Caveolin-1 phosphorylation regulates vascular endothelial insulin uptake and is impaired by insulin resistance in rats.
OBJECTIVE
As insulin entry into muscle interstitium is rate-limiting for its overall peripheral action, defining the route and regulation of its entry is critical. Caveolin-1 is required for caveola formation in vascular endothelial cells (ECs) and for EC insulin uptake. Whether this requirement reflects simply the need for caveola availability or involves a more active role for caveolae/caveolin-1 is not known. Here, we examined the role of insulin-stimulated tyrosine 14 (Tyr(14))-caveolin-1 phosphorylation in mediating EC insulin uptake and the role of cellular Src-kinase (cSrc), TNF-α/IL-6 and high fat diet (HFD) in regulating this process.


METHODS
Freshly isolated ECs from normal or HFD-fed rats and/or cultured ECs were treated with FITC-labelled or regular insulin with or without a Src or phosphotidylinositol-3-kinase inhibitor, TNF-α or IL-6, or transfecting FLAG-tagged wild-type (WT) or mutant (Y14F) caveolin-1. Tyr(14)-caveolin-1/Tyr(416) cSrc phosphorylation and FITC-insulin uptake were quantified by immunostaining and/or western blots.


RESULTS
Insulin stimulated Tyr(14)-caveolin-1 phosphorylation during EC insulin uptake. Inhibiting cSrc, but not phosphotidylinositol-3-kinase, reduced insulin-stimulated caveolin-1 phosphorylation. Furthermore, inhibiting cSrc reduced FITC-insulin uptake by ∼50%. Overexpression of caveolin-1Y14F inhibited, while overexpression of WT caveolin-1 increased, FITC-insulin uptake. Exposure of ECs to TNF-α or IL-6, or to 1-week HFD feeding eliminated insulin-stimulated caveolin-1 phosphorylation and inhibited FITC-insulin uptake to a similar extent.


CONCLUSIONS
Insulin stimulation of its own uptake requires caveolin-1 phosphorylation and Src-kinase activity. HFD in vivo and proinflammatory cytokines in vitro both inhibit this process.
Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, 450 Ray C. Hunt Drive, Box 801410, Charlottesville, VA, 22908, USA, Hw8t@virginia.edu.
Hong
H
Wang
Aileen X
AX
Wang
Kevin
K
Aylor
Eugene J
EJ
Barrett
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D051242:Caveolin 1 / Q000378:metabolism*; D059305:Diet, High-Fat; D042783:Endothelial Cells / Q000166:cytology*; D007328:Insulin / Q000378:metabolism; D007333:Insulin Resistance; D015850:Interleukin-6 / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000378:metabolism*
C495537:Cav1 protein, rat; D051242:Caveolin 1; D007328:Insulin; D015850:Interleukin-6; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases