Measurement of the increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals.
Measurement of the increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals.
复制标题
测量肥胖，耐胰岛素耐药性个体脂肪组织中内质网应激相关蛋白和基因的增加。
DOI：
10.1016/b978-0-12-385116-1.00004-2
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发表时间：
2011
影响因子：
--
通讯作者：
Merali, Salim
中科院分区：
文献类型：
--
作者：
Boden, Guenther;Merali, Salim
在这里，我们提供了蛋白质组学、蛋白质印迹和 RT-PCR 分析的详细描述，这些分析用于检查瘦胰岛素敏感和肥胖胰岛素抵抗非糖尿病个体的脂肪活检样本，以获取内质网 (ER) 应激的证据。从六名瘦人和六名肥胖非糖尿病受试者的大腿上部获取皮下脂肪活检。脂肪匀浆用于蛋白质组学（二维凝胶 (2DE) 和 MALDI-TOF/TOF）、蛋白质印迹和 RT-PCR 分析。蛋白质组学分析揭示了肥胖受试者脂肪中 19 种差异上调的蛋白质。其中三种蛋白质是内质网应激相关的未折叠蛋白反应 (UPR) 蛋白钙网蛋白、蛋白二硫键异构酶 A3 和谷胱甘肽-S-转移酶 P；蛋白质印迹显示其他几种 UPR 应激相关蛋白的上调，包括钙联蛋白（一种膜结合分子伴侣）和磷酸化 c-jun NH2 末端激酶 (JNK)-1（一种 ER 应激下游效应蛋白）； RT-PCR 分析揭示了 X-box 结合蛋白-1s 剪接形式的上调，X-box 结合蛋白-1s 是一种有效的转录因子，也是近端 ER 应激传感器肌醇需求酶 1 通路的一部分。这些发现证明了肥胖人类受试者皮下脂肪组织中 UPR 的激活。由于 JNK 可以抑制胰岛素作用并激活促炎途径，因此 JNK 的 ER 应激激活可能是肥胖、胰岛素抵抗和炎症之间的联系。
Here, we provide a detailed description of proteomic, Western blot and RT-PCR analyses performed to examine fat biopsy samples from lean insulin-sensitive and obese insulin-resistant nondiabetic individuals for evidence of endoplasmic reticulum (ER) stress. Subcutaneous fat biopsies were obtained from the upper thighs of six lean and six obese nondiabetic subjects. Fat homogenates were used for proteomic (two-dimensional gel (2DE) and MALDI-TOF/TOF), Western blot, and RT-PCR analysis. Proteomic analysis revealed 19 differentially upregulated proteins in fat of obese subjects. Three of these proteins were the ER stress-related unfolded protein response (UPR) proteins calreticulin, protein disulfide-isomerase A3, and glutathione-S-transferase P; Western blotting revealed upregulation of several other UPR stress-related proteins, including calnexin, a membrane-bound chaperone, and phospho c-jun NH2-terminal kinase (JNK)-1, a downstream effector protein of ER stress; RT-PCR analysis revealed upregulation of the spliced form of X-box-binding protein-1s, a potent transcription factor and part of the proximal ER stress sensor inositol-requiring enzyme-1 pathway. These findings demonstrate of UPR activation in subcutaneous adipose tissue of obese human subjects. As JNK can inhibit insulin action and activate proinflammatory pathways, ER stress activation of JNK may be a link between obesity, insulin resistance, and inflammation.