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.     

CGI-58在动物脂类代谢中的作用

细胞中脂滴(Lipid droplets, LDs)表面存在多个调控脂肪储存和分解的蛋白, 这些蛋白对机体的脂肪代谢起着很重要的调控作用。CGI-58(Comparative gene identification-58)分布在LDs表面, 属于α/β水解酶折叠家族, 是脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)和依赖酰基辅酶A溶血磷脂酸酰基转移酶(Lysophosphatidic acid acyltransferase, LPAAT)的激活剂。在脂肪分解过程中, CGI-58结合PAT蛋白家族成员之一的脂滴包被蛋白(Perlipin)和ATGL, 促进脂肪分解, 同时CGI-58对ATGL的激活功能受脂滴包被蛋白家族成员间蛋白质与蛋白质相互作用的影响。文章结合国内外研究热点, 针对CGI-58在动物脂类代谢中的作用进行了综述。     

PAT家族蛋白在细胞内脂滴代谢过程中的作用

哺乳动物细胞内的甘油三酯是以脂滴的形式贮存的,现在有很多证据表明,脂滴参与多种代谢过程,因而被看作胞内有功能的细胞器。脂滴含有甘油三酯构成的脂质核心,脂核表面覆盖有单层磷脂,在单层磷脂内镶嵌着在结构上具有相关性的PAT家族蛋白,包括perilipin、ADRP、TIP47和S3—12。本文就这些蛋白在甘油三酯水解和脂滴合成中的调节作用加以综述。     

Perilipin5基因功能结构域载体的构建及亚细胞定位分析

目的:依据perilipin5的功能结构域,构建含perilipin5截断体的真核表达载体,并研究它们的亚细胞定位。方法:以小鼠肝脏cDNA文库为模板,PCR扩增出perilipin5的全长及功能结构域,将之分别装载入真核表达载体PCMV5中,并引入HA标签。酶切和测序鉴定,脂质体法将构建的质粒转染293T细胞,Western blot验证表达,免疫荧光检测标记HA,于荧光显微镜下观察perilipin5各结构域的亚细胞定位。结果:构建的质粒序列正确,转染细胞后可检测到HA-perilipin5融合蛋白的表达,免疫荧光显示含有1-188aa结构域的perilipin5截断体可定位于脂滴表面,1-188aa一旦缺失perilipin5的截断体则弥散于胞内。结论:包含perilipin5功能结构域的真核表达载体构建成功,perilipin5的1-188aa与其脂滴定位密切相关。     

Forskolin引起大鼠脂肪细胞内脂滴重塑(英文)

脂肪组织是人体的主要能量储库,甘油三酯贮存在细胞内脂滴(lipid droplets, LDs)中,越来越多的研究表明脂滴是细胞内代谢活跃的细胞器。本研究旨在探讨forskolin长时间刺激脂肪分解过程中脂滴形态和脂滴表面perilipin家族蛋白的改变。以Sprague-Dawley (SD)大鼠附睾脂肪垫来源的分化脂肪细胞为研究对象,给予1μmol/L forskolin慢性刺激24 h,采用比色方法测定培养基中甘油的浓度;采用尼罗红染色观察细胞内脂滴形态的变化;采用荧光定量PCR检测perilipin家族蛋白mRNA水平的改变;采用免疫印迹和免疫荧光染色观察蛋白水平以及蛋白的亚细胞定位。结果表明,1μmol/L forskolin孵育24 h可以持续刺激脂肪分解。伴随着脂肪分解的进行,细胞内脂滴形态逐渐发生改变,细胞内聚集存在的大脂滴逐渐减少,位于细胞周边的小脂滴逐渐增加,最终细胞内大脂滴全部消失,取而代之的是在细胞质中弥散存在的微小脂滴。在脂肪分解过程中,perilipin家族蛋白水平也发生明显变化。分化成熟的脂肪细胞几乎没有Plin2蛋白表达,而forskolin慢性刺激可以显著增加Plin2蛋白以及mRNA的水平,增加的Plin2蛋白特异性结合在脂滴表面。Forskolin慢性刺激对Plin3的mRNA水平无显著影响,但可以显著降低Plin1、Plin4和Plin5的mRNA水平。以上结果提示,在forskolin慢性持续刺激脂肪分解过程中,脂滴形态和perilipin家族蛋白均发生显著改变。     

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。     

高内涵脂滴三维影像定量分析研究细胞内的脂质储存

摘要 目的：研究细胞内脂滴含量的变化对肥胖、糖尿病等代谢性疾病发生发展的影响。方法：建立高内涵脂滴三维成像和定量分析系统,获得脂滴三维动态表型参数,例如细胞内脂滴的总体积量、脂滴平均体积、单一细胞内脂滴平均数量等指标。选择HeLa、AML-12、COS-7和3T3-L1四种细胞系进行油酸、基因沉默、酶活性抑制剂的处理,量化处理后四种细胞内的脂滴数量与大小的表型差异。结果：在加入油酸情况下,细胞随油酸浓度增加而生成更多、更大的脂滴,但AML-12细胞只有展现增加脂滴数量的变化表型;在HeLa细胞中进行19种中性脂合成通路上关键基因的转录表达沉默,发现需要同时双敲降两种甘油三酯合成酶DGAT1和DGAT2才能显着降低细胞内脂滴总体积储存量,但在COS-7细胞中只需要单敲降DGAT1即可降低脂滴存量;进一步使用了DGAT1/2抑制剂处理四种细胞后,发现对抑制剂响应可区分为两类细胞分组（HeLa、AML-12与COS-7、3T3-L1）的脂滴存量表型差异,其原因是DGAT1和DGAT2的转录表达谱在这两类细胞分组中的不同。结论：建立了高内涵脂滴三维成像和定量分析系统,量化了四种细胞系的脂滴数量与大小的表型差异,揭示了细胞的脂滴脂储存方式与蛋白酶表达谱的关系。     

PAT蛋白抑制饥饿诱导的脂滴快速融合

目的:脂滴快速融合是增大脂滴直径的方式之一,但其研究相对少。本研究旨在建立脂滴快速融合的细胞模型,以便对其进行深入的生物学研究。方法:本研究使用大鼠肾成纤维细胞系NRK和小鼠前脂肪细胞系3T3-L1两种细胞系,先用油酸诱导细胞内产生大量脂滴,再使用饥饿缓冲液培养细胞,利用显微镜实时观测技术跟踪脂滴动态变化,建立脂滴快速融合的模型。而后在此模型中,加入自噬抑制剂或者以过表达CCT为阳性对照,过表达PAT蛋白(PLIN1、ADRP和TIP47),来探究它们在调控脂滴快速融合方面的功能。结果:饥饿缓冲液处理约3小时可诱导细胞发生脂滴快速融合,其融合速率很快,从脂滴接触到融合完成可发生在20秒内,显然不同于CIDE蛋白调控的缓慢脂滴融合过程。自噬抑制剂可以抑制自噬,但是并没有显著影响脂滴快速融合,说明饥饿诱导的脂滴快速融合不依赖于自噬。另发现,与过表达GFP相比,过表达定位于脂滴的GFP-CCT、GFP-PLIN1、GFP-ADRP或GFP-TIP47均能显著性抑制快速融合导致的脂滴变大的现象。结论:本研究建立了饥饿缓冲液诱导脂滴发生快速融合的细胞模型,并证明PAT蛋白(PLIN1、ADRP、TIP47)能抑制脂滴快速融合。     

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.     

Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.     

Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation

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 beta-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.     

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.     

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.     

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.     

Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

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.     

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.     

Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis

The majority of eukaryotic cells synthesize neutral lipids and package them into cytosolic lipid droplets. In vertebrates, triacylglycerol-rich lipid droplets of adipocytes provide a major energy storage depot for the body, whereas cholesteryl ester-rich droplets of many other cells provide building materials for local membrane synthesis and repair. These lipid droplets are coated with one or more of five members of the perilipin family of proteins: adipophilin, TIP47, OXPAT/MLDP, S3-12, and perilipin. Members of this family share varying levels of sequence similarity, lipid droplet association, and functions in stabilizing lipid droplets. The most highly studied member of the family, perilipin, is the most abundant protein on the surfaces of adipocyte lipid droplets, and the major substrate for cAMP-dependent protein kinase [protein kinase A (PKA)] in lipolytically stimulated adipocytes. Perilipin serves important functions in the regulation of basal and hormonally stimulated lipolysis. Under basal conditions, perilipin restricts the access of cytosolic lipases to lipid droplets and thus promotes triacylglycerol storage. In times of energy deficit, perilipin is phosphorylated by PKA and facilitates maximal lipolysis by hormone-sensitive lipase and adipose triglyceride lipase. A model is discussed whereby perilipin serves as a dynamic scaffold to coordinate the access of enzymes to the lipid droplet in a manner that is responsive to the metabolic status of the adipocyte.     

Perilipin targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive lipase

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.