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 (V PDH ) to tricarboxylic acid flux (V TCA ) in livers of awake rats with lipid-induced hepatic insulin resistance. V PDH /V TCA flux was estimated from the [4-13 C]glutamate/[3-13 C]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 V PDH /V TCA 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 V PDH / V TCA 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 V PDH /V TCA was reduced by 95% in rats with hepatic insulin resistance (from 17.2 6 1.5% to 1.3 6 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase V PDH /V TCA flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did V PDH /V TCA flux increase (44.6 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 V PDH / V TCA flux in vivo and that this flux is severely diminished in rats with chronic lipidinduced hepatic insulin resistance. (HEPATOLOGY 2011;53:1175-1181) P yruvate dehydrogenase (PDH) is a multienzyme complex, located in the mitochondrial matrix, and is responsible for the oxidative decarboxylation of pyruvate into acetyl-CoA with concomitant reduction of nicotinamide adenine dinucleotide (NAD þ ) to NADH. This reaction has a particularly important role in the regulation of fuel selection because it controls the entry of glucose carbons into the tricarboxylic acid (TCA) cycle. PDH is allosterically inhibited by high [acetyl-CoA]/[CoA] and [NADH]/[NAD þ ] and activated by high pyruvate concentrations. Regulation of PDH is also Abbreviations: DAG, diacylglycerol; GIR, glucose infusion rate; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase; PKC, protein kinase C; POCE, proton-observed carbon-edited; PPARa, peroxisome proliferator-activated receptor a; V PDH , flux through pyruvate dehydrogenase; V TCA , flux through tricarboxylic acid cycle.