Insulin stimulates glucose uptake in muscle cells via activation of protein kinase B (PKB). The protein tyrosine phosphatase (PTP) inhibitor vanadate, is a known insulin mimetic agent but the mechanism whereby vanadate exerts its effect is not clearly understood. Vanadate also has beneficial effects in the diabetic myocardium. The aim of this study was to correlate insulin stimulation of glucose uptake and PKB activation with that induced by vanadate in adult ventricular myocytes from lean and obese Zucker fa/fa rats. In lean Zucker rats, 100 nM insulin and 5 mM vanadate stimulated myocardial 2-deoxy-D-[3]glucose (2-DG) uptake from 27.17 +/- 1.72 to 96.52 +/- 10.87 and 43.86 +/- 4.02 pmole/mg protein p/30 min respectively while a combination of insulin and vanadate could not improve the maximal response of insulin. In obese Zucker hearts, basal as well as insulin and vanadate stimulated glucose uptake were severely impaired (15.49 +/- 1.44 vs 25.51 +/- 3.11 and 20.11 +/- 1.68 pmole/mg protein/30 min respectively). A combination of insulin and vanadate, resulted in a response significantly improved from the maximal response of insulin. This stimulation of 2-DG uptake was, in all instances, blocked by the PI 3-kinase inhibitors wortmannin and LY 294002. Insulin could not activate PKB, as measured by the Ser473 phosphorylated form of the enzyme, in the obese Zucker rats to the same extent as in lean controls. Similar to glucose uptake, activation of PKB by vanadate plus insulin was significantly more than that accomplished by insulin alone in obese rats. Both insulin and vanadate activation of PKB was prevented by wortmannin and LY 294002. Thus, the present study demonstrates that: (i) in cardiomyocytes from lean and obese Zucker rats, both insulin and vanadate stimulate glucose uptake and PKB activation through a PI 3-kinase sensitive pathway. (ii) In obese Zucker rats, neither insulin nor vanadate could induce glucose uptake or activation of PKB to the same extent as in lean controls. (iii) A combination of insulin with vanadate may be beneficial to increase glucose uptake in diabetic hearts, as this gives a better response than insulin alone.