was supported by Grants in Aid from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare of Japan.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

Lipid droplet-organelle interactions; sharing the fats

Lipid droplets (LDs) are key cellular organelles involved in lipid storage and mobilisation. While the major signalling cascades and many of the regulators of lipolysis have been identified, the cellular interactions involved in lipid mobilisation and release remain largely undefined. In non-adipocytes, LDs are small, mobile and interact with other cellular compartments. In contrast, adipocytes primarily contain very large, immotile LDs. The striking morphological differences between LDs in adipocytes and non-adipocytes suggest that key differences must exist in the manner in which LDs in different cell types interact with other organelles. Recent studies have highlighted the complexity of LD interactions, which can be both homotypic, with each other, and heterotypic, with other organelles. The molecules involved in these interactions are also now emerging, including Rab proteins, key regulators of membrane traffic, and caveolin, an integral membrane protein providing a functional link between the cell surface and LDs. Here we summarise recent insights into the cell biology of the LD particularly focussing on the homotypic and heterotypic interactions in both adipocytes and non-adipocytes. We speculate that these interactions may involve inter-organelle membrane contact sites or a hemi-fusion type mechanism to facilitate lipid transfer.     

Identification of the lipid droplet targeting domain of the Cidea protein

Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domains. Expression of full-length Cidea in undifferentiated 3T3-L1 cells or COS-1 cells increases total cellular triglyceride and strikingly alters the morphology of lipid droplets by enhancing their size and reducing their number. Remarkably, both lipid droplet binding and increased triglyceride accumulation are also elicited by expression of only the carboxy-terminal 104 amino acids, indicating this small domain directs lipid droplet targeting and triglyceride shielding. However, unlike the full-length protein, expression of the carboxy-terminus causes clustering of small lipid droplets but not the formation of large droplets, identifying a novel function of the N terminus. Furthermore, human Cidea promotes lipid storage via lipolysis inhibition, as the expression of human Cidea in fully differentiated 3T3-L1 adipocytes causes a significant decrease in basal glycerol release. Taken together, these data indicate that the carboxy-terminal domain of Cidea directs lipid droplet targeting, lipid droplet clustering, and triglyceride accumulation, whereas the amino terminal domain is required for Cidea-mediated development of enlarged lipid droplets.     

Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes

The spatiotemporal dynamics of triglyceride (TG) storage in unilocular adipocytes are not well understood. Here we applied ex vivo technology to study trafficking and metabolism of fluorescent fatty acids in adipose tissue explants. Live imaging revealed multiple cytoplasmic nodules surrounding the large central lipid droplet (cLD) of unilocular adipocytes. Each cytoplasmic nodule harbors a series of closely associated cellular organelles, including micro–lipid droplets (mLDs), mitochondria, and the endoplasmic reticulum. Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG. This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte. This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.     

The role of mannosylated enzyme and the mannose receptor in enzyme replacement therapy

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal-/-) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide-type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)-positive J774E cells. After intraperitoneal injection into lal-/- mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal-/- mice produced normalization of hepatic color, decreased liver weight (50%-58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal-/-;MMR-/-) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor-independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR.     

Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes

Previous reports attributed cholesteryl ester transfer protein (CETP)-mediated HDL cholesteryl ester (CE) selective uptake to the CETP-mediated transfer of CE from HDL to newly secreted apolipoprotein B-containing lipoproteins, which are then internalized by the LDL receptor (LDL-R). CETP has also been implicated in the remodeling of HDL, which renders it a better substrate for selective uptake by scavenger receptor class B type I (SR-BI). However, CETP-mediated selective uptake of HDL3-derived CE was not diminished in LDL-R null adipocytes, SR-BI null adipocytes, or in the presence of the receptor-associated protein. We found that monensin treatment or energy depletion of the SW872 liposarcoma cells with 2-deoxyglucose and NaN3 had no effect on CETP-mediated selective uptake, demonstrating that endocytosis is not required. This is supported by data indicating that CETP transfers CE into a compartment from which it can be extracted by unlabeled HDL. CETP could also mediate the selective uptake of HDL3-derived triacylglycerol (TG) and phospholipid (PL). The CETP-specific kinetics for TG and CE uptake were similar, and both reached saturation at approximately 5 microg/ml HDL. In contrast, CETP-specific PL uptake did not attain saturation at 5 microg/ml HDL and was approximately 6-fold greater than the uptake of CE. We propose two possible mechanisms to account for the role of CETP in selective uptake.     

Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites

Lipid droplets (LDs) are dynamic cellular organelles that control many biological processes. However, molecular components determining LD growth are poorly understood. Genetic analysis has indicated that Fsp27, an LD-associated protein, is important in controlling LD size and lipid storage in adipocytes. In this paper, we demonstrate that Fsp27 is focally enriched at the LD-LD contacting site (LDCS). Photobleaching revealed the occurrence of lipid exchange between contacted LDs in wild-type adipocytes and Fsp27-overexpressing cells but not Fsp27-deficient adipocytes. Furthermore, live-cell imaging revealed a unique Fsp27-mediated LD growth process involving a directional net lipid transfer from the smaller to larger LDs at LDCSs, which is in accordance with the biophysical analysis of the internal pressure difference between the contacting LD pair. Thus, we have uncovered a novel molecular mechanism of LD growth mediated by Fsp27.     

Fat-specific Protein 27 Undergoes Ubiquitin-dependent Degradation Regulated by Triacylglycerol Synthesis and Lipid Droplet Formation

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with β-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during β-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.     

Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets

The lipid fraction ("fat cake") of rat epididymal adipocytes contains a prominent phosphoprotein (62 kDaapp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) that is multiply phosphorylated by cAMP-dependent protein kinase in vivo, at which point it migrates as a 65/67-kDaapp doublet by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is by far the most heavily radiolabeled protein in the cell. Western blot analysis of various tissues with immunopurified antibodies purified from antisera raised against the 62-kDa species suggests that the protein is specific for adipocytes. This protein, which we term perilipin, is found in differentiated cultured 3T3-L1 adipocytes, but not in their precursor 3T3-L1 fibroblasts. Immunocytochemical studies with specific antiserum shows that the perilipin is closely associated with the periphery of lipid storage droplets in cultured adipocytes. Given its adipocyte specificity, acute regulation by hormones, and subcellular location, we speculate that perilipin plays a role in the specialized lipid storage function of adipocytes.     

ASP enhances in situ lipoprotein lipase activity by increasing fatty acid trapping in adipocytes

Acylation-stimulating protein (ASP) increases triglyceride (TG) storage (fatty acid trapping) in adipose tissue and plays an important role in postprandial TG clearance. We examined the capacity of ASP and insulin to stimulate the activity of lipoprotein lipase (LPL) and the trapping of LPL-derived nonesterified fatty acid (NEFA) in 3T3-L1 adipocytes. Although insulin increased total LPL activity (secreted and cell-associated; P < 0.001) in 3T3-L1 adipocytes, ASP moderately stimulated secreted LPL activity (P = 0.04; 5% of total LPL activity). Neither hormone increased LPL translocation from adipocytes to endothelial cells in a coculture system. However, ASP and insulin increased the V(max) of in situ LPL activity ([(3)H]TG synthetic lipoprotein hydrolysis and [(3)H]NEFA incorporation into adipocytes) by 60% and 41%, respectively (P 相似文献   

Endogenous adipocyte apolipoprotein E is colocalized with caveolin at the adipocyte plasma membrane

Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Recently, endogenous expression of apoE in adipocytes has been shown to play an important role in adipocyte lipoprotein metabolism and gene expression consistent with a nonsecreted cellular itinerary for apoE. We designed studies to evaluate if adipocyte apoE was retained as a constituent protein in adipocytes and to identify a cellular retention compartment. Using confocal microscopy, coimmunoprecipitation, and sucrose density cellular fractionation, we establish that endogenous apoE shares a cellular itinerary with the constituent protein caveolin-1. Altering adipocyte caveolar number by modulating cellular cholesterol flux or altering caveolin expression regulates the distribution of cellular apoE between cytoplasmic and plasma membrane compartments. A mechanism for colocalization of apoE with caveolin was established by demonstrating a noncovalent interaction between an aromatic amino acid-enriched apoE N-terminal domain with the caveolin scaffolding domain. Absent apoE expression in adipocytes alters caveolar lipid composition. These observations provide evidence for an interaction between two proteins involved in cellular lipid metabolism in a cell specialized for lipid storage and flux, and rationalize a biological basis for the impact of adipocyte apoE expression on adipocyte lipoprotein metabolism.     

The molecular mechanism of acylation stimulating protein regulation of adipophilin and perilipin expression: involvement of phosphoinositide 3-kinase and phospholipase C

The novel adipokine acylation stimulating protein (ASP) is involved in lipid metabolism and obesity‐related disorders. Adipophilin and perilipin, two members of the lipid droplet protein family, participate not only in fat storage within adipocytes, but also in ectopic lipid deposition in the form of cytoplasmic triglyceride (TG) droplets within many types of mammalian cells. During differentiation to mature adipocytes, mechanisms controlling the synthesis and turnover of these lipid droplet proteins are only partially understood, the mechanisms regulating gene/protein expression as yet unidentified. In our previous study, ASP has been shown to regulate adipophilin and perilipin expression to facilitate TG synthesis during 3T3‐L1 cell differentiation. Our aim in this study was to provide insight into the physiological importance of phosphoinositide 3‐kinase (PI3K) and phospholipase C (PLC) in ASP‐triggered alteration of adipophilin and perilipin expression. We found that acute (2.5 h) inhibition of PLC or PI3K results in a decrease in mRNA and protein of perilipin and adipophilin at any time during differentiation. The fact that there is such a rapid change even with mRNA levels suggests a rapid turnover of both mRNA and protein independent of a direct ASP effect. Also, the presence of these inhibitors blocked the ASP stimulatory effects with a maximal decrease in gene and protein expression of adipophilin (?45% and ?60%, respectively, P < 0.01) and perilipin (?96% and ?63%, respectively, P < 0.01 and P < 0.05). These findings provide further understanding of the adipogenic properties of ASP in adipocytes. J. Cell. Biochem. 112: 1622–1629, 2011. © 2011 Wiley‐Liss, Inc.     

Intracellular role of exchangeable apolipoproteins in energy homeostasis,obesity and non‐alcoholic fatty liver disease

Exchangeable apolipoproteins play an important role in systemic lipid metabolism, especially for lipoproteins with which they are associated. Recently, emerging evidence has suggested that exchangeable apolipoproteins, such as apolipoprotein A4 (apoA4), apolipoprotein A5 (apoA5), apolipoprotein C3 (apoC3) and apolipoprotein E (apoE), also exert important effects on intracellular lipid homeostasis. There is a close link between lipid metabolism in adipose tissue and liver because the latter behaves as the metabolic sensor of dysfunctional adipose tissue and is a main target of lipotoxicity. Given that the energy balance between these two major lipogenic organs is intimately involved in the pathogenesis of obesity and non‐alcoholic fatty liver disease (NAFLD), we here review recent findings concerning the intracellular function of exchangeable apolipoproteins in triglyceride metabolism in adipocytes and hepatocytes. These apolipoproteins may act as mediators of crosstalk between adipose tissue and liver, thus influencing development of obesity and hepatosteatosis. This review provides new insights into the physiological role of exchangeable apolipoproteins and identifies latent targets for therapeutic intervention of obesity and its related disorders.     

Fat-specific Protein 27 (FSP27) Interacts with Adipose Triglyceride Lipase (ATGL) to Regulate Lipolysis and Insulin Sensitivity in Human Adipocytes

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120–220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120–220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.     

The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism

Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK) signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL) and acetyl-CoA carboxylase (ACC), in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT), suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA), which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through AMPK and/or PKA pathway(s) and improves glucose intolerance caused by obesity.     

CGI-58 facilitates the mobilization of cytoplasmic triglyceride for lipoprotein secretion in hepatoma cells

Comparative Gene Identification-58 (CGI-58) is a member of the alpha/beta-hydrolase family of proteins. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman syndrome, a rare autosomal recessive genetic disease in which excessive triglyceride (TG) accumulation occurs in multiple tissues. In this study, we investigated the role of CGI-58 in cellular lipid metabolism in several cell models and discovered a role for CGI-58 in promoting the packaging of cytoplasmic TG into secreted lipoprotein particles in hepatoma cells. Using both gain-of-function and loss-of-function approaches, we demonstrate that CGI-58 facilitates the depletion of cellular TG stores without altering cellular cholesterol or phospholipid accumulation. This depletion of cellular TG is attributable solely to augmented hydrolysis, whereas TG synthesis was not affected by CGI-58. Furthermore, CGI-58-mediated TG hydrolysis can be completely inhibited by the known lipase inhibitors diethylumbelliferyl phosphate and diethyl-p-nitrophenyl phosphate, but not by p-chloro-mercuribenzoate. Intriguingly, CGI-58-driven TG hydrolysis was coupled to increases in both fatty acid oxidation and secretion of TG. Collectively, this study reveals a role for CGI-58 in coupling lipolytic degradation of cytoplasmic TG to oxidation and packaging into TG-rich lipoproteins for secretion in hepatoma cells.     

Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells.