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Forskolin引起大鼠脂肪细胞内脂滴重塑

刘梅芳^1,*, 杜从阔², 苏雪莹²

¹济宁医学院药学院，日照 276826；²北京大学基础医学院生理与病理生理学系，北京 100191

摘要

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1 μmol/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，1 μmol/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。

关键词： Forskolin; 脂肪细胞; 脂滴; perilipin家族蛋白

分类号：R329.55

Forskolin induced remodeling of lipid droplets in rat adipocytes

LIU Mei-Fang^1,*, DU Cong-Kuo², SU Xue-Ying²

¹College of Pharmacy, Jining Medical University, Rizhao 276826, China；²Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Beijing 100191, China

Abstract

Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 μmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.

Key words: Forskolin; adipocyte; lipid droplet; perilipin family proteins

收稿日期：2018-08-03　　录用日期：2018-11-08

通讯作者：刘梅芳　　E-mail: lmf_bjmu@163.com

DOI: 10.13294/j.aps.2019.0038

引用本文：

刘梅芳, 杜从阔, 苏雪莹. Forskolin引起大鼠脂肪细胞内脂滴重塑[J]. 生理学报 2019; 71 (3): 379-387.

LIU Mei-Fang, DU Cong-Kuo, SU Xue-Ying. Forskolin induced remodeling of lipid droplets in rat adipocytes. Acta Physiol Sin 2019; 71 (3): 379-387