【作者】 张婷婷；

【导师】 郭晓蕙； 徐国恒；

【作者基本信息】 北京大学 ， 内科学， 2008， 博士

【摘要】 目的:研究二甲双胍对异丙基肾上腺素和高浓度葡萄糖刺激脂肪分解的抑制效应,并探讨其分子机制,进一步阐明二甲双胍降低游离脂肪酸改善胰岛素抵抗的作用机理,同时也为深入了解脂肪分解调控机制提供了资料。方法:本研究以SD大鼠原代附睾脂肪细胞为对象。细胞分离后设对照组,二甲双胍(250或500μmol/L)干预组,异丙基肾上腺素(100 nmol/L或1μmol/L)或高浓度葡萄糖(25 mmol/L)干预组,以及二甲双胍加异丙基肾上腺素或高浓度葡萄糖共同干预组。细胞培养后比色法测定培养液中甘油的累积量或释放量作为评价脂肪分解的指标。用Western blot方法检测不同组别细胞中perilipin表达及其磷酸化水平、ERK1和磷酸化ERK1/2、HSL和ATGL表达。用酶化学法测定胞浆脂肪分解酶活性。分别用125I和32P标记的放射免疫方法测定cAMP水平及PKA活性。用Western blot方法和免疫荧光方法检测HSL的转位情况。实验数据应用GraphPad Prism 4.0软件进行统计学分析。结果:1、二甲双胍抑制异丙基肾上腺素刺激的脂肪分解。1μmol/L的异丙基肾上腺素使甘油释放量增加3.8倍(P <0.001);250μmol/L和500μmol/L的二甲双胍分别使异丙基肾上腺素刺激的甘油释放量降低43.6%(P <0.01)和56.7%(P <0.001),这种抑制作用从二甲双胍孵育4 h开始持续到24 h。异丙基肾上腺素作用后细胞cAMP水平和PKA活性分别增加了1.5倍(P <0.05)和3.0倍(P <0.01),二甲双胍干预后分别使其降低50.3%(P <0.001)和70.5%(P <0.001)。异丙基肾上腺素使ERK1/2磷酸化增加了2倍(P <0.01),二甲双胍使其减少54.7%(P <0.001)。二甲双胍还能够减少异丙基肾上腺素引起的perilipin磷酸化。异丙基肾上腺素刺激后胞内脂肪分解酶活性升高1.96倍(P <0.01),二甲双胍使其降低44.5%(P <0.01),但二甲双胍不影响HSL和ATGL的蛋白表达以及HSL转位。2、二甲双胍抑制高浓度葡萄糖刺激的脂肪分解。25 mmol/L葡萄糖增加基础脂肪分解1.72倍(P <0.001),这种效应从16 h开始明显并持续到24 h,而10 mmol/L葡萄糖则对基础脂肪分解无明显影响。此外,在胰岛素存在的条件下,高浓度葡萄糖进一步增强异丙基肾上腺素刺激的脂肪分解,但胰岛素不影响高浓度葡萄糖本身的刺激脂肪分解效应。250μmol/L和500μmol/L的二甲双胍分别降低高浓度葡萄糖刺激的脂肪分解31.8%(P <0.001)和69.1%(P <0.001),另外500μmol/L二甲双胍还可以明显抑制基础状态下的脂肪分解和高糖高胰岛素状态下异丙基肾上腺素更强的促脂肪分解效应。高浓度葡萄糖和二甲双胍可以影响perilipin磷酸化水平但不影响其总蛋白表达,即高浓度葡萄糖使磷酸化perilipin与总perilipin的比值增加1.53倍(P <0.05),二甲双胍干预后使其降低41.1%(P <0.01)。高浓度葡萄糖升高胞内脂肪分解酶活性1.59倍(P <0.05),二甲双胍使其降低35.8%(P <0.05)。高浓度葡萄糖和二甲双胍均使HSL蛋白表达明显增加,且有协同作用,但二者均不影响ATGL蛋白表达。结论:二甲双胍能够抑制异丙基肾上腺素和高浓度葡萄糖刺激的脂肪分解,也能够抑制高糖高胰岛素状态异丙基肾上腺素刺激的更强的脂肪分解。二甲双胍通过降低异丙基肾上腺素引起的cAMP水平升高和PKA活性增强,减少perilipin磷酸化,同时减少ERK磷酸化和胞内脂肪分解酶活性增加,从而抑制异丙基肾上腺素的刺激脂肪分解效应。二甲双胍通过抑制perilipin磷酸化增加和胞内脂肪分解酶活性增强从而减少高浓度葡萄糖刺激的脂肪分解。提示二甲双胍可能通过抑制异丙基肾上腺素和糖尿病状态下高浓度葡萄糖刺激的脂肪分解,减少FFA释放,从而改善胰岛素敏感性。更多还原

【Abstract】 Objective: To investigate the inhibitory effects of metformin on isoproterenol or high-concentration glucose induced lipolysis in primary rat adipocytes and further elucidate the underlying mechanisms, for better understanding of the improvement of insulin sensitivity by metformin.Methods: Adipocytes were isolated from epididymal fat pads of Sprague-Dawley (SD) rats. After isolation and digestion, packed adipocytes were incubated in the presence or absence of excess glucose or/and metformin at the concentrations as planed, washed and then treated for 30 min after the addition of isoproterenol or not. Glycerol released into the media was determined by use of a colorimetric assay and served as an index of lipolysis. The expressions of perilipin and its phosphorylation state, phosphorylated-extracellular signal-related kinase (p-ERK1/2) and total ERK1, hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were examined by Western blot. Adipose lipases activity was assayed by using an enzymatic assay. Cyclic adenosine monophosphate (cAMP) level and protein kinase A (PKA) activity were examined by immunoradioassay using 125I and 32P respectively. HSL translocation was detected by Western blot and immunofluorescence. Data were statistically analyzed by GraphPad Prism version 4.0. Results: 1. Metformin inhibits isoproterenol-stimulated lipolysis in primary adipocytes. Isoproterenol at 1μmol/L induced a 3.8-fold (P < 0.001) increase in glycerol production over the 30 min control. The antilipolytic action of metformin at 500μmol/L was more effective than that at 250μmol/L. They inhibited the isoproterenol-stimulated lipolysis by 56.7%(P <0.001)and 43.6%(P <0.01)respectively. The time course study depicted that the inhibition began as early as 4 h after the addition of 500μmol/L metformin and lasted to 24 h incubation. The addition of isoproterenol elevated intracellular cAMP level and PKA activity by 1.5-fold (P<0.05)and 3.0-fold(P <0.01)approximately, while metformin caused a 50.3%(P <0.001)and 70.5% (P <0.001)inhibition respectively. Metformin greatly inhibited the double augment of isoproterenol-mediated ERK1/2 phosphorylation by 54.7%(P <0.001), and also reduced the increment of perilipin phosphorylation caused by isoproterenol. Isoproterenol enhanced the total lipolytic lipases activity to round 200% (P <0.01) over that in control, while treatment with metformin reduced the lipases activity by 44.5% (P <0.01). In addition, metformin had no effect on the expression of HSL and ATGL as well as HSL translocation. 2. Metformin restricts the chronic lipolytic action upon high-concentration glucose. High-concentration glucose stimulated lipolysis; glucose at 25 mmol/L elevated glycerol release by 1.72-fold(P <0.001), and such lipolytic response occurred apparently at 16 h and remained high after 24 h incubation, whereas glucose at 10 mmol/L did not produce stimulative effect on basal lipolysis. Additionally, in the presence of insulin, the incubation of adipocytes with high-concentration glucose further enhanced the lipolytic response upon adrenergic stimulation of isoproterenol, while insulin did not affect the intrinsic lipolytic action of high-concentration glucose. Metformin at 500μmol/L showed more potent antilipolytic effect than that at 250μmol/L. They could reduce high glucose-induced lipolysis by 69.1%(P <0.001)and 31.8%(P <0.001)separately, meanwhile, metformin at 500μmol/L markedly inhibited basal and promoted lipolysis induced by isoproterenol in the combination of high-concentration glucose and insulin. High-concentration glucose increased the phosphorylation of perilipin, however metformin significantly attenuated it. Neither high glucose nor metformin altered the expression of perilipin. Exposure of the adipocytes to a medium with high glucose led to an increase by a factor of 1.59-fold (P <0.05) in adipose lipases activity, whereas pretreatment with metformin caused a reduction of 35.8% (P <0.05). Both high glucose and metformin upregulated the expression of HSL with cooperative effect in combination, though neither of them affected the expression of ATGL. Conclusion: Metformin reduces lipolysis in primary rat adipocytes stimulated by isoproterenol or high-concentration glucose. It also restrains the lipolytic stimulation of isoproterenol enhanced by the combination of high-concentration glucose and insulin. Metformin attenuates isoproterenol-mediated lipolysis by inhibiting the elevation of cellular cAMP level and PKA activity, suppressing phosphorylation of ERK1/2 and perilipin and also abrogating the increased activity of adipose lipases. Metformin restricts high glucose-induced lipolysis through eliminating perilipin phosphorylation and decreasing the augment of lipolytic lipases activity. This study provides novel evidences that metformin reduces plasma free fatty acid (FFA) through antagonizing isoproterenol- and high glucose-induced lipolysis in adipocytes that leads to the improvement of insulin sensitivity.更多还原