Methionine Restriction Improves Glucose Metabolism And Cognitive Ability Through H19 In Middle-Aged Mice

Slowed glucose metabolism and enhanced oxidative stress in middle-aged populations can induce neurodegenerative diseases.Methionine restriction（MR）has been shown to regulate glucose metabolism,alleviate oxidative stress and inflammation,and improve cognitive ability in middle-aged populations.However,the mechanisms by which MR improves glucose metabolism and cognitive ability in middle-aged people still need to be investigated.Lnc RNA H19（H19）is a key regulator of insulin sensitivity and glucose metabolism,which regulates cell proliferation,apoptosis,and neurological disorders.Highland barley（HB）is a low-methionine content grain that not only provides the body with high-quality protein,but also has improved glucose metabolism,antioxidant and anti-inflammatory properties.Therefore,the aim of this study was to investigate the effects of MR-regulated glucose metabolism on cognitive performance,explore the potential mechanism of H19 in it,assess the feasibility of HB as a dietary protein source for MR diets,design a low methionine diet based on HB whole grains,and validate the effect of MR on H19 expression in the HB diet.The contents and results of this study are as follows:1.MR ameliorated pancreatic cell proliferation and apoptosis and promoted insulin secretion in HFD middle-aged mice via the H19/Pi3k/Akt pathway.Middle-aged mice（C57BL/6J）were given a control diet（CON,0.86%methionine+4.2%fat）,a CON+MR diet（CMR,0.17%methionine+4.2%fat）,a high-fat diet（HFD,0.86%methionine+24%fat）,or an HFD+MR diet（HFMR,0.17%methionine+24%fat）for 25 weeks.The mouse isletβ-cell lineβ-TC6 cells were used to investigate the effect of MR onβ-cell proliferation and apoptosis.The results showed that MR increased the expression of B-cell lymphoma 2（Bcl-2）and decreased the expression of Bcl-2 associated protein X（Bax）and cleaved cysteinyl aspartate specific proteinase-3（Cleaved Caspase-3）in the pancreas;promoted the expression of pancreatic Cyclin D1（Ccnd1）,Cyclin D2（Ccnd2）,and cyclin-dependent kinase 4（Cdk4）;promoted the expression ofβ-cell development-related genes pancreatic and duodenal homeobox-1（Pdx-1）and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A（Maf A）,increased the number of pancreatic cells,and promoted insulin secretion fromβ-TC6cells.Meanwhile,MR promoted the expression of H19 inβ-TC6 cells and pancreas by inhibiting reactive oxygen species（ROS）and decreasing H19 methylation,and increased the protein phosphorylation levels of phosphatidylinositol-3-kinase（Pi3k）and protein kinase B（Akt）.However,these results were reversed after knockdown of H19 inβ-TC6 cells.2.MR enhances skeletal muscle insulin-dependent glucose uptake and utilization via the H19/IRS/Akt pathway,thereby improving glucose homeostasis and insulin resistance in middle-aged mice with HFD.Middle-aged mice（C57BL/6J）were given a control diet（CON,0.86%methionine+4.2%fat）,a CON+MR diet（CMR,0.17%methionine+4.2%fat）,a high-fat diet（HFD,0.86%methionine+24%fat）,or an HFD+MR diet（HFMR,0.17%methionine+24%fat）for 25 weeks.The mouse myogenic cell line C2C12 cells were used to establish an insulin resistance model.The results showed that MR promoted the expression of H19 in C2C12 cells and skeletal muscle by inhibiting ROS and decreasing H19 methylation;MR increased the insulin receptor substrate-1/insulin receptor substrate-2（IRS-1/IRS-2）ratio,promoted the phosphorylation of Akt and glycogen synthase kinase 3β（GSK3β）,and the expression of hexokinase 2（HK2）,thereby promoting C2C12 cells glucose uptake and skeletal muscle cell glycogen synthesis as well as glycolysis.However,these results were reversed after knockdown of H19 in C2C12 cells.3.MR can protect neurons from HFD,HG,or H₂O₂-induced injury and apoptosis by improving hippocampal glucose metabolism to alleviate oxidative stress and improving the H19/let-7/Caspase-3 pathway.Middle-aged mice（C57BL/6J）were given a control diet（CON,0.86%methionine+4.2%fat）,a high-fat diet（HFD,0.86%methionine+24%fat）,or an HFD+MR diet（HFMR,0.17%methionine+24%fat）for 25 weeks.The mouse hippocampal neuronal cell line HT22 cells were used to establish a high glucose（HG）-induced early apoptosis model.The results showed that in vitro,MR significantly improved HT22 cell viability,inhibited ROS production,and suppressed HG-induced HT22 cell apoptosis in a gradient-dependent manner;in vivo,MR alleviated insulin resistance,improved hippocampal glucose metabolism,reduced hippocampal oxidative stress,inhibited HFD-induced hippocampal neuronal damage and apoptosis,and enhanced cognitive ability.In addition,MR could inhibit HFD,HG,or H₂O₂-induced overexpression of H19 and Caspase-3,and promote the expression of let-7a,b,and e.4.HBHF ameliorated hippocampal neuronal damage through the MR strategy by improving hippocampal glucose metabolism,alleviating oxidative stress and inflammation,and inhibiting H19 expression,which in turn improved learning memory capacity and alleviated anxiety in middle-aged mice with HFD.Middle-aged mice（C57BL/6J）were given a control diet（CON,0.86%methionine+4.2%fat）,a high-fat diet（HFD,0.86%methionine+24%fat）,a highland barley whole grain high-fat diet（HBHF,0.17%methionine+24%fat）,or a HBHF+methionine diet（HBHFmet,0.82%methionine+24%fat）for 25 weeks.The results showed that HBHF improved HFD-induced increases in body weight,fasting glucose,insulin,HOMA-IR,and lipid levels,decreased plasma inflammation and oxidative stress levels,reduced fat accumulation,and promoted energy expenditure in middle-aged mice.Compared with the HFD group,HBHF increased the expression of H19,Bcl-2,Maf A,and Pdx-1 and decreased the expression of Cleaved Caspase-3 and Bax in the pancreas.HBHF promoted the expression of H19,the phosphorylation of Akt,and the expression of p-GSK3β,HK2,pyruvate kinase M2（PKM2）,and phosphofructokinase 1（PFK1）in skeletal muscle,which in turn improved skeletal muscle glycogen synthesis as well as glycolysis.The above results suggest that HBHF inhibits apoptosis of pancreatic cells,improves insulin secretion,and improves skeletal muscle glucose metabolism,thereby alleviating HFD-induced glucose elevation and insulin resistance.However,these effects were attenuated in the HBHFmet group.The reduction of blood glucose and systemic insulin resistance levels was beneficial in alleviating the hippocampal neuronal damage caused by HFD.The results showed that HBHF alleviated HFD-induced learning memory impairment and anxiety in middle-aged mice.HBHF promoted the gene expression of hippocampal IRS-1 and glucose transporter 1（GLUT1）,activated the phosphorylation of Akt,which in turn promoted the protein expression of HK2 and p-GSK3β;promoted the expression of nuclear factor erythroid 2-related factor 2（Nrf2）,heme oxygenase-1（HO-1）,and NADPH quinone oxidoreductase-1（NQO-1）;promoted total antioxidant capacity（T-AOC）and interleukin-10（IL-10）levels,and decreased malondialdehyde（MDA）and interleukin-1β（IL-1β）levels.HBHF inhibited the expression of hippocampal H19,Caspase-3,and Bax,promoted the expression of Bcl-2,alleviated HFD-induced hippocampal neuronal injury,and promoted the learning and memory related gene expression（CAMK2A,BDNF,Trk B,SYNPO,and CREB）.In summary,MR promoted pancreatic cell proliferation,inhibited pancreatic cell apoptosis,improved pancreatic insulin secretion function,and also improved skeletal muscle glucose uptake,glycogen synthesis,and glycolysis through H19,which in turn lowered blood glucose levels and improved body glucose tolerance and insulin resistance.And MR ameliorated cognitive impairment caused by glucose metabolism disorders by improving hippocampal glucose metabolism and oxidative stress,inhibiting H19 expression,and alleviating hippocampal neuronal damage and apoptosis.The above findings were verified in HB diets,and it was found that HB diets have MR effects,providing new ideas and theoretical support for the development of MR diets.