Pyruvate dehydrogenase plays a critical role in the regulation of hepatic glucose and fatty acid oxidation; however, surprisingly little is known about its regulation in vivo. In this study we examined the individual effects of insulin and substrate availability on the regulation of pyruvate dehydrogenase flux (VPDH) to tricarboxylic acid flux (VTCA) in livers of awake rats with lipid-induced hepatic insulin resistance. VPDH/VTCA flux was estimated from the [4-13C]glutamate/[3-13C]alanine enrichments in liver extracts and assessed under conditions of fasting and during a hyperinsulinemic-euglycemic clamp, whereas the effects of increased plasma glucose concentration on VPDH/VTCA flux was assessed during a hyperglycemic clamp in conjunction with infusions of somatostatin and insulin to maintain basal concentrations of insulin. The effects of increases in both glucose and insulin on VPDH/VTCA were examined during a hyperinsulinemic-hyperglycemic clamp. The effects of chronic lipid-induced hepatic insulin resistance on this flux were also examined by performing these measurements in rats fed a high-fat diet for 3 weeks. Using this approach we found that fasting VPDH/VTCA was reduced by 95% in rats with hepatic insulin resistance (from 17.2 ± 1.5% to 1.3 ± 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase VPDH/VTCA flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did VPDH/VTCA flux increase (44.6 ± 3.2%, P < 0.0001 versus basal) in low-fat fed animals but not in rats with chronic lipid-induced hepatic insulin resistance. Conclusion: These studies demonstrate that the combination of both hyperinsulinemia and hyperglycemia are required to increase VPDH/VTCA flux in vivo and that this flux is severely diminished in rats with chronic lipid-induced hepatic insulin resistance. (HEPATOLOGY 2011.)