| Objective:Small intestinal bypass(SIB)has a better lipid-lowering effect while improving glucose tolerance and insulin sensitivity;we also found that elevated levels of bile acids(BA)are closely associated with improved glucose metabolism.However,the mechanism of how SIB regulates blood glucose through BA metabolism is not well understood.Combined with the results of previous studies,we hypothesize that the alteration of intestinal flora after SIB interacts with BA metabolism to regulate hepatic gluconeogenesis in diabetes mellitus(DM)rats,which in turn improves glucose metabolism.In this study,we selected streptozotocin(STZ)-induced diabetes mellitus(STZ-DM)rats as the study subjects and used middle small intestinal bypass(MSIB)as the surgical model to clarify the role of intestinal flora in regulating BA metabolism after MSIB and elucidate the effect of BA on inhibiting hepatic gluconeogenesis.To clarify the role of intestinal flora in regulating BA metabolism after MSIB,and to elucidate the key mechanism of BA inhibition of liver gluconeogenesis.To provide new ideas and strategies for the clinical treatment of DM.Methods:Male Sprague-Dawley(SD)rats of 4-6 weeks of age,weighing 180-220 g,were selected to establish a diabetic rat model by one-time intrapitoneal injection of STZ(60mg/Kg).The model rats(random blood glucose >16.7mmol/L)were selected as further experimental subjects 3 days later.STZ-DM rats were randomly divided into two groups with 8 rats in each group,and MSIB and Sham(sham operation group)were performed,respectively.Body weight(BW),food Intake and fasting blood glucose(FBG)of the rats were measured before surgery and at 1,2,3,4,5 and 6 weeks after surgery.Oral glucose tolerance test(OGTT)and Insulin tolerance test(ITT)were performed before surgery and at 2 and 6 weeks after surgery,respectively.The rats were executed 6 weeks after surgery,and blood was collected from the portal vein to determine the levels of lipids and total bile acids(TBA).The serum levels of Insulin,glucagon-like peptide-1(GLP-1),peptide YY(PYY),and Leptin were measured by enzyme linked immune-sorbent assay(ELISA).Liver specimens were collected,and liver glycogen was measured by Periodic acid-Schiffs(PAS)staining,and glycogen content was determined by optical density analysis.Gene expression of gluconeogenetic key enzymes and transcription factors in liver tissue was measured by q PCR.Fecal specimens were collected and analyzed for intestinal flora in rat feces using 16 s r RNA sequencing.BA composition and content in rat stool were determined by targeted metabolomics.The expression level of bile acid metabolism related proteins in terminal ileum was determined by Western blot(WB).Results:1.The survival rate of the rats in both groups: one rat in the MSIB group died in the first week after surgery,one rat in the Sham group died in the second and fifth weeks after surgery,and the rest of the rats survived until the end of the experiment.2.BW,food intake and FBG level: There was no statistical difference in body weight and food intake between the MSIB group and the Sham group before surgery(P > 0.05).Body weight was lower in the MSIB group than in the Sham group from 1to 2 weeks after surgery(P < 0.05),while there was no statistical difference in body weight between the two groups from 3 to 6 weeks after surgery(P > 0.05).From 1 to 6weeks after surgery,the MSIB group ate less than the Sham group(P < 0.05),and the FBG levels were significantly lower than those of the Sham group(P < 0.01).3.OGTT: There was no statistical difference between the MSIB group and the Sham group before surgery(P > 0.05).At the 2nd and 6th week after surgery,glucose tolerance of rats in MSIB group was significantly improved compared with that in Sham group,and the peak blood glucose in OGTT(30min or 60min)was significantly decreased compared with that in Sham group(P < 0.01).In addition,AUCOGTT of MSIB group was significantly lower than that of Sham group at 2 and 6 weeks after surgery(P < 0.001).4.ITT: At the second week after surgery,the rate of blood glucose decrease in MSIB group was higher than that in Sham group at 45 min after intraperitoneal injection of insulin(P < 0.05).At the 6th week after surgery,the rate of blood glucose decrease in MSIB group was higher than that in Sham group at 60 min after intraperitoneal injection of insulin(P < 0.05).At the second week after surgery,AUCITT of MSIB group was lower than that of Sham group(P < 0.05),but at the sixth week after surgery,there was no statistical difference between the two groups(P > 0.05).5.Changes in serum total bile acids and lipid levels: At 6 weeks postoperatively,serum fasting TBA levels were higher in the MSIB group than in the Sham group(P <0.05).fasting serum levels of TG,CHOL,HDL,LDL,NHDL and FFA were lower in the MSIB group than in the Sham group(P < 0.05).6.Changes in serum hormone levels: At 6 weeks after surgery,the fasting serum Insulin and Leptin levels in the MSIB group were higher than those in the Sham group(P < 0.05),and the serum Ghrelin levels in the MSIB group were lower than those in the Sham group(P < 0.05),and the serum GLP-1,PYY,and FGF-21 levels in the two groups were not statistically different(P > 0.05).7.Hepatic glycogen content: At 6 weeks after surgery,PAS staining of the liver was observed microscopically in both groups,and the liver tissue structure was clear in both groups.glycogen granules were abundant and purple in the MSIB group,and glycogen granules remained better in situ,while there were more vacuoles in the hepatocytes of the Sham group,with more glycogen loss and lighter staining.glycogen optical density values were significantly higher in the MSIB group than in the Sham group(P < 0.05).8.Fluorescence quantitative PCR: At week 6 postoperatively,the relative expression levels of liver gluconeogenic key enzymes PCK1 and G6 PC gene m RNA were significantly lower in the MSIB group compared with the Sham group(P < 0.01);the levels of transcription factors FOXO1 and PGC-1α were lower in the liver tissue of the MSIB group compared with the Sham group(P < 0.05);there was no statistical difference between the two groups in CREB levels(P > 0.05).9.Changes in the intestinal flora in the stool: At 6 weeks postoperatively,stool of rats in both groups was collected and 16 s r RNA sequencing was used to analyze the intestinal flora in stool of rats.The intestinal flora of STZ-DM rats changed significantly after MSIB,and the diversity of flora in the MSIB group was higher than that in the Sham group(P < 0.05).At the level of genus,the abundance of Bacteroides,Clostridium,Escherichia,Lactobacillus,and Shigella was elevated in the MSIB group compared to the Sham group(P < 0.05).10.Changes in fecal bile acid composition: At 6 weeks postoperatively,the feces of rats in both groups were collected,and each bile acid content component in the feces was determined by GC/MS.TBA levels: increased in the MSIB group compared to the Sham group(P < 0.05).Primary bile acid levels: glycocholic acid(GCA),taurocholic acid(TCA),glycocholic goose deoxycholic acid(GCDCA)levels decreased in the MSIB group compared with the Sham group(P < 0.05),and goose deoxycholic acid(CDCA)levels increased in the MSIB group compared with the Sham group(P < 0.05).Secondary bile acids levels: the levels of deoxycholic acid(DCA),glycocholic deoxycholic acid(GDCA),lithodeoxycholic acid(LCA),glycocholic acid(GLCA),and ursodeoxycholic acid(UDCA)in the fecal tissues of rats in the MSIB group were elevated compared with the Sham group(P < 0.05).11.Correlation analysis: Clostridium abundance in the feces of rats after MSIB was significantly and positively correlated with serum total bile acid TBA content,significantly and positively correlated with fecal LCA and GLCA content,and negatively correlated with rat feeding,FBG,OGTT and PCK1 expression.Fecal secondary bile acid content was negatively correlated with the amount of food eaten,FBG,OGTT and the expression of PCK1 and G6 PC in rats.12.Changes of FXR and TGR5 levels in ileum: 6 weeks after surgery,the ileal tissues of rats in both groups were collected and the expression levels of FXR and TGR5 in the terminal ileum were measured by WB method.the expression levels of FXR and TGR5 in the terminal ileum of MSIB group were higher than those of Sham group(P < 0.05).Conclusion:1.MSIB reduces the expression of hepatic gluconeogenic enzymes PCK1 and G6PC;2.elevated Clostridium Clostridium after MSIB is closely associated with decreased blood glucose;3.MSIB improves blood glucose by elevating secondary bile acids(LCA and GLCA)and inhibiting hepatic gluconeogenesis. |
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