Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation

Abstract

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.

Pyruvate inhibition of pyruvate dehydrogenase kinase

“…In mitochondria oxidising 2-oxoglutarate+malate, the effect of added pyruvate to increase the proportion of PDHa is greatly reduced when the mitochondria are prepared from 48-h-starved or diabetic animals [4,[8][9][10]. In addition, the percentage of PDHa in rat heart mitochondria oxidising 2-oxoglutarate+malate is much lower in mitochondria from starved rats, than with those from fed rats [4].…”

The impact of increased dietary lipid on the regulation of glucose uptake and oxidation by insulin in brown- and a range of white-adipose-tissue depots in vivo

“…Delayed PDHC reactivation in oxidative tissues, including liver, on re-feeding after prolonged starvation, has been attributed, at least in part, to substantial (2-to 4-fold) stable increases in PDK activity. 28,46 A reduced response of BAT PDHC to an intravenous glucose load has been noted in overnight-starved rats, previously maintained on a high-fat diet. 10 In the present experiments, the provision of the high-fat diet for 28 d led to a modest, but nevertheless signi®cant (P`0.01), 1.4-fold stable increase in the activity of PDK, measured in extracts of isolated mitochondria (control, 0.141 AE 0.017 min 71 (n 14); high-fat-fed, 0.226AE 0.013 min 71 (n 6); P`0.01).…”

“…A unit of PDH a or citrate synthase activity is de®ned as that which converts 1 mmol of substrate into productamin at 30 C. PDK activity was computed as the apparent ®rst-order rate constant for ATP-dependent PDH a inactivation. 28 …”

“…PDK activities were determined in mitochondrial extracts by the rate of ATP-dependent inactivation of PDH a as described in Ref. 28. The incubation mixture contained 0.5 mM ATP, 1 mM MgCl2, 36 mgaml oligomycin B and 150 ± 350 m-units of active PDH a per ml.…”

“…It is assumed that the rapid removal and prompt freeze clamping of tissues from anaesthetised animals, followed by extraction in a buffer containing EDTA, assures that the initial pyruvate dehydrogenase activity in the extracts accurately re¯ects the proportion of active form in the tissue in vivo. Total PDHC activities 27 and PDK activities 28 were assayed in extracts of mitochondria. In brief, mitochondria were incubated for 30 min at 30 C without respiratory substrate and in the presence of carbonyl cyonide p-(tri¯uoromethyoxy) phenylhydrazone (FCCP) (15 mM) to effect PDHC activation.…”