Modulation of the phosphorylation state of rat liver pyruvate kinase by allosteric effectors and insulin.
The regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone. In this case, flux through pyruvate kinase was increased. The increase in flux correlated with an elevation in fructose bisphosphate levels but not with P-enolpyruvate levels which were unchanged. Pyruvate kinase was activated and its affinity for P-enolpyruvate was increased 7-fold in hepatocyte homogenates. Precipitation of the enzyme from homogenates with ammonium sulfate removed fructose 1,6-bisphosphate and activation was no longer observed. These results indicate that flux through and activity of pyruvate kinase can be controlled by the intracellular level of fructose 1,6-bisphosphate. The effect of elevated fructose 1,6-bisphosphate levels on the ability of glucagon to inactivate pyruvate kinase was also studied where only covalent enzyme modification is observed. Inactivation by maximally effective hormone concentrations was unaffected by elevated levels of fructose 1,6-bisphosphate, but the half-maximally effective concentration was increased from 0.3 to 0.8 nM. Activation of the cyclic AMP-dependent protein kinase by 0.3 nM glucagon was unaffected, but the initial rate of pyruvate kinase inactivation was suppressed. These results suggest that alterations in the level of fructose 1,6-bisphosphate can affect the ability of physiological concentrations of glucagon to inactivate pyruvate kinase by opposing phosphorylation of the enzyme. Consistent with this view was the finding that physiological concentrations of fructose 1,6-bisphosphate inhibited in vitro phosphorylation of purified pyruvate kinase. Inactivation of pyruvate kinase by 0.3 nM glucagon or 1 microM phenylephrine was also suppressed by 10 nM insulin. Insulin did not act by increasing fructose 1,6-bisphosphate levels. The antagonism to glucagon correlated well with the ability of insulin to suppress activation of the cyclic AMP-dependent protein kinase. However, no such correlation was observed with phenylephrine in the absence or presence of insulin. Thus, insulin can enhance pyruvate kinase activity by both cyclic AMP-dependent and independent mechanisms.
D000494:Allosteric Regulation; D000818:Animals; D004098:Dihydroxyacetone / Q000494:pharmacology*; D005635:Fructosediphosphates / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology; D005943:Gluconeogenesis / Q000187:drug effects; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010656:Phenylephrine / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism*; D051381:Rats; D014306:Trioses / Q000494:pharmacology*
D005635:Fructosediphosphates; D007328:Insulin; D014306:Trioses; D010656:Phenylephrine; D010728:Phosphoenolpyruvate; D005934:Glucagon; D011494:Protein Kinases; D011770:Pyruvate Kinase; D004098:Dihydroxyacetone