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Insulin Signaling Stimulates Insulin Transport By Bovine Aortic Endothelial Cells

¹Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, VA 22908, USA

Objective: In vivo evidence suggests that insulin entry into skeletal muscle is rate-limiting for its overall metabolic action. Though there has been controversy regarding whether insulin crosses the endothelium by a passive (transcellular or paracellular) or mediated process, accumulating data favors the latter. Here, we addressed whether insulin signaling within the endothelial cell is required for the first step of trans-endothelial insulin transport, its uptake by the endothelial cell.

Research Design and Methods: Bovine aortic endothelial cells (bAECs) were incubated in serum-free media for 6 hours prior to addition of 50nM fluoroisothiocyanate (FITC)-labeled insulin for 30 min and uptake of FITC-insulin was quantified by confocal immunocytochemistry.

Results: Cellular insulin uptake was temperature dependent, being greater at 37 versus 4°C (p<0.05). Inhibiting PI-3 kinase (wortmannin), MEK (PD98059), the cSrc-family tyrosine kinase (PP1) or the insulin receptor tyrosine kinase (genistein) markedly diminished FITC-insulin uptake (p<0.05 for each). In contrast, inhibiting the phosphotyrosine phosphatase PTP1B further stimulated insulin uptake (p<0.05). Addition of the inflammatory cytokine TNF (5ng/ml) for six hours prior to adding 50 nM FITC-insulin diminished insulin uptake significantly (p<0.05). This inhibitory effect of TNF could be partially reversed by a specific p38 MAPK inhibitor (SB203580).

Conclusions: Insulin uptake by bAECs requires intact insulin signaling via both the PI-3-kinase and MEK signaling cascades as well as cSrc-family tyrosine kinases and that endothelial cell insulin uptake is sensitive to cytokine-induced insulin resistance.

Key Words: insulin signaling pathway • proinflammatory cytokine • insulin action • caveolin-1 • endothelial cells • insulin uptake • immunocytochemistry

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