背景:肥胖患者脂肪组织中过多的脂质积累改变了脂肪细胞的内分泌和能量储存功能。脂肪细胞脂滴代表在这些细胞中协调脂质储存和动员的关键细胞器。最近,我们发现了小的GTPase,Rab34,在脂肪细胞的脂滴卵白组中。在这里,我们对分布进行了表征,细胞内运输,以及这种GTP酶对脂肪细胞生理及其在肥胖中的调节的潜在贡献。
方法:在体外分化3T3-L1和人原代前脂肪细胞,并使用细胞区室标记分析Rab34的分布和运输。用表达载体和/或Rab34siRNA转染3T3-L1脂肪细胞,并评估其分泌活性。脂质积累和调节脂质代谢的蛋白质的表达。蛋白质组学和蛋白质相互作用分析用于Rab34相互作用组的鉴定。这些研究与功能分析相结合,揭示了GTP酶在脂肪细胞中的作用,重点关注Rab34相互作用蛋白传达的行为。最后,还评估了响应于肥胖的Rab34调节。
结果:我们的结果表明Rab34定位于前脂肪细胞的高尔基体。在脂滴生物生成过程中,Rab34从高尔基体转移到内质网相关区室,然后到达脂肪细胞脂滴表面。Rab34发挥与其细胞内位置相关的不同功能。因此,在高尔基,Rab34调节水箱完整性以及脂联素运输和寡聚化。在脂滴处,这种GTP酶通过与E1-泛素连接酶的相互作用来控制脂质积累和脂解,UBA1诱导脂肪酸转运蛋白和Rab34相互作用体成员的泛素化和蛋白酶体降解,FABP5.最后,脂肪组织和脂肪细胞中的Rab34水平因肥胖和相关的致病性损伤而受到调节(即,纤维化)。
结论:Rab34在脂肪细胞分化过程中发挥相关作用,包括来自低聚的调节(即,生物活性)和分泌具有胰岛素增敏作用的主要脂肪因子,脂联素,脂质储存和从脂滴动员。肥胖中的Rab34失调可能导致脂肪因子分泌和脂质代谢的改变,这是过度肥胖条件下脂肪细胞功能障碍的特征。
BACKGROUND: Excessive lipid accumulation in the adipose tissue in obesity alters the endocrine and energy storage functions of adipocytes. Adipocyte lipid droplets represent key organelles coordinating lipid storage and mobilization in these cells. Recently, we identified the small GTPase,
Rab34, in the lipid droplet proteome of adipocytes. Herein, we have characterized the distribution, intracellular transport, and potential contribution of this GTPase to adipocyte physiology and its regulation in obesity.
METHODS: 3T3-L1 and human primary preadipocytes were differentiated in vitro and Rab34 distribution and trafficking were analyzed using markers of cellular compartments. 3T3-L1 adipocytes were transfected with expression vectors and/or
Rab34 siRNA and assessed for secretory activity, lipid accumulation and expression of proteins regulating lipid metabolism. Proteomic and protein interaction analyses were employed for the identification of the Rab34 interactome. These studies were combined with functional analysis to unveil the role played by the GTPase in adipocytes, with a focus on the actions conveyed by Rab34 interacting proteins. Finally,
Rab34 regulation in response to obesity was also evaluated.
RESULTS: Our results show that
Rab34 localizes at the Golgi apparatus in preadipocytes. During lipid droplet biogenesis, Rab34 translocates from the Golgi to endoplasmic reticulum-related compartments and then reaches the surface of adipocyte lipid droplets.
Rab34 exerts distinct functions related to its intracellular location. Thus, at the Golgi, Rab34 regulates cisternae integrity as well as adiponectin trafficking and oligomerization. At the lipid droplets, this GTPase controls lipid accumulation and lipolysis through its interaction with the E1-ubiquitin ligase, UBA1, which induces the ubiquitination and proteasomal degradation of the fatty acid transporter and member of Rab34 interactome, FABP5. Finally, Rab34 levels in the adipose tissue and adipocytes are regulated in response to obesity and related pathogenic insults (i.e., fibrosis).
CONCLUSIONS: Rab34 plays relevant roles during adipocyte differentiation, including from the regulation of the oligomerization (i.e., biological activity) and secretion of a major adipokine with insulin-sensitizing actions, adiponectin, to lipid storage and mobilization from lipid droplets. Rab34 dysregulation in obesity may contribute to the altered adipokine secretion and lipid metabolism that characterize adipocyte dysfunction in conditions of excess adiposity.