Regulation of Pyruvate Dehydrogenase in Rat Heart

1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity, of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mm) or dichloroacetate (1m) increased the proption of active (dephosphorylated) pyruvate debydrogenase in perfused rat heart, presumably by inhibiting the pyrtvate dehydrogenase kinase, reaction. Alloxan-diabetes markedly decreased-the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity ofpyruvatedehydrogease in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oyoglutarate plus malate increased Al? and NADH concentrations and decreased the proportion of active pyruvate dhlydrogenase. The decrease in active -ehydrogenase was somewhat greater in mitochondria prepared from hearts -of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10mM) or dichloroacetate (4-50#M) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition.of the pyruvate dehydrogenase kinase reactio;t. They were much lessffective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of nondiabetic rats (approx. 16-fold at 10piM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-'4C]pyruvate by rat heart mitochondria (6.85nmol/min per mg of protein withi 5Ompyruvate) was approx. 46% of the Vma.. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoAI[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changlng the proportion of active pyruvate dehydrogenase. It is concluded that,inhibition of pyruvate delydrogenase by the increased ratio of [acetyl-CoA]/[CoAJdid not enhance phosphorylatiQn anid inactivation of the dehydrogenase under these conditions, perhaps because the cins was fully activated by the high ratio of INADH]/[NAD+] (see point 8). 7. A wide vaiety of respiratory substrates increased ATP concentration and lowered pyruvate dehydrogenase activity, (active form) in rat heart mitochondria. In general ATP concentration and pyruvate dehydrogenase activity were inversely correlated, with the following important exceptions. Succinate (0.25 or 5mM) generated equivalent ATP concentrations, but pyruvate dehydrogenase activity was lowest at 5mM. As palmitoylcarnitine concentration was increased from 10 to 50,M, pyruvate dehydrogenase activity fell, whereas ATP concentration was little changed. Octanoate (5OpM) decreased pyruvate dehydrogenase