The Effect Of Chronic Exercise And Butyrate Supplementation On Microbiota Composition And Lipid Metabolism In Mice

Objective:Nowadays,more than 500 million people are obese worldwide leading to considerable economical costs as well as public health challenge.Recent evidence that the gut microbiota can affect nutrient acquisition,energy regulation and fat storage.These findings raise the possibility that the gut microbiota plays a role in regulating host energy metabolism and may contribute towards the development of obesity and associated metabolic diseases.Indeed,gut ecosystem development and its stability can be influenced by an existing dynamic balance between intrinsic and extrinsic factors such as diet and exercise,which in turn can impact host health.Choi et al.showed that a total of 2510 taxa of bacteria showed differences between the exercise group and the sedentary group.Mice that performed physical exercise showed more abundance of the Lactobacillales order,presenting up to 24 times than sedentary mice,which could enhance the absorption of vitamins,has anti-obese and anti-inflammatory effects.Food is the substrate for the growth of microbiota and directly affects its composition.A high-fat diet(HFD)induces obesity in isogenic C57BL/6J mice partially by a progressive loss of diversity and dropout of major bacterial groups.While feeding rodents a high level of dietary fiber protects against the high-fat-diet-induced increases in body weight and fat mass.SCFAs are small molecular weight compounds derived from the intestinal microbiota through fermentation of the fibrous diet.The major SCFAs are acetate,propionate and butyrate.Among these SCFAs,butyrate,which is the primary source and has been widely documented with regard to human health.Li et al showed that butyrate consumption could improve lipid and glucose metabolism and alter gut microbiota composition.To our knowledge,this is the first study to explore the combined interaction of gut microbiome,butyrate and exercise.We aimed to compare the effect of exercise and butyrate on host lipid metabolism and to what extent the two factors contribute to the beneficial metabolic and microflora population.At the same time,we also investigate the underlying mechanism between intestinal microbiota and metabolic health.Methods:C57BL/6 male 4-week old mice were divided into normal diet(ND)group and high-fat diet(HFD)group.Mice received an NCD with(exercise group;NE)or without exercise training(control group;NC)for 8 weeks.Under high-fat diet conditions,mice ab libitum and underwent one of four training regimes:(1)high-fat diet control group(HC)(2)HC with oral sodium butyrate group(HC+Na B)(3)HC with chronic exercise group(HE),(4)HE with oral sodium butyrate group(HE+Na B)for 8 weeks.Food intake studies were performed in individually caged mice by measuring diet consumed every day after normalizing diet spilled in the cage.Body weight and food intake were assessed every day.After 8 weeks,mice were euthanised and test the body composition,including FM,FFM,BMI;mouse feces were used for microbial community analysis;At the end of the study,mice were euthanised via cervical dislocation,after which liver and abdominal visceral fat were excised and weighed and intestinal content was sampled.Fecal and blood SCFAs were measured using gas chromatography(GC);inflammation and lipid profiles were detected;The protein levels of Sestrin2,CRTC2,ACC,p-ACCSer79,FAS,CPT1,PPARα and PPARγ were determined by western blot in mice liver;Hep G2 cells were treated with LPS followed by butyrate for 24 h and the protein levels of Sestrin2,CRTC2,ACC,p-ACCSer79,FAS,CPT1,PPARα and PPARγ were determined by western blot;In order to further explore the relationship between Sestrin2 and CRTC2,we constructed Ad-SESN2 and si RNA CRTC2 in vitro Hep G2 cell,the protein levels of Sestrin2,CRTC2,ACC,p-ACCSer79,FAS,CPT1,PPARα and PPARγ were determined by western blot.Results:(1)Exposure to HFD for 8 weeks showed an increase in body weight gain(P < 0.05)as compared to NC group,while we found no significant difference between NC and NE group.Mice fed HFD supplemented with butyrate(HC+Na B)or HFD Ex mice(HE)had significantly restrain the increasing of body weight gain(the improvement effect of HE group was more obvious).Combined therapy(HE+Na B)followed the same trend with HCB & HE but showed no statistical significance.Within HFD groups,chronic oral butyrate supplementation caused a sustained reduction in food and energy intake during the 8-week intervention period(p<0.05 for all weeks),while HE showed no difference compared with HC group.(2)ELISA results showed that HC group accompanied with the highest levels of LPS and pro-inflammatory cytokines of TNF-α and IL-1β.All treatments tend to improve the serum levels of LPS and inflammation to different degree.Among them,the improvement effect of HE group was the most obvious.Gas chromatography results showed exercise and chronic butyrate consumption did not alter the levels of butyrate in peripheral blood,but significantly increased in fecal particularly in HE group.However,HE+Na B did not increase the level of butyrate.The results indicate that exercise may increase abundance of butyrate-producing fecal microbiome,and butyrate supplement may have an effect on microbiota composition.(3)By analyzing specific composition of microbiota,we observed that Firmicutes,Bacteroidetes,Actinobacteria and Proteobacteria are the four most important bacteria.16 S ribosomal RNA gene sequencing of the colonic and fecal microbiome has linked obesity to an altered balance between the two dominant bacterial phyla,with a relatively low proportion of Bacteroidetes and a correspondingly higher proportion of Firmicutes.Within LFD,exercise markedly increased phylum levels of Bacteroidetes and decreased Firmicutes/Bacteriodetes compared with NC group.Within HFD,HC showed a sharp increase in the ratio of Firmicutes/Bacteriodetes and the phylum levels of Firmicutes.All treatments tend to improve the microbiota composition to different degree.Among them,HE increased colonization of butyrate-producing bacteria,while chronic butyrate intervention weakened exercise beneficial effects on microflora population in HE+Na B.(4)Sestrin2 and CRTC2 functioned as important mediators to maintain lipid homeostasis in mice liver.HE and HC+Na B both promote the protein expression of Sestrin2 and CRTC2 in mice liver,and the protein expression were positive significantly correlated with levels of butyrate.Hep G2 cells were treated with LPS followed by butyrate.The inflammatory cytokines including IL-1β and TNF-ɑ was strikingly promoted in LPS-treated group while significantly inhibited in LPS-butyrate group.In addition,LPS inhibited protern expression of Sestrin2,CRTC2 and lipolysis protein(PPARα,PPARγ)while promoting the expression of lipid synthesis protein(p-ACC,FAS).However,butyrate reverses the effects of LPS on Hep G2 cells.Conclusions:(1)Both exercise and butyrate can protect against diet-induced obesity,inflammation and dyslipidemia,but HE was more effective than HC+Na B.Butyrate at least partially regulates body weight by inhibiting food intake.(2)Gut ecosystem development and its stability can be influenced by an existing dynamic balance between intrinsic and extrinsic factors that in turn can impact host health,such as diet and exercise.Chow diet and exercise can increase the proportion of probiotics in the intestine.HFD is associated with gut microbiota dysbiosis,which leads to increased blood levels of LPS toxins and pro-inflammatory cytokines.Both exercise and butyrate programs were able to prevent this dysbiosis but were more effective in HE group and had only minor effects in the combined therapy group.This suggests that butyrate is a double-edged sword regarding health.Excess butyrate may reduce the proportion of intestinal butyrate-producing bacteria,influence the regulation of energy metabolism.(3)Exercise promotes the expression of Sesn2 and CRTC2,which also showed increased expression upon butyrate treatment.And the altered gut microbiota stimulates differential production of SCFAs(like butyrate)that in turn promote the expression of Sesn2 and CRTC2 to improve metabolic health and protect against obesity.This suggests that butyrate may play a key role in the regulation of Sestrin2 and CRTC2 in the regulation of lipid metabolism by intestinal flora changes.