Measurement of the increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals

Methods Enzymol. 2011;489:67-82. doi: 10.1016/B978-0-12-385116-1.00004-2.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipose Tissue / metabolism*
  • Blotting, Western
  • Calreticulin / metabolism
  • Electrophoresis, Gel, Two-Dimensional / methods
  • Endoplasmic Reticulum / metabolism*
  • Glutathione S-Transferase pi / metabolism
  • Heat-Shock Proteins / metabolism*
  • Humans
  • Insulin Resistance / physiology
  • Obesity / physiopathology*
  • Protein Disulfide-Isomerases / metabolism
  • Proteomics / methods
  • Reverse Transcriptase Polymerase Chain Reaction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Unfolded Protein Response

Substances

  • Calreticulin
  • Heat-Shock Proteins
  • Glutathione S-Transferase pi
  • Protein Disulfide-Isomerases
  • PDIA3 protein, human