Carboxylation of Pyruvate by Isolated Rat Adipose Tissue Mitochondria

Mitochondria were isolated from rat epididymal adipose tissue and shown to be functionally intact as judged from both the P:O and respiratory control ratios, and structurally intact as determined by electron microscopy. In the presence of pyruvate, ATP, inorganic phosphate, and magnesium ions, these mitochondria incorporated H14CO3- into organic acids. Citrate and malate were the main radioactive products, with citrate containing about two-thirds of the radioactivity fixed. With the use of a rapid syringe filtration method the efflux of the newly formed intermediates from the mitochondria was shown. The observed high rates of pyruvate carboxylation and citrate formation have been further supported by the levels of pyruvate carboxylase and citrate synthase in mitochondria. The deletion of inorganic phosphate from the incubation medium reduced the H14CO3- fixation presumably due to the accumulation of ADP. Pyruvate carboxylation was also inhibited by ADP even in the presence of ATP. Mitochondria were shown to metabolize exogenous labeled citrate and malate, and pyruvate caused a 5-fold increase in the formation of 14C-citrate from 14C-malate. The results suggest that pyruvate carboxylation is regulated, in part, by the levels of pyruvate and ATP in adipose tissue mitochondria. Our observations lend experimental evidence supporting the role of the mitochondria in supplying the precursors for the citrate cleavage and glyceroneogenic pathways in rat adipose tissue, and emphasize the important role of pyruvate carboxylase in lipogenesis.