We investigated the effects of branched-chain amino acids on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Of the branched-chain amino acids, only leucine (10(-5)-10(-3) M) induced hepatocyte DNA synthesis and proliferation in a time-and dose-dependent manner. The addition of valine or isoleucine on its own had no significant effects on the hepatocyte DNA synthesis and proliferation. When combined, isoleucine competitively antagonized leucine-stimulated hepatocyte mitogenesis. U73122 (10(-6) M), AG1478 (10(-7) M), wortmannin (10(-7) M), PD98059 (10(-6) M) and rapamycin (10 ng/ml) inhibited the ability of leucine to stimulate the hepatocyte DNA synthesis and proliferation, suggesting that phospholipase C, tyrosine kinase, phosphatidylinositol 3-kinase, mitogen-activated protein (MAP) kinase, and p70 S6 kinase are involved in leucine signaling. The mitogenic effects of leucine are completely abolished by the addition of anti-transforming growth factor-a (TGF-alpha) antibody to the culture medium. Furthermore, leucine stimulated TGF-a secretion into the culture medium and the leucine effect was inhibited by U73122. Isoleucine alone had no significant effect on TGF-a secretion but this agent blocked leucine-induced TGF-a secretion. The results suggest that leucine triggers TGF-a secretion through a putative leucine receptor. The secreted TGF-a then stimulates hepatocyte DNA synthesis and proliferation through activation of TGF-a receptor to induce tyrosine kinase/MAP kinase activity and other downstream growth-related signal transducers. (c) 2005 Published by Elsevier B.V.