一种调控脂解的重要蛋白——围脂滴蛋白(Perilipin)

围脂滴蛋白（perilipin）是脂滴相关蛋白家族的核心成员之一,是定位于脂滴表面的高磷酸化的蛋白,对脂肪组织中甘油三酯的代谢有双重调节作用,既可通过阻止脂肪酶接近脂滴降低基础状态下的脂解,又可促进激素刺激的脂肪分解.Perilipin在脂代谢中发挥重要作用,其表达调控可能与肥胖及其相关代谢疾病如糖尿病、胰岛素抵抗等有重要关系.本文主要介绍了perilipin的发现、命名、结构特征以及激素和转录因子对perilipin的调控,并阐述了其与相关脂肪酶间的相互作用.目前的研究主要集中于围脂滴蛋白（perilipin）和激素敏感脂肪酶（HSL）之间,与新近发现的脂肪酶脂肪三酰甘油脂酶（ATGL）的相互作用则有待于进一步研究.     

脂肪甘油三酯脂肪酶（ATGL）的生物学功能及调控机制

脂肪甘油三酯脂肪酶(ATGL)是近年来研究发现的启动脂肪动员的又一关键脂肪酶. ATGL能特异性地水解甘油三酯(TAG)的第一酯键,被认为是TAG水解过程的限速酶. ATGL在脂肪组织和非脂肪组织脂代谢过程中都发挥着重要作用,其活性和表达在细胞内受到转录水平、翻译后水平等调控.ATGL介导的脂解过程可能与肥胖、糖尿病、脂肪肝等代谢疾病存在关联.本文主要就ATGL的结构特征、生物学功能及其调控机制进行综述,并对今后的研究方向和应用进行了展望.     

脂蛋白脂酶基因与代谢综合征关系的研究进展

脂蛋白脂酶是脂质代谢的关键酶之一,是血浆中清除甘油三酯的限速酶,主要催化乳糜微粒和极低密度脂蛋白核心中的甘油三酯水解,使得机体能够利用由食物摄取和肝脏合成的脂肪,同时释放出脂肪酸和单酰甘油.脂蛋白脂酶基因突变可以影响脂蛋白脂酶的活性,从而导致脂代谢紊乱,与肥胖、胰岛素抵抗、2型糖尿痛和高血压的发病风险相关.本文综述了脂蛋白脂酶的结构、功能、基因结构及其多态性与血脂和代谢综合征关系的研究进展.     

POMC神经元自噬与脂质体自噬

正机体脂肪动员产热,不仅依赖于脂肪酶系统,"脂质体自噬"(lipophagy)亦密切参与其中。机体重要的脂肪酶包括:脂肪甘油三酯脂酶(adipose triglyceride lipase,ATGL)和激素敏感脂肪酶(hormone-sensitive lipase,HSL),二者均可促进"脂滴动员"(lipid droplet mobilization),提高脂质分解代谢水平。最近,美国爱因斯坦医学院Nuria Martinez-Lopez等研究人员发现:"自噬相关蛋白"介导的脂质体自噬亦参与脂滴动员,而且,该过程受下丘脑代谢神经元的调控。     

激素敏感脂酶基因研究进展

在脂肪的沉积过程中,激素敏感酯酶(HSL)是脂肪分解的限速酶,动物体内激素敏感酯酶的多寡、活性的高低对动物脂肪的沉积具有重要的影响。近年来,国内外大量研究报道了激素敏感酯酶对脂肪分解代谢的调控。本文将从激素敏感酯酶基因的功能与作用机理;日粮、激素、添加剂及动物生理阶段等方面对激素敏感酯酶基因的表达调控进行综述。     

Leptin对猪原代脂肪细胞脂解及其关键脂酶mRNA表达的影响

Leptin是由脂肪组织分泌的内源因子,在调节机体能量平衡过程中起重要作用.Leptin促进脂解的研究由来已久,但其作用机理尚不完善.本研究旨在通过系统检测关键脂酶mRNA的表达变化来探讨Leptin促进脂解的分子机理.运用形态学观察,油红O染色和RT-PCR鉴定培养的猪原代脂肪细胞;甘油测定试剂盒和游离脂肪酸(FFA)测定试剂盒分别检测甘油和FFA的释放;半定量RT-PCR检测关键脂酶mRNA的表达.结果显示:100 nmol/L的Leptin可显著上调ATGL、TGH-2、HSL、MGL和LPL mRNA的表达(P<0.01),但同时下调Perilipin mRNA的表达(P<0.01);Leptin呈浓度依赖性促进甘油的释放(P<0.01),但对FFA的释放影响不显著(P>0.05).以上结果提示,Leptin可能主要通过上调ATGL、MGL、LPL和下调Perilipin的表达促进猪原代脂肪细胞的脂解;同时推测,FFA释放的相对稳定可能是由Leptin通过上调UCPs的表达而增加FFA的消耗引起的.     

Fsp27基因沉默载体的构建及其对细胞脂解的影响研究

构建脂肪特异性蛋白27(Fat-specific protein of 27,Fsp27)基因沉默载体,研究沉默Fsp27基因表达对3T3-L1细胞脂解的影响,并对其作用机制进行探究。采用RNAi技术,构建Fsp27基因真核干扰载体,下调Fsp27基因的表达。“鸡尾酒”法诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞。脂质体转染脂肪细胞,油红O染色脂滴,酶法测定细胞中甘油及甘油三酯的含量。Western blot法检测细胞中Fsp27、HSL、ATGL和PPARγ的蛋白表达。Western blot结果显示:阳性sh-Fsp27干扰载体均能有效下调Fsp27的表达,且伴随细胞内ATGL和PPARγ的表达量升高(P<0.05),其中sh-Fsp27-2的沉默效果最好;酶学方法检测结果显示:阳性sh-Fsp27干扰组细胞中甘油三酯含量下降,甘油含量升高(P<0.05);油红O染色结果发现:空白对照组与阴性对照组均有大脂滴堆积,阳性sh-Fsp27组小脂滴分布广泛,未见明显的大脂滴。sh-Fsp27-2组基因沉默载体的沉默效果最好,Fsp27基因沉默可以加快3T3-L1细胞的脂解速率,其主要是通过抑制脂滴融合和增强ATGL酶的水解来完成对脂解的调控。     

下调Perilipin 1基因表达对3T3-L1细胞脂解的影响

目的:研究下调围脂滴蛋白基因(PLIN1)表达对3T3-L1细胞脂解的影响。方法:采用RNA干扰技术,构建3组阳性及1组阴性sh-PLIN1重组载体,并进行菌液PCR和DNA测序鉴定。Western blot测定PLIN1A蛋白表达,评价载体下调效果。细胞转染有效载体2天后,Bodipy 493/503染色脂滴;酶学方法测定细胞中甘油三酯和甘油含量;Western blot检测甘油三酯脂肪酶(ATGL)、激素敏感性脂肪酶(HSL)及其磷酸化蛋白(p-HSL)的表达。酶联免疫吸附法(ELISA)测定细胞中环磷酸腺苷(c AMP)和蛋白激酶A(PKA)的浓度。结果:各sh-PLIN1干扰载体构建成功,且3组阳性载体均能显著下调PLIN1A蛋白的表达(P0.05)。转染有效载体后,与阴性转染组相比,sh-PLIN1转染组细胞中脂滴减小,甘油三酯含量降低,甘油含量升高,ATGL和HSL相对表达量显著升高(P0.05),p-HSL相对表达量及c AMP、PKA的浓度无显著性差异(P0.05)。结论:下调PLIN1基因表达可加快3T3-L1细胞脂解速率,其可能通过上调ATGL和HSL的表达而实现,c AMP/PKA信号通路对其无明显调节作用。     

脂肪甘油三酯水解酶的研究进展

胰岛素抵抗等代谢疾病的发生与脂代谢紊乱密切相关。细胞中的脂肪主要储存在一个以中性脂为核的细胞器——脂滴(lipid droplet,LD)中。脂肪甘油三酯水解酶(adiposetriglyceride lipase,ATGL)是在脂滴上发现的水解甘油三酯的脂肪酶。除脂肪组织外,ATGL也广泛存在于骨骼肌等多种非脂肪组织中,并发挥着重要的生理功能。越来越多的研究表明,ATGL与中性脂质贮存异常、胰岛素抵抗等代谢疾病密切相关。运动可以通过改变ATGL的表达起到调控脂代谢的作用,进而在防治胰岛素抵抗等代谢疾病中发挥作用。     

Fsp27通过抑制HSL的脂滴定位调控脂肪水解

Fsp27是CIDE蛋白家族的一员,其特异性地在脂肪组织中表达并定位于脂滴表面,促进脂滴融合增大和脂肪积累.Fsp27敲除小鼠表现出胰岛素敏感性增强,有较高的能量消耗,并且可以抵抗高脂食物引起的肥胖,但Fsp27是否直接参与脂肪水解的调控过程并不清楚.本研究发现,在3T3-L1脂肪细胞中基因沉默Fsp27导致脂肪水解速率上升,并且这种上升是由激素敏感型脂肪酶(HSL)所介导.进一步在3T3-L1前脂肪细胞中过表达Fsp27以及HSL,对其定位的观察结果显示,Fsp27可以显著地抑制HSL在脂滴表面的定位.本研究表明,在脂肪组织中,Fsp27能够直接影响HSL在脂滴表面的定位,进而抑制脂肪水解速率,导致脂类积累.     

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.     

Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A

Adipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycerides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the protein–protein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites.     

Several agents and pathways regulate lipolysis in adipocytes

Adipose tissue is the only tissue capable of hydrolyzing its stores of triacylglycerol (TAG) and of mobilizing fatty acids and glycerol in the bloodstream so that they can be used by other tissues. The full hydrolysis of TAG depends on the activity of three enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase, each of which possesses a distinct regulatory mechanism. Although more is known about HSL than about the other two enzymes, it has recently been shown that HLS and ATGL can be activated simultaneously, such that the mechanism that enables HSL to access the surface of lipid droplets also permits the stimulation of ATGL. The classical pathway of lipolysis activation in adipocytes is cAMP-dependent. The production of cAMP is modulated by G-protein-coupled receptors of the Gs/Gi family and cAMP degradation is regulated by phosphodiesterase. However, other pathways that activate TAG hydrolysis are currently under investigation. Lipolysis can also be started by G-protein-coupled receptors of the Gq family, through molecular mechanisms that involve phospholipase C, calmodulin and protein kinase C. There is also evidence that increased lipolytic activity in adipocytes occurs after stimulation of the mitogen-activated protein kinase pathway or after cGMP accumulation and activation of protein kinase G. Several agents contribute to the control of lipolysis in adipocytes by modulating the activity of HSL and ATGL. In this review, we have summarized the signalling pathways activated by several agents involved in the regulation of TAG hydrolysis in adipocytes.     

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.     

Adipose triglyceride lipase expression and fasting regulation are differently affected by cold exposure in adipose tissues of lean and obese Zucker rats

Adipose triglyceride lipase (ATGL) hydrolyzes triacylglycerols to diacylglycerols in the first step of lipolysis, providing substrates for hormone-sensitive lipase (HSL). Here we studied whether ATGL messenger RNA (mRNA) and protein levels were affected by 24-h cold exposure in different white adipose tissue depots and in interscapular brown adipose tissue of lean and obese Zucker rats submitted to feeding and 14-h fasting conditions. HSL mRNA expression was also studied in selected depots. In both lean and obese rats, as a general trend, cold exposure increased ATGL mRNA and protein levels in the different adipose depots, except in the brown adipose tissue of lean animals, where a decrease was observed. In lean rats, cold exposure strongly improved fasting up-regulation of ATGL expression in all the adipose depots. Moreover, in response to fasting, in cold-exposed lean rats, there was a stronger positive correlation between circulating nonesterified fatty acids (NEFA) and ATGL mRNA levels in the adipose depots and a higher percentage increase of circulating NEFA in comparison with control animals not exposed to cold. In obese rats, fasting-induced up-regulation of ATGL was impaired and was not improved by cold. The effects of obesity and cold exposure on HSL mRNA expression were similar to those observed for ATGL, suggesting common regulatory mechanisms for both proteins. Thus, cold exposure increases ATGL expression and improves its fasting-up-regulation in adipose tissue of lean rats. In obese rats, cold exposure also increases ATGL expression but fails to improve its regulation by fasting, which could contribute to the increased difficulty for mobilizing lipids in these animals.     

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.     

ATGL调节细胞内脂质代谢和代谢相关疾病

脂肪组织甘油三酯水解酶(adipose triglyceride lipase, ATGL)是一种催化甘油三酯第一步水解的重要脂肪酶,在机体能量代谢调节中发挥重要作用.本文介绍了ATGL的基因和蛋白质结构,并详细综述了ATGL的功能调控和与其相关联疾病的研究进展,最后通过与激素敏感脂肪酶(HSL)比较,对ATGL的特征进行总结.     

Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.     

Breed difference and regulation of the porcine adipose triglyceride lipase and hormone sensitive lipase by TNFα

Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are major novel triglyceride lipases in animals. The aim of this study was to determine if there are differences in the porcine ATGL ( pATGL ) and HSL genes between Jinhua pigs (a fatty breed) and Landrace pigs (a leaner breed). In addition, the effect of TNFα and pATGL-specific siRNA ( pATGL-siRNA ) on the expression of pATGL and HSL in porcine adipocytes was also examined. Compared with Landrace pigs, the body weight ( BW ) of Jinhua pigs was lower ( P <  0.01), while intramuscular fat content (in the longissimus dorsi muscle), as well as the back fat thickness and body fat content were higher ( P <  0.01). The expression of pATGL and HSL mRNA in Jinhua pigs was lower ( P <  0.01) in subcutaneous adipose tissue, and greater ( P <  0.01) in longissimus dorsi muscle compared with Landrace pigs. In vitro treatment of porcine adipocytes with TNFα decreased ( P <  0.01) the glycerol release and the gene expression of pATGL , HSL and PPARγ in porcine adipocytes. Furthermore, transfection with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL , while it had no effect on the expression of HSL . Treatment with 25 ng/ml TNFα in conjunction with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL and HSL in cultured porcine adipocytes. These results provide useful information to further the understanding of the function of pATGL and HSL in porcine lipid metabolism, which should be applicable to the regulation of fat deposition and improvement of meat quality.     

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.