Metformin inhibits citric acid cycle activity in isolated mitochondria. Mitochondria were incubated with [U-13C]pyruvate (m + 3) and unlabeled malate in the presence of ddH2O (control) or metformin (5 mM) for 30 minutes. (A) Schematic depicting stable isotope tracer experiment where substrates used are uniformly labeled [U-13C]pyruvate and unlabeled malate. The metabolites analyzed in B-E are placed into gray boxes where the isotopic enrichment is written as m + k where k is the number of 13C (black circles). (B) Enrichment of lactate (m + 3), (C), citrate (m + 2), (D) alpha-ketoglutarate (m + 2) and (E) succinate (m + 2) as evaluated by GC/MS analysis of mass distributions. Data are expressed as normalized ion amount which represents values obtained from mass isotopomer distribution (MID) × corrected area. Data are presented as means ± SEM. n = 3. *P <0.05, Student’s t-test (m + 2 or m + 3). #P <0.05, Student’s t-test (m + 0).