Effects Of Methionine Restriction On Lipid Metabolism And Redox State By Endogenous Hydrogen Sulfide In High-fat Diet-fed Mice

Purpose: High fat-diet（HFD） can induce lipid metabolism disorder and oxidative stress.Dietary methionine restriction reduces adiposity and oxidative stress in HFD,however,the underlying mechanisms of benefits remains largely unresolved.H₂S is increasingly being considered as an important regulator of lipid metabolism and redox state.The purpose of this article was to investigate the effects of methionine restriction on lipid metabolism,oxidative stress and endogenous H₂S.We provided the potential mechanism of methionine restriction in improving lipid metabolism and regulating transsulfuration pathway（TSP）.Methods: Sixty male C57BL/6 mice were randomly equally divided into control diet group（CON,4% fat,0.86% methionine）,high-fat diet group（HF,20% fat,0.86% methionine）,highfat with methionine restriction diet group（MR,20% fat,0.17% methionine）,respectively.The body weight was monitored per week,the metabolic status of mice were measured by comprehensive laboratory animal monitoring system.Ten mice were selected randomly from each group and sacrificed with fasting for 8 hours at the end of eleven and twenty-two weeks respectively.The hepatic morphological analysis was performed by microscopy,plasma lipid profiles and hepatic lipids were measured.Redox status-related indicators in plasma and liver were tested.The levels of endogenous H₂S in plasma and liver,the activity of enzymes related to the TSP were measured.The expression of genes related to lipid metabolism and the TSP were conducted by q PCR.The expression of protein related to TSP in liver were determined.The expression of mi RNAs in liver were evaluated by high-throughput sequencing.The expression of mi RNAs were assayed by q PCR and target relationship between mi R-328-3p and CSE was carried out by dual-luciferase report gene assay.Results: When C57BL/6 mice were fed for 11 weeks,HF mice had higher final body weight than CON mice.MR mice on HFD,however,had significantly lower body weight compared to HF mice.Compared to HF,MR significantly decreased the concentrations of TG and TC in plasma and liver,the levels of MDA in plasma,and the expression of FAS m RNA in liver.MR remarkedly increased the expression of CPT1 m RNA and PPARα m RNA in liver,the activity of GSH-Px in plasma and liver,the levels of endogenous H₂S in liver,the expression of CSE m RNA and protein in liver.Meanwhile,the levels of mi R-328-3p,mi R-872-5p and mi R-30c-5p m RNA in liver were downregulated.When C57BL/6 mice were fed for 22 weeks,we found that HFD significantly increased the body weight.Compared to HF,MR significantly decreased the body weight,the concentrations of TG and TC in plasma and liver,the expression of FAS,ACC1 and SREBP1 c m RNA in liver,the levels of MDA in liver and plasma.MR significantly increased the expression of CYP7A1,CPT1 and PPARα m RNA in liver,T-AOC and the activity of GSH-Px in plasma and liver,the ratio of GSH/GSSG in plasma.Moreover,the levels of endogenous H₂S in plasma and liver,the activity of CSE,the expression of CSE m RNA and protein in liver were upregulated.The levels of mi R-328-3p and mi R-194-5p m RNA were downregulated in liver.The results of dual-luciferase assay system showed that the relative luciferase activity had no significant difference in mi R-328-3p group compared to NC group in the experiments of transfection with mutant plasmid vector.In the experiments of transfection with recombinant plasmid containing the CSE 3’-UTR,the relative luciferase activity was apparently inhibited in mi R-328-3p group compared to NC group.These results suggest that mi R-328-3p could directly bind to CSE 3’-UTR.Conclusions: The HFD induced redox imbalance,inhibited the production of endogenous H₂S,led to interruption of lipid metabolism related genes and lipid metabolism disorder.mi R-328-3p could directly bind to CSE 3’-UTR.Dietary methionine restriction upregulated the expression of CSE protein by mi R-328-3p,and attenuated high-fat diet induced adiposity and oxidative stress possibly through increasing the production of endogenous H₂S.