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促酰化蛋白对3T3-L1 脂肪细胞分化进程中adipophilin 和 perilipin 表达的影响

吴静, 卢慧玲^*, 胡秀芬, 梁晓燕, 林汉华, 王宏伟, 范晓蔚

华中科技大学同济医学院附属同济医院儿科，武汉430030

摘要

本文旨在通过观察前脂肪细胞分化过程中促酰化蛋白(acylation stimulating protein, ASP)对脂滴相关蛋白adipophilin 和perilipin 表达的影响，探讨adipophilin 和perilipin 在介导ASP 促成脂过程中的作用。体外培养小鼠来源的3T3-L1 前脂肪 细胞，经典激素鸡尾酒法诱导分化，根据实验室前期工作验证的adipophilin 和perilipin 基因和蛋白水平的时序性变化规律 选取几个代表性的时间点(0 d, 3 d, 6 d, 9 d)，设立ASP (1 μmol/L)刺激组、胰岛素(100 nmol/L)刺激组及空白对照组，[3H]油 酸掺入法测定脂肪细胞甘油三酯合成率，[3H]-2- 脱氧葡萄糖([3H]-2-DG)掺入法测定脂肪细胞葡萄糖转运率，RT-PCR 和 Western blot 方法分别检测adipophilin 和perilipin mRNA 和蛋白表达。结果显示：(1)脂肪细胞分化第3 天和第6 天，ASP 组甘油三酯合成率显著高于空白对照组(P<0.05, P<0.01)，胰岛素组各检测时间点甘油三酯合成率与正常对照组之间无显著 性差异；(2)脂肪细胞分化第6 天和第9 天，ASP 组和胰岛素组葡萄糖转运率均较同期空白对照组显著增加(P<0.05, P<0.01)， 胰岛素组增加效应较ASP 组更为显著(P<0.05, P<0.001)；(3)与空白对照组相比，ASP 组adipophilin 基因和蛋白表达水平从 0 天起显著升高(P<0.05, P<0.001)，第4 天以后无显著统计学差异，ASP 组perilipin 基因和蛋白表达水平从第3 天起显著升 高(P<0.05, P<0.001)，直至完全分化成熟(第9 天)仍显著高于同期对照组(P<0.05)；(4)胰岛素组adipophilin 和perilipin 基因 和蛋白表达水平均与同期对照组相比无显著统计学差异。以上结果提示，ASP 组perilipin 及adipophilin 的表达与脂肪细胞 分化过程中ASP 促进甘油三酯合成之间具有显著相关性，ASP 可能通过调节adipophilin、perilipin 基因和蛋白的表达影响 脂肪细胞内脂质聚积和脂滴形成。

关键词： 促酰化蛋白; 脂滴相关蛋白; 脂代谢; 脂肪细胞

分类号：R363；R335+.9 ；R589.2

[Effect of acylation stimulating protein on the perilipin and adipophilin expression during 3T3-L1 preadipocyte differentiation.] [Ariticle in Chinese]

WU Jing, LU Hui-Ling^*, HU Xiu-Fen, LIANG Xiao-Yan, LIN Han-Hua, WANG Hong-Wei, FAN Xiao-Wei

Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Abstract

Perilipin and adipophilin, two significant lipid droplet (LD)-specific proteins, participate in storing fat or ectopic lipid deposition and fat mobilization in many types of mammalian cells. Acylation stimulating protein (ASP) is a novel adipocyte-derived hormone known for a major determinant for triglyceride synthesis (TGS) and lipid metabolism. The present study was aimed to investigate: (1) whether ASP, rather than insulin, is a powerful potentiator which could physiologically and directly influence TGS during 3T3-L1 preadipocyte differentiation; (2) whether ASP exposure at indicated time points during 3T3-L1 preadipocyte differentiation could influence the gene/protein expression of adipophilin and perilipin. 3T3-L1 preadipocytes were differentiated by traditional hormone cocktail and divided into control, ASP and insulin groups according to the treatment of ASP (1 μmol/L) or insulin (100 nmol/L). ASP-stimulated and insulin-stimulated TGS rate at indicated time points (0 d, 3 d, 6 d, 9 d) were assayed by measuring the incorporation of [3H]-oleic acid into TG, and the corresponding glucose transport was assayed by [3H]-2-DG uptake. The effects of ASP or insulin on gene/protein expression of adipophilin and perilipin at indicated time points were evaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The results obtained were as follows: (1) on the 3rd and 6th day of differentiation, ASP dramatically enhanced TGS rate compared with control group (P<0.05, P<0.01); There was no significant difference in TGS rate between insulin group and control group; (2) on the 6th and 9th day of differentiation, both ASP and insulin promoted glucose uptake (P<0.05, P<0.01), and the promoting effect in ASP group was greater than that in insulin group; (3) ASP elevated adipophilin gene and protein expression at the very early stage of differentiation (P<0.05, P<0.001) and had no significant effect from the 4th day of differentiation. Perilipin gene and protein expression increased throughout preadipocyte differentiation and its expression was up-regulated following ASP stimulation from the 3rd day of differentiation (P<0.05, P<0.001) to the end of differentiation (P<0.05); (4) Insulin did not affect gene and protein variation pattern of adipophilin and perilipin. Taken together, this study provides evidence that ASP-evoked changes in gene and protein expression of adipophilin and perilipin correlate with ASPstimulated TGS acceleration, and adipophilin and perilipin are involved in the molecular mechanism of ASP-induced adipogenesis and LD formation.

Key words: acylation stimulating protein; lipid droplets associated proteins; lipid metabolism; adipocyte

收稿日期：2008-07-24　　录用日期：2008-10-28

通讯作者：卢慧玲　　E-mail: huilinglu@yahoo.cn

引用本文：

吴静, 卢慧玲, 胡秀芬, 梁晓燕, 林汉华, 王宏伟, 范晓蔚. 促酰化蛋白对3T3-L1 脂肪细胞分化进程中adipophilin 和 perilipin 表达的影响[J]. 生理学报 2009; 61 (1): 56-64.

WU Jing, LU Hui-Ling, HU Xiu-Fen, LIANG Xiao-Yan, LIN Han-Hua, WANG Hong-Wei, FAN Xiao-Wei. [Effect of acylation stimulating protein on the perilipin and adipophilin expression during 3T3-L1 preadipocyte differentiation.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (1): 56-64 (in Chinese with English abstract).