Induction of insulin resistance in primary cultured adult cardiac myocytes.

Primary cultured cardiac myocytes from adult rats were used to elucidate the role of insulin and catecholamines in the development of insulin resistance in this tissue. Cardiomyocytes exhibited a stable response toward insulin up to at least 48 h in serum-free culture, as determined by measuring the effect of the hormone on initial rates of 2-deoxyglucose uptake. Culturing of cells in the absence of insulin for 6 and 19 h, respectively, resulted in a loss of insulin sensitivity and a reduced (33%) maximal responsiveness after 19 h of insulin deficiency. This was paralleled by a decrease in [14C]phenylalanine incorporation and an unaltered level of insulin binding. Insulin action was completely lost in cells cultured in the presence of cycloheximide for 19 h. When added to the culture medium for 4 h, both isoproterenol and (Bu)2cAMP decreased insulin binding by about 50%. Under these conditions maximal insulin responsiveness was not affected by isoproterenol but was reduced by 46% by (Bu)2cAMP. Nifedipine antagonized the inhibitory action of (Bu)2cAMP, but was ineffective when the culture period was extended to 19 h. Cardiomyocytes cultured in the presence of palmitate exhibited a largely reduced (67%) insulin responsiveness, which was only partly restored by inhibition of fatty acid oxidation. From these data we conclude that: 1) insulin deficiency induces insulin resistance due to decreased protein synthesis; 2) sustained, prolonged elevation of cAMP modulates insulin action by both Ca(++)-dependent and Ca(++)-independent mechanisms; and 3) free fatty acids antagonize insulin action by both metabolic and nonmetabolic pathways.