Michael G. Clark

Co-ordinated regulation of muscle glycolysis and hepatic glucose output in exercise by catecholamines acting via α-receptors

Recent findings indicate that glucose uptake by contracting hindlimb [Acta Physiol. Scand. (1982) 116, 215‐222] and heart [Biochem. Biophys. Res. Commun. (1982) 108, 124–131] of the rat is stimulated by epinephrine acting through α‐adrenergic mechanisms. Since in exercise hepatic glucose output may be increased markedly by activation of α‐adrenergic receptors and matched by the increase in muscle glucose uptake (maintaining blood glucose levels relatively constant), it is now proposed that a general coordination of glucose metabolism may operate via α‐adrenergic receptor mechanisms. The basis for this proposal is discussed.

Epinephrine activation of phosphofructokinase in perfused rat heart: Regulation via α-adrenergic receptor mechanism independent of phosphorylation

Epinephrine treatment of the perfused rat heart leads to the activation of phosphofructokinase (PFK) via an a-adrenergic receptor mechanism, independent of changes in the intracellular concentration of cyclic AMP [ 1 ]. The activation is characterized by a loss in sensitivity to the inhibitors, ATP [ 1 ] and citrate [2], and is stable to gel-filtration [2,3]. Reconversion of the activated form to the non-activated form is catalyzed in heart extracts but does not appear to involve phosphoprotein phosphatase [2]. Here, the enzyme assay from [ 1,4] and SDS-polyacrylamide gel electrophoresis of immunoprecipitates of PFK were used to examine the relationship between c~-receptormediated activation and phosphorylation. 5 vol. 100 mM Tris-HC1 (pH 7.4) containing 1 mM dithiothreitol, 100 mM phosphate and 30 mM NaF. Supernatants (100 000 × g for 15 min) were mixed with an equal volume of reconstituted antiserum (100 mg/ml Tris-HCl-dithiothreitol) and allowed to stand at 4°C for 17 h. Immunoprecipitates (8000 × g for 10 min) were washed twice with isotonic saline and solubilized by incubation for 10 min at 95°C in 200/21 63 mM Tris-HC1 (pH 7.0), 1% SDS, 2.5% 2-mercaptoethanol, 5% glycerol and 0.02% bromophenol blue. Electrophoresis on 6% gels (10 cm) was done as in [8]. The specific radioactivity of [,),.32p]. ATP in heart powders was determined as in [9]. The activity ratio of PFK [ 1 ] was determined in hearts to which [32p] phosphate had not been added. Other experimental details were as in [ 1,3,4].

Adrenergic regulation of pyruvate kinase and gluconeogenesis in hepatocytes from phosphorylase kinase-deficient (gsdgsd) rats

Abstract Hepatocytes were prepared from a strain of rats deficient in hepatic phosphorylase b kinase and were used to assess the role of this enzyme in the adrenergic regulation of pyruvate kinase and gluconeogenesis. Epinephrine (10 μM) stimulated glucose output and gluconeogenesis from 1.8 mM lactate but did not significantly affect the concentration of hepatocyte glycogen. In addition epinephrine treatment led to an inhibition of pyruvate kinase. The stimulation of gluconeogenesis and the inhibition of pyruvate kinase by epinephrine were blocked by both α- and β-antagonists: similar effects with epinephrine were observed in cells from control animals. It is concluded that mechanisms for the adrenergic regulation of pyruvate kinase and gluconeogenesis are similar in hepatocytes from both phosphorylase kinase-deficient and normal rats.

Intracellular redox state and stimulation of gluconeogenesis and glycogenolysis in isolated hepatocytes from the pig (Sus scrofa)

Abstract 1. 1. Isolated pig hepatocytes were prepared and the effects of changes in the cytoplasmic [NADH]/[NAD + ] ratio on the efficacy of glucagon to alter rates of metabolism were examined. 2. 2. With hepatocytes from fed pigs 1 μM-glucagon stimulated glucose output. The response to glucagon was similar in magnitude regardless of whether 10 mM-lactate or 10 mM-pyruvate was present in incubations. 3. 3. With hepatocytes from 72-hr fasted pigs, glucagon (1 μM) increased the rate of gluconeogenesis from 10 mM-pyruvate but was without effect on the rate from 10 mM-lactate. These results differed from those obtained using rat hepatocytes where 1 μM-glucagon increased gluconeogenesis from 10 mM-lactate and inhibited gluconeogenesis from 10 mM-pyruvate. 4. 4. Intracellular concentrations of lactate and pyruvate were measured following 10 min incubations of pig hepatocytes with 10 mM-lactate or 10 mM-pyruvate. Comparisons with similar experiments conducted using rat hepatocytes indicated that both lactate and pyruvate entered the cells of both species and significantly altered the lactate/pyruvate ratio. 5. 5. Properties of the membrane-bound low K m cyclic AMP phosphodiesterase from pig and rat liver were compared. The activity of the enzyme in each species was similar and was inhibited to the same extent by NADH. 6. 6. The inability of pyruvate to inhibit the stimulatory effect of glucagon on glucose output and gluconeogenesis in pig hepatocytes does not appear to result from differences in the permeation of substrate into the cells or the sensitivity of cyclic AMP phosphodiesterase to altered cytoplasmic [NADH]/[NAD + ] ratio mediated by pyruvate or lactate addition.