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 NH(2)-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.
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