Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes

Molecular and Cellular Endocrinology
Karin G StenkulaF H Nystrom

Abstract

Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% +/- 5 (mean +/- S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.

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Citations

Jun 4, 2010·Antioxidants & Redox Signaling·Leanne M Redman, Eric Ravussin
Nov 10, 2004·Current Opinion in Clinical Nutrition and Metabolic Care·Julie V SmithEric Ravussin
Apr 6, 2006·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Karen K DealE Michelle Southard-Smith
Jun 19, 2007·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Anita OstPeter Strålfors

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