The Roles Of Bile Acids In Treating T2DM After Duodenal-jejunal Bypass In A Diabetic Rat Model

Part Ⅰ The effects of bile acids on amelioration of type 2 diabetes mellitus after duodenal-jejunal bypass in type 2 diabetic ratsBackgroundDiabetes mellitus, of which type 2 diabetes mellitus (T2DM) accounts for over 90%, is a metabolic disease characterized by chronic increasing of blood glucose. Currently there are 415 million adults who are estimated to have diabetes, and the relevant expense is up to USD 673 billion. If this rise is not halted, by 2040 there will be 642 million people living with the disease. Enormous diabetic patients and relevant expenses have made diabetes a global public health issue leading to serious health outcomes. Traditional treatment for T2DM can improve glucose metabolism within a short period but the long-term efficacy is compromised. Bariatric/metabolic surgery, which was initially designed to treat morbid obesity and now has become one of the officially recommended treatments by CDS, ADA and IDF, has the capacity to induce rapid and durable improvement in glucose tolerance. Duodenal-jejunal bypass (DJB) represents one kind of metabolic surgeries, and has been confirmed to be effective in improving glucose metabolism in T2DM without reducing body weight. However, its mechanism remains largely unknown. DJB rearranges the gut in a Roux-en-Y fashion, leading to alterations of bile flow and elevated serum total bile acid levels. Accumulating evidence has shown that bile acids play a role in regulating metabolism as signaling molecules. Whether bile acids play a role after DJB in treating T2DM needs to be elucidated.ObjectivesBile diversion (BD), DJB and SHAM procedures were performed and compared in a diabetic rat model induced by high-fat diet (HFD)/streptozotocin (STZ). The primary objectives included:(1) to investigate whether BD could simulate the effect of DJB on serum total bile acids; (2) to investigate whether BD per se is sufficient to induce T2DM improvement; (3) to investigate whether bile acids play a role after DJB in treating T2DM.MethodsT2DM rats induced by HFD/STZ were allocated to undergo BD, DJB and SHAM procedures (n=10 each). BD procedure diverted bile flow directly to the distal jejunal where the jejunojejunal anastomosis was performed in DJB, mimicking the altered bile flow following DJB. Body weight, energy intake, blood glucose, serum hormones, insulin sensitivity, lipid profiles, as well as systemic total bile acids were measured at week 2 and 8 postsurgery.Results1. The body weight was significantly lower with BD than SHAM at week 8 postsurgery (P< 0.05), without significant difference between DJB and SHAM groups (P> 0.05). Energy intake was comparable between all groups (P> 0.05).2. Prior to surgery, neither fasting blood glucose or the AUC in response to intragastric glucose tolerance test (IGGTT) was different between all groups (P> 0.05). At week 2 and 8 postsurgery, both fasting blood glucose and the AUCIGGTT were significantly lower with DJB and BD compared to SHAM (P< 0.05 both), and the fasting blood glucose and AUCIGGTT were less at week 8 after DJB vesus BD (P< 0.01).3. At week 2 postsurgery, serum CHO concentrations during fasting and in response to Ensure gavage did not differ between three groups (P> 0.05); while both serum TG and NEFA concentrations during fasting and in response to Ensure gavage were lower in BD group, compared with DJB and SHAM groups, without significant difference between the latter two. At week 8, serum concentrations of CHO and NEFA during fasting and in response to Ensure gavage remained similar to those at week 2. In contrast, both fasting and postprandial TG concentrations did not differ from those at week 2 postsurgery with SHAM and BD (being lower in the latter group), but decreased with DJB. However, serum TG concentrations of DJB group were still higher, when compared to BD group.4. At week 2 and 8 postsurgery, serum insulin concentrations during fasting and in response to Ensure gavage did not differ between three groups (P>0.05). HOMA-IR remained unchanged after SHAM and BD postsurgery, but was significantly reduced after DJB at weeks 2 and 8 (P<0.05 both).5. At week 2 postsurgery, serum total GLP-1 concentrations during fasting and after Ensure gavage were similar between the three groups (P>0.05).At week 8, fasting total GLP-1 and the AUC for GLP-1 after Ensure gavage remained unchanged with SHAM, but were increased with both DJB and BD (P<0.05 both).6. At week 2 postsurgery, fasting total bile acid concentrations were higher after BD than SHAM (P<0.05), and tended to be higher after DJB than SHAM (P=0.10), without significant difference between BD and DJB. At week 8 postsurgery, fasting total bile acid concentrations remained unchanged with SHAM, but were increased with both DJB and BD (P<0.05 both), with concentrations being highest in BD group.7. At week 2 postsurgery, serum ALB, ALT, AST, or TBIL did not differ between the three groups (P>0.05). However, serum ALP concentrations were lower in DJB and BD groups compared to those in SHAM group (P<0.05 both), without significant difference between the former two (P>0.05). At week 8, not only ALP, but also ALT and AST concentrations were lower in DJB than SHAM group (P<0.05 both). Similarly, both ALP and AST concentrations were lower in BD than SHAM group (P <0.05 both). In addition, serum ALP was lower in DJB than BD group (P<0.05).Conclusions1. BD can simulate the effects of DJB on bile acids in a diabetic rat model, leading to increase of serum bile acid concentrations.2. BD has the capacity to induce T2DM improvement as well. However, it’s less prominent when compared to DJB.3. Bile acids represent important components of DJB in restoring glucose and lipid homeostasis in diabetic state. However, other mechanisms associated with DJB (eg. lipid sensing, incretin hormones) also appear to make complementary contributions to metabolic regulation.Part Ⅱ The effects of duodenal-jejunal bypass on serum bile acid composition and gut microbiota in type 2 diabetic ratsBackgroundBariatric/metabolic surgery has been confirmed as both prominent and durable. However, the intrinsic mechanisms remain unknown. Bile acids, formerly known as the "intestinal soap", are now important signaling molecules. Bile acids can activate membrane TGR5 receptor, enhancing GLP-1 secretion and energy expenditure. Bile acids are also natural ligands for nuclear receptor, FXR, via which hepatic glucose production and lipid de novo synthesis can be repressed. Besides, serum bile acid level is elevated following all bariatric surgeries except gastric banding, indicating that bile acids, at least partly, mediate the benefits of bariatric/metabolic surgery. Bile acids represent a compound that contains over 20 individual species. Traditional method (3a-enzyme cycling method) for serum bile acid measurement only detects total bile acids without further classification. Nevertheless, during bariatric/metabolic surgery, bile acid component analysis may be of great significance, as different bile acid species have diverse effects on metabolism regulation. For example, chenodeoxycholic acid (CDCA) is a strong agonist of FXR with cholic acid (CA) a less potent one while MCA and UDCA are antagonists. Compared with unconjugated and glycine-conjugated bile acids, taurine-conjugated bile acids are more potent in activating TGR5. Additionally, Bile acids are closely related to gut microbiota. Secondary bile acid generation requires the 7a-dehydroxylation function of certain gut bacteria, and bile acid gavage has the capacity of altering gut microbiota. One recent study even found that feces transplantation did help improve metabolic disorders. Taken together, investigation into the postsurgical serum bile acid composition and related gut microbiota after bariatric/metabolic surgery may help us better understand the intrinsic mechanisms of bariatric/metabolic surgery.ObjectivesDuodenal-jejunal bypass (DJB) and SHAM procedures were performed and compared based on HFD/STZ-induced diabetic rat model. The main objectives included:(1) to investigate the change of serum bile acid composition after DJB; (2) to investigate the gut microbiota after DJB.MethodsT2DM rats induced by HFD/STZ were allocated to undergo DJB and SHAM procedures (n=10 each). The outcomes of DJB were evaluated at week 2 and 12 postsurgery. Serum bile acid composition was analyzed by high-pressure liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), and gut microbiota from dried fecal pellets were detected using Illumina MiSeq platform at week 2 and 12 postsurgery.Results1. Prior to surgery, body weight, energy intake and IGGTT did not differ between DJB and SHAM groups (P>0.05). At week 11 and 12 postsurgery, energy intake was higher with SHAM than DJB groups (P<0.05). DJB induced better fasting and postprandial blood glucose, improved insulin sensitivity and enhanced GLP-1 secretion (P<0.05). No difference was detected between two groups in postsurgical insulin secretion or body weight (P>0.05).2. Total bile acid concentrations were higher in the distal ileum and colon after DJB than SHAM (P<0.05). At week 2 and 12 postsurgery, serum total bile acid concentrations were significantly higher after DJB than SHAM (P<0.01), without significant difference in fecal bile acids (P>0.05).3. Cholic acid (CA) accounted for over 50% of total bile acids in SHAM group while this percentage was less than 40% after DJB at week 2 and 12 (P< 0.05). Though the absolute value of some bile acid species elevated at week 2, the relative percentages were unchanged at all (P> 0.05). In contrast, at week 12, the percentages of taurine-conjugated bile acids, except taurodeoxycholic acid (TDCA) and taurochenodeoxycholic acid (TCDCA), were significantly higher in DJB group than SHAM (P< 0.05).4. The fecal bacteria population in both groups was dominated by Bacteroidetes, Firmicutes, and Proteobacteria at the phylum level. In SHAM group, Bacteroidetes was most abundant at both week 2 and 12. However, after DJB, both Firmicutes and Proteobacteria expanded, at the expense of Bacteroidetes. At an FDR of 5%, compared with SHAM, Bacteroidetes showed decreased abundance at both week 2 and 12 in DJB group (P< 0.05 both) with Firmicutes increased at only week 2 (P= 0.019). No alteration was observed in other phyla. At an FDR of 25%, in addition to Bacteroidetes, Firmicutes was found increased at both week 2 and 12 (P=0.019 and 0.186).5. In the correlation analyses, we found a negative correlation between mean total bile acid levels in the colon and Bacteroidetes abundance (r=-0.73,P<0.001). Also, a positive correlation was found between the mean colonic total bile acid levels and Proteobacteria abundance (r=0.62, P< 0.001).Conclusions1. DJB preferentially elevates serum taurine-conjugated bile acids.2. Gut microbiota is altered following DJB, with Firmicutes and Proteobacteria expanding at the expense of Bacteroidetes.3. The alterations of gut microbiota after DJB might be related to mean total bile acid concentrations within the colon.Part Ⅲ Mechanisms of bile acid alterations after duodenal-jejunal bypass in type 2 diabetic ratsBackgroundBile acids, synthesized from cholesterol within hepatocytes, are main component of bile. Primary bile acids include cholic acid (CA) and Chenodeoxycholic acid (CDCA), which are directly secreted by hepatocytes within the liver, and can be deconjugated and dehydroxylated to secondary bile acids (eg. Deoxycholic acid, Lithocholic acid and Ursodeoxycholic acid). Bile acids can be further classified into unconjugated and conjugated bile acids as per their conjugation with taurine or glycine. Following secretion of bile acids into the duodenum, most (-95%) are reabsorbed in the distal ileum and recycled by hepatocytes. It is notable that the hepatic recovery of bile acids from the portal vein is incomplete, thus accounting for the presence of low levels of bile acids in the peripheral circulation of humans and rodents. Our previous study found that serum bile acids were elevated following DJB, with preferential increase of taurine-conjugated bile acids. However, the intrinsic mechanisms still remain unclear. DJB reconstructs the small intestine in a Roux-en-Y fashion, leading to higher bile acid concentrations in the distal gut, which might faciliate bile acid reabsorption. However, serum bile acids represent net effects of bile acid synthesis, bile acid reabsorption and hepatic uptake. Elevated serum bile acid concentrations might be due to a complicated alteration of bile acid metabolism. To date, no report has been published regarding alterations of bile acid metabolism after DJB.ObjectivesThis study was to investigate the mechanisms underlying the elevation of serum total and taurine-conjugated bile acids following DJB.MethodsHepatic samples reserved from previous study (Part II) were utilized by Western blot to analyze the expression of CYP7A1, CYP27A1 and CYP8B1, the rate-limiting enzymes in the synthesis of bile acids, as well as BACS and BAAT, the rate-limiting enzymes of bile acid conjugation. Intestinal and hepatic samples from previous study (Part Ⅱ) were utilized by RT-PCR to analyze mRNA levels of BSEP, NTCP, ASBT, ILBP, OSTa, FXR and SHP, the genes related to bile acid transport within the enterohepatic circulation.Results1. At week 12 postsurgery, the expression of CYP7A1 was lower after DJB than SHAM (P<0.05). The expression of CYP27A1 and CYP8B1 did not differ between DJB and SHAM groups (P>0.05).2. At week 12 postsurgery, the expression of BACS and BAAT in DJB group was enhanced compared to SHAM group (P<0.05).3. At week 12 postsurgery, bile acid transporters, ASBT, ILBP, and OSTa, showed increased expression in DJB group (P<0.05 each), which might indicate more active BA reabsorption in the ileum after DJB. The expression of NTCP and BSEP was not altered (P>0.05 both).4. At week 12 postsurgery, hepatic SHP and ileal FXR were increased with DJB group compared to SHAM group (P<0.05 both). No change was observed in heaptic FXR or ileal SHP (P>0.05 both).Conclusions1. CYP7A1 expression is repressed, while the mRNA levels of ASBT, ILBP and OSTa are increased after DJB, indicating decreased bile acid synthesis and increased bile acid absorption after DJB, which might account for the elevation of serum bile acids postsurgery.2. The expression of BACS and BAAT is enhanced following DJB, suggesting a more active bile acid conjugation process postsurgery.3. The mRNA levels of hepatic SHP are increased while the ileal FXR is repressed. The decreased synthesis of bile acids after DJB might be explained by negative feedback through hepatic "FXR-SHP" pathway.4. DJB has diverse effects on hepatic and ileal FXR, suggesting possible different compositions of bile acids within the liver and small intestine.