Hexose metabolism in pancreatic islets: pyruvate carboxylase activity

The anaplerotic hypothesis for insulin release postulates that an increased generation of malonyl-CoA, acyl residues and diacylglycerol in nutrient-stimulated pancreatic islets may couple the catabolism of nutrient secretagogues to more distal events in the secretory sequence. In the light of this hypothesis, pyruvate carboxylase activity was measured in rat pancreatic islets using two distinct radioisotopic procedures. The first procedure is based on the conversion of oxalacetate generated from pyruvate to 14C-labelled citrate in the presence of [1-14C]acetyl-CoA and citrate synthase. The second technique involves the conversion of 14C-labelled oxalacetate generated from [1-14C]pyruvate to radioactive aspartate in the presence of L-glutamate and glutamate-oxalacetate transaminase. Pyruvate carboxylase activity amounted to 10 pmol/min per islet, was restricted to mitochondria, displayed a Km for pyruvate close to 0.4 mM, and demonstrated dependency towards ATP (apparent Ka close to 0.1 mM), Mg2+ and acetyl-CoA. It is proposed that pyruvate carboxylase activity accounts for the generation of 14C-labelled amino acids other than alanine in islets exposed to D-[3,4-14C]glucose and participates to the pyruvate/citrate shuttle for the transport of acetyl-CoA out of the mitochondria in nutrient-stimulated islets.