Branched-chain amino acids (BCAA) supplementation has been reported to suppress the incidence of liver cancer in obese patients with liver cirrhosis or in obese and diabetic model animals of carcinogenesis. Whether BCAA directly suppresses cell proliferation of hepatic tumor cells under hyperinsulinemic condition remain to be defined. The aim of this study was to investigate the effects of BCAA on insulin-induced proliferation of hepatic tumor cells and determine the underlying mechanisms. BCAA suppressed insulin-induced cell proliferation of H4IIE, HepG2 cells. In H4IIE cells, BCAA did not affect cell cycle progression but increased apoptosis by suppressing expressions of anti-apoptotic genes and inducing pro-apoptotic gene via inactivation of PI3K/Akt and NF-κB signaling pathways. Further studies demonstrated that BCAA inhibited PI3K/Akt pathway not only by promoting negative feedback loop from mammalian target of rapamycin complex 1 (mTORC1)/S6K1 to PI3K/Akt pathway, but also by suppressing mTORC2 kinase activity toward Akt. Our findings suggest that BCAA supplementation may be useful to suppress liver cancer progression by inhibiting insulin-induced PI3K/Akt and subsequent anti-apoptotic pathway, indicating the importance of BCAA supplementation to the obese patients with advanced liver disease.

Obesity and related metabolic abnormalities, including adipocytokine dysbalance, are risk factors for hepatocellular carcinoma (HCC). Visfatin, an adipocytokine that is highly expressed in visceral fat, is suggested to play a role in the progression of human malignancies. Branched-chain amino acids (BCAA) reduce the incidence of HCC in obese patients with liver cirrhosis and prevent obesity-related liver carcinogenesis in mice. In this study, we investigated the possible role of visfatin on HCC progression and the effects of BCAA on visfatin-induced proliferation of HCC cells. In patients with HCCs, serum visfatin levels were significantly correlated with stage progression and tumor enlargement. Visfatin preferentially stimulated the proliferation of HepG2, Hep3B, and HuH7 human HCC cells compared with Hc normal hepatocytes. Visfatin phosphorylated extracellular signal-regulated kinase (ERK), Akt, and GSK-3b proteins in HepG2 cells. LY294002 [a phosphoinositide-3-kinase (PI3K) inhibitor], PD98059 [a MAP/ERK 1 kinase (MEK1) inhibitor], CHIR99021 (a GSK-3b inhibitor), and BCAA significantly inhibited visfatin-induced proliferation in HepG2 cells. BCAA also inhibited phosphorylation of GSK-3b, increased cellular levels of p21 CIP1 , caused cell-cycle arrest in G 0 /G 1 phase, and induced apoptosis in HCC cells in the presence of visfatin. These findings suggest that visfatin plays a critical role in the proliferation of HCC cells and may be associated with the progression of this malignancy. In addition, BCAA might inhibit obesity-related liver carcinogenesis by targeting and, possibly, by overcoming the stimulatory effects of visfatin.

Many animal models of nonalcoholic steatohepatitis have been reported. While these models exhibit mild onset of hepatitis and fibrosis, induction is often slow. For faster screening of drug candidates, there is a compelling need for convenient animal models of steatohepatitis and nonalcoholic steatohepatitis in which fatty liver and hepatitis are stably induced within a short period. Here, we analyzed the hepatic lipid composition in nonalcoholic steatohepatitis, and used this information to successfully establish a murine model where steatohepatitis is induced within only 1 week using a novel diet (steatohepatitis‐inducing high‐fat diet, STHD‐01) high in saturated fatty acids and cholesterol. After receiving STHD‐01 for 1 week, normal mice (C57BL/6J) showed elevated markers of fatty liver and hepatitis, including hepatic triglycerides and plasma alanine aminotransferase; the administration of angiotensin receptor blockers reduced these symptoms. Furthermore, we confirmed that STHD‐01 administration for 36 weeks induced not only sustained elevation of hepatic triglyceride and plasma alanine aminotransferase levels, but also fibrosis and tumor formation. Pretreatment with the carcinogen diethylnitrosamine accelerated tumor formation, and hepatic lesions were observed within 30 weeks of STHD‐01 feeding following diethylnitrosamine pretreatment. Finally, branched‐chain amino acids, known to reduce the risk for hepatocellular carcinoma in preclinical models, were effective in reducing the progression of liver fibrosis induced by STHD‐01 feeding after diethylnitrosamine pretreatment. We concluded that STHD‐01 administration successfully induces steatohepatitis within a short period of time. The proposed murine model is suitable for studying the long‐term effects of pharmaceutical agents targeting steatohepatitis, fibrosis, and tumor formation.

Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy.