Regulatory Mechanism of Ketogenesis by Glucagon and Insulin in Isolated and Cultured Hepatocytes

The ketogenic effect of glucagon and its mechanism have been studied in isolated and cultured hepatocytes. The effects of l-carnitine and insulin were also studied. Glucagon at lOOng/ml consistently stimulated ketogenesis by 70-80%within lh with 0.3 mM palmitate in hepatocytes isolated from rats on regular laboratory chow. Glucagon failed to exhibit a ketogenic effect, but inhibited the esterification of [U-14C]palmitate in the presence of (+)octanoyl-d-carnitine (0.5 mM). (+)Octanoyl-d-carnitine inhibited the ketogenesis by 50+, but the inhibition was completely reversed by 3.3 mM l-carnitine. This indicates that the site of inhibition and the stimulatory step of ketogenesis by glucagon are at the carnitine palmitoyl-trans-ferase (CPT) 1 reaction. In mitochondria isolated from hepatocytes treated with glucagon for 30 min, ketogenesis from added palmitate was increased by 50-55+. CPT 1 activity determined in these mitochondria was found to be increased by 25-50% with increased affinity for palmityl CoA over normal (Km = 13 μm versus 25 μM). When cultured hepatocytes were supplemented with glucagon for 24 h, ketone body formation in the media was increased by 44 %. In mitochondria isolated from these cells, the CPT level was elevated by 83 %. Insulin antagonized the ketogenic effect of a submaximal dose of glucagon in isolated hepatocytes, shifting the glucagon concentration which gives one-half maximal effect on ketogenesis from 5-7ng/ml to 10-20 ng/ml. In cultured hepatocytes, insulin inhibited ketone body formation in the media and also significantly suppressed CPT activity by 34%.Although l-carnitine enhanced ketogenesis in isolated hepatocytes, it had no effect in the cultured hepatocytes. These observations indicate that glucagon stimulates ketogenesis through early activation of CPT activity, possibly through cyclic AMP. Also, glucagon seems to induce the enzyme, while insulin suppresses CPT as a long-term regulation mechanism.