高浓度胰岛素、葡萄糖对HepG2细胞蛋白激酶C活性的影响

高浓度胰岛素、葡萄糖对HepG2细胞蛋白激酶C活性的影响 2011-01-16 叶娟 李果 陈家伦 【摘要】 目的 研究高浓度胰岛素、葡萄糖对HepG2细胞蛋白激酶C(PKC)活性的影响。 方法建立具胰岛素抵抗特性受体下调的HepG2(DR-HepG2)细胞模型，测定10-7mol/L胰岛素、 25 mmol/L葡萄糖对未处理HepG2(NDR-HepG2)细和DR-HepG2细胞膜PKC、胞浆PKC活性 的影响。结果DR-HepG2细胞未受高浓度胰岛素、葡萄糖刺激时膜和胞浆PKC活性均高于 NDR-HepG2细胞(P,0.05)。10-7mol/L胰岛素刺激NDR-HepG2细胞或DR-HepG2细胞，30秒 和5,10分时膜PKC活性增高，胞浆PKC活性降低(P,0.01)。25mmol/L葡萄糖引起二种不 同状态下HepG2细胞膜PKC活性二相增高，胞浆PKC活性增高(NDR-HepG2细胞)或降低 (DR-HepG2细胞)(P,0.01)。10-7mol/L胰岛素和25 mmol/L葡萄糖同时刺激细胞，膜和 胞浆PKC活性的变化与25mmol/L葡萄糖单独刺激时基本一致。结论 PKC上调与DR-HepG2 细胞的胰岛素抵抗状态有关。10-7mol/L胰岛素或25 mmol/L葡萄糖刺激二种不同状态下 的HepG2细胞，引起膜PKC活性二相增高，但对胞浆PKC活性的影响不完全相同;二者同时 刺激主要通过葡萄糖介导的机制激活PKC。 【关键词】 蛋白激酶C 胰岛素 葡萄糖 HepG2细胞 Effects of high concentrations of insulin and glucose on the protein kinase C activities of HepG2 cells YE Juan,LI Guo,CHEN Jialun Shanghai Institute of Endocrinology,Shanghai,200025 【Abstract】 Objective To study the effec?s of high concentrations of insulin and glucose on the prot?in kinase C (PKC) activities of HepG2 cells. Methods A model of receptor down-regulated HepG2 (DR-HepG2) cells with characteristics of insulin resistance was developed. The membrane and cytosolic PKC activities of NDR-HepG2 cells and DR-HepG2 cells stimulated by 10-7mol/L insulin and 25mmol/L glucose were measured. Results Without stimulation by 10-7mol/L insulin or 25mmol/L glucose, the PKC activities of membrane and cytosol of the DR-HepG2 cells were higher than those observed in NDR-HepG2 cells (P,0.05). 10-7mol/L insulin induced biphasic increases of membrane PKC activities by NDR-HepG2 cells and DR-HepG2 cells (P,0.01), the initial increase being at 30 sec and the secondary at 5,10min. Meanwhile, the activities of cytosolic PKC reduced markedly. 25mmol/L glucose induced biphasic increases of the membrane PKC activities by NDR-HepG2 cells and DR-HepG2 cells (P,0.01). At the same time the activities of cytosolic PKC increased slightly by NDR-HepG2 cells or decreased by DR-HepG2 cells. The effects of 25mmol/L glucose were parallel to those when 10-7mol/L insulin and 25mmol/L glucose were treated simultaneously. Conclusion The up-regulation of PKC may be related to the insulin resistance of DR-HepG2 cells. 10-7mol/L insulin or 25mmol/L glucose induced biphasic increases of membrane PKC activities of NDR-HepG2 cells and DR-HepG2 cells. But the effects on cytosolic PKC were different. PKC may be activated through the glucose-induced mechanisms mainly when insulin and glucose were treated s multaneously. 【Key words】 Protein kinase C Insulin Glucose HepG2 cells (Chin J Endocrinol Metab, 1999,15:294-297) 蛋白激酶C(PKC)是一族普遍存在于细胞内的专一性蛋白质丝氨酸/苏氨酸磷酸化激酶，广泛参与细胞生长分化、基因达调控、激素和神经递质的分泌、跨膜信号传递、受体失敏和介导免疫反应等〔1〕。胰岛素、葡萄糖或其它激动剂通过二酰甘油(diacylglycerol, DG)途径激活PKC，后者通过其蛋白质磷酸化作用产生一系列生物效应。但是，一种或多种PKC亚型的过度激活可导致胰岛素受体信号传递的减弱，如磷酸化胰岛素受体β亚单位丝氨酸/苏氨酸残基使其酪氨酸激酶活性降低、抑制糖原合成酶活性等，与胰岛素抵抗密切相关〔2，3〕;过度激活的PKC还可能通过增加肾小球内皮细胞通透性、刺激肾系膜细胞合成基质蛋白等机制参与糖尿病肾病的病理改变〔4〕;高糖介导的PKC激活可改变一些与血管功能有关的酶或蛋白质的活性和基因表达，导致大小血管功能紊乱，是糖尿病慢性并发症的发病因素之一〔5，6〕。 受体下调HepG2(DR-HepG2)细胞既具有肝细胞的代谢特点，又有胰岛素受体和受体后功能缺陷〔7，8〕，是研究糖尿病及胰岛素抵抗的理想细胞模型。本研究以未处理HepG2(ND-HepG2)细胞和(DR-HepG2)细胞为研究对象，观察10-7mol/L胰岛素或25mmol/L葡萄糖及二者同时刺激对二种不同状态下HepG2细胞膜和胞浆PKC活性的影响，探讨PKC在糖尿病和胰岛素抵抗中的潜用。 材料和方法 一、材料 HepG2细胞株购于日本筑波细胞库。Ac-MBP(4-14)、PKC(19-36)、PMA、磷酸纤维素薄膜等购于Gibco公司。32P-ATP、14C-葡萄糖购于Dupont公司。125I-胰岛素购于Amersham公司。其余试剂购于Sigma公司。 主要仪器:超净工作台(苏州净化设备厂);CO2温箱(Napco,5410型);超速离心机(Beckman,Optima)等。 二、方法 1.细胞培养:HepG2细胞培养于细胞培养板，10%小牛血清的DMEM培养液、37?、5%CO2温箱中孵育。(DR-HepG2)细胞模型的建立:单层贴壁HepG2细胞置于含10-7mol/L胰岛素的DMEM培养液中孵育24小时。弃培养液，于4?条件下用预冷的pH 4.0的DMEM培养液和0.01mol/L磷酸盐缓冲液(PBS)分别洗涤细胞5次，即成为DR-HepG2细胞。 2.残余125I-胰岛素结合率测定:NDR-HepG2细胞或DR-HepG2细胞置于含10-9mol/L或10-7mol/L胰岛素的DMEM培养液中孵育24小时。弃培养液，于4?条件下，用预冷的p4.0的DMEM培养液及0.01mol/L PBS分别冲洗5次。每孔中加2ml含3μCi125I-胰岛素的DMEM培养液，4?孵育3小时。弃培养液用0.01mol/L PBS 冲洗5次，每孔中加入0.25%胰酶1ml，室温消化25分钟，用含15%小牛清的DMEM培养液终止反应。吹打细胞并移入测试管中，用1ml PBS洗5次，洗液一并加入测试管中，2000rpm离心10分钟。弃上清，测沉淀物放射计数。用10-7mol/L胰岛素与125I-胰岛素竞争结合后的残余125I-胰岛素结合量作为非特异性结合。未用胰岛素刺激的HepG2细胞残余125I-胰岛素结合作为对照。