Dietary Methionine Restriction Activates Metabolism By Improving Thyroid Function In Middle-aged Mice

In the present society,the change of dietary structure,aging of the population and a surge in chronic diseases are prominent.To explore the key factors in dietary structure is helpful to prevent the occurrence and development of aging and metabolic diseases.The reduction of energy metabolism is a typical feature of chronic metabolic diseases and aging.Methionine restriction?MR?diet,reducing dietary methionine by 80%,could activate material and energy metabolism,promoting a variety of health benefits,and even prolonging life.The thyroid gland,as the largest endocrine tissue,releases thyroid hormone,which is one of the important regulatory factors of energy metabolism.However,whether MR activates energy metabolism through thyroid function,and how it regulates age-induced characteristics,has not been reported.Therefore,this project intends to reveal the role of MR intervention in activating energy metabolism through thyroid function,and its influence on protein turnover metabolism,behavioral ability,and intestinal function.Based on these studies,the suitable ranges of MR in middle-aged mice with different energy diets were also evaluated.This present research will provide some theoretical basis for recommending appropriate methionine requirements and providing guidance on feasible and healthy dietary for middle-aged people with different diets.To investigate the role of MR in activating energy metabolism through thyroid function and its effect on protein metabolism,4-week-old C57BL/6J mice were used to establish a high-fat obesity-induced obesity model.After 22-week of MR intervention,blood thyroid hormone T3 and T4 were increased,while thyroid-stimulating hormone?TSH?was decreased,and thyroid secretion synthesis and REDOX status indexes were improved in obese mice.Moreover,the gene expressions of thyroid hormone receptor??THR??and thyroid hormone receptor??THR??,as well as type 1 deiodinase?DIO1?and type 2 deiodinase?DIO2?in the liver were up-regulated.These variations promoted gene levels of liver mitochondrial synthesis of PGC-1,NRF1,and TFAM,up-regulating hepatic glycogen level and down-regulating the area of hepatic fat droplets to activate energy metabolism.Furtherly,to determine the protein synthesis and degradation,L-[¹⁵N]-phenylalanine was selected as the tracer and the flooding method was used to inject the stable isotopes.The results showed that protein fractional synthesis rate?FSR?and degradation rate?FDR?in the liver of obese mice upon MR were only reduced by 13.5%and 16.1%respectively,and deposition efficiency?PRE?was considerately enhanced.Compared with the whole amino acid diet,the protein synthesis efficiency of the MR diet was increased by about 83%.Moreover,MR reduced the gene expression of protein synthesis pathways m TORC1/S6K1 and e IF2B,but significantly upregulated the eukaryotic translation initiation factor1?4EBP1?,the eukaryotic translation initiation factor?e IF2??,and activated transcription factor?ATF4?.Meanwhile,MR reduced the gene expression of ubiquitin-proteasome in the protein degradation pathway but activated the gene expression of the autophagy pathway.Besides,MR relieves oxidative stress in obese mice by activating energy metabolism and endogenous H₂S production,thereby attenuating endoplasmic reticulum stress and reducing the production of ineffective and incorrect proteins by regulating indicators such as XBP-1,Chop,IRE1,and Bip.These results suggested that MR improved the activation of energy metabolism via thyroid function to provide sufficient energy for the upregulation of translation initiation factor and activating transcription factor of protein synthesis,and improvement of efficiency of protein synthesis,which satisfied the basic needs of protein metabolism of the body at a low methionine level.To study the effect of activation of energy metabolism through thyroid function on the behavioral ability upon MR in middle-aged mice,8-month-old C57BL/6J male mice were randomly divided into 12 groups for 16-week diet intervention,according to low-,medium-,and high-fat diet?CON,MF,and HF?and different MR gradients 0%,40%,60%and 80%?MR0,MR40,MR60,and MR80?.Several indexes were researched such as thyroid function,body weight,white fat,feed and water intake,whole-body heat production,voluntary movement,fat accumulation and oxidative stress in liver,oxidative stress and inflammation in blood.These results found that MR improved thyroid function,leading to the activation of energy metabolism in a dose-dependent manner.Moreover,open field test,pole-climbing test,holding power,tail suspension,novel object recognition,elevated plus maze and Morris water maze test were used to evaluate the free activities,coordinated bodily movements,muscle strength,depression-like behaviors,nonspatial and spatial learning and memory.These data explained that MR improved the behavior ability in a dose-dependent manner in middle-aged mice with different energy diets.Furthermore,the effect of thyroid hormone on behavioral ability was explored,which showed that MR enhanced the thyroid hormone-related indexes in hippocampus,such as hippocampal thyroid hormone receptor THR?,monocarboxylate transporter-8?MCT-8?,deiodinase DIO2,thyroid hormone targeted genes including neurogranin?RC3?and neuromodulin/growth-associated protein?Gap-43?,as well as improved the hippocampal redox indicators.Based on these regulations,some learning,memory,and antidepressant indicators depending on the hippocampus and being regulated by thyroid hormone were increased,including 5-hydroxytryptamine?5-HT?,brain-derived neurotrophic factor?BDNF?,c AMP response element-binding protein?CREB?and Ca²⁺/calmodulin-dependent protein kinase II?Ca MKII?.These results showed that the MR,in a dose-dependent manner,improved thyroid function to enhance its targeting effect on the hippocampus,to promote hippocampal learning,memory and antidepressant indexes regulated by thyroid hormone,thus improve the behavioral ability in middle-aged mice with different energy diets.Besides,it was also observed that MR40 had no effect on thyroid function,energy metabolism and behavioral ability in middle-aged mice,and MR80 had significant regulatory effect,while MR60 only showed significant regulatory effect in high-fat diet indicating that high-energy diet promoted the significance of low-gradient MR for beneficial regulation of the body.The effects of MR on intestinal mucosal function and intestinal flora in middle-aged mice were studied.MR promoted the production of intestinal short-chain fatty acids?SCFAs?and reduced serum endotoxin?LPS?through enhancing the intestinal thyroid-hormone-related receptors TRH?and deiodinase DIO2,and reducing thyroid-stimulating hormone receptor?TSH-R?and thyrotropin-releasing hormone receptor?TRH-R?in middle-aged mice with different energy diets.Based on the variation of SCFAs and LPS levels,MR increased the ratio of villi height to crypt depth,and the gene expression of transmembrane proteins Claudin-3,Occludin,and tight junction proteins ZO-1 to improve the mucosal barrier function,as well as reduced systemic inflammation through the LPS/LBP/CD14/TLR4 pathway.Furtherly,16s ribosomal RNA gene sequencing was used to verify the effect of MR on gut microbes.These results showed that MR increased cecum biodiversity and upregulated the abundance of SCFAs-producing bacterium Bacteroides,Faecalibaculum,Roseburia,and downregulated the LPS-producing or pro-inflammatory bacterium Desulfovibrio and Escherichia-Shigella.It was also observed that due to the influence of aging to a certain extent,compared with the normal diet,the moderating effect of the medium fat diet was not significant,and some indexes of high fat diet were significant.In conclusion,this study found that MR could activate energy metabolism to promote protein metabolism efficiency by improving thyroid function;enhance free activities,coordinated bodily movements,muscle strength,depression-like behaviors,nonspatial and spatial learning and memory;improve intestinal mucosal barrier function and flora diversity in middle-aged mice.Moreover,this study revealed that the middle-aged mice with a low-or medium-fat diet could choose MR60-MR80,because MR60 had a certain regulatory effect but not significant,while MR80 had a significant effect.While the middle-aged group with a high-fat diet could adopt the MR60-MR80 diet,which could significantly promote health.Therefore,choosing a low methionine diet is an effective way to improve the metabolic characteristics and chronic diseases caused by aging in the middle-aged group.