Stimulation of malic enzyme formation in hepatocyte culture by metabolites: Evidence favoring a nonglycolytic metabolite as the proximate induction signal

Recent studies have shown that the addition of increasing concentrations of glucose to the medium of primary adult rat hepatocyte cultures results in the progressive induction of malic enzyme. We have undertaken experiments to determine (1) whether metabolism of glucose was an essential prerequisite for such induction, and (2) whether a specific glycolytic intermediate could be shown to constitute the proximate carbohydrate signal triggering such induction. In line with these objectives we investigated the ability of various sugars and glycolytic metabolites to induce malic enzyme in this system and assessed the influence of insulin, glucagon, and thyroid hormone (triiodothyronine, T₃) on this process. Our results show that only those sugars capable of entering the cell and being metabolized induce malic enzyme (glucose, fructose, and galactose). The nonmetabolizable sugars 3-O-methylglucose and 2-deoxyglucose are ineffective. Incubation with 20 mmol/L lactate, pyruvate, dihydroxyacetone, or glycerol resulted in malic enzyme induction, whereas incubation with acetate, citrate, and α-ketoisocaproate was without effect. The induction by all sugars and metabolites required presence of insulin. As previously reported for glucose, addition of T₃, under all metabolic conditions, resulted in a constant 3.6-fold increase in the rate of malic enzyme induction and further supports the proposal that T₃ acts to multiply the effect of a common carbohydrate-generated signal. Glucagon administration led to a dose-dependent inhibition of the carbohydrate effect with a half-maximal effect and maximal effect at 2 and 100 nmol/L, respectively. None of the glycolytic metabolites tested could reverse the glucagon inhibition completely. Our results indicate that metabolism of glucose is essential for induction of malic enzyme and are most consistent with the view that the signal arises from the metabolism of pyruvate at a step before the formation of cytsolic acetyl-CoA. Since these processes are mitochondrial in location, it appears likely that this organelle is the site of origin of the inducing signal. Of some interest in this connection was the finding that dichloroacetate led to a major induction of malic enzyme, even in the absence of insulin and in the presence of glucagon. The ability of dichloroacetate to bypass the glucagon block thus further supports a nonglycolytic origin of the inducing signal.