| The prevalence of obesity and overweight has become a global public health problem,about one-third of the populations in the world are obese since 2013. Obestity is an independent risk factor for several clinical chronic diseases, including diabetes, hypertension, hyperlipidemia and cardiovascular diseases and has increased morbidity of these associated metabolic diseases and economic costs.It is well known that obesity results from an imbalance between energy intake and energy expenditure, thus reducing energy intake while increasing energy consumption was the most fundamental way. Recently, several researches have showed that supplementation of branched chain amino acid, especially Leucine, can play an important role in energy metabolism. However, the effect of increasing dietary leucine on lipid metabolic and weight change was controversial in animals. Therefore, we need to investigate more about the underlying mechanisms of the relationship between leucine supplementation and energy metabolism.ObjectiveTo investigate the mechanism of how leucine could modify energy metabolism and body weight in mice fed with a high-fat/cholesterol diet.MethodSixty C57BL/6J mice were fed a normal chow diet(NC), high-fat/cholesterol diet(Research diets D1245 + 1% cholesterol)(HFC), HFC supplemented with 1.5% leucine(H+1.5% Leu group), or 3% leucine(H+3.0%Leu group) for 24 weeks.The body weight, food intake and energy intake was measured every week. Blood samples was taken from the angular vein every eight weeks(weeks 0, 8, 16, 24), separated and stored until further analysis of glucose, TG and cholesterol. After 24 week of feeding, blood samples was taken and serum FFAs, insulin, adiponectin and inflammatory factors(IL-6, TNF-α) were analyzed.At the end of the experiment, the liver, epididymal WAT(White adipose tissue) and interscapular BAT(Brown adipose tissue) were immediately collected for further analysis. Histological examination of the liver, WAT and BAT samples were carried out with HE staining method. Lipid levels of TG, TC and glycerol in the liver were analyzed. Protein levels of acetyl-coenzyme A carboxylase(ACC), fatty acid synthase(FAS), sterol regulatory element binding protein-1(SREBP-1), and liver X receptorα(LXRα) in liver was determined by Western bolting.Expect for the histological examination of the WAT, we observed the effect of leucine on lipolysis by cAMP-PKA-HSL and leptin signaling pathway and related proteins were measured including cAMP-dependent protein kinase(PKA), atriglyceride lipase(ATGL), Perilipin-A, hormone sensitive lipase(HSL), phospho-HSL563(p-HSL563), phosph-HSL660(p-HSL660), lepin receptoer(ob-R), activators of transcription 3(STAT3), Janus protein tyrosine kinase2(JAK2) and suppressors of cytokine signaling 3(SOCS3) in WAT by Western blotting. Meanwhile, expressions of β3 adrenergic receptors(β3AR), uncoupling protein 1(UCP1), peroxisome proliferator-activated receptor coactivator-1α+β(PGC-1α+β), and fibroblast growth factor 21(FGF-21) were measured in epididymal and interscapular adipose tissues.Results1. Effect of leucine on food intake, body weight, glucose, TG, FFAs, and cholesterol:(1) Food and energy intake was not changed during the experiment among the 4 groups. Body weight and liver index was increased significantly induced by high-fat/cholesterol diet compared with mice fed with normal diet. The mice in the two leucine groups had less weight gain and liver index than the HFC group.(2) Serum Glucose, TG, insulin and HOMA-IR was significantly higher in the HFC group than in the NC group. Leucine supplementation significantly decreased serum Glucose and TG at weeks 8 and 16. At the end of the experiment, serum insulin and HOMA-IR was obviously reduced compared with the HFC group.(3) Serum total cholesterol and LDL-C was was significantly increased in the HFC group and those was significantly decreased in leucine supplement groups.2. Effect of leucine on lipid metabolism in the liver tussues:(1) Histological examination showed that high-fat/cholesterol diet caused obvious steatosis in liver tissues. Leucine supplementation alleviated hepatic lipid accumulation.(2) Hepatic TC and glycerol levels were significantly increased by HFC, and significantly decreased to a level similar to the NC group by supplemented with 1.5% and 3.0%.leucine.(3) Key protein expressions of lipogenesis including ACC, FAS, LXR-α and SREBP-1c was measured in the liver tissues. As a result, HFC increased FAS, ACC, LXR-α and SREBP-1 expression and the overexpression was reverted by leucine supplementation.3. Effect of leucine on lipolysis, thermogenic effect and WAT browning:(1)Histological analysis of WAT showed that HFC enlarged the adipocyte size of WAT. However, adipocyte size was markedly reduced by leucine supplementation.(2) ATGL and HSL are two major lipases in adipocyte lipolysis, the protein expression levels related were measured in the WAT tissues. ATGL and phosphorylated HSL was significantly decreased in HFC group, and was markedly increased by leucine supplementation. We also analyzed the involved protein expressions of PKA pathway including CAMP, β3-AR and PKA and found that the decreased expression by HFC was restored by leucine intervention.(3) Mice in the HFC group had no difference in expression levels of β3-AR、UCP1、FGF-21、PGC-1α which were involved in the thermogenic program in BAT. Leucine supplementation remarkedly increased the expression of those proteins compared with the HFC group. However, HFC caused the decreased expression of the four proteins in WAT, and leucine supplementation still increased the expression of those proteins.4. Effect of leucine on adipokines and leptin resistance:(1) HFC significantly increased serum levels of letpin, IL-6 and TNF-α, and decreased the levels of adiponectin. Both the 1.5% Leu and 3.0% Leu group was significantly decreased the levels of leptin, insulin and HOMA-IR. IL-6 and TNF-α levels were significantly decreased in the 3.0% Leu group.(2) Mice on the HFC group decreased the Ob-R expression, additional leucine supplementation obviously upregulated the expressions of Ob-R, JAK2, downregualted STAT3 expression compared with the HFC group.ConclusionsOur findings demonstrated that chronic lecuine supplementation prevents high-fat/cholesterol diet-induced obesity by improving the energy metabolism. The beneficial effects of leucine might ascribe to suppressing hepatic lipogenesis, enhanceind lipolysis, thermogenesis, WAT bowning and improving leptin resistance. |
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