Preventive effects of branched-chain amino acid supplementation on the spontaneous development of hepatic preneoplastic lesions in C57BL/KsJ- db/db obese mice

Obesity and its associated disorders, such as non-alcoholic steato- hepatitis, increase the risk of hepatocellular carcinoma. Branched-chain amino acids (BCAA), which improve protein malnutrition in patients with liver cirrhosis, reduce the risk of hepatocellular carcinoma in these patients with obesity. In the present study, the effects of BCAA supplementation on the spontaneous development of hepatic premalignant lesions, foci of cellular alteration, in db/db obese mice were examined. Male db/db mice were given a basal diet containing 3.0% of either BCAA or casein, a nitro- gen-content-matched control of BCAA, for 36 weeks. On killing the mice, supplementation with BCAA significantly inhibited the development of foci of cellular alteration when compared with casein supplementation by inhibiting cell proliferation, but induc- ing apoptosis. BCAA supplementation increased the expression levels of peroxisome proliferator-activated receptor- γ , p21 CIP1 and p27 KIP1 messenger RNA and decreased the levels of c- fos and cyclin D1 mRNA in the liver. BCAA supplementation also reduced both the amount of hepatic triglyceride accumulation and the expression of interleukin (IL)-6, IL-1 β , IL-18 and tumor necrosis factor- α mRNA in the liver. Increased macrophage infiltration was inhibited and the expression of IL-6, TNF- α , and monocyte chemoattractant protein-1 mRNA in the white adipose tissue were each decreased by BCAA supplementation. BCAA supplementation also reduced adipocyte size while increasing the expression of peroxisome proliferator-activated receptor- α , per- oxisome proliferator-activated receptor- γ and adiponectin mRNA in the white adipose tissue compared with casein supplementa- tion. These findings indicate that BCAA supplementation inhibits the early phase of obesity-related liver tumorigenesis by attenu- ating chronic inflammation in both the liver and white adipose tissue. BCAA supplementation may be useful in the chemopre- vention of liver tumorigenesis in obese individuals.


Introduction
Obesity is a serious health problem worldwide since it often causes a number of medical disorders, including metabolic syndrome and type 2 diabetes mellitus. Recent evidence also indicates that obesity and its related metabolic abnormalities are associated with an increased risk of developing hepatocellular carcinoma (HCC (1)(2)(3)(4)(5)). Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome and a subset of patients with this disease can progress to non-alcoholic steatohepatitis (NASH), which involves the risk of developing chronic hepatitis, cirrhosis and HCC (6)(7)(8). Obesity and diabetes mellitus have been shown to increase the risk of developing HCC also in patients with viral hepatitis (3,5). A state of chronic inflammation caused by insulin resistance and hepatic steatosis is considered to play a critical role in the development of HCC in several obesity-related pathophysiological conditions (2,(6)(7)(8)(9)(10). Therefore, obese patients, especially those with complications of NASH or chronic viral hepatitis, are at high risk for developing HCC, and targeting chronic inflammation might be an effective strategy for preventing obesity-related liver carcinogenesis (11).
Branched-chain amino acids (BCAA), which are a group of three essential amino acids comprising valine, leucine and isoleucine, are used clinically to improve protein malnutrition in patients with liver cirrhosis (12,13). Oral supplementation with BCAA prevents progressive hepatic failure and improves event-free survival in patients with chronic liver diseases (14,15). Moreover, a multicenter, randomized controlled trial has reported that long-term oral BCAA supplementation reduced the risk of developing HCC in patients with chronic viral hepatitis; however, the effect was evident only in the patients who are obese (3). The results seen in that clinical trial are considered to be associated with the improvement of insulin resistance achieved by BCAA supplementation (13,16). In fact, BCAA supplementation inhibited the development of carcinogen-induced liver and colorectal carcinogenesis in obese mice by improving insulin resistance (17,18). Treatment with BCAA also suppressed insulin-induced proliferation of HCC cells by antagonizing the anti-apoptotic function of insulin (19).
In addition to improving protein malnutrition and glucose metabolism, BCAA supplementation has been reported to reduce lipid deposition in the liver in recent rodent studies (17,20). Supplementation with BCAA also retarded excess weight gain and reduced epididymal white adipose tissue (WAT) weight in mice that fed a high-fat diet (20). Because chronic low-grade systemic inflammation produced by excess lipid storage in WAT and liver is involved in both the development of NASH and the obesity-related liver tumorigenesis (2,6-10), BCAA supplementation may prevent the development of liver neoplasms in obese mice by reducing excess fat accumulation in WAT and by improving liver steatosis, thereby attenuating inflammation in these organs.
The spontaneous development of hepatic preneoplastic lesions, foci of cellular alteration (FCA), have been previously reported to be enhanced in obese and diabetic C57BL/KsJ-db/db (db/db) mice, when compared with C57B6 or C57BL/KsJ-+/+ mice, genetic controls for db/db mice (17). In the present study, we examined the effects of BCAA supplementation on the spontaneous development of FCA in db/db mice while focusing on the attenuation of inflammation in both the liver and the WAT. In addition, we investigated whether BCAA supplementation alters adipocyte size and the expression of peroxisome proliferator-activated receptor (PPAR)-α, PPAR-γ and adiponectin, which are key regulators of inflammatory signaling in obese adipose tissue (21)(22)(23)(24)(25), in the WAT of db/db mice.

Mice and diets
Male db/db mice (4 weeks old) were obtained from Japan SLC (Shizuoka, Japan) and humanely maintained at Gifu University Life Science Research Center in accordance with Institutional Animal Care Guidelines. BCAA and casein were obtained from Ajinomoto (Tokyo, Japan). The BCAA composition (2:1:1.2 = leucine:isoleucine:valine) was set at the clinical dosage used for the treatment of decompensated liver cirrhosis in Japan (3,14).
Carcinogenesis vol.33 no.12 pp.2499-2506, 2012 doi:10.1093/carcin/bgs303 Advance Access publication October 2, 2012 Experimental procedure The experimental protocol was approved by the Institutional Committee of Animal Experiments of Gifu University. At 5 weeks of age, a total of 10 db/db mice were divided into two groups. The mice in Group 2 (n = 5) were given a basal diet (CRF-1, Oriental Yeast, Tokyo, Japan) supplemented with 3.0% BCAA (w/w) through the end of the experiment, whereas the mice in Group 1 (n = 5) were given a basal diet supplemented with 3.0% casein (w/w) that served as a nitrogen-content-matched control for the BCAA-treated group. At 41 weeks of age (after 36 weeks of supplementation with the experimental diet), all of the mice were killed using CO 2 asphyxiation and the development of FCA was analyzed.
Histopathology and measurement of adipocyte size Maximum sagittal sections of each liver lobe (six sublobes) and WAT obtained from the periorchis were used for histological examination. The tissue specimens were fixed in 10% buffered formaldehyde and then embedded in paraffin. The sections (4 µm thick) were cut from the tissue blocks and stained with hematoxylin and eosin (H&E). The presence of FCA, which are phenotypically altered hepatocytes showing swollen and basophilic cytoplasm and hyperchromatic nuclei, was determined according to the criteria described previously (26). The multiplicity of the FCA was assessed on a per unit area basis (per cm 2 ). Fatty metamorphosis (% of fatty degeneration) was determined on the H&E-stained liver section using the BZ-Analyzer-II software (KEYENCE, Osaka, Japan (27)).
To evaluate adipocyte size, 10 adipocytes from each stained section (a total of 50 adipocytes) in each group were analyzed using a fluorescence microscope BZ-9000 (KEYENCE). Adipocyte size was measured and averaged using the BZ-Analyzer-II (KEYENCE). The unit of mean adipocyte size was square micrometers (µm 2 ).

Immunohistochemical analysis of proliferating cell nuclear antigen and F4/80
Immunohistochemical staining of proliferating cell nuclear antigen (PCNA), a G 1 -to-S phase marker, was performed to estimate the cell proliferative activity of FCA using an anti-PCNA antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). On the PCNA-immunostained sections, the cells with intensively reacted nuclei were considered to be positive for PCNA, and the indices (%) were calculated in 20 FCA randomly selected from each group (28).
Immunohistochemical staining to detect F4/80, a mature macrophage marker, was also performed to estimate the presence of macrophage infiltration in the WAT. After endogenous peroxidase activity was blocked with H 2 O 2 , the sections were incubated with a F4/80 primary antibody (1:50; AbD Serotec, Oxford, UK) for 30 min at 37°C. Subsequently, the sections were incubated with biotinylated secondary antibodies against the primary antibodies (Dako, Carpinteria, CA, USA) and then incubated with avidin-coupled peroxidase. The sections were then developed with 3,3′-diaminobenzidine using Dako Liquid DAB Substrate-Chromogen System (Dako) and counterstained with hematoxylin.

Hepatic lipid analysis
Approximately 200 mg of frozen liver was homogenized, and the lipids were extracted using a chloroform:methanol (2:1 v/v) solution, as described by Folch et al. (29). The levels of triglycerides in the livers of the mice were measured using the triglyceride E-test kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer's protocol (17).

Statistical analysis
All data were expressed as the mean ± the standard error mean (SEM). Differences between the two groups were analyzed using Student's t-test. All analyses were conducted using JMP 8.0 (SAS Institute Inc., Cary, NC, USA). Values with P < 0.05 were considered to be significant.

General observations
Body, liver, kidney and fat (WAT of the periorchis and retroperitoneum) weights and hepatic triglyceride levels of the two groups measured at the end of the study are listed in Table II. The mean liver weight and mean level of triglycerides in the livers of the mice in the BCAA supplementation group were found to be significantly less than those in the mice in the casein-treated group (P < 0.05). BCAA supplementation also improved macrovesicular steatosis, which was observed in the casein-fed mice (P < 0.05, Figure 1A), suggesting that BCAA supplementation inhibits hepatomegaly by improving the accumulation of lipids in the liver. Other measurements did not differ significantly between the two groups. All of the mice remained healthy, and no clinical signs indicating toxicity of BCAA were observed during the experiment. Histopathologically, there were no   with BCAA significantly decreased the number of FCA when compared with casein supplementation (P < 0.05, Figure 1B). An immunohistochemical analysis to detect PCNA showed the mean PCNA-labeling index for FCA in the BCAA-supplemented mice to be significantly lower than that in the casein-supplemented mice (P < 0.05, Figure 1C). In the whole liver, BCAA supplementation also inhibited the expression levels of PCNA and c-fos messenger RNA (mRNA) in comparison with casein supplementation  (P < 0.05, Figure 1D). These findings suggest that BCAA supplementation prevents the development of FCA, at least in part, by reducing cell proliferation.

Effects of BCAA supplementation on the expression levels of IL-6, IL-1β, IL-18 and TNF-α mRNA in the livers of the db/db mice
Chronic inflammation induced by the excessive production of storage lipids plays a role in obesity-related liver carcinogenesis (2,6-10). Therefore, the effects of BCAA supplementation on the expression levels of proinflammatory cytokines IL-6, IL-1β, IL-18 and TNF-α mRNA, which are central mediators of chronic inflammatory diseases (2,6-10), in the livers of the db/db mice were determined. Quantitative real-time RT-PCR revealed that in comparison with the casein-supplemented mice, the experimental mice showed significantly decreased expression levels of mRNA in the liver following BCAA supplementation (P < 0.05, Figure 2). These findings suggest that BCAA supplementation attenuates chronic inflammation in the livers of obese and diabetic db/ db mice.

Effects of BCAA supplementation on macrophage infiltration and the expression level of IL-6, TNF-α and MCP-1 mRNA in the WAT of the db/db mice
Macrophages play important roles in inflammation in obese adipose tissue (21,22). Therefore, whether BCAA supplementation attenuates chronic inflammation or inhibits increased infiltration of macrophages in WAT was examined. Immunohistochemical analysis performed with an antibody to F4/80 revealed the presence of apparent macrophage infiltration in the periorchis WAT of the casein-supplemented db/db mice; however, the infiltration was markedly inhibited by BCAA supplementation ( Figure 3A). The expression levels of IL-6 and TNF-α mRNA in the WAT were also reduced by BCAA supplementation. Additionally, supplementation with BCAA significantly inhibited the expression of MCP-1 mRNA (P < 0.05, Figure 3B), which plays a role in the recruitment of macrophages into obese adipose tissue (30,31). These findings suggest that inhibition of macrophage infiltration and subsequent attenuation of chronic inflammation in WAT by BCAA supplementation are, at least in part, associated with the suppression of MCP-1 expression.

Effects of BCAA supplementation on adipocyte size and expression levels of PPAR-α, PPAR-γ, and adiponectin mRNA in the WAT of the db/db mice
The induction of inflammation in obese adipose tissue is associated with increased adipocyte size (21,22). Therefore, whether BCAA supplementation alters the histology of WAT was next examined. Histological analysis showed that in addition to the inhibition of macrophage infiltration, BCAA supplementation reduced the size of adipocyte ( Figure 4A). The average adipocyte size observed in the BCAA-supplemented mice was significantly smaller than that observed in the casein-supplemented mice (P < 0.05, Figure 4B).

BCAA inhibits liver tumorigenesis in obese mice
Moreover, BCAA supplementation increased the expression of PPAR-α mRNA, which can be a key regulator of inflammatory signaling (23,24), in the WAT of the db/db mice. Furthermore, the expression levels of PPAR-γ, a master regulator of adipocyte differentiation, and its downstream adiponectin mRNA, which also possesses the ability to suppress proinflammatory signaling (24,25), in the WAT were both significantly upregulated by BCAA supplementation (P < 0.05, Figure 4C).
Effects of BCAA supplementation on the expression levels of PPAR-α, PPAR-γ, Bax, Bcl-2, p21 CIP1 , p27 KIP1 and cyclin D1 mRNA in the livers of the db/db mice Recent studies have revealed the activation of PPAR-γ to exert a beneficial effect against HCC by inducing apoptosis and cell-cycle arrest (32,33). Therefore, in addition to the WAT ( Figure 4C), whether BCAA supplementation also increases the expression levels of PPAR-γ in the liver was next examined. The expression levels of PPAR-γ mRNA in the liver were found to be significantly increased by BCAA supplementation (P < 0.05), whereas this agent did not alter the levels of PPAR-α mRNA ( Figure 5A). Supplementation with BCAA increased the levels of Bax mRNA, which accelerates apoptosis, and decreased the levels of Bcl-2 mRNA, an anti-apoptotic member of the Bcl-2 family, in the livers of the experimental mice ( Figure 5B, P < 0.05). There were also significant increases in the expression levels of p21 CIP1 and p27 KIP1 mRNA and decreases in the levels of cyclin D1 mRNA in the livers of the mice supplemented with BCAA ( Figure 5C, P < 0.05).

Discussion
Obesity, which is implicated in the development of NAFLD and NASH, has been shown to increase the risk of developing HCC (1)(2)(3)(4)(5). The present study, a NAFLD mice model in which obesity and severe steatosis were developed, clearly indicates that dietary supplementation with BCAA effectively prevents the spontaneous development of liver preneoplastic lesions in db/db mice through the inhibition of cell proliferation. These findings are consistent with our recent report that BCAA supplementation suppresses the chemically induced liver tumorigenesis in obese mice by improving insulin resistance (17). We considered that the results of the present study showed an equivalent significance to those of the previous experiment (17) because NAFLD that has not yet progressed to NASH can induce hepatocyte proliferation and hepatic hyperplasia, both of which initiate the hepatic neoplastic process in obesity (34). Therefore, targeting NAFLD, a hyperproliferative field, through intervention using specific agents such as BCAA supplementation might be an effective strategy for preventing obesity-related liver carcinogenesis.
In various obesity-related metabolic disorders, substantial evidence has shown that chronic inflammation caused by obesity contributes to the progression of NAFLD to NASH and finally to HCC (2,6-10). Hepatic steatosis, which is a source of inflammation, also promotes the development of HCC (8,9). Therefore, reduction of lipid accumulation and attenuation of chronic inflammation in the liver achieved by BCAA supplementation play a critical role in the suppression of the spontaneous development of hepatic neoplastic lesions in obese mice. The inhibition of the expression of IL-6 and TNF-α by BCAA supplementation is particularly important in the suppression of the spontaneous development of hepatic neoplastic lesions because increases in these proinflammatory cytokines, which are accompanied by lipid accumulation in the liver, are critically involved in obesity-related liver carcinogenesis (2,(6)(7)(8)(9)(10). The preventive effects of obesity-related liver tumorigenesis by targeting IL-6 and TNF-α expression and liver steatosis are also demonstrated in other rodent studies (28,35,36). In addition, the alleviation of hepatic steatosis with BCAA supplementation, which might be associated with the effects of improving insulin resistance (13), is consistent with previous reports (17,20).
In addition to the benefits observed in the liver, the present study also showed that BCAA supplementation significantly attenuates chronic inflammation in the WAT of db/db mice. Macrophage infiltration into WAT, which is accompanied by IL-6 and TNF-α production, is an early contributing event for the development of chronic low-grade systemic inflammation (21,22). MCP-1 plays a crucial role in the recruitment of macrophages into obese adipose tissue (30,31). MCP-1 is also capable of inducing steatosis in hepatocytes, indicating that secretion of this chemokine by adipose tissue may induce steatosis not only by recruiting macrophages but also by acting directly on hepatocytes (37). In addition, upregulation of IL-6, TNF-α and MCP-1 in WAT is critically involved in the induction of systemic insulin resistance (21,22), which is a key factor for accelerating obesity-related liver carcinogenesis (2,(6)(7)(8)(9)(10). Therefore, the inhibition of enhanced adipose tissue inflammation, that is increased macrophage infiltration and IL-6, TNF-α and MCP-1 expression, by BCAA supplementation is important in preventing the development of steatosis and subsequent liver tumorigenesis in obese mice.
The present study demonstrated that adipocyte size in BCAAsupplemented mice is much smaller than that in control mice. This finding might be associated with the effects of BCAA on the induction of PPAR-α and PPAR-γ in WAT because activation of these nuclear receptors significantly prevents adipocyte hypertrophy (24,38). An increase in the number of small adipocytes induces adiponectin and its receptors, which downregulates the production of IL-6 and TNFα, thereby reducing obesity-related inflammation in adipose tissue (24,25). A lack of adiponectin enhances the progression of hepatic steatosis and tumor formation in a mice model of NASH (39), whereas this adipokine alleviates hepatic steatosis by decreasing TNF-α production (40). Moreover, the induction of adiponectin plays a role in the suppression of chemically induced liver tumorigenesis in obese mice (28). Therefore, in the present study, the effects of BCAA on the upregulation of PPAR-α, PPAR-γ and adiponectin achieved by inhibiting adipocyte hypertrophy may contribute to preventing obesity-related liver tumorigenesis.
In addition to the WAT, the present study also showed the first evidence that BCAA supplementation increases the mRNA level of PPAR-γ, but not that of PPAR-α, in the livers of obese mice. The precise mechanisms underlying the upregulation of the expression of PPAR-γ in the liver by BCAA have not yet been clarified. However, these findings are significant when considering the prevention of liver carcinogenesis because PPAR-γ is regarded to be an antitumorigenic factor in HCC, whereas the role of PPAR-α in HCC development is contradictory (32,33,41). The overexpression of PPAR-γ suppresses the growth of HCC cells by reducing cell proliferation and inducing apoptosis (32). The activation of PPAR-γ by its ligand also inhibits the proliferation of HCC cells by upregulating the p21 CIP1 and p27 KIP1 expression, which thus leads to the G 1 arrest of the cell cycle (33). These reports (32,33), together with the results of the present study showing that BCAA supplementation increases the expression of PPAR-γ, Bax, p21 CIP1 and p27 KIP1 mRNA and decreases the expression of Bcl-2 and cyclin D1 mRNA, suggest that the induction of apoptosis and regulation of cell-cycle progression induced by BCAA via the upregulation of PPAR-γ in the liver may also help to inhibit the development of FCA.
Finally, it should be noted again that improved insulin resistance achieved from BCAA supplementation, which has been demonstrated in several basic and clinical studies (13,16), is critical to suppress the development of neoplasms in both the liver and the colon of obese mice (17,18). Because chronic inflammation occurring in WAT plays a role in systemic insulin resistance (30,31), BCAA supplementation might prevent the spontaneous development of hepatic preneoplastic lesions via the attenuation of adipocyte inflammation and the subsequent improvement of insulin resistance. These findings suggest that in addition to the liver, as shown in the present and previous studies (17,42), WAT might be a critical target for BCAA to exert chemopreventive properties in obesity-related liver carcinogenesis.
In conclusion, supplementation with BCAA may be an effective strategy for the chemoprevention of HCC, especially in obese patients who are at an increased risk of developing HCC. The results of the present study further strengthen our hypothesis that targeting obesityinduced pathologic conditions, such as chronic inflammation, might be effective for preventing liver carcinogenesis in obese individuals (11).