高浓度葡萄糖刺激脂肪细胞脂肪分解的效应及其机制

目的:研究高浓度葡萄糖刺激脂肪细胞脂肪分解升高血浆游离脂肪酸(free fatty acid, FFA)水平的效应及其机制,进一步阐明高糖导致胰岛素抵抗的作用机制.方法:以Sprague-Dawley(SD)大鼠附睾原代脂肪细胞为研究对象,设为正常葡萄糖(5mmol/L)对照组和高浓度葡萄糖(25mmol/L)干预组.将细胞孵育一定时间直接或加入异丙基肾上腺素刺激后测定培养基中甘油含量作为评价脂肪分解的指标.脂肪分解数据表示为每升脂肪细胞压积的毫摩尔甘油释放量(mmol/L packed cell volume, mmol/L PCV).用Western blot方法分别检测总的及磷酸化的脂滴包被蛋白(perilipin)含量以及激素敏感性脂肪酶(hormone-sensitive lipase, HSL)和脂肪组织甘油三酯水解酶(adipose triglyceride lipase, ATGL)的含量.用酶化学法测定胞浆脂肪分解酶活性.结果:高浓度葡萄糖明显刺激大鼠原代脂肪细胞脂肪分解.将细胞在高糖中孵育24h,甘油累积量从4.4mmol/L PCV增加至6.4mmol/L PCV,即增加1.5倍(P<0.01);30min甘油释放量从0.11mmol/L PCV增加至0.24 mmol/L PCV,即增加2.1倍(P<0.01).高糖刺激脂肪分解的作用从细胞孵育16h开始持续到24h,且25mmol/L葡萄糖的效应较10mmol/L葡萄糖强.高糖明显增加perilipin磷酸化,但不影响该蛋白表达.高糖使胞内脂肪分解酶活性升高1.74倍并显著上调HSL蛋白表达,但不影响ATGL蛋白含量.异丙基肾上腺素刺激的脂肪分解在高糖环境下进一步增强.结论:高浓度葡萄糖通过增加perilipin磷酸化、上调HSL蛋白表达和升高脂肪分解酶活性从而直接刺激脂肪细胞脂肪分解.这提示高浓度葡萄糖单独即可以使FFA从脂肪组织向血浆中释放增加,循环FFA水平升高从而导致胰岛素抵抗.

The mechanisms of stimulated lipolysis by high concentration of glucose in primary rat adipocytes

OBJECTIVE: To investigate the cellular mechanisms of adipocyte lipolytic response to a high concentration of glucose, which liberates free fatty acids (FFA) efflux from adipose cells to plasma. METHODS: Adipocytes were isolated from epididymal fat pads of Sprague-Dawley (SD) rats, and were incubated in the presence or absence of excess glucose (25 mmol/L). Glycerol released in the culture medium was determined by use of a colorimetric assay and served as an index of lipolysis. The data are expressed as micromoles glycerol per liter packed cell volume (PCV) of adipocytes. The protein levels of native and phosphorylated perilipin, hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were examined by immunoblotting. Adipose lipases activity was determined by performing an enzymatic assay. RESULTS: A high concentration of glucose at 25 mmol/L caused a significant increase in lipolysis in primary rat adipocytes. A 24 h incubation with 25 mmol/L glucose elevated glycerol accumulation from 4.4 mmol/L PCV to 6.4 mmol/L PCV, by 1.5 fold (P<0.01) and promoted glycerol release from 0.11 mmol/L PCV to 0.24 mmol/L PCV, by 2.1 fold (P<0.01). The lipolytic response mediated by high glucose occurred apparently at the end of 16 h and remained high after 24 h incubation. The lipolytic action of glucose at 25 mmol/L was more effective than that at 10 mmol/L. To elucidate the mechanism by which high glucose stimulated lipolysis, we showed that high glucose promoted the phosphorylation of perilipin but did not alter its protein level. Further, we showed that high glucose caused a significant increase in adipose lipases activity by 1.74 fold. The protein level of HSL but not ATGL was significantly upregulated by high glucose. Moreover, the acute lipolytic response to isoproterenol was further enhanced by high glucose. CONCLUSION: High glucose directly stimulates the releases of glycerol and FFA from adipocytes; the cellular mechanisms of this lipolytic action involve promoted perilipin phosphorylation, augmented HSL expression and increased adipose lipases activity under high glucose conditions. These results suggest that a high concentration of glucose alone can directly liberate FFA from adipocyte to plasma, hence increasing systemic FFA levels and impairing insulin sensitivity.