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。     

脂肪分化相关蛋白的研究进展

细胞内脂滴是一种代谢活跃的细胞器,脂滴表面蛋白在脂滴的代谢调节中起到了重要作用。ADRP是一种重要的脂滴表面蛋白,在机体组织和细胞内广泛表达。脂肪肝、动脉粥样硬化、糖尿病等均伴随脂质的异常蓄积,近年来的研究表明ADRP参与这些疾病的发生发展。本文就ADRP在各组织和器官正常的生理功能以及对疾病状态的调控加以综述。     

促酰化蛋白对脂肪细胞分化中脂滴相关蛋白TIP47表达的影响

研究促酰化蛋白（acylation stimulating protein, ASP）在3T3-L1脂肪细胞分化中对脂滴相关蛋白TIP47(tail-interacting protein 47 kD)表达的影响,从而探讨ASP在成脂方面的重要意义．用免疫荧光染色法观察3T3-L1前脂肪细胞中TIP47的表达定位;采用经典激素鸡尾酒法诱导分化3T3-L1前脂肪细胞,用RT-PCR和Western 印迹方法检测诱导分化的3T3-L1脂肪细胞中TIP47 mRNA和蛋白表达;在分化过程中不同时点,对诱导分化中的3T3-L1脂肪细胞分别给予胰岛素和ASP处理,并设立相应空白对照,用RT-PCR和Western印迹方法检测TIP47 mRNA和蛋白表达． 结果显示,3T3-L1前脂肪细胞中TIP47主要在胞浆内表达;诱导分化过程中的3T3-L1脂肪细胞TIP47 mRNA和蛋白的表达水平呈时间依赖性降低;ASP对诱导分化的3T3-L1脂肪细胞中TIP47 mRNA和蛋白表达有显著的上调作用,但随着分化至48 h,其上调作用已不明显;胰岛素仅在分化的0 d对脂肪细胞中TIP47 mRNA和蛋白表达有上调作用,之后基本无影响．结果提示,ASP促成脂作用可能与其调节脂滴相关蛋白TIP47的表达密切相关,从而为认识及防治肥胖症开拓新的思路．     

脂滴在丙肝病毒生命周期中的作用

脂滴是细胞内中性脂的主要储存细胞器。最近许多研究都发现脂滴与丙型肝炎病毒(Hepatitis C virus,HCV)有着密切的关系。脂滴对HCV的生命周期发挥着重要作用,影响HCV的感染、复制、组装和分泌一系列生命过程。本文就脂滴在病毒生命周期中的作用的最新进展进行综述。     

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。     

雷帕霉素对小鼠原代肝细胞脂滴形态和脂滴表面蛋白表达的影响

目的:探讨雷帕霉素(Rapamycin)对小鼠原代肝细胞脂滴形态和脂滴表面蛋白表达的影响。方法:采用胶原酶灌注方法分离和培养小鼠原代肝细胞,采用100μM油酸诱导肝细胞内脂肪的合成。采用0、10、20、50μM的雷帕霉素处理肝细胞12 hr后,利用中性脂肪染料Bodipy493/503对肝细胞内的脂滴进行染色,荧光显微镜下观察细胞脂滴形态和数量。定量试剂盒检测细胞内甘油三酯(TG)的含量利用Western blot检测不同浓度雷帕霉素处理的小鼠原代肝细胞脂滴表面蛋白ADRP的表达水平。结果:成功分离和培养了小鼠原代肝细胞,使用油酸处理能够明显增加原代肝细胞内脂滴的数量。随着体外雷帕霉素处理浓度的增加,荧光显微镜下观察发现原代肝细胞内脂滴的数量呈现明显的下降趋势,甘油三酯的含量也呈见明确的下降趋势,在20μM浓度下就表现出显著性差异。Western blot结果显示雷帕霉素能够在抑制肝细胞内脂肪储积的同时降低脂滴表面蛋白ADRP的表达水平,并且随着雷帕霉素处理浓度的增加,其对ADRP表达的抑制越明显。结论:雷帕霉素能够抑制肝细胞内中性脂肪的储积,同时降低脂滴表面蛋白ADRP的表达水平。也间接说明了mTOR信号通路能够影响肝细胞内脂肪的储积,也为脂肪肝的防治提供了一个新的实验基础。     

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。     

脂滴与动脉粥样硬化的研究进展

脂肪组织是哺乳动物最重要的能量储存库,脂滴(LDs)是脂肪细胞的基本单位,是生物体胞浆中重要的亚细胞器,广泛存在于真核生物胞浆中,参与机体能量储存、甾体激素合成、应激等生理过程。LDs不仅存在于脂肪组织,在心肌、肝脏、骨骼肌、睾丸、肾脏等器官和组织中均有表达,其功能不尽相同。近年来研究表明心肌细胞脂滴代谢与动脉粥样硬化斑块发生与发展密切关联,本文将对脂滴与动脉粥样硬化的功能联系及其临床意义的研究进展进行总结。     

PAT family proteins pervade lipid droplet cores

The PAT family proteins, named after perilipin, adipophilin, and the tail-interacting protein of 47 kDa (TIP47), are implicated in intracellular lipid metabolism. They associate with lipid droplets, but how is completely unclear. From immunofluorescence studies, they are reported to be restricted to the outer membrane monolayer enveloping the lipid droplet and not to enter the core. Recently, we found another kind of lipid droplet-associated protein, caveolin-1, inside lipid droplets. Using freeze-fracture immunocytochemistry and electron microscopy, we now describe the distributions of perilipin and caveolin-1 and of adipophilin and TIP47 in lipid droplets of adipocytes and macrophages. All of these lipid droplet-associated proteins pervade the lipid droplet core and hence are not restricted to the droplet surface. Moreover, lipid droplets are surprisingly heterogeneous with respect to their complements and their distribution of lipid droplet-associated proteins. Whereas caveolin-1 is synthesized in the endoplasmic reticulum and is transferred to the lipid droplet core by inundating lipids during droplet budding, the PAT proteins, which are synthesized on free ribosomes in the cytoplasm, evidently target to the lipid droplet after it has formed. How the polar lipid droplet-associated proteins are accommodated among the essentially hydrophobic neutral lipids of the lipid droplet core remains to be determined.     

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface. The authors Yuki Ohsaki and Takashi Maeda have contributed equally to this work.     

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.     

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.     

A proposed model of fat packaging by exchangeable lipid droplet proteins

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3-12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.     

PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3–12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.     

Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins

The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the Human Genome Organization Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each gene member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.     

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.