Alanine Metabolism in Rat Liver Mitochondria

TLDR

The study examined whether l‑cycloserine enters mitochondria and inhibits alanine metabolism. Rat liver mitochondria were incubated with alanine, 2‑oxoglutarate, bicarbonate, Pi, and ATP to assess alanine metabolism. Alanine metabolism in rat liver mitochondria produced glutamate, aspartate, malate, and citrate, with low steady‑state pyruvate; octanoate stimulated the pathway, while l‑cycloserine strongly inhibited it; inhibitors of pyruvate transport did not affect alanine metabolism, indicating mitochondrial specificity; and cellular studies suggest alanine is largely converted to pyruvate within mitochondria. Chem.

Abstract

In the presence of 2‐oxoglutarate, bicarbonate, P i and ATP, rat liver mitochondria were found to metabolize alanine. The main products were glutamate, aspartate, malate and citrate. Pyruvate did not accumulate but reached a low steady‐state concentration. Addition of octanoate caused a strong stimulation of alanine metabolism and a concomitant decrease in pyruvate concentration. NO inhibition of alanine metabolism was obtained in the presence of inhibitors of mitochondrial pyruvate transport, α‐cyano‐4‐hydroxy‐cinnamate and α‐cyano‐cinnamate, showing that alanine was not transaminated by a contamination of the mitochondria by cytosolic alanine aminotransferase. In the presence of l ‐cycloserine, alanine metabolism in mitochondria was strongly inhibited indicating that in contrast to earlier reports l ‐cycloserine can enter the mitochondria. In isolated liver cells, addition of α‐cyano‐cinnamate caused a strong pyruvate accumulation and a nearly complete inhibition of gluconeogenesis with serine as substrate, whereas with alanine no pyruvate increase and a weak inhibition of gluconeogenesis was observed. From a comparison of the employed substrate concentrations and of the observed rates of alanine transamination in intact mitochondria with those under physiological conditions in cellular systems, it is concluded that cellular conversion of alanine to pyruvate most likely occurs to a great extent within the mitochondria as has been proposed earlier by DeRosa and Swick (1975) J. Biol. Chem. 250 , 7961‐7967.