Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of perilipin null mice (Peri-/- MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri Adelta1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri Adelta1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri Adelta1-6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri-/- MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri-/- MEF adipocytes expressing Peri Adelta1-6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes.     

Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes

In adipocytes, lipid droplet (LD) size reflects a balance of triglyceride synthesis (lipogenesis) and hydrolysis (lipolysis). Perilipin A (Peri A) is the most abundant phosphoprotein on the surface of adipocyte LDs and has a crucial role in lipid storage and lipolysis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major rate-determining enzymes for lipolysis in adipocytes. Each of these proteins (Peri A, ATGL, and HSL) has been demonstrated to regulate lipid storage and release in the adipocyte. However, in the absence of protein kinase A (PKA) stimulation (basal state), the lipases (ATGL and HSL) are located mainly in the cytoplasm, and their contribution to basal rates of lipolysis and influence on LD size are poorly understood. In this study, we utilize an adenoviral system to knockdown or overexpress ATGL and HSL in an engineered model system of adipocytes in the presence or absence of Peri A. We are able to demonstrate in our experimental model system that in the basal state, LD size, triglyceride storage, and fatty acid release are mainly influenced by the expression of ATGL. These results demonstrate for the first time the relative contributions of ATGL, HSL, and Peri A on determination of LD size in the absence of PKA stimulation.     

Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.     

Serum amyloid A induces lipolysis by downregulating perilipin through ERK1/2 and PKA signaling pathways

Serum amyloid A (SAA) is not only an apolipoprotein, but also a member of the adipokine family with potential to enhance lipolysis. The purpose of this study was to explore how SAA facilitates lipolysis in porcine adipocytes. We found that SAA increased the phosphorylation of perilipin and hormone-sensitive lipase (HSL) after 12-h treatment and decreased perilipin expression after 24-h treatment, and these effects were prevented by extracellular signal-regulated kinase (ERK) or protein kinase A (PKA) inhibitors in primary adipocyte cell culture. SAA treatment decreased HSL and adipose triglyceride lipase (ATGL) expression. SAA treatment also activated ERK and PKA by increasing the phosphorylation of these kinases. Moreover, SAA significantly increased porcine adipocyte glycerol release and lipase activity, which was inhibited by either ERK (PD98059) or PKA (H89) inhibitors, suggesting that ERK and PKA were involved in mediating SAA enhanced lipolysis. SAA downregulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) mRNA, which was reversed by the ERK inhibitor. We performed a porcine perilipin promoter assay in differentiated 3T3-L1 adipocytes and found that SAA reduced the porcine perilipin promoter specifically through the function of its PPAR response element (PPRE), and this effect was reversed by the ERK inhibitor. These findings demonstrate that SAA-induced lipolysis is a result of downregulation of perilipin and activation of HSL via ERK/PPARγ and PKA signaling pathways. The finding could lead to developing new strategies for reducing human obesity.     

脂肪组织甘油三酯水解酶参与脂肪分解调控

循环中游离脂肪酸增高与肥胖、胰岛素抵抗和2型糖尿病密切相关,其主要来源于脂肪细胞内甘油三酯水解.调控脂肪分解的脂肪酶主要包括激素敏感脂肪酶(hormone-sensitive lipase,HSL)和最近发现的脂肪组织甘油三酯水解酶(adipose triglyceride lipase,ATGL),后者主要分布在脂肪组织,特异水解甘油三酯为甘油二酯,其转录水平受多种因素调控.CGI-58(属于α/β水解酶家族蛋白),可以活化ATGL,基础条件下该蛋白和脂滴包被蛋白(perilipin)紧密结合于脂滴表面,蛋白激酶A激活刺激脂肪分解时,CGI-58与perilipin分离,进而活化ATGL.     

雌激素受体关联受体α 调节脂肪细胞甘油三酯分解

雌激素受体关联受体a (Estrogen-related receptor a,ERRα) 是调控机体能量代谢的关键转录调控因子,也是脂肪生成的关键调控者。为研究ERRα对脂肪细胞甘油三酯分解的影响及其分子机制,分化的猪脂肪细胞在PKA (Protein kinase A) 或/和ERK (Extracellular signal-related kinase) 抑制剂预处理和不处理的情况下,再用Ad-ERRα侵染或XCT790处理48 h。通过测定脂肪细胞中甘油三酯浓度和培养液中的甘油释放量分析脂肪细胞的脂解变化;Western blotting方法检测PPARγ (Peroxisome proliferator-activated receptor γ,PPARγ)、perilipin A、p-perilipin A、HSL (Hormone sensitive lipase,HSL) 和ATGL (Adipose triglyceride lipase,ATGL) 蛋白表达。结果显示,ERRα显著促进猪脂肪细胞分化及甘油三酯积累,同时促进了甘油三酯水解;分别及同时阻断PKA和ERK通路并不影响ERRα对脂肪细胞甘油释放的促进作用;ERRα显著上调HSL、ATGL、PPARγ及perilipin A蛋白表达,但p-perilipin A水平并未发生变化。推测过量表达ERRα可能导致HSL和ATGL蛋白表达上调并促进甘油三酯水解,从而为脂肪细胞分化提供更多的游离脂肪酸 (Free fat acid,FFA) 作为甘油三酯合成周转的底物。     

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.     

Cytoplasmic receptor-interacting protein 140 (RIP140) interacts with perilipin to regulate lipolysis

Receptor-interacting protein 140 (RIP140) is abundantly expressed in mature adipocyte and modulates gene expression involved in lipid and glucose metabolism. Protein kinase C epsilon and protein arginine methyltransferase 1 can sequentially stimulate RIP140 phosphorylation and then methylation, thereby promoting its export to the cytoplasm. Here we report a lipid signal triggering cytoplasmic accumulation of RIP140, and a new functional role for cytoplasmic RIP140 in adipocyte to regulate lipolysis. Increased lipid content, particularly an elevation in diacylglycerol levels, promotes RIP140 cytoplasmic accumulation and increased association with lipid droplets (LDs) by its direct interaction with perilipin. By interacting with RIP140, perilipin more efficiently recruits hormone-sensitive lipase (HSL) to LDs and enhances adipose triglyceride lipase (ATGL) forming complex with CGI-58, an activator of ATGL. Consequentially, HSL can more readily access its substrates, and ATGL is activated, ultimately enhancing lipolysis. In adipocytes, blocking cytoplasmic RIP140 accumulation reduces basal and isoproterenol-stimulated lipolysis and the pro-inflammatory potential of their conditioned media (i.e. activating NF-κB and inflammatory genes in macrophages). These results show that in adipocytes with high lipid contents, RIP140 increasingly accumulates in the cytoplasm and enhances triglyceride catabolism by directly interacting with perilipin. The study suggests that reducing nuclear export of RIP140 might be a useful means of controlling adipocyte lipolysis.     

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.     

Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes

Lipolysis is primarily regulated by protein kinase A (PKA), which phosphorylates perilipin and hormone-sensitive lipase (HSL), and causes translocation of HSL from cytosol to lipid droplets in adipocytes. Perilipin coats lipid droplet surface and assumes to prevent lipase access to triacylglycerols, thus inhibiting basal lipolysis; phosphorylated perilipin facilitates lipolysis on PKA activation. Here, we induced lipolysis in primary rat adipocytes by inhibiting protein serine/threonine phosphatase with specific inhibitors, okadaic acid and calyculin. The incubation with calyculin promotes incorporation of 32Pi into perilipins, thus, confirming that perilipin is hyperphosphorylated. The lipolysis response to calyculin is gradually accompanied by increased accumulation of phosphorylated perilipin A in a concentration- and time-responsive manner. When perilipin phosphorylation is abrogated by the addition of N-ethylmaleimide, lipolysis ceases. Different from a considerable translocation of HSL upon PKA activation with isoproterenol, calyculin does not alter HSL redistribution in primary or differentiated adipocytes, as confirmed by both immunostaining and immunoblotting. Thus, we suggest that inhibition of the phosphatase by calyculin activates lipolysis via promoting perilipin phosphorylation rather than eliciting HSL translocation in adipocytes. Further, we show that when the endogenous phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin to the hyperphosphorylated species, and induces enhanced lipolysis. Apparently, as PKA phosphorylates perilipin and stimulates lipolysis, the phosphatase acts to dephosphorylate perilipin and attenuate lipolysis. This suggests a two-step strategy governed by a kinase and a phosphatase to modulate the steady state of perilipin phosphorylation and hence the lipolysis response to hormonal stimulation.     

Relative contribution of adipose triglyceride lipase and hormone-sensitive lipase to tumor necrosis factor-α (TNF-α)-induced lipolysis in adipocytes

TNF-α potently stimulates basal lipolysis in adipocytes, which may contribute to hyperlipidemia and peripheral insulin resistance in obesity. Recent studies show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) act sequentially in catalyzing the first two steps of adipose lipolysis in response to β-adrenergic stimulation. Here, we sought to determine their functional roles in TNF-α-induced lipolysis. Silencing of ATGL expression in adipocytes almost completely abolished basal and TNF-α-induced glycerol release. In comparison, the glycerol release under the same conditions was only partially decreased upon reduction in expression of either HSL or the ATGL coactivator CGI-58. Interestingly, overexpression of ATGL restored the lipolytic rates in cells with silenced HSL or CGI-58, indicating a predominant role for ATGL. While expression of ATGL, HSL and CGI-58 remains mostly unaffected, TNF-α treatment caused a rapid abrogation of the ATGL inhibitory protein G0S2. TNF-α drastically decreased the level of G0S2 mRNA, and the level of G0S2 protein could be maintained by inhibiting proteasomal protein degradation using MG-132. Furthermore, coexpression of G0S2 was able to significantly decrease TNF-α-stimulated lipolysis mediated by overexpressed ATGL or CGI-58. We propose that the early reduction in G0S2 content is permissive for TNF-α-induced lipolysis.     

α-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on α-melanocyte-stimulating hormone (α-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the α-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of α-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in α-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed.     

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.     

Cardiotrophin-1 stimulates lipolysis through the regulation of main adipose tissue lipases

Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.     

Role of PAT proteins in lipid metabolism

One of the central reactions in bodily energy metabolism is lipolysis in adipocytes, the reaction that governs the release of stored fatty acids from the adipocyte triacylglycerol pool, which constitutes the major energy reserve in animals. These fatty acids are then transported by serum albumin to various tissues to supply their energy requirements. This reaction was previously thought to result from phosphorylation and activation of hormone-sensitive lipase by protein kinase A (PKA) but is now known to be governed by a translocation of the lipase from the cytosol to the surface of the intracellular lipid droplet that houses the reservoir of TAG. This droplet is coated with perilipin A, which is also phosphorylated by PKA in response to lipolytic stimuli, and phosphorylation of perilipin A is essential for HSL translocation and stimulated lipolysis.     

Chronic TNFα and cAMP pre-treatment of human adipocytes alter HSL, ATGL and perilipin to regulate basal and stimulated lipolysis

We examined the effects of chronic TNFα and dibutyryl-cAMP (Db-cAMP) pre-treatment on the lipolytic machinery of human hMADS adipocytes. TNFα decreased adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein content and triglycerides (TG)-hydrolase activity but increased basal lipolysis due to a marked reduction in perilipin (PLIN) protein content. Conversely, Db-cAMP increased ATGL and HSL protein content but prevented PLIN phosphorylation, the net result being accentuated basal lipolysis. In forskolin-stimulated conditions, TNFα and Db-cAMP pre-treatment decreased stimulated TG-hydrolase activity and impaired PLIN phosphorylation. Together, this resulted in a severely attenuated response to forskolin-stimulated lipolysis.     

Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Lipolysis in hMADS Adipocytes

Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes.Adipose tissue fat stores in humans are mainly dependent upon fatty acid (FA)² supply, FA esterification to triglycerides (TG), and TG breakdown, or lipolysis. Adipose tissue lipolysis is governed by three lipases. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) both have the capacity to initiate TG degradation by cleaving the first ester bond, but HSL is unique in its capacity to break down the second ester bond, converting diglycerides (DG) to monoglycerides (1–3). The non-rate-limiting monoglyceride lipase completes lipolysis by cleaving the last ester bond from a monoglyceride molecule, leading to glycerol release (4). Adipose tissue lipolysis has received much attention over the past 10 years because of its altered regulation in obesity (5).HSL resides freely in the cytosol and can associate with lipid droplets (LD). It is regulated by hormones such as catecholamines, insulin, and natriuretic peptides. Catecholamines bind to β-adrenoceptors on adipocyte cell membranes and activate cyclic AMP-dependent protein kinase. Similarly, natriuretic peptides bind to type A receptors and activate cyclic GMP-dependent protein kinase (6). The protein kinase action in stimulated lipolysis is 2-fold: 1) phosphorylation of HSL, leading to its translocation from the cytosol to LD (7, 8), and 2) phosphorylation of perilipin A (6, 9, 10), the predominant perilipin isoform in adipocytes, enhancing interaction between HSL and LD. The importance of HSL activity in stimulating complete lipolysis is indisputable, particularly given its unique capacity to hydrolyze DG. However, lipolysis is not exclusively dependent upon HSL because HSL null mice revealed residual TG lipase activity in adipose tissue (2, 11). Another adipose tissue lipase was identified (3, 12, 13). ATGL, also known as desnutrin or patatin-like phospholipase domain-containing protein 2, shows affinity toward TG only (3, 14). ATGL is activated by CGI-58, an esterase/thioesterase/lipase subfamily protein devoid of TG hydrolase enzymatic activity (15, 16). The role of HSL and ATGL has been investigated in murine fat cell lipolysis, but the relative importance of these lipases in basal and protein kinase A-stimulated human fat cell lipolysis has remained elusive.Increased fat mass is associated with defects in adipose tissue metabolism. In obesity, resistance to catecholamine-induced lipolysis is observed (17–19). This inhibition of lipolysis may be naturally occurring as an adaptive protective mechanism to minimize FA release and its deleterious consequences on metabolism. Indeed, decreased expression of HSL and ATGL has been observed in isolated adipocytes and differentiated preadipocytes of obese subjects and adipose tissue of insulin-resistant subjects, respectively (20–23). However, by virtue of its mass, adipose tissue basal lipolysis elevates circulating levels of FAs in obese subjects, thereby increasing the risk of insulin resistance. Therefore, the use of pharmacological lipid-lowering agents that act through inhibition of lipolysis has been a promising research avenue leading to the development of several series of HSL inhibitors (24).Herein, we sought to examine the respective contributions of HSL and ATGL to lipolysis and re-esterification in fat cells derived from human adipose tissue derived-multipotent stem cells (termed hMADS cells). These cells, which exhibit at a clonal level normal karyotype, self-renewal ability, and no tumorigenicity, are able to differentiate into functional adipocytes (25, 26). We investigated the localization of HSL and ATGL in basal and stimulated lipolytic conditions and studied lipase activities and whole cell lipolysis in adipocytes with altered expression levels of HSL, ATGL, and its coactivator CGI-58. Our results provide novel insights into ATGL localization and its critical role with coactivator CGI-58 in DG provision to HSL during basal and stimulated lipolysis.     

Medium‐Chain Fatty Acids Attenuate Agonist‐Stimulated Lipolysis,Mimicking the Effects of Starvation

Objective: To test the hypothesis that incorporation of medium‐chain fatty acids (FAs) into adipocyte triglycerides alters intracellular lipolysis. Research Methods and Procedures: 3T3‐L1 adipocytes were pretreated with octanoate for various incubation periods. After the removal of exogenous FAs, cells were incubated with different lipolytic agonists. To determine the effects on lipolysis, we measured the following: the release of glycerol and FAs, lipase activity, protein levels of hormone‐sensitive lipase (HSL), and perilipin A; translocation of HSL; phosphorylation of perilipin A; and levels of cellular adenosine triphosphate, cyclic adenosine monophosphate, and H₂O₂. To compare the effects of starvation with those caused by octanoate pretreatment, we measured glycerol release and H₂O₂ generation in rat adipocytes of starved donors. Results: Pretreatment of adipocytes with octanoate in vitro increased basal lipolysis but decreased the cellular response for agonists. The same effects were seen in starvation in vivo. Preincubation with octanoate for 48 hours did not affect basal lipase activity, HSL, and perilipin protein levels, but it reduced agonist‐stimulated perilipin phosphorylation and HSL translocation toward fat droplets. This was associated with a reduction in basal cellular adenosine triphosphate levels and agonist‐stimulated cyclic adenosine monophosphate generation. Starvation and octanoate pretreatment both increased intracellular H₂O₂ concentrations, which might also contribute to the inhibition on agonist‐stimulated lipolysis. Discussion: Pretreatment with octanoate seems to induce changes in adipocyte lipolysis in a pattern mimicking the effects of starvation. Such changes could contribute, in part, to weight loss in animals and humans associated with dietary medium‐chain FAs.     

Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes

Our previous studies have demonstrated that natriuretic peptides (NPs), peptide hormones with natriuretic, diuretic, and vasodilating properties, exert a potent control on the lipolysis in human adipocytes via the activation of the type A guanylyl cyclase receptor (1, 2). In the current study we investigated the intracellular mechanisms involved in the NP-stimulated lipolytic effect in human preadipocytes and adipocytes. We demonstrate that the atrial NP (ANP)-induced lipolysis in human adipocytes was associated with an enhanced serine phosphorylation of the hormone-sensitive lipase (HSL). Both ANP-mediated lipolysis and HSL phosphorylation were inhibited in the presence of increasing concentrations of the guanylyl cyclase inhibitor LY-83583. ANP did not modulate the activity of the cAMP-dependent protein kinase (PKA). Moreover, H-89, a PKA inhibitor, did not affect the ANP-induced lipolysis. On primary cultures of human preadipocytes, the ANP-mediated lipolytic effect was dependent on the differentiation process. On differentiated human preadipocytes, ANP-mediated lipolysis, associated with an increased phosphorylation of HSL and of perilipin A, was strongly decreased by treatment with the inhibitor of the cGMP-dependent protein kinase I (cGKI), Rp-8-pCPT-cGMPS. Thus, ANP-induced lipolysis in human adipocytes is a cGMP-dependent pathway that induces the phosphorylation of HSL and perilipin A via the activation of cGKI. The present study shows that lipolysis in human adipocytes can be controlled by an independent cGKI-mediated signaling as well as by the classical cAMP/PKA pathway.