Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect.
Previous studies have shown that the combination of vanadate and H2O2 generates peroxide(s) of vanadate (pervanadate) that is able to mimic insulin in stimulating lipogenesis or protein synthesis and inhibiting lipolysis in rat adipocytes. Here we report that pervanadate is a potent trigger of 3-O-methylglucose transport in rat adipocytes, with an effective concentration of 5 microM and a maximum at 20 microM. Moreover, pervanadate produced an additional activation of approximately 60% on glucose influx in cells treated with maximally activating concentrations of insulin. Vanadate was ineffective in potentiating insulin-stimulated glucose uptake. Quercetin, a bioflavonoid that inhibits insulin receptor tyrosine kinase, blunted this effect of pervanadate. Treatment of adipocytes with pervanadate inhibited protein phosphotyrosyl phosphatase activity of cell extracts in a dose-dependent manner, with an ID50 of 5 microM and complete inhibition at 80 microM. In contrast, vanadate (1-800 microM) did not appreciably inhibit cell phosphotyrosyl phosphatases. The inhibitory effect of pervanadate correlated with the increase in protein phosphotyrosine accumulation, as determined by Western blotting with antiphosphotyrosine antibodies. The most prominent phosphotyrosine-containing band detected in pervanadate-treated adipocytes was that of autophosphorylated insulin receptor, identified by immunoblotting or immunoprecipitation with antiinsulin receptor antibodies. The addition of insulin to pervanadate-treated adipocytes (20 microM) caused a further increase (approximately 70%) in receptor autophosphorylation. In a cell-free system using partially purified insulin receptor devoid of tyrosine phosphatase activity, pervanadate did not stimulate the receptor autophosphorylation or interfere with the stimulating effect of insulin. These results suggest that 1) pervanadate triggers glucose uptake by increasing autophosphorylation of insulin receptor, preventing its dephosphorylation; 2) under physiological conditions, cellular protein phosphotyrosyl phosphatase activity is high, thereby significantly opposing insulin-mediated hexose transport; and 3) pervanadate has the unique ability to markedly increase maximal cell responsiveness in stimulating glucose transport achieved at a saturating insulin concentration. These findings suggest a possible clinical application in the management of glucose uptake in pathological conditions of insulin resistance and hyperinsulinemia.
Shisheva
A
A
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Shechter
Y
Y
D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D004357:Drug Synergism; D005947:Glucose / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics*; D007328:Insulin / Q000494:pharmacology*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*
D007328:Insulin; C076715:pervanadate; D014638:Vanadates; D011794:Quercetin; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases; D005947:Glucose