脂滴包被蛋白(perilipin)调控脂肪分解

脂滴包被蛋白（perilipin）包被在脂肪细胞和甾体生成细胞脂滴表面。基础状态下perilipin可减少甘油三酯水解,使其贮备增加;脂肪分解时磷酸化的perilipin能促进甘油三酯水解,而且该蛋白对激素敏感脂酶从胞浆向脂滴转位是必需的。据推测,perilipin可能在脂肪分解调控中起到“分子开关”的作用。蛋白激酶A（PKA）、细胞外信号调节激酶（ERK）等信号转导通路参与了脂肪分解。肿瘤坏死因子仅（TNFα）、过氧化物酶体增殖物激活受体γ（PPAγ）激动剂、瘦素（leptin）均可以影响perilipin的表达。新近研究表明,perilipin可通过蛋白酶体途径来调节其蛋白量的表达。脂肪分解调控中的关键蛋白perilipin可以和2型糖尿病、肥胖、动脉粥样硬化等多种代谢性疾病及心血管疾病联系起来。     

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.     

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。     

脂滴膜转运蛋白Rab18与脂质代谢的关系研究

脂滴是动物细胞内储存脂质的一种亚细胞器。脂滴表面存在多种脂滴周围相关蛋白,参与脂质动态平衡的调节,防止脂质代谢异常的发生。其中,Rab18作为脂滴周围相关蛋白中的一种,在脂质代谢的调节、信号的转导、膜运输等多种生理功能中发挥重要作用。对于Rab18与脂质代谢之间关系的研究,为动脉粥样硬化、糖尿病、非酒精性脂肪肝及肥胖等多种代谢性疾病的防治发挥重要作用,本文就Rab18与细胞脂质代谢之间的关系研究作一综述。     

脂滴包被蛋白2在动脉粥样硬化中的作用

动脉粥样硬化性心血管疾病严重威胁着人类生命健康,其中脂质代谢异常和炎症反应是其重要的发病机制。脂滴是细胞内储存脂质的一种亚细胞器,其表面存在多种脂滴包被蛋白,参与调控脂质动态平衡。脂滴包被蛋白2(Plin2)作为脂滴包被蛋白的一种,在脂质代谢的调节、脂肪酸的氧化及炎症反应等多种生理功能中发挥重要作用。近年来,越来越多的研究发现Plin2在动脉粥样硬化的发生发展中扮演着重要的角色。因此,本文主要综述Plin2在胆固醇代谢、脂质合成、自噬和炎症反应等过程中发挥的作用,进一步阐述其与动脉粥样硬化之间的关系。     

脂滴与线粒体相互作用的研究进展

肥胖和多种代谢类疾病的发生有着密切的关系,而导致肥胖的脂肪多以中性脂的形式储存于细胞的一种细胞器——脂滴中。越来越多的研究表明,脂滴能够和其它细胞器发生相互作用,而它和线粒体的相互作用可能与Ⅱ型糖尿病的形成密切相关:非正常的脂滴和线粒体的相互作用有可能是导致细胞胰岛素抵抗的重要原因。我们通过对脂滴表面蛋白质组学、脂滴与线粒体的空间位置,以及相关蛋白等研究的总结,结合本实验室的研究结果,对脂滴与线粒体相互作用的物质基础及可能方式、受骨骼肌有氧运动的影响,及其与骨骼肌胰岛素抵抗发生的关系等,进行了讨论。     

蛋白激酶和蛋白磷酸脂酶的抑制剂

蛋白激酶和蛋白磷酸脂酶的抑制剂裴钢（中国科学院上海细胞生物学研究所２０００３１）夏国宏（上海生命科学研究中心２０００３１）蛋白磷酸化和去磷酸化是世间一切生命现象中最基本调控机制之一。在细胞内蛋白磷酸化是由蛋白激酶完成的,而蛋白的磷酸化可被蛋白磷酸脂酶...     

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。     

脂滴与其他细胞器互作调控脂稳态的分子机制

随着影像技术的发展,越来越多的研究表明细胞器之间存在广泛的直接相互作用,其主要功能是参与物质运输、细胞器新生与生长、细胞器分裂与融合等.细胞器间的互作主要由定位于这些膜器表面的蛋白质分子相互作用介导,磷脂也在其中发挥作用.脂滴作为储存中性脂的细胞器,是细胞内脂质代谢的中心,同时对机体脂稳态的维持起着至关重要的作用.从脂...     

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.     

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.     

Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins

Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.     

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.     

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.     

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。     

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.     

Perilipin A and the control of triacylglycerol metabolism

Perilipin A is the most abundant protein associated with the lipid droplets of adipocytes and functions to control both basal and stimulated lipolysis. Under basal or fed conditions, perilipin A shields stored triacylglycerols from cytosolic lipases, thus promoting triacylglycerol storage. When catecholamines bind to cell surface receptors to initiate signals that activate cAMP-dependent protein kinase (PKA), phosphorylated perilipin A facilitates maximal lipolysis. Mutagenesis studies have revealed that central sequences of moderately hydrophobic amino acids are required to target nascent perilipin A to lipid droplets and provide an anchor into the hydrophobic environment of lipid droplets. Sequences of amino acids in the unique carboxyl terminus of perilipin A and those in amino terminal sequences flanking the first hydrophobic stretch are required for the barrier function of perilipin A in promoting triacylglycerol storage. Site-directed mutagenesis studies of serine residues within six PKA consensus sites of perilipin A reveal functions for phosphorylation of at least three of the sites. Phosphorylation of one or more of the serines within three amino terminal PKA sites is required to facilitate hormone-sensitive lipase access to lipid substrates. Phosphorylation of serines within two carboxyl terminal sites is also required for maximal lipolysis. Phosphorylation of serine 492 (site 5) triggers a massive remodeling of lipid droplets, whereby large peri-nuclear lipid droplets fragment into myriad lipid micro-droplets that scatter throughout the cytoplasm. We hypothesize that perilipin A binds accessory proteins to provide assistance in carrying out these functions.     

SREBP Activation by Antipsychotic- and Antidepressant-Drugs in Cultured Human Liver Cells: Relevance for Metabolic Side-Effects?

Despite the critical roles of intracellular lipid droplets (LDs) in lipid storage and metabolism, little is known about the molecular mechanisms of their functions. Several protein components associated with the surface of LDs have been identified. A major one is perilipin in adipocytes and steroidogenic cells, whereas ADRP in most other cell types. They are loosely grouped as a small protein family sharing a common N-terminal motif, called the PAT domain. Perilipin regulates the breakdown of triacylglycerol in LDs via its phosphorylation. ADRP is characterized as a fatty acid binding protein and involved in lipid uptake and LD formation. For examining the functions of perilipin and ADRP at the molecular level, we performed yeast two-hybrid screening in this study, to find their functional partners. We identified CGI-58, a product of the causal gene of Chanarin-Dorfman syndrome (CDS), as an interactor for both perilipin and ADRP. Specific interaction between CGI-58 and perilipin was confirmed in a GST-pulldown assay and supported by fluorescence microscopic analyses. We further demonstrated that CGI-58 is principally located at the surface of LDs in 3T3-L1 cells, together with perilipin, and its expression is upregulated upon stimulation for adipocyte differentiation. Other than CGI-58, we also identified in yeast two-hybrid screening HSP86 and D52 tumor proteins as binding partners of perilipin, and IRG-47 of ADRP. These factors might be cooperated with perilipin and ADRP, and hence involved in membrane dynamics of LDs as well as the regulation of lipolysis on the surface of LDs.