Leucine and Branched‐Chain Amino Acids Modulate Translation in Rat Primary Hepatocytes

Abstract

We have previously demonstrated that high amino acids (AA) and insulin are required for stimulation of translation in hepatocytes. Three hepatic sensing pathways are regulated by dietary protein, i.e. mTOR, GCN2 and AMPK. However, the nature of the AA signal is still unknown. We aimed to identify this signal and clarify the relationship between the transduction pathways using primary hepatocyte culture. We examined the role of branched‐chain amino acids (BCAA) and L‐leucine (Leu) in these pathways. With similar findings to the ones we obtained upon high AA treatment, BCAA or Leu stimulated phosphorylation of mTOR, 4E‐BP1 and S6K1 and decreased phosphorylation of AMPK and GCN2. We further characterized the roles of mTOR and AMPK in the pathway by using AICAR, an activator of AMPK, and rapamycin, an inhibitor of mTOR. Our results show that AICAR not only induced AMPK phosphorylation with or without insulin, but also decreased mTOR phosphorylation. Rapamycin treatment further decreased mTOR phosphorylation. Surprisingly, 4E‐BP1, a well known target of mTOR was dramatically decreased by AICAR, but not by rapamycin. These results suggest that BCAA and Leu are the key amino acids sensed by hepatocytes, and that AMPK may act as a general switch during high AA thereby controlling protein synthesis. To characterize the hierarchy of the system more in detail we next aim to identify the protein phosphatases induced by high AA.