Dietary supplementation with branched-chain amino acids suppresses diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice

Abstract

Obesity and related metabolic abnormalities, including insulin resistance, are risk factors for hepatocellular carcinoma in non-alcoholic steatohepatitis as well as in chronic viral hepatitis. Branched-chain amino acids (BCAA), which improve insulin resistance, inhibited obesity-related colon carcinogenesis in a rodent model, and also reduced the incidence of hepatocellular carcinoma in obese patients with liver cirrhosis. In the present study, we determined the effects of BCAA on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in obese C57BL/KsJ-db/db (db/db) mice with diabetes mellitus. Male db/db mice were given tap water containing 40 ppm DEN for an initial 2 weeks and thereafter they received a basal diet containing 3.0% of BCAA or casein, which served as a nitrogen content-matched control of BCAA, throughout the experiment. Supplementation with BCAA significantly reduced the total number of foci of cellular alteration, a premalignant lesion of the liver, and the expression of insulin-like growth factor (IGF)-1, IGF-2, and IGF-1 receptor in the liver when compared to the casein supplementation. BCAA supplementation for 34 weeks also significantly inhibited both the development of hepatocellular neoplasms and the proliferation of hepatocytes in comparison to the basal diet or casein-fed groups. Supplementation with BCAA improved liver steatosis and fibrosis and inhibited the expression of alpha-smooth muscle actin in the DEN-treated db/db mice. The serum levels of glucose and leptin decreased by dietary BCAA, whereas the value of the quantitative insulin sensitivity check index increased by this agent, indicating the improvement of insulin resistance and hyperleptinemia. In conclusion, oral BCAA supplementation improves insulin resistance and prevents the development of liver tumorigenesis in obese and diabetic mice.

Figure 1

Macroscopic (a) and microscopic (b–d) analyses of liver neoplasms in diethylnitrosamine‐treated db/db mice. (a) Macroscopically, white tumors (hepatocellular carcinoma; indicated by arrowheads) were detected in the liver of diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. (b–d) Paraffin‐embedded sections were stained with H&E. Representative photomicrographs show adenoma (b), hepatocellular carcinoma (c), and foci of cellular alteration (d) in liver of experimental mice.

Figure 2

Effect of branched‐chain amino acid (BCAA) supplementation on the development of foci of cellular alteration (FCA) and on the expression of insulin‐like growth factor (IGF)‐1, IGF‐2 and IGF‐1 receptor (IGF‐1R) mRNAs in the liver of diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. Livers were excised from treated mice supplemented with casein or BCAA for 16 weeks. (a) Paraffin‐embedded liver sections were stained with H&E and the total numbers of FCA were counted. Values are the means ± SD (n = 4). (b–d) Total RNA was isolated from the removed liver and the expression of IGF‐1 (b), IGF‐2 (c), and IGF‐1R (d) genes were examined by quantitative real‐time RT‐PCR. The expression of each gene was normalized to β‐actin expression. Each experiment was done in triplicate. *P < 0.05; **P < 0.01.

Figure 3

Effect of branched‐chain amino acid (BCAA) supplementation on the serum levels of BCAA, alanine aminotransferase (ALT), and leptin in diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. After mice were killed, blood samples were collected and the serum levels of BCAA (a), ALT (b), and leptin (c) were then assayed. Values are the means ± SD (n = 8). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Effect of branched‐chain amino acid (BCAA) supplementation on hepatic steatosis in diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. (a) Frozen sections of basal diet (CRF‐1)‐fed, casein‐supplemented, or BCAA‐supplemented treated mice were stained with Sudan III stain to show steatosis. (b) Hepatic lipids were extracted from the frozen livers and the levels of triglyceride were then measured. Values are the means ± SD (n = 8). *P < 0.01; **P < 0.001.

Figure 5

Effect of branched‐chain amino acid (BCAA) supplementation on hepatic fibrosis in diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. (a) Paraffin‐embedded sections of basal diet (CRF‐1)‐fed, casein‐supplemented, or BCAA‐supplemented treated mice were stained with Azan stain to show fibrosis. (b) Hepatic hydroxyproline contents were quantified colorimetrically. Values are the means ± SD (n = 8). *P < 0.05; **P < 0.01.

Figure 6

Effect of branched‐chain amino acid (BCAA) supplementation on the expression of α‐smooth muscle actin (α‐SMA) in diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. (a) Immunohistochemical expression of α‐SMA in the liver of basal diet (CRF‐1)‐fed, casein‐supplemented, or BCAA‐supplemented treated mice. (b) Total protein was extracted from the liver of experimental mice and the expression of α‐SMA protein was examined by Western blot analysis. An antibody to GAPDH served as a loading control. Repeat Western blots gave similar results. The results obtained were quantitated by densitometry and are shown in the right‐hand panels. Values are the means ± SD (n = 5). *P < 0.05; **P < 0.01.

Figure 7

Effect of branched‐chain amino acid (BCAA) supplementation on insulin sensitivity and the serum level of glucose in diethylnitrosamine‐treated C57BL/KsJ‐db/db mice fed basal diet (CRF‐1), or supplemented with casein or BCAA. (a) The value of the quantitative insulin sensitivity check index (QUICKI), was calculated to evaluate the insulin sensitivity. (b) The serum concentration of glucose was measured by the hexokinase method. Values are the means ± SD (n = 8). *P < 0.01; **P < 0.001.

Figure 8

Effect of branched‐chain amino acid (BCAA) supplementation on hepatic cell proliferation in diethylnitrosamine‐treated C57BL/KsJ‐db/db mice. (a) Immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in the liver of basal diet (CRF‐1)‐fed, or casein‐ or BCAA‐supplemented treated mice. (b) PCNA‐labeling index in non‐lesional hepatocytes was determined by counting the PCNA‐positive nuclei in the hepatocytes. *P < 0.05; **P < 0.01.