The mitochondrial carnitine palmitoyltransferase (CPT) system is composed of two proteins, CPT-I and -II, which, together with carnitine acylcarnitine translocase, are involved in the transport of fatty acids into the mitochondrial matrix for beta-oxidation. In the liver, CPT-I and its inhibition by malonyl-CoA are sensitive to hormonal (10(-9) M) levels of insulin; however, a similar effect of insulin on heart CPT is controversial. In cultured neonatal rat cardiac myocytes, tissue culture concentrations (1.7 microM) of insulin increase CPT and cytochrome oxidase activities as well as mitochondrial protein synthesis, suggesting that a growth mechanism may be involved. Because insulin at high concentrations may interact with the insulin-like growth factor (IGF-I) receptor, the consequences of insulin's action on heart cells in culture may be mediated through the IGF pathway. Consistent with an IGF-mediated pathway for the effect of insulin, incorporation of radioactivity into immunoprecipitated CPT-II from insulin-treated cardiac myocytes is dramatically increased over control cells. The amount of immunoreactive CPT-I is also increased in insulin-treated cells. Moreover, an IGF-I analogue that inhibits the autophosphorylation of the IGF-I receptor blunts the insulin-mediated increase in CPT-I and -II activities by > 70%. At low physiologically relevant concentrations (10 ng/ml), IGF-I significantly increases the activities of both CPT-I and -II, and the IGF-I analogue eliminates the IGF-I response. This is the first study to suggest involvement of the IGF-I pathway in the regulation of mitochondrial CPT synthesis and activities in the heart.