Alan L. Kerbey

Cyclic AMP and free fatty acids in the longer-term regulation of pyruvate dehydrogenase kinase in rat soleus muscle.

Starvation increased pyruvate dehydrogenase (PDH) kinase activity in extracts of freshly excised rat soleus 2.2-fold (from 0.6 min-1 in fed rats to 1.31 min-1 in 48-h-starved rats). In fed rats, activities were unchanged following 24 h of culture in medium 199, but increased 2.1-fold on 24 h of culture with 50 microM dibutyryl cAMP plus 1 mM n-octanoate and 1.6-1.7-fold with either agent alone. Approx. 70% of the increase in PDH kinase induced by starvation was lost following 24 h of culture in medium 199; the loss was prevented by 50 microM dibutyryl cAMP plus 1 mM n-octanoate. cAMP concentrations in fresh soleus muscle were 1 nmol/g (fed rats) and 1.6 nmol/g (starved rats). After 20-60 min of culture the fed-starved difference disappeared and [cAMP] fell to 0.4 nmol/g. Calcitonin-gene-related peptide (CGRP) increased cAMP 3-fold; the increase was maintained throughout 24 h of culture, but was readily reversed at 30 min or 24 h of culture by 60-min incubation with CGRP-free medium. Starvation of the rat (48 h) had no effect on the sensitivity of soleus towards the [cAMP]-increasing effect of CGRP. It is concluded that culture may reverse effects of starvation on PDH kinase activity by lowering cAMP and by removal from the in vivo effects of circulating free fatty acids; and that starvation and CGRP had no detectable long-term effects on the cAMP system in soleus muscle.

The roles of intrinsic kinase and of kinase/activator protein in the enhanced phosphorylation of pyruvate dehydrogenase complex in starvation

Extracts of heart mitochondria from fed and from 48 h starved rats subjected to gel filtration on Sephacryl S‐300 gave 4 major protein peaks. Pyruvate dehydrogenase complex eluted in the void volume and was assayed for intrinsic pyruvate dehydrogenase kinase activity which was increased approximately 3‐fold by 48 h starvation of the rat. A second fraction, containing peaks 2 and 3 which overlapped, enhanced the activity of the intrinsic kinase and corresponds to kinase/activator protein described previously. Its activity was increased 1.5‐fold by starvation.

Thermolabile factor accelerates pyruvate dehydrogenase kinase reaction in heart mitochondria of starved or alloxan-diabetic rats

The PDH complex of animal tissues is converted into PDHP complex and inactivated by PDH kinase (utilizing MgATP) and reactivated by PDHP phosphatase [I]. The kinase and phosphatase reactions constitute a cycle with the proportions of PDH and PDHP complexes depending on their relative rates. Alloxandiabetes or starvation in the rat markedly decrease the concentration of active PDH complex by conversion into inactive PDHP complex [2]. The mechanism by which this is brought about is not completely understood. There is evidence that diabetes and starvation activate the kinase reaction and inhibit reactivation by the phosphatase. The PDH kinase reaction is activated by increasing concentration ratios of ATP/ADP, NADH/NAD’ and acetyl CoA/CoA [3-51. Oxidation of lipid fuels in diabetes and starvation may increase the concentration ratio of acetyl CoA/CoA and thus activate the kinase reaction. However, changes in the concentrations of effecters of the PDH kinase reaction do not explain fully the decreased proportion of active PDH complex in heart mitochondria in diabetes and starvation [6,7]. Evidence for a further mechanism activating PDH kinase in extracts of heart mitochondria from diabetic or starved rats was given in [8]. This was a stable mechanism which persisted through isolation, incubation and extraction of mitochondria. Inhibition of reactivation by PDHP phosphatase (EC

Long term culture of rat soleus muscle in vitro : its effects on glucose utilization and insulin sensitivity

Rat soleus muscle strips cultured for 24 h in medium 199 were well preserved in terms of electron microscopy; ATP and creatine phosphate concentrations; rates of glucose utilization, glycogen and protein synthesis, and effects of insulin thereon. Culture led to modest changes in fluid spaces and intracellular (K+); increased basal glucose utilization up to two‐fold; had no effect on the maximum response to insulin; and had no effect on sensitivity to insulin except in the presence of adenosine deaminase. Thus in vitro neither denervation nor absence of insulin had any marked effects in 24 h to decrease responses to insulin.

Role of multi-site phosphorylation in regulation of pig heart pyruvate dehydrogenase phosphatase.

Pig Heart PDH complex is phosphorylated (with MgATP) and inactivated by PDH kinase intrinsic to the complex; and reactivated by PDHP phosphatase. Fully phosphorylated PDHP complex ((w,P&) contams three phosphorylated serine residues recoverable in two tryptic phosphopeptides of known amino acid sequence [ 11. Inactivation is correlated with phosphorylation of one serine residue (site 1); phosphorylation of the other two serine residues inhibits reactivation by the phosphatase [l-3]. This function of the other two sites of phosphorylation has recently been questioned [4]. We define here the optimum conditions for demonstrating different rates of reactivation of pig heart PDHP (oP.&) and PDHP (o2P&) by ox heart or pig heart phosphatase and app. Km values for PDHP complexes (substrate) and Mg*+ and Ca*+ (activators).

Hepatic pyruvate metabolism during liver regeneration after partial hepatectomy in the rat

Hepatic pyruvate kinase (PK) and pyruvate dehydrogenase (PDHa) specific activities were decreased after partial hepatectomy or sham operation. The decreases were more marked and sustained after partial hepatectomy. These activity changes ensure that hepatic carbon flux after partial hepatectomy is predominantly in the direction of gluconeogenesis. The decrease in PK specific activity observed after partial hepatectomy was associated with a decreased PK activation ratio (activity measured at 0.15 mM PEP: activity measured at 5.0 mM PEP), and hepatic concentrations of PEP were increased. The low hepatic PDHa activity observed at the first day after partial hepatectomy occurred concomitantly with an increased fatty acid concentration. PDHa activity increased after inhibition of lipolysis. The results indicate that carbohydrate utilization is unimportant for hepatic energy supply during liver regeneration. There was no evidence that the control of PK or PDH in the regenerative liver after partial hepatectomy differed from that observed in the liver of the unoperated rat.

Inactivation of pig heart pyruvate dehydrogenase complex by adenosine-5′-O(3-thiotriphosphate)

Porcine and bovine PDH complexes are inactivated and phosphorylated with MgATP by a kinase intrinsic to the complex. Fully phosphorylated PDHP complex (+P&) contains 3 phosphorylated serine residues. These are recovered after tryptic digestion in a tetradecapeptide (site 1, Ser 5; site 2, Ser 12) and a nonapeptide (site 3, Ser 6) of known amino acid sequence [ 1,2]. Discontinuous phosphorylation by incremental additions of a limiting amount of ATP leads to selective phosphorylation of site 1 yielding PDHPcomplex (CUP . a/3,) [ 1,3], This and other methods [2,4] have shown that inactivation is correlated with phosphorylation of site 1. Phosphorylation of sites 2 and 3 inhibits reactivation of the complex by PDHP phosphatase [S ,6]. Investigation of the physiological significance of site 2 and site 3 phosphorylations would be facilitated if one or more sites of phosphorylation could be blocked by groups which are resistant to attack by PDHP phosphatase. Conversion of phosphorylase b to phosphorylase a with phosphorylase kinase has been accomplished with ATPyS. The resultant (presumed) thiophosphoryl phosphorylase is resistant to a-b conversion by phosphorylase phosphatase [7]. We describe here inactivation of pig heart PDH complex by ATPyS,