The Role Of Bile Acids And Bile Acid Receptors In Piglet Gut Health And Their Regulation

Bile acids,including primary and secondary bile acids,have been suggested to be important signaling molecules,and their biological functions are closely correlated with the activation of their receptors.Recent studies have revealed the important roles of bile aicds and their receptors in intestinal physiology.Farnesoid X receptor(FXR)and Takeda G protein coupled receptor 5(TGR5)are important bile acids receptor associated with multiple biological functions including regulating their own synthesis,cell proliferation,immune responses as well as energy metabolism.However,studies focused on investigating bile acids and their receptors in pigs are scarce.Intestinal microbiome is responsible for the biotransformation of bile acids especially the production of secondary bile acids.Therefore,factors affecting intestinal microbiome may lead to altered bile acid metabolism and thus have impacts on the biological function of bile acids and their receptors in intestine.Accompanied with changes of nutrition source and nutrition intake,abrupt changes in gut microbiome also occur in piglets during weaning transition periods.During this critical period,how bile acid metabolism changes and its potential effects on intestinal physiology properties including intestinal cell proliferation and mucosa immune and barrier function is still obscure.Therefore,the following four experiments were designed.Weaning piglets,piglets supported by total parental nutrition(TPN),piglets with short bowel syndrome(SBS)and IPEC-J2 cells were used as models to investigate the roles of FXR and TGR5 in the modulation of bile acids metabolism and intestine physiology and the potential mechanisms and to explore nutritional strategies in the regulation of bile acids metabolism.The main results of our research are as followes.Experiment 1 Effects of nutrition on gut microbiome,bile acids metabolism and expression of their receptors in pigletsTo explore the crosstalk between nutrition,gut microbiome and bile acids metabolism in piglets,the first experiment investigated the composition of intestinal microbial community,bile acids profile and crucial genes in FXR signaling in sow suckled piglets at day 21(S21)or28 days of age(S28)and 28 day old weaning piglets with undernutrition(UN)or normal nutrition(NN)status.There were no differences in portal bile acids composition among S21,S28 and NN piglets.Compared with S21,S28 and NN piglets,UN piglets had higher portal concentrations of Lithocholic acid(LCA),deoxycholic acid(DCA),GDCA and TDCA whereas the portal concentrations of HCA,THCA and CDCA in UN group were less than 1/3 of those in the other groups.Theses results indicated that nutrition intake(adequate vs deficiency)outweigh age of suckling piglets(21-day vs 28-day)and nutrition sources(sow milk vs feeds)in inducing changes of bile acid metabolism.The lower primary bile acids concentration in portal blood suggested suppressed bile acids synthesis.Accordingly,portal FGF19 concentrations and hepatic SHP were increased,whereas hepatic CYP7A1 was decreased in UN compared with NN piglets.In accordance with the changes in portal blood,The UN group had the highest LCA and DCA level among all groups,which were 14 and 24 fold respectively of these in NN group.This could partly explained the increased secrection of FGF19,the marker of FXR activation,and might also suggested the potential role of gut microbiome in bile acids metabolism.Compared with UN group,NN group showed lower relative abundance of proteobacteria while higher relative abundance of lactobacillus indicating the potential role of lactobacillus in bile acids metabolism.Experiment 2 Effects of secondary bile acids on proliferation,tight junction protein expression and immune responses in IPEC-J2 cells.In experiment 1,fold increase of LCA and DCA were observerd in undernutrition piglets,and LCA and DCA are well known for their cytotoxicity among all natural bile acids.To determine the potential impacts of LCA and DCA on cell proliferation,barrier and immune function of porcine enterocytes,LCA,DCA and combination of LCA and LPS treatments were administered in IPEC-J2 cells.In our study,the lowest concentration to decrease the proliferation of IPEC-J2 cells was 5?M of LCA and 25?M of DCA.The gene expression or protein abundances of tight junction proteins ZO-1,Occludin and Claudin-1 could also be inhibited by 5?M of LCA and 25?M of DCA.5?M of LCA also stimulated the secretion of IL-8 and the gene expression of IL-6 and IL-8 and decreased the expression of antioxidant genes CAT and SOD1.Intriguingly,LCA synergize with LPS in the regulation of nutrient transporter gene.Compared with LCA treatment alone,coadministration of LCA and LPS decreased the gene expression of GLUT2.Compared with LPS treatment alone,coadministration of LCA and LPS decreased the gene expression of SGLT1 and PEPT1.Our results indicate the detrimental effects of secondary bile acids on cell proliferation,barrier and immune function and antioxidant property in porcine enterocytes.LCA may synergize with LPS to depress nutrients transportation.Experiment 3 Supplementation of lactobacillus changes bile acid profile in weaning piglets by modifying gut microbiome.Experiment 2 confirmed the detrimental effects of LCA and DCA in porcine enterocytes.Experiment 1 indicated that the increased production of LCA and DCA might be associated with lower relative abundance of lactobacillus.Evidences have suggested the beneficial effects of probiotic in improving growth and intestinal health of weaning piglets.Whether the beneficial role of probiotics is correlated with bile acid metabolism has not been investigated.Therefore,in this study,we investigated the effects of lactobacillus supplementation on intestinal microbiome,bile acids profile in ileum and expression of genes related to bile acids metabolism in weaning piglets.We also detected the vaiation of prandial plasma glucose concentration by implanting jugular cathethers in Piglets.Compared with control group,lactobacillus group had lower relative abundances of Bacteroides,Clostridium?sensu?stricto?1,Parabacteroides,Ruminococcus?1 and Desulfovibrio.Piglets supplemented with lactobacillus showed lower total LCA level in ileum.In control group,the elevation of LCA,the most potent endogenous Takeda G-protein-coupled receptor 5(TGR5)ligand,was companied by increased plasma GLP-1 level probably indicating that the higher LCA level increased the activation of TGR5.Accordingly,the lower plasma GLP-1 level in piglets supplemented with lactobacillus was accompanied by increased prandial blood glucose level at 1 hour after feeding and AUC at3 hours after feeding.These results suggested that lactobacillus supplementation changed gut microbiome related to bile acids metabolism,decrease LCA production and GLP-1 secrection and increase the metabolism of glucose.Experiment 4 Differential Action of TGR5 Agonists on GLP-2 Secretion and Promotion of Intestinal Adaptation in Piglet Short Bowel ModelThe third experiment showed that the variation in the concentration of LCA,potent TGR5 ligand,led to altered plasma GLP-1 level.Given that GLP-1 and GLP-2 are derived from the proglucagon gene and always co-secreted from enteroendocrine L cells and GLP-2 is an trophic hormone,we hypothesized that enteral activation of TGR5 expressed in enteroendocrine L cells could augment endogenous GLP-2 secretion and increase the intestinal adaptation responses following intestine resection.Therefore,TPN piglet model and SBS piglet model were created to assess the efficacy of different TGR5 agonists to stimulate GLP-2 secretion and intestinal adaptation.In study1,parenterally-fed,neonatal pigs(n=6/group)were gavaged with vehicle,olive extract(OE)at 10 or 50 mg/kg,or ursolic acid(UA)10 mg/kg and plasma GLP-2 were measured for 6 hr.In study 2,neonatal pigs(n=6-8/group)received either transection or 80%mid small intestine resection and after 2 d assigned to treatments for 10 d as follows: 1)transection +vehicle(Control),2)resection + vehicle(SBS),3)resection +(30 mg UA)(SBSUA),4)resection +(180 mg/kg OE)(SBS-OE).Plasma samples were collected for the measurement of GLP-2.On day 9 of the experiment,stable isotop labled amino acids were injected for 4 hours and plasma samples were collected to at 0,3,3.5 and 4h.Piglets were slaughtered on day 10 of the experiment to collect intestine and liver sample.We measured plasma GLP-2,intestinal histology,cell proliferation,gene expression as well as whole body citrulline-arginine kinetics and bile acid profiles.In study 1,GLP-2 secretion was increased by UA and tended to be increased with OE.In study 2,SBS alone but not additional treatment with either TGR5 agonist resulted in increased crypt cell proliferation and villus height in remnant jejunum and ileum sections.SBS increased biliary,ileal and colon concentration of bile acids and expression of inflammatory and FXR-target genes,but these measures were suppressed by UA treatment.In conclusion,UA is an effective oral GLP-2 secretagogue in parenterally-fed pigs but was not capable of augmenting GLP-2 secretion nor the intestinal adaptation response after massive small bowel resection.To sum up,the main conclusions are as follows.1.The decrease of primary bile acids including HCA and CDCA induced by nutrition insufficiency may be associated with enhanced FXR-FGF19 signaling.The elevation of LCA and DCA may be related to changes of gut microbiome especially the decreased relative abuncance of lactobacillus genera.High levels of LCA and DCA can suppress cell proliferation,increase secretion and gene expression of inflammatory cytokines and down-regulte expression of genes related to antioxidant and nutrient transportation.2.The decreased total LCA in piglets supplemented with lactobacills may be associated with decreased bacteria genera including Bacteroides,Clostridium?sensu?stricto?1,Parabacteroides and Ruminococcus?1.The higher prandial blood glucose in piglets supplemented with lactobacills is related to decreased secrection of GLP-1,marker of TGR5 activation.3.In TPN piglets,Ursolic acid,a TGR5 ligands,is capable of stimulating GLP-2 secrection.In SBS piglets,ursolic acid can improve bile acids metabolism and decrease inflammatory gene expression in intestine and liver but had no effects on GLP-2 secretion and intestinal adaptation.