Study On The Metabolic Effects And Lipid-lowering Mechanism Of Methionine Restriction In Obese Mice

The prevalence of obesity has become a critical global health challenge.Therefore,how to effectively prevent and control obesity has become an urgent problem for human beings to solve.Many previous studies have consistently demonstrated that a methionine-restricted diet（MRD）has many beneficial health effects,including reducing fat deposition,increasing insulin sensitivity,improving oxidative stress and prolonging the life span of many species.However,the effects of MRD on systemic metabolism and the mechanism responsible for reduced lipid accumulation are still unknown.The aim of this study was to investigate the effects of MRD on systemic metabolism and its lipid-lowering mechanism in obese mice by using metabonomics,transcriptome,microbiome,histomorphology,and molecular biology techniques.The main methods,results,and conclusions are as follows:1.The effects of MRD on metabolites in plasma,urine and colon contents of mice fed with high-fat diet（HFD）were analyzed using NMR-based metabolomics.The results showed that MRD reduced body weight,improved plasma lipid levels,increased plasmaβ-hydroxybutyrate and acetoacetate levels,decreased plasma lipids（–CH₂–,CH₂C=C,and=CCH₂C=）and unsaturated lipid levels,increased urinaryβ-hydroxybutyrate,acetoacetate and acetone levels in HFD mice,suggesting that MRD promoted lipid catabolism and fatty acid oxidation metabolism.MRD decreased plasmaα-glucose andβ-glucose levels,increased plasma citrate level,increased urinary lactate,succinate andα-ketoglutarate levels in HFD mice,indicating that MRD promoted glycolysis and tricarboxylic acid cycle metabolism.MRD increased amino acid catabolism,decreased plasma 1-methylhistidine and3-methylhistidine levels,reduced muscle protein catabolism.MRD increased energy expenditure,reduced plasma lipid levels,body fat rate and body weight in HFD mice.MRD increased the decomposition and fermentation of carbohydrates and the production of short-chain fatty acids（SCFAs）in the colon,increased bile acids metabolism and the absorption of amino acids such as methionine,increased choline bioavailability,decreased the absorption of trimethylamine and ethanol,and improved the intestinal environment and tissue damage in HFD mice.MRD decreased the levels of inflammatory protein O-acetyl glycoprotein and N-acetyl glycoprotein in plasma,allantoin and indoxyl sulfate in urine and urocanate in colon contents,thus indicating that MRD improved inflammation and oxidative stress in HFD mice.2.Metabonomics and transcriptome techniques were combined to investigate the effects of MRD on liver metabolism in HFD mice.The results showed that MRD decreased liver weight and index,hepatic fatty vacuoles area ratio,and hepatic TG、TC and FFA levels,inhibited hepatic fatty acid synthesis,promoted fatty acid oxidation,ketone synthesis and degradation,glycolysis and TCA cycle metabolism,and amino acid catabolism,up-regulated oxidative phosphorylation pathway,and decreased hepatic lipid accumulation in HFD mice.MRD decreased hepatic methionine and cysteine metabolism,increased the utilization of taurine and betaine,activated autophagy,up-regulated DNA replication,RNA polymerase,spliceosome,and ribosome pathways during the process of protein synthesis to maintain hepatic protein synthesis in HFD mice.MRD decreased hepatic purine metabolism,AND allantoin and TMAO production,increased the levels of T-AOC and H₂S in plasma and liver,decreased the levels of ROS in plasma and liver,suggesting that MRD reduced hepatic oxidative stress in HFD mice.MRD increased glutathione metabolism,pyrimidine metabolism,bile secretion and primary bile acid synthesis,and decreased ubiquitin-mediated proteolysis,adipocytokines,aging-related diseases and cancer signaling pathways in HFD mice.MRD changed the expression of many miRNA in the liver,these target genes regulated by miRNA are mainly involved in the regulation of fatty acid synthesis,browning of white adipose tissue,autophagy,glycolysis,tricarboxylic acid cycle,hydrogen sulfide production,inflammation,myocardial damage,and longevity.3.The effects of MRD on hepatic lipid metabolism,autophagy and browning of adipose tissue in obese mice were investigated by using a 10-week HFD induced obesity model in mice.The results showed that MRD decreased body weight,plasma lipid levels,liver weight,hepatic fatty infiltration area ratio,and hepatic TG and TC levels,down-regulated hepatic expression of the lipid synthesis-related genes SREBP-1c,FAS,ACC-1 and SCD1 and that of the protein FAS,and up-regulated the hepatic expression of the lipid catabolism-related genes CrAT,FABP,PPARα,CPT-1,MCAD,FATP1,Acox-1,HSL,LPL,PKA,PerilipinA,ATGL,CYP7A1,and CD36 and the protein expression of HSL and p-HSL in obese mice,thus indicating that MRD decreased lipid synthesis,promoted lipid catabolism and fatty acid oxidation,and reduced hepatic lipid accumulation.MRD decreased fat mass and percent fat mass,the weight and adipocyte volume of iWAT and BAT,promoted the expression of browning genes UCP1,PGC-1α,Prdm16 and Cidea in iWAT and BAT,as well as the protein expression of UCP1,thus promoting browning of adipose tissue and reducing lipid accumulation in obese mice.MRD up-regulated hepatic expression of the autophagy-related genes LC3A,LC3B,Atg4b,Atg7,ULK1,ULK2,Fip200,Beclin1,Lamp2α,Gabarap,Gabarapl1,Atg5,Atg8 and Atg12,the protein expression of LC3B,the protein ratio of LC3BII/LC3BI,and the protein expression of Beclin1,down-regulated the protein expression of mTOR and p62,and increased the number of autophagic vacuoles in the cytoplasm of liver cells,thus suggesting that MRD promoted hepatic autophagy in obese mice.Obese mice increased the proportion of liver protein and maintained the same amount of lean mass under MRD conditions.4.Obese mice fed a MRD diet were injected intraperitoneally with chloroquine（CQ）,an autophagy inhibitor,to demonstrate that MRD reduced hepatic lipid accumulation by activating autophagy.The results showed that CQ increased the gene and protein expression of LC3B and the protein ratio of LC3BII/LC3BI,down-regulated the protein expression of mTOR,up-regulated the protein expression of Beclin1,resulted in the accumulation of p62protein and autophagic vacuoles in the liver of MRD mice,thus indicating that CQ inhibited the hepatic autophagy of MRD mice.CQ increased plasma lipid levels,liver weight and index,hepatic fatty infiltration area ratio,and hepatic TG and TC levels,up-regulated hepatic expression of the lipid synthesis-related genes and protein,and down-regulated the hepatic expression of the lipid catabolism-related genes and proteins in obese mice,thus indicating that MRD increased lipid synthesis,reduced lipid catabolism and fatty acid oxidation,and increased hepatic lipid accumulation.MRD decreased fat mass and percent fat mass,the weight and adipocyte volume of iWAT and BAT,up-regulated the expression of browning genes UCP1,PGC-1αand Prdm16 in iWAT,and UCP1 and PGC1-αin BAT,as well as the protein expression of UCP1 in iWAT and BAT,thus suggesting that MRD promoted browning of adipose tissues and reduced lipid accumulation in obese mice.CQ inhibited hepatic autophagy,which likely led to the overexpression of SIRT1-FGF21 pathway,promoted the browning of adipose tissues,and then reduced fat mass and body fat rate in MRD mice.These results suggested that MRD promoted the browning of adipose tissue not by activating autophagy,but by promoting the expression and secretion of FGF21 in the liver.CQ led to the decrease of hepatic protein content and lean mass,which indicated that the activation of autophagy under MRD condition is necessary for hepatic protein content and lean mass to remain unchanged.5.The effects of MRD on the composition of intestinal bacteria in obese mice,and the effects of fecal bacteria from MRD mice on gut microbiota,hepatic lipid metabolism,autophagy and browning of adipose tissues in obese mice were studied by a method of faecal transplantation.The results showed that MRD increased the abundance of beneficial bacteria Bifidobacterium,Lactobacillus,Faecalibaculum,and butyrate-producing bacteria Lachnospiraceae_NK4A136_group,Roseburia,Rikenellaceae_RC9_gut_groupand Subdoligranulum,decreased the abundance of harmful bacteria Helicobacter and Erysipelatoclostridium,and increased the production of SCFAs in cecal contents of obese mice.Faecal transplantation from MRD mice decreased body weight and plasma lipid levels of obese mice.Faecal transplantation from MRD mice increased the abundance of Bifidobacterium and butyrate-producing bacteria Lachnospiraceae_NK4A136_group,Roseburia,[Ruminococcus]_torques_group,and Intestinimonas,and increased the production of short-chain fatty acids,especially butyrate in cecal contents of obese mice.Faecal transplantation from MRD mice increased the protein ratio of LC3BII/LC3BI and protein expression of Beclin1,down-regulated the protein expression of mTOR and p62,and increased the number of autophagic vacuoles in the cytoplasm of liver cells,thus suggesting that faecal transplantation from MRD mice promoted hepatic autophagy in obese mice.Faecal transplantation from MRD mice decreased liver weight,hepatic fatty infiltration area ratio,and hepatic TG and TC levels,down-regulated hepatic expression of the lipid synthesis-related genes SREBP-1c,ACC-1 and SCD1,and the protein expression of FAS,up-regulated the hepatic expression of the lipid catabolism-related genes PPARα,LPL and CYP7A1,and the protein expression of HSL and p-HSL,and decreased plasma lipids（–CH₂–）levels,increased plasmaβ-hydroxybutyrate and acetoacetate levels in obese mice,thus indicating that faecal transplantation from MRD mice decreased lipid synthesis,promoted lipid catabolism and fatty acid oxidation,and reduced hepatic lipid accumulation.Faecal transplantation from MRD mice reduced the weight and adipocyte volume of iWAT and BAT,up-regulated the gene and protein expression of UCP1 in iWAT and BAT,promoted browning of adipose tissues in obese mice.Faecal transplantation from MRD mice up-regulated the hepatic SIRT1-autophagy pathway,promoted autophagy and lipid metabolism,and reduced hepatic lipid accumulation in obese mice.Faecal transplantation from MRD mice up-regulated the hepatic SIRT1-FGF21 pathway,increased the synthesis and secretion of FGF21,promoted the browning of adipose tissues,and reduced the weight of adipose tissues in obese mice.Butyrate and its producing bacteria Lachnospiraceae_NK4A136_group and Roseburia may have contributed to the occurrence of these processes.In summary,MRD promoted hepatic autophagy,lipid metabolism and browning of adipose tissues by remodeling gut microbiota in obese mice,thereby reducing lipid accumulation in liver and body.