Increased MAPK activation and impaired insulin signaling in subcutaneous microvascular endothelial cells in type 2 diabetes: the role of endothelin-1. Diabetes 2009;58:2238–2245

OBJECTIVE—To establish a method for isolation and culture of subcutaneous microvascular endothelial cells (MVEC) from small human tissue biopsies to compare gene and protein ex-pression of insulin signaling molecules in MVEC from insulin-resistant and healthy control subjects. RESEARCH DESIGN AND METHODS—Stromavascular cells from subcutaneous needle biopsies of type 2 diabetic and control subjects were expanded in culture and the endothelial cells selected with magnetic immune separation. Western blots and RT-PCR were used for protein and gene expression assays. RESULTS—At least 99 % of the expanded primary MVEC could be characterized as endothelial cells. The expression of insulin receptors was low, but insulin increased tyrosine phosphoryla-tion of both the insulin receptor and insulin receptor substrate (IRS)-1 and activated protein kinase B (PKB). The IRS-1 protei