Elsevier

Hepatology Research

Volume 33, Issue 1, September 2005, Pages 27-32
Hepatology Research

Oral administration of branched-chain amino acids activates the mTOR signal in cirrhotic rat liver

https://doi.org/10.1016/j.hepres.2005.07.001Get rights and content

Abstract

BCAA granules (a mixture of branched-chain amino acids) have been used to reverse the hypoalbuminemia of decompensated liver cirrhotic patients in Japan. Our previous studies showed that BCAA promoted albumin secretion through the mTOR signal transduction pathway in rat primary hepatocyte culture [Ijichi C, Matsumura T, Tsuji T, Eto Y. Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system. Biochem Biophys Res Commun 2003;303:59–64]. However, the mTOR-activating effect of BCAA in the experimental cirrhotic animals presenting with hypoalbuminemia has not yet been examined. The purpose of this study is to assess whether oral administration of BCAA induces mTOR activity in the livers of normal rats and CCl4-induced cirrhotic rats (CCl4 rats). Biochemical analysis of liver extracts isolated from several rats showed that oral administration of BCAA (0.75 g/kg body weight (BW)) induced phosphorylation of 4E-BP1 and stimulated the enzymatic activity of p70 S6K. Both of these molecules act downstream of mTOR. From the results, we conclude that orally administrated BCAA augments albumin synthesis in the liver, not only by supplementation of material substrates for protein synthesis, but also by induction of an mTOR signal that is critical for translational initiation. Furthermore, we conclude that induction of mTOR signaling is one of the major pharmacological mechanisms by which BCAA granules reverse the hypoalbuminemia of cirrhotic patients.

Introduction

An imbalance of plasma amino acids, including a decrease of branched-chain amino acids (BCAA) and an increase in aromatic amino acids (AAA), frequently occurs in patients with liver cirrhosis [2], [3]. In these patients, according to a report by Muto et al., the serum albumin concentration significantly correlates with the BCAA/AAA molar ratio (Fischer's ratio) [4]. Therefore, the decrease of Fischer's ratio observed in these patients seems to cause their low protein nutritional state [4], [5], [6]. A mixture of BCAA was developed to correct this plasma amino acid imbalance and to alleviate the hypoalbuminemia that occurs in liver cirrhosis patients [5], [6], [7].

When cirrhotic patients presenting with hypoalbuminemia received BCAA granules, known as LIVACT granules (Ajinomoto Co. Ltd., Tokyo, Japan), orally, their concentrations of plasma albumin improved [7], [8]. The granules consisted of l-isoleucine (0.952 g), l-leucine (1.905 g) and l-valine (1.143 g), and they were taken three times daily (a total dose of 12 g/day). Recent reports have indicated that l-Leu, one of the BCAA components, can induce mTOR signaling that is critical for protein synthesis at the translational initiation level. This was demonstrated by showing phosphorylation of 4E-BP1 and activation of p70 S6K, which are downstream of mTOR, in response to l-Leu administration, both in vitro and in vivo [1], [9], [10], [11], [12]. Shigemitsu et al. showed the p70 S6K activation ability of various amino acids by screening, using a rat hepatoma cell line, H4IIE. They concluded that the p70 S6K activation ability of l-Leu was the highest among the amino acids tested [10]. In our previous report, we showed that BCAA promoted albumin secretion, and that this followed activation of the mTOR signaling pathway in rat primary hepatocytes. We also showed that l-Leu had the highest activity among the BCAA components [1]. Furthermore, Yoshizawa et al. have reported that oral administration of l-Leu caused phosphorylation of 4E-BP1 and p70 S6K in liver and skeletal muscle of normal rats [11], [12]. However, the mTOR-activating effect of the BCAA mixture, or each BCAA alone, after oral administration has not yet been examined. More importantly, the effect of orally administered BCAA on mTOR signal induction using liver cirrhotic animal models has also not been examined.

We initiated this study in order to clarify whether the oral administration of BCAA activates mTOR signaling in vivo, using normal rats as well as CCl4-induced liver cirrhotic rats presenting hypoalbuminemia.

Section snippets

Animals

Male Sprague-Dawley rats purchased from Charles River Japan (Yokohama, Japan) were maintained at 23 ± 3 °C under a 12-h light/12-h dark cycle. Rats had free access to a standard diet (CRF-1, Oriental Yeast, Tokyo, Japan). Liver cirrhosis was induced by repeated injections of carbon tetrachloride (CCl4, Wako Pure Chemical, Osaka, Japan) according to the standard method [13], [14]. CCl4-induced cirrhotic rats (CCl4 rats) with values of plasma albumin 2.8–3.2 g/dl, and presenting with hypoalbuminemia,

Results

At the beginning of the study, in order to elucidate the activation of the mTOR signal in liver after oral administration of a BCAA mixture (l-Ile:l-Leu:l-Val = 1:2:1.2, Ajinomoto Co. Ltd.), we determined optimal conditions using normal rats. First, we performed time course experiments. BCAA was administered orally at 0.75 g/kg BW to the 7-week-old normal rats. The phosphorylation of 4E-BP1 was examined by immunoblotting, and the activation of p70 S6K by a 32P-uptake enzyme assay using liver

Discussion

In a previous study using rat primary hepatocyte culture, we reported that addition of BCAA mixture to the culture medium accelerated activation of the mTOR signal pathway and increased albumin secretion in a dose-dependent manner [1]. It is known that oral administration of BCAA improves the condition of liver cirrhosis patients [4], [5], [6], [7] and a similar effect was seen in a cirrhotic animal model [13]. However, there is no evidence as to whether oral administration of BCAA activates

Acknowledgements

We are grateful to Dr. Kenta Hara and Dr. Kazuyoshi Yonezawa for technical advice, and we also thank Mr. Tetsuo Kobayashi for technical assistance.

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