Role of Bile Acids and GLP-1 in Mediating the Metabolic Improvements of Bariatric Surgery

Abstract

Background & aims: Bile diversion to the ileum (GB-IL) has strikingly similar metabolic and satiating effects to Roux-en-Y gastric bypass (RYGB) in rodent obesity models. The metabolic benefits of these procedures are thought to be mediated by increased bile acids, although parallel changes in body weight and other confounding variables limit this interpretation.

Methods: Global G protein-coupled bile acid receptor-1 null (Tgr5^-/-) and intestinal-specific farnesoid X receptor null (Fxr^Δ/E) mice on high-fat diet as well as wild-type C57BL/6 and glucagon-like polypeptide 1 receptor deficient (Glp-1r^-/-) mice on chow diet were characterized following GB-IL.

Results: GB-IL induced weight loss and improved oral glucose tolerance in Tgr5^-/-, but not Fxr^Δ/E mice fed a high-fat diet, suggesting a role for intestinal Fxr. GB-IL in wild-type, chow-fed mice prompted weight-independent improvements in glycemia and glucose tolerance secondary to augmented insulin responsiveness. Improvements were concomitant with increased levels of lymphatic GLP-1 in the fasted state and increased levels of intestinal Akkermansia muciniphila. Improvements in fasting glycemia after GB-IL were mitigated with exendin-9, a GLP-1 receptor antagonist, or cholestyramine, a bile acid sequestrant. The glucoregulatory effects of GB-IL were lost in whole-body Glp-1r^-/- mice.

Conclusions: Bile diversion to the ileum improves glucose homeostasis via an intestinal Fxr-Glp-1 axis. Altered intestinal bile acid availability, independent of weight loss, and intestinal Akkermansia muciniphila appear to mediate the metabolic changes observed after bariatric surgery and might be manipulated for treatment of obesity and diabetes.

Keywords: Glucagon-Like Polypeptide 1 (Glp-1); Gut Microbiome; Lymph Fistula; Metabolic Surgery.

Figure 1.. Effects of bile diversion to the ileum in HFD-fed, obese global Tgr5^−/− and intestinal Fxr deficient (Fxr^Δ/E) mice.

(A) Surgical schematic of bile diversion operations. Mice underwent surgical bile diversion to the ileum (GB-IL) or no operation (Chow). Average body weight (B, E), interval body composition assessments (C, F), daily food intake (D, G), oral glucose excursions (H and J) and glucose area under the curve (AUC; I and K) in high-fat diet- (HFD-) fed obese Tgr5^−/− and Fxr^Δ/E mice after bile diversion to the ileum (GB-IL). (Tgr5^−/−: n=14 Con HFD, 5 GB-IL; Fxr^Δ/E n=5 Con HFD, 7 GB-IL). Data are represented as the mean ± SEM. Statistical analysis using Student’s t-test. ***P<0.001; ****P<0.0001.

Figure 2.. Whole body and tissue specific responses to chronic bile diversion in lean chow-fed mice.

C57BL/6J mice underwent surgical bile diversion to the ileum (GB-IL), or no operation (Chow). (A) Body weight, (B) average daily and (C) cumulative food intake were measured for the entirety of the study (only every other day are shown in the graphs above for clarity). (D) Non-invasive body composition was measured at 2, 4, 6 and 8 weeks. (E) Fecal lipids were quantified from total feces over a 6-hour time period 4 weeks postop. (F) Total plasma bile acids (BAs). (G) The 12α/non-12α-hydroxylated bile acid ratio. Data are represented as the mean ± SEM. Kruskal-Wallis test of Chow, GB- D (data not shown), GB-IL, adjusted for multiplicity, was used on sample sizes of (A-C) n=10 GB-IL, 12 Chow; (D, F and G) 9 GB-IL, 12 Chow, (E) 10 GB-IL, 12 Chow. **P<0.01.

Figure 3.. Glucose metabolism in lean, chronic bile diversion mice.

Chronic bile diversion mice underwent clamp studies at 4 weeks postoperative for whole body and tissue-specific assessment of insulin sensitivity and glucose kinetics. (A) Blood glucose was clamped at 5h fasted levels, with a (B) constant insulin infusion to elevate plasma insulin concentrations over food-restricted levels and a (C) variable glucose infusion to maintain euglycemia among the groups. (D) Chow and GB-IL mice also underwent standard oral glucose tolerance tests (OGTT; 2 mg/kg body weigh) at 4 weeks postoperatively; and (E) the corresponding area under curve (AUC_0–120) glucose was calculated. (F) Ratios of insulin to glucose at baseline (t = 0 min) and following glucose gavage (t = 20 min) were assessed. (G) Regression lines obtained when comparing the glucose and insulin concentrations at basal fasting (shaded gray) and 20 min after glucose stimulation during an OGTT. Slopes among all lines are not significantly different, while the intercept for GB-IL compared to Chow is higher (P<0.01). (H) 3-[³H]-O-methylglucose (3-OMG) counts in peripheral circulation after administering glucose supplemented with 3-OMG tracer during OGTT. (I) Liver glycogen in 5h fasted GB-IL and Chow controls. Data are represented as the mean ± SEM. Kruskal-Wallis test of Chow, GB-D (data not shown), GB-IL, adjusted for multiplicity, was used on sample sizes of (A-C) 6 GB-IL, 8 Chow. (D-E) 13 GB-IL, 16 Chow; (F, G) 7 GB-IL, 14 Chow; (H) 6 GB-IL, 7 Chow; (I) 9 GB-IL, 12 Chow. *P<0.05, **P<0.01.

Figure 4.. Improvement in oral glucose tolerance after bile diversion to the ileum enhance lymphatic GLP-1 tone.

Bile diversion or control mice underwent mesenteric lymphatic cannulation at four weeks postop. Intestinal lymph samples were collected hourly before and after a nutrient bolus delivered at the indicated time as described in the ‘Methods’. Lymph concentrations of (A) GLP-1 and (D) GIP from the mesenteric lymph over time, as well as (B, E) basal (t = 0) and nutrient stimulated (t = 1h) and (C, F) 0 to 1 h changes are shown above. Bile diversion mice underwent hyperglycemic clamp studies at 4 weeks postoperative for assessments of glucose-stimulated insulin secretion independent of the gastrointestinal tract. (G) Blood glucose was clamped at 250 mg/dl in 5 h fasted mice, with a (H) variable glucose infusion to assess (I) insulin release. Data are represented as the mean ± SEM. Kruskal-Wallis test of Chow, GB-D (data not shown), GB-IL, adjusted for multiplicity, was used on sample sizes of (A-F) 7 GB-IL, 8 Chow; (G-I) 6 GB-IL, 4 Chow. **P<0.01.

Figure 5.. Pharmacologic and genetic blockade of GLP-1R blunt the glucoregulatory effects of elevated bile acids after GB-IL.

Bile diversion to the ileum (GB-IL) and Chow mice underwent oral glucose tolerance testing. (A) Plasma glucose during OGTT after 50 µg exendin-9 (Ex-9) was administered by intraperitoneal injection into 5h fasted mice. (B) Glucose AUC_0–120 in mice with and without Ex-9 pretreatment. Post-prandial blood glucose excursion (C) and glucose AUC_0–120 (D) in GB-IL mice after pre-treatment with the bile acid sequestrant, cholestyramine (500 mg/kg p.o., daily for 3 days). Serum bile acid profiles in GB-IL and Chow mice after cholestyramine treatment (E). Serum bile acid profiles in GB-IL mice with and without cholestyramine treatment (F). Effects of GB-IL in mice lacking the Glp-1 receptor (Glp-1R^−/−). Average body weight (G), and (H) oral glucose excursions in low-fat diet-(LFD-) fed, lean Glp-1R^−/− mice after GB-IL. (Glp-1R^−/−; n=7 Chow, 8 GB-IL). Data are presented the mean ± SEM. Statistical analysis using Student’s t-test. *P<0.05, **P<0.01.

Figure 6.. Bile diversion to the ileum is associated with increased cecal Akkermansia muciniphila content.

(A) LeFSe cladrogram illustrating the taxa with the greatest differences in abundance among GB-IL and Chow mice. Control-enriched taxa (green) and GB-IL enriched taxa (magenta) are indicated by color. Various shades of background highlighting indicate changes at several taxonomic levels. Individual colored circles represent single OTUs and the size of each circle is proportional to the abundance of that given OTU as implemented in the LeFSe software. (B) Stacked bar graph illustrating relative OTU abundance by intervention of the thirty most highly abundant taxa among the groups (6 GB-IL, 6 Chow). Bar graphs illustrating OTU abundance differences, at the family level, in fecal (C) Verrucomibociaceae, of which Akkermansia muciniphila is the only member, (D) Lactobacillaceae, (E) Clostridiales, (F) Streptococcaceae, (G) Ruminococcaceae and (H) Lachnospiraceae (Roseburia sp.).