The Mechanism Underlying The Improvement Of T2DM In Rats By Modified BA Profiles After Bariatric Surgery

Part 1:Bile Acid Profiles within the Enterohepatic Circulation in a Diabetic Rat Model after Bariatric SurgeriesBackground:Last decades,the world has seen an alarming prevalence of type 2 diabetes mellitus(T2DM).Medical interventions,including healthy lifestyle and/or medications,cannot keep effective and long-lasting amelioration,which has concerned both doctors and patients.Fortunately,bariatric surgery provides a promising therapeutic option for obese diabetic patients with its significant effects on sustaining weight loss,improving glucose tolerance and long-lasting amelioration for T2DM.Whereas,the underlying mechanisms are still far from completely elucidated.Bile acids(BAs),synthesized in the liver and cycled in the enterohepatic circulation has been recognized as a signal molecule by activating its receptors in the intestine and liver.Serum taurine-conjugated BAs have been shown to be elevated after bariatric surgeries,whereas the postoperative BA profiles within the enterohepatic circulation have not been investigated.To clarify this can help to explain the mechanisms by which bariatric surgery leads to BA profile alternations and subsequent metabolic effects.Objectives:In this study,we performed sleeve gastrectomy(SG),duodenal-jejunal bypass(DJB),and SHAM procedures in an obese diabetic rat model and mainly investigated 3 important issues:(1)To confirm the anti-diabetic effects of SG and DJB.(2)To detect the total BA(TBA)levels and BA profiles in peripheral circulation and enterohepatic circulation of T2DM rats after SG and DJB.(3)To explore the underlying mechanisms through which bariatric surgery regulates the BA metabolism.Methods:The obese diabetic rat models were induced by high-fat-diet(HFD)and streptozotocin(STZ)and matched into SHAM group(n=10),DJB group(n=10)and SG group(n=10).The weight-loss and anti-diabetic effects of SG and DJB were evaluated at week 12 post-surgery.After the detection of TBA levels in peripheral serum,portal vein and bile,we measured the BA profiles of these three kinds of samples by High-Pressure Liquid Chromatography Coupled With Tandem Mass Spectrometry(HPLC-MS/MS).Then,expression levels of the key enzymes for hepatic BA synthesis(CYP7A1)and conjugation(BRCS and BAAT),the BA transporters within enterohepatic circulation(BSEP?NTCP?ASBT?OSTa)as well as the key regulator for BA synthesis(FGF15)were examined by RT-PCR and Western Blot.Results:1.No significant difference of body weight and food intake was observed between DJB and SHAM group.However,the body weight and food intake of SG group increased significantly slower since week 4 compared with DJB and SHAM groups.Correspondingly,the body weight gain was also notably lower in SG group than that in DJB and SHAM groups at week 12 postsurgery.2.Compared with SHAM group,glucose tolerance of T2DM rat was obviously improved in both SG and DJB groups based on OGTT and AUCOGTT results at week 12 after surgery.In addition,AUCITT and HOMA-IR were both lower in SG and DJB groups.3.The secretion of GLP-1 of T2DM rats were stimulated by SG and DJB at week 12 post-surgery compared with SHAM group.Whereas,no difference of glucose-stimulated insulin secretion was observed among these groups.4.At week 12 after operation,TBA levels in the serum,portal vein and bile were significantly higher within DJB and SG groups than that in SHAM group.For subgroup analysis,the ratio of primary and secondary BAs showed no difference between operation groups and SHAM group in each kind of samples.The percentage of conjugated species in DJB and SG group was significantly higher than SHAM group in both serum and portal vein,whereas,no difference of conjugated percentage was observed between operation groups and SHAM group in bile samples.5.According to the BA profiles at week 12 after surgery,CA was the most abundant BA specie in both serum and portal vein,while in bile the most abundant was TCA.In peripheral serum,all the taurine-conjugated BAs plus aMCA were more abundant in both DJB and SG group than that in SHAM group.Furthermore,PMCA,CDCA and UDCA were also elevated significantly in DJB group.In portal vein samples,almost all the BA species were increased in DJB and SG group compared with SHAM group except CA and HDCA.Unlike in the serum and portal vein,the amount of every unconjugated BAs was limited in bile,among which only CDCA and DCA were increased after DJB and SG.Compared with SHAM group,all the conjugated species were also elevated in DJB and SG group.6.CYP7A1,the rate-limiting enzyme for BA synthesis,was inhibited in both DJB and SG groups at week 12 post-surgery,while the expressions of BACS and BAAT(key enzymes catalyzing the conjugation with taurine of BAs)were significantly higher in DJB and SG groups at both protein and transcriptional level.Moreover,the expression of ileal FGF15 was also increased in both DJB and SG groups.7.At week 12 after surgery,the ileal expressions of ASBT and OSTa(main transporters for ileal reabsorption of BAs)were significantly elevated in DJB and SG group compared with SHAM group,while no difference was observed for hepatic BESP and NTCP expressions among the three groups.Conclusion:1.SG could induce sustained weight loss,whereas DJB has no weight loss effect.2.Both DJB and SG significantly improved glucose metabolism for T2DM rats.3.The TBA levels of T2DM rats were significantly elevated after DJB and SG,especially the taurine-conjugated species.4.The depressed expression of key enzyme for hepatic BA synthesis together with the elevated expression of ileal FGF15 indicated that the elevation of BA levels within DJB-and SG-operated rats was not the result of the stimulated hepatic synthesis.5.The increased expression levels of hepatic BACS and BAAT reflected the stimulated conjugation of BAs in liver,which also explained the reason for elevation of taurine-conjugated BAs.6.The elevated expression levels of ileal BA transporters indicated that the elevation of BA levels after DJB and SG was mainly because of the increased ileal reabsorption.Part 2:The mechanism underlying the amelioration of T2DM in rats by elevated BAs after SGBackground:Bariatric surgery is currently the most effective procedure for obese diabetic patients,of which Roux-en-Y gastric bypass(RYGB)and sleeve gastrectomy(SG)represent the two most commonly performed procedures.Moreover,SG has replaced RYGB as the first choice for the majority of surgeons all over the world.Our previous studies have demonstrated that bariatric surgeries can elevate the levels of serum BAs,which have been implicated as potential mediators of the weight-independent effects of bariatric surgery because of the considerable significance of their receptors,TGR5 and FXR.Then we need to further investigate the underlying mechanisms through which the elevated BAs after bariatric surgery improve glucose metabolism.By activating the farnesoid-X receptor(FXR)in the small intestine,BAs could stimulate the expression and secretion of FGF15/19,which conversely regulates the BA homeostasis in an endocrine pattern.Moreover,FGF15/19 also plays a role in regulating hepatic glucose metabolism by repressing gluconeogenesis and promoting glycogen synthesis in the liver.Therefore,whether the increase of FGF15/19 is induced by BAs,and whether elevated FGF15/19 participates in the improvement of hepatic glucose metabolism after bariatric surgery needs further investigation.Objectives:In this study,we performed sleeve gastrectomy(SG)and SHAM procedures in an obese diabetic rat model and mainly investigated the following issues:(1)To confirm the weight loss and anti-diabetic effects of SG;(2)To detect the hepatic glycogen content and gluconeogenesis activity after SG;(3)To examine the expression levels of ileal FXR and FGF15 as well as the levels of TBA and related BA species within peripheral serum and portal vein after SG.(4)To explore the activity of FGFR4 and its corresponding signal pathways involved in regulating hepatic glucose metabolism.Methods:The obese diabetic rat models were induced by high-fat-diet(HFD)and streptozotocin(STZ)and matched into SHAM group(n=10)and SG group(n=10).The weight-loss and anti-diabetic effects of SG were evaluated at week 12 post-surgery.Then we examined the levels of TBA together with the FXR-agonistic BA subspecies in systemic serum and portal vein at week 12 post-surgery.After sacrificing,we stained and detected the the hepatic glycogen content by PAS Stain Kit and Glycogen Assay kit,and examined the expression levels of key enzymes for hepatic gluconeogenesis(G6Pase and PEPCK)by RT-PCR.Finally,the histological expression of ileal FXR?FGF15 and hepatic FGFR4 with its corresponding signal pathways involved in glucose metabolism(ERK-GS pathway and CREB-PGC-1? pathway)were detected.Results:1.At week 12 after surgery,the glycogen content and the percentage of PAS-positive cells in liver were significantly higher in SG-operated rats than in SHAM group.Moreover,mRNA levels of G6Pase and PEPCK were significantly declined in SG group.2.TBA levels of serum and portal vein in SG group were significantly higher than in SHAM group at week 12 post-surgery.In addition,Both CDCA and LCA levels in these two kinds samples were elevated and the portal vein DCA level was also higher in SG group.3.According to the RT-PCT and immunohistochemistry results,both mRNA and protein expression levels of ileal FGF15 and hepatic FGFR4 were significantly higher in SG group at week 12 after surgery.Also,FXR mRNA level was also considerably elevated in SG-operated rats compared with the SHAM group.4.At week 12 post-surgery,the western blot results showed that the phosphorylation of ERK1/2 was increased and the phosphorylation of GS was depressed in SG-operated rats,which indicates the activation of ERK-GS pathway.In addition,the declined phosphorylation degree of CREB and decreased expression of PGC-1? illustrated the inhibition of CREB-PGC-1? pathway in SG group.Conclusion:1.The TBA levels of T2DM rats were significantly elevated after SG,especially the FXR-agonistic subspecies.Together with theelevated expression of ileal FXR and FGF15 indicated that the changes of BA levels and profiles after SG activated the ileal FXR-FGF15 pathway.2.The hepatic FGFR4 was significantly promoted after SG,which indicated the activation of FGF15-FGFR4 and its corresponding signal pathways in T2DM rats.3.After SG,the FGF15-induced hepatic glycogen-synthesis regulating signal pathway ERK-GS was significantly promoted.Whereas,the CREB-PGC-lapathway which is responsible for gluconeogenesis process was depressed in T2DM rats.In conclusion,elevated BAs after SG induced FGF15 expression in distal ileum by activating their receptor FXR.Moreover,the action of promoted FGF15 on hepatic glycogen synthesis and gluconeogenesis further contributes to the anti-diabetic effects of SG.