Regulation of glucose synthesis in hormone-sensitive isolated rat hepatocytes

A simplified procedure was developed for isolation of intact, hormone-sensitive liver cells in a high and reproducible yield. These cells produce glucose from various precursors at rates comparable to those achieved in isolated perfused liver. Glucagon enhanced glucose synthesis from pyruvate, dihydroxyacetone, fructose, or xylitol more effectively at low than at high substrate concentration. At high pyruvate concentrations (>2 mM), glucagon or adenosine 3':5'-cyclic monophosphate (0.1 mM) exerts a curious inhibition of gluconeogenesis that can be reverted to stimulation on addition of ethanol. It is suggested that glucagon and cyclic AMP inhibit pyruvate dehydrogenase and thus limit the supply of reducing equivalents needed for glucose formation. Supporting evidence for hormonal control of pyruvate dehydrogenase in isolated liver cells is provided by the fact that glucagon decreases and insulin increases decarboxylation of [1-(14)C]pyruvate. Calcium salts (1.3 mM) enhance glucose formation from pyruvate but greatly enhance the inhibition exerted by the divalent cationophore, A23187. Inhibition by glucagon of glucose synthesis from pyruvate is additive with the effects of A23187 + Ca(++). However, with dihydroxyacetone as substrate, glucagon partially reverses the inhibition exerted by A23187 + Ca(++). The results are consistent with glucagon effecting an inhibition of pyruvate dehydrogenase and a stimulation of hexosediphosphatase activities.