Branched-chain amino acids prevent hepatic fibrosis and development of hepatocellular carcinoma in a non-alcoholic steatohepatitis mouse model

Oral supplementation with branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in patients with liver cirrhosis potentially suppresses the incidence of hepatocellular carcinoma (HCC) and improves event-free survival. However, the detailed mechanisms of BCAA action have not been fully elucidated. BCAA were administered to atherogenic and high-fat (Ath+HF) diet-induced nonalcoholic steatohepatitis (NASH) model mice. Liver histology, tumor incidence, and gene expression profiles were evaluated. Ath+HF diet mice developed hepatic tumors at a high frequency at 68 weeks. BCAA supplementation significantly improved hepatic steatosis, inflammation, fibrosis, and tumors in Ath+HF mice at 68 weeks. GeneChip analysis demonstrated the significant resolution of pro-fibrotic gene expression by BCAA supplementation. The anti-fibrotic effect of BCAA was confirmed further using platelet-derived growth factor C transgenic mice, which develop hepatic fibrosis and tumors. In vitro, BCAA restored the transforming growth factor (TGF)-β1-stimulated expression of pro-fibrotic genes in hepatic stellate cells (HSC). In hepatocytes, BCAA restored TGF-β1-induced apoptosis, lipogenesis, and Wnt/β-Catenin signaling, and inhibited the transformation of WB-F344 rat liver epithelial stem-like cells. BCAA repressed the promoter activity of TGFβ1R1 by inhibiting the expression of the transcription factor NFY and histone acetyltransferase p300. Interestingly, the inhibitory effect of BCAA on TGF-β1 signaling was mTORC1 activity-dependent, suggesting the presence of negative feedback regulation from mTORC1 to TGF-β1 signaling. Thus, BCAA induce an anti-fibrotic effect in HSC, prevent apoptosis in hepatocytes, and decrease the incidence of HCC; therefore, BCAA supplementation would be beneficial for patients with advanced liver fibrosis with a high risk of HCC.


Pdgf-c Tg model
The generation and characterization of Pdgf-c Tg mice have been described previously [1]. Non-transgenic WT and Pdgf-c Tg mice on a C57BL/6J background were maintained in a pathogen-free animal facility under a standard 12-h/12-h light/dark cycle. After weaning at week 8, male mice were divided randomly into the following 2 groups: (i) Pdgf-c Tg or WT mice fed a basal diet (CRF-1; Charles River Laboratories Japan K.K., Yokohama, Japan) with 3% casein, and (ii) Pdgf-c Tg or WT mice fed CRF-1 supplemented with 3% BCAA. The mice were killed at week 28 to analyze the progression of hepatic fibrosis and development of hepatic tumors.
All animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Takara-machi Campus of Kanazawa University, Japan.

Cell culture
Human hepatic stellate cells (Lx-2; kindly provided by Dr. Scott Friedman, Mount Sinai School of Medicine, New York, NY) and Huh-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin (normal medium). Amino acid-free medium (ZERO medium) was prepared by mixing 5.81 g nutrition-free DMEM (Nacalai Tesque, Kyoto, Japan), 1.85 g NaHCO 3 , 1 g glucose, and 0.5 mL of 1 M (mol/L) sodium pyruvate in 500 mL Milli-Q water, then sterilizing with a 0.22-μm filter (Millipore, Billerica, MA). Low amino acid medium (×1/5 DMEM) was prepared by diluting ×1 DMEM with ZERO medium. Powdered BCAA (leucine-isoleucinevaline, 2:1:1.2) (Ajinomoto Pharma, Tokyo, Japan) were freshly dissolved in distilled water at 100 mmol/L, and then applied to the culture medium at 4, 8, or 16 mmol/L. A total of 5.0 × 10 4 Lx-2 cells were seeded in normal medium at 24 h before performing the experiments. The medium was changed to low amino acid medium and maintained for up to 24 h, and then the medium was replaced with low amino acid medium containing 3 ng/ mL recombinant human transforming growth factor (TGF)-β1 (R&D, Minneapolis, MN) with or without BCAA. After incubation for 24 h, the cells were harvested for analysis.

Isolation and culture of mouse hepatic stellate cells
Hepatic stellate cells (HSC) were isolated from C57BL/6J mice. Pronase-collagenase liver digestion was used to isolate HSC from mice. All experiments were replicated at least twice. Freshly isolated HSC suspended in culture medium were seeded in uncoated 24-well plates and incubated at 37°C in a humidified atmosphere of 5% CO 2 for 72 h in normal medium. Non-adherent cells were removed with a pipette and the culture medium was replaced with medium containing 10 ng/mL recombinant mouse TGF-β1 (R&D) with or without BCAA. The cells were harvested for analysis after incubation for 24 h. Cell viability was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay after 24 h stimulation by TGF-β1 with or without BCAA.

Western blotting
Whole-cell lysates from mouse liver were prepared and lysed using the CelLytic MT Cell Lysis Reagent (Sigma-Aldrich Japan K.K., Tokyo, Japan) containing Complete Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science). Cytoplasmic and nuclear protein extracts were prepared using an NE-PER Nuclear Extraction Reagent Kit (Pierce Biotechnology, Rockford, IL). Lx-2 cells were washed in phosphatebuffered saline (PBS) and lysed in RIPA buffer containing Complete Protease Inhibitor Cocktail and PhosSTOP (Roche Applied Science). The membranes were blocked in Blocking One-P (Nacalai Tesque). The following primary antibodies were used: mTOR (1:1,000 dilution), p-mTOR (1:1,000 dilution), p70S6K (1:1,000 dilution), p-p70S6K

Immunofluorescence staining
For immunofluorescence staining, the cells were fixed with methanol and then permeabilized with 0.1% Triton-X 100 in PBS. The primary anti-collagen 1 antibody (1:100 dilution; Abcam) was used at a final concentration of 2 μg/mL in PBS containing 2% fetal bovine serum at 4°C for 16 h. Incubation with the Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA) at a 500-fold dilution in PBS containing 2% fetal bovine serum antibody was performed for 4 h, and the cells were stained with Hoechst 33258 to visualize nuclear DNA (Vector Laboratories, Burlingame, CA).

Knockdown experiments
Lx-2 or Huh-7 cells were transfected with control (Stealth RNAi Negative Control Low GC Duplex #2; Invitrogen, Carlsbad, CA) or target (Raptor and NFYA) small interfering RNA (Thermo Fisher Scientific K.K., Yokohama, Japan) using the Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's instructions. After 24 h, the culture medium was replaced with medium containing 3 ng/mL recombinant human TGF-β1 (R&D) with or without BCAA. The cells were harvested for analysis after incubation for 24 h.

Transfection
Lx-2 cells were transfected with negative control or plasmids encoding the constitutively active form of Ras homolog enriched in brain (Rheb) protein [4] using Lipofectamine 2000 (Invitrogen). After 24 h, the culture medium was replaced with medium containing 3 ng/ mL recombinant human TGF-β1 (R&D). The cells were harvested for analysis after incubation for 24 h.

Generating recombinant lentivirus expressing TGF-β1
A cDNA fragment encoding TGF-β1 was generated by PCR from mouse liver tissuederived cDNA using the forward primer 5′-GGTG GTATACTGAGACACCTTGGTG-3′ and reverse primer 5′-TGTATTTAAGGACACCTGCACCCCC-3′. The TGF-β1 fragment was inserted into the pLVSIN-CMV Pur Vector and cloned in DH5α competent cells. pLVSIN-CMV-TGF-β1 vector or control empty vector, and the three packaging plasmids were co-transfected into Lenti-X 293T cells in 10-cm dishes to generate the recombinant lentiviruses LVSIN-TGF-β1 and LVSINcont, respectively. After 48 h, the virus-containing medium was harvested and kept at -80°C until used. TGF-β1 expression levels were evaluated by western-blotting.

Spheroid assay
The rat liver progenitor cell-like cell line WB-F344 (purchased from Japanese Collection of Research Bioresources Cell Bank of the National Institutes of Biomedical Innovation, Health, and Nutrition) was cultured in DMEM (Invitrogen) containing 10% fetal bovine serum (Gibco, Invitrogen). WB-F344 cells were pre-seeded in a 6-well plate, cultured overnight, and then infected with 2 mL lentivirus for 8 h followed by the addition of 2 mL DMEM/F12 medium. After 48 h, the infected cells were plated in 6-well ultra-low attachment culture dishes at 1.0 × 10 6 cell per well with fresh DMEM containing 5 μg/mL puromycin with or without BCAA (16 mmol/L). After 3 weeks, the number of spheroids was counted.

Gene expression profiling
Gene expression profiling of mouse liver was performed using a GeneChip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA). Liver tissue from mice fed the basal, Ath+HF, or Ath+HF diet containing 3% BCAA for 30 or 60 weeks was obtained. The expression data were deposited in the Gene Expression Omnibus database (NCBI accession no.: GSE57290). Pathway analysis was conducted using MetaCore (Thomson Reuters, New York, NY). Functional ontology enrichment analysis was conducted to compare the Gene Ontology process distribution of the differentially expressed genes.

Promoter assay
DNA fragments from -1000 to +56 bp relative to the transcription initiation site of TGFβR1 were inserted into pGL4-Basic (Promega Corporation, Madison, WI) using KpnI and XhoI sites. Point mutations in the seed region of predicted NFY binding sites were generated using Multisite-QuikChange (Agilent Technologies, Inc., Santa Clara, CA) according to the manufacturer's protocol. All constructs were confirmed by sequencing. Lx-2 cells were cultured for 24 h in 24-well plates, and 200 ng reporter plasmids were co-transfected with 2 ng Renilla luciferase expression vector (pSV40-Renilla) using Lipofectamine 2000 (Invitrogen). The medium was changed to low amino acid medium and maintained for up to 24 h, and the medium was replaced with medium with or without BCAA. After incubation for 24 h, the cells were harvested for analysis.

Statistical analysis
The results are expressed as the mean ± standard deviation. Significance was tested by one-way analysis of variance with Bonferroni's method, and differences were considered statistically significant at a P < 0.05.