High-fat (HF) and high-sucrose (SU) diets increase gluconeogenesis. The present study was designed to determine the contributions of pyruvate dehydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate kinase fluxes to this accelerated gluconeogenesis (GNEO) in the absence and presence of fatty acids. Male Sprague-Dawley rats were fed an HF, SU, or starch (ST) diet for 1 wk, and hepatocytes or mitochondria were isolated. In the absence of palmitate, the tracer estimated rates of GNEO (nmol. min(-1). mg(-1)) were elevated in hepatocytes isolated from SU (32.3 +/- 1.8) and HF (35.4 +/- 1.8) vs. ST (22.8 +/- 1.5). Pyruvate carboxylase and PEPCK flux rates (nmol. min(-1). mg(-1)) were increased in the SU (47.5 +/- 2.2 and 34.8 +/- 1.5) and HF (49.4 +/- 1.8 and 38.2 +/- 1.8) groups compared with the ST group (32.8 +/- 3.2 and 44.3 +/- 2.0). Palmitate (250-1,000 microM) stimulation of these fluxes was not significantly different among groups. Bromopalmitate, an inhibitor of fat oxidation, abolished differences in GNEO, pyruvate carboxylase, and PEPCK fluxes in HF and SU vs. ST. In isolated mitochondria, pyruvate carboxylation and palmitoyl carnitine oxidation were not significantly different among groups. The results of this study suggest that the increased gluconeogenic flux observed with HF and SU diets is associated with an increased pyruvate flux through pyruvate carboxylase and PEPCK. Moreover, the ability of bromopalmitate to normalize gluconeogenic fluxes suggests that endogenous fatty acids contribute to diet-induced increases in GNEO.