Effect Of Leucine On Improving Nonalcoholic Fatty Liver Disease And The Underlying Mechanisms

The global prevalence of nonalcoholic fatty liver disease(NAFLD) is increasing at an alarming rate. NAFLD is a component of the metabolic syndromes and its underlying mechanisms is still not completely clear. Day put forward “two-hit”theory, including insulin resistance, leptin resistance and oxidative stress. There are no ideal drugs for prevention and treatment. Research showed that branched chain amino acids(leucine, isoleucine, and valine) levels in patients with chronic liver disease were significantly lower than those in the control group. This suggested that the branched chain amino acids have improving effects on NAFLD. Therefore, further study of branched chain amino acids, especially leucine and its effects on NAFLD is very necessary.ObjectiveTo elucidate the effects of different doses of leucine on NAFLD and to further investigate the underlying mechanism by which the factors of “two-hit” theory play an important role in NAFLD.Methods1. Effect of leucine on hepatic steatosis in model rats: Forty-eight 5 wk male SD rats fed with high fat diet(HFD) contained 60.3% calories from fat, were randomly divided into 4 groups: high-fat group and leucine groups added 1.5%, 3% and 4.5% leucine, respectively. During the experiment, body weight and food intake were recorded weekly. After 16 weeks, all the rats were sacrificed. The pathological changes in rat liver were observed, and the serum alanine aminotransferase(ALT), aspartate transferase(AST) were measured.2. Effect of leucine on glucose and lipid metabolism, oxidative stress, and inflammatory factors: At the end of the experiment, the serum fasting blood glucose(FBG) and fasting insulin(FINS) were measured. Homeostasis model assessment of insulin resistance(HOMA-IR) was calculated according to FBG and FINS. On the other hand, the serum triglycerides(TG), total cholesterol(TC), low density lipoprotein cholesterol(LDL-C), high density lipoprotein cholesterol(HDL-C), and the liver hepatic total anti-oxidation capacity(T-AOC), total superoxide dismutase(T-SOD), reduced glutathione hormone(GSH-Px), malondialdehyde(MDA), Interleukin-6(IL-6), Tumor necrosis factor-α(TNF-α), and C reactive protein(CRP) were measured. The protein expressions of SREBP1 in liver and PPARγ in adipose tissues were detected by Western blot.3. Effects of leucine on leptin signaling pathway: At the end of the experiment, serum leptin level was determined. Leptin receptor(Lep R) in the hypothalamus and liver was examined by immunohistochemistry. The protein expressions of Lep R and leptin signaling pathway in adipose tissues were detected by Western blot.Results1. Effect of leucine on hepatic steatosis in model rats:(1) Body weight and food intake are slightly lower in 1.5% and 3.0% leucine groups, but ther e was no significant difference among 4 groups.(2) The livers of rats in the HFD group showed obvious steatosis. Thus, the model of NAFLD was established by HFD. Leucine supplementation improved steatosis.(3) Serum levels of ALT, AST in leucine groups were significantly lower than that in the HFD group.2. Effect of leucine on glucose and lipid metabolism, oxidative stress, and inflammatory factors:(1) Serum levels of FINS, and HOMA-IR in leucine groups were significantly lower than the HFD group.(2) The serum TC, TG and LDL-C were significantly lower in three leucine groups than in the HFD group. The T-SOD and GSH-PX in the liver were significantly higher in 1.5% and 3.0% leucine groups and MDA was lower in 4.5% leucine group.(3) The expression of SREBP1 in liver was significantly lower, and that of PPARγ in adipose tissues was significantly higher in the three leucine groups than in the HFD group.3. Effects of leucine on leptin signaling pathway:(1) Serum levels of leptin was 2.31±0.23ng/m L, 1.69±0.26ng/m L, 1.87±0.27ng/m L and 1.91±0.29 ng/m L in HFD and three leucine groups, respectively was significantly lower in the leucine groups than the HFD group.(2) Lep R expression in the liver tissue was 22.70±3.21, 54.33±3.06, 39.50±2.08 and 36.00±3.51, with significantly higher expression in the three leucine groups than in the HFD group. Similar with liver tissue, the Lep R expressed significantly higher in the leucine groups than in the HFD group.(3) The expressions of JAK2 and STAT3(activated by Ob R) were significantly higher, and that of SOCS3(inhibits leptin signaling) was significantly lower in the three leucine groups than in the HFD group.Conclusions1. Chronic leucine supplementation decreased liver steatosis of rats induced by high fat diet, resulting in the improvement of NAFLD.2. Leucine supplementation improved glucose and lipid metabolism, antioxidant capacity and inflammatory response.3. Leucine supplementation decreased the circulating leptin level and increased the expression of Lep R, and leptin signaling related JAK2 and STAT3 in central and peripheral tissues. At the same time, SOCS3 was decreased. Leptin resistance induced by high-fat diets was improved by leucine.4. Thus, the beneficial effects of leucine on NAFLD were linked to the inhibition of “two-hit” theory to liver.