肿瘤坏死因子-α上调大鼠分化脂肪细胞脂肪分化相关蛋白水平及其机制

Tumor necrosis factor-α upregulates the protein levels of adipose differentiation-related protein in rat differentiated adipocytes

目的:研究肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)对大鼠原代分化脂肪细胞内脂肪分化相关蛋白(adipose differentiation-related protein,ADRP)表达的影响及其机制。方法:以SD大鼠附睾来源的原代分化脂肪细胞为对象,设正常对照组和TNF-α(25μg/L)干预组。细胞孵育后测定培养基中甘油含量作为评价脂肪分解的指标。Western blot检测脂滴包被蛋白perilipin和ADRP的蛋白表达。用免疫荧光染色以及脂滴染色的方法,观察脂肪细胞内脂滴的形态变化以及蛋白在细胞内的定位。结果:在脂肪细胞分化5～8d,TNF-α可显著刺激其脂肪分解,该作用在孵育8h开始出现并持续到48h。Western blot结果显示TNF-α增加ADRP蛋白表达,减少perilipin蛋白并增加其磷酸化水平。形态学结果显示TNF-α引起脂滴碎裂,增加的ADRP与perilipin一起包被在细胞内碎裂脂滴表面。结论:TNF-α慢性刺激脂肪分解过程中引起脂滴碎裂,增加脂滴表面包被蛋白ADRP,而该蛋白表达增加可能与脂滴碎裂相关。

Objective ：To investigate the effect and basis of tumor necrosis factor-α （TNF-α） on the lipid droplet associating protein adipose differentiation-related protein （ADRP） in rat differentiated adipo- cytes. Methods： Differentiated rat adipocytes derived from epididyrnal fat pads of Sprague-Dawley rats were incubated in the presence or absence of TNF-α （25 μg/L）. Glycerol level released in the culture medium was determined by a colorimetrie assay and served as an index of lipolysis. The protein levels of two lipid droplet associating proteins, perilipin and adipose differentiation-related protein, were examined by immunoblotting analysis. The intracellular lipid droplets were stained by nile red and the localizations of perilipin and ADRP on the lipid droplets were determined by immunostaining. Results： On day 5 or day 8 after the differentiation of adipocytes, TNF-α significantly stimulated lipolysis. The lipolysis response to TNF--α occurred apparently at hour 8 and remained high 48 h after the stimulation. TNF-α increased the protein levels of ADRP, downregulated perilipin proteins but promoted its phosphory lation level. TNF-α caused the lipid droplet dispersion, whereas ADRP and perilipin proteins were remained to associate with the surface of the dispersed lipid droplets. Conclusion： Chronic stimulation of lipolysis by TNF-α resulted in the dispersion of the lipid droplets and therefore upregulated the levels of ADRP proteins.