Stimulation of mitochondrial functions by glucagon treatment. Evidence that effects are not artifacts of mitochondrial isolation

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Abstract
Activation of rat liver mitochondrial functions following glucagon treatment was demonstrated in mitochondria that had not been isolated by the conventional technique of differential centrifugation and washing in sucrose solutions. Crude liver homogenates in 0.3 M sucrose or 0.15 M KCl prepared from rats treated with glucagon showed stimulation of State 3 and uncoupled respiration, carboxylation of pyruvate, and citrulline synthesis comparable with those previously reported in isolated mitochondria. During the isolation procedure of mitochondria the hormonal stimulations of pyruvate carboxylation and citrulline formation were not enhanced by sequential washing. Mitochondria isolated from glucagon-treated rats by differential centrifugation and washing in 0.3 M mannitol/1 mM EGTA [ethylene glycol-bis(.beta.-aminoethyl ether) N,N,N'',N''-tetraacetic acid], pH 7.0, exhibited a mean rate of citrulline synthesis that was greater than twice that of the control. Liver homogenates prepared in 0.3 M sucrose or 0.3 M mannitol showed identical rates of State 3 respiration and percentage stimulations of respiration by glucagon treatment. Addition of glucagon led to a rapid accumulation of malate and aspartate and decreased the amounts of glutamate and citrate in isolated hepatocytes incubated with L-lactate. When gluconeogenesis was inhibited at the phosphoenolpyruvate carboxykinase (EC 4.1.1.32) reaction these phenomena were accentuated, lending support to the interpretation that they are the direct result of stimulation of carboxylation and oxidation reactions in the mitochondria. The mitochondrial effects of glucagon treatment apparently do not result from a stabilization of mitochondria to detrimental effects of sucrose during their isolation. The mean hormonal stimulation of pyruvate carboxylation in mitochondria isolated in 0.3 M sucrose was .apprx. 2.5-fold when assayed either at 37.degree. or 25.degree. C. In contrast, on the basis of similar experiments, others concluded that the effects of glucagon on hepatic mitochondria are not characteristic or a true hormonal stimulation. The data indicate this conclusion to be unjustified.