Methionine Restriction Ameliorates High-fat Diet Induced Cognitive Dysfunction Via Mechanisms Linking Microbiome Diurnal Rhythmicity And Metabolic Rhythm

With the change of modern life style and the continued elevation of the standard of living,the high-fat diet(HFD)has become a common dietary pattern in the world.Growing evidence has implicated HFD in the elicitation of obesity and a variety of health problems,such as insulin resistance,metabolic rhythm disorder and cognitive dysfunction,and damage to gut microbiome.In recent years,the exploration of dietary pattern that may improve metabolism and cognitive function has become a popular research direction in food nutrition field.Methionine restriction(MR)has been demonstrated to improve obesity and insulin resistance induced by HFD,reduce ROS levels and inflammatory response,however,the effects of MR on HFD-induced metabolic rhythm disorder and cognitive dysfunction remains unclear.With this background,the aim of the current study was to investigate the protective effect and the potential underlying mechanisms of MR on HFD-induced cognitive impairments via inproving microbiome diurnal rhythmicity and metabolic rhythm.The main investigations and results are as follows:(1)To investigate the effects of MR on glucose and lipid metabolism in HFD-induced obese mice,8-week-old male C57BL/6J mice were fed a low-fat diet or HFD for 4 weeks,followed with a full diet(0.86 % methionine,w/w)or a methionine-restricted diet(0.17 %methionine,w/w)for 8 weeks.Results revealed that after 8 weeks of MR intervention,the bodyweight of HFD-induced obese mice could be effectively reduced without restricting the energy intake.Next,we performed GTT and ITT to evaluate the glucose intolerance and insulin resistance.The results showed that MR could reduce the fasting blood glucose level and HOMA-IR of HFD-induced obese mice.Histological staining results showed that MR significantly improved lipid accumulation and adipocyte hypertrophy in the liver of HFD-induced obese mice.Therefore,MR can significantly improve HFD-induced glucose and lipid metabolism disorders in mice.(2)To investigate the protective effects of MR on HFD-induced cognitive dysfunction.The behavior experiments,histological staining and molecular biological methods were used to evaluate the cognitive function and nerve damage of HFD-induced obese mice.The results of open field test,Y maze test and Morris water maze test indicated that MR could effectively improve the decrease of working memory and spatial memory ability triggered by HFD.H&E staining and synaptic ultramicro images showed that MR ameliorated synaptic structural damage and the neuronal cells damages triggered by HFD.The expression of PSD-95 was detected by q PCR,the results showed that MR increased the expressions of synaptosomal functional proteins PSD-95.Moreover,MR activated the ERK/CREB/BDNF signaling and upregulated the expressions of neurotrophic-factor.MR significantly increased the expressions of mitochondrial complex related proteins and autophagy related genes Atg3 and Lc3.Furthermore,MR effectively improved HFD-induced neuroinflammation by inhibiting the expressions of inflammatory factors IL-1? and Tnf-?.In addition,MR improved the expressions of clock related genes Bmal1,Per1 and Cox2 in the brain of HFD-induced obese mice.(3)To examine the mechanism by which MR improves the gut barrier function and gut microbiome in cognitive dysfunction in HFD-induced obese mice,the gut tissues and gut microbiome were selected to examine the effects of MR on HFD-induced changes on gut barrier function and disturbance of microbiome diurnal rhythmicity.Results indicated that MR could improve the gut barrier damage in obese mice by increasing the expressions of intestinal tight junction protein Claudin-1 and Occludin.Moreover,MR reduced the levels of LPS and inflammatory factors in the colon of HFD-induced obese mice.Using16 S r RNA sequencing to evaluate the effects of MR on the composition of gut microbiome in obese mice,and PICRUSt was further used to predict the function of gut microbiome.The results showed that MR dampened the diurnal oscillation of LPS synthesis pathway.In addition,MR restored HFD-disrupted the cyclical fluctuations of the gut microbiome,which led to time-specifically decreased the abundance of inflammation-related microbiome and dampened the diurnal oscillation of inflammation-related microbiome,and restored the diurnal oscillation of short-chain fatty acids producing bacteria,increased the levels of short-chain fatty acids.Moreover,MR improved the expressions of clock genes Per2 and Rev-erb? in the gut of HFD-induced obese mice.(4)To investigate the mechanism by which MR improves metabolic rhythm in cognitive dysfunction in HFD-induced obese mice,the liver tissues were selected to examine the effects of MR on HFD-induced hepatic lipid metabolism and bile acids metabolism disorder.Results showed that MR could significantly improve the rhythmic disturbance of TC,TG,HDL-C,and LDL-C in serum of HFD-induced obese mice.Moreover,MR led to a time-specific enhancement of the expression of FGF21 in the liver and serum,and activated the AMPK/PGC-1? signaling,improved HFD-induced energy metabolism disorder.In addition,MR restored the HFD-disrupted cyclical fluctuations of lipidolysis genes Ppar?,Ppar?,and Srebp1.Using UPLC-MS/MS to evaluate the levels of bile acids in serum,the results showed that MR upregulated the cyclical levels of cholic acid and chenodeoxycholic acid,and downregulated the cyclical level of deoxycholic acid in the dark phase.Moreover,q PCR was used to examine the expressions of bile acids metabolism-related genes,and the results showed that MR significantly restored the rhythmic oscillations of Cyp7a1 and Cyp27a1.Furthermore,MR improved the expressions of clock genes Bmal1 and Cry1 in the liver and the brown adipose tissue of HFD-induced obese mice.In conclusion,MR might regulate gut barrier function and microbiome diurnal rhythmicity,improve metabolic rhythm,and systemic inflammatory responses induced by HFD,and ultimately improve cognitive dysfunction.The present study found that the mechanism of microbiome-metabolic rhythm mediated by MR could provide a theoretical basis for the application of MR as a new dietary pattern and could provide new strategies for optimizing dietary pattern to intervene cognitive dysfunction induced by high-calorie diets.