Incorporation of ³²P_i into pyruvate dehydrogenase phosphate in mitochondria from control and insulin-treated adipose tissue

Abstract

PYRUVATE dehydrogenase complexes (EC 1.2.4.1) isolated from mammalian sources have been shown to be inactivated by phosphorylation of the aα subunit of the pyruvate decarboxylase component. This covalent modification is brought about by a tightly bound, ATP-requiring kinase, which is activated by NADH and acetyl Co A and inhibited by pyruvate, Ca²⁺, thiamine pyrophosphate and ADP. Reactivation is catalysed by a specific phosphatase which requires both Mg²⁺ and Ca²⁺ (refs 1–6). In rat epididymal fat pads, the proportion of the complex in the active non-phosphorylated form is increased following brief exposure of pads to insulin^6–8 and this effect persists during preparation and subsequent incubation of mitochondria with oxidisable substrates such as oxoglutarate and malate^9,10. The mechanism by which the interaction of insulin with the cell membrane results in the changes of this mitochondrial enzyme system has not been established. It is possible that insulin may act through activation of the phosphatase by an increase in mitochondrial Ca²⁺ concentration and this possibility has been explored extensively in this laboratory^3,9,11. In contrast, other workers^7,8 have argued that the effect of insulin is brought about by an inhibition of the kinase caused by a lowering of the mitochondrial concentration ratio ATP:ADP. We report here evidence against the latter hypothesis.