Protein degradation in hepatocyte monolayers. Effects of glucagon, adenosine 3':5'-cyclic monophosphate and insulin

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Abstract
Hepatocytes were isolated by collagenase perfusion of livers from fed rats and established in stationary monolayer culture. Degradation of intracellular protein was measured in these monolayers after labeling for 16 h with [3H]Leu followed by a 3 h chase period in medium containing 2 mM-Leu. Proteolysis in this system was stimulated by physiological concentrations of glucagon and also by added dibutyryl cyclic AMP. The effects of these 2 agents were not additive, which is consistent with the view that they act by the same mechanism. A close correlation was found between intracellular cyclic AMP concentrations generated by glucagon and the degree of stimulation of proteolysis elicited by the hormone. Insulin reduced glucagon-stimulated proteolysis, but not glucagon-elevated intracellular cyclic AMP concentrations. The continual presence of either insulin or glucagon was necessary for the full expression of their effects on proteolysis. In the presence of cycloheximide, proteolysis was normally responsive to glucagon but not to insulin. Proteolysis was not responsive to either hormone in the presence of ammonia, an agent that blocks the final lysosomal step of protein breakdown. In hepatocyte monolayers glucagon may act via cyclic AMP to increase cellular autophagy and thus increase proteolysis, whereas insulin inhibits these processes independently of cyclic AMP.