A surgical model in male obese rats uncovers protective effects of bile acids post-bariatric surgery

Abstract

Bariatric surgery elevates serum bile acids. Conjugated bile acid administration, such as tauroursodeoxycholic acid (TUDCA), improves insulin sensitivity, whereas short-circuiting bile acid circulation through ileal interposition surgery in rats raises TUDCA levels. We hypothesized that bariatric surgery outcomes could be recapitulated by short circuiting the normal enterohepatic bile circulation. We established a model wherein male obese rats underwent either bile diversion (BD) or Sham (SH) surgery. The BD group had a catheter inserted into the common bile duct and its distal end anchored into the middistal jejunum for 4-5 weeks. Glucose tolerance, insulin and glucagon-like peptide-1 (GLP-1) response, hepatic steatosis, and endoplasmic reticulum (ER) stress were measured. Rats post-BD lost significantly more weight than the SH rats. BD rats gained less fat mass after surgery. BD rats had improved glucose tolerance, increased higher postprandial glucagon-like peptide-1 response and serum bile acids but less liver steatosis. Serum bile acid levels including TUDCA concentrations were higher in BD compared to SH pair-fed rats. Fecal bile acid levels were not different. Liver ER stress (C/EBP homologous protein mRNA and pJNK protein) was decreased in BD rats. Bile acid gavage (TUDCA/ursodeoxycholic acid [UDCA]) in diet-induced obese rats, elevated serum TUDCA and concomitantly reduced hepatic steatosis and ER stress (C/EBP homologous protein mRNA). These data demonstrate the ability of alterations in bile acids to recapitulate important metabolic improvements seen after bariatric surgery. Further, our work establishes a model for focused study of bile acids in the context of bariatric surgery that may lead to the identification of therapeutics for metabolic disease.

Figure 1.

A, Illustration of BD. Black arrow points to polyethylene catheter. B, Body weight after surgery. Rats in BD had catheter inserted 15 cm distal to ligament of Treitz. This group lost more body weight compared with rats in the SH and Naïve groups. N: BD = 12; SH = 8; Naive = 5 (repeated measures ANOVA: *, P < .05 BD vs SH and Naïve). C, Fasting insulin at 4 weeks. Plasma insulin levels were lower in the BD surgery group compared with rats in the SH surgery and surgery Naïve groups. N: BD = 12; SH = 8; Naive = 5 (ANOVA, post hoc Tukey's; *, P < .05 SH vs BD; **, P < .01 Naïve vs BD). D, Body weight after surgery with different catheter insertion sites. Rats had either SH surgery, BD catheter inserted into the jejunum 15 cm from the ligament of Treitz (BD-15) or 30 cm from the ligament of Treitz surgery (BD-30). The BD-15 and BD-30 groups lost more body weight compared with rats in the SH surgery group. N: SH = 5, BD-15 = 8, BD-30 = 9 (repeated measures ANOVA: *, P < .05 BD-15 and BD-30 vs SH). E, Body composition estimation of fat mass change by magnetic resonance. The weight gained by the SH group had an increase in their proportion of fat mass whereas BD-15 and BD-30 surgery groups lost fat mass over the same time period. N: SH = 5; BD-15 = 8; BD-30 = 9 (]ANOVA, post hoc Tukey's: **, P < .01 for BD-15 and BD-30 vs SH). F, Serum bile acid levels after surgery. Fasting total serum bile acid levels were higher in BD-15 and BD-30 rats compared with SH group. N: SH = 5; BD-15 = 7; BD-30 = 8 (ANOVA, post hoc Tukey's *, P < .05 for BD-15 and BD-30 vs SH). G, Plasma GLP-1 levels after surgery. Postprandial plasma GLP-1 levels (15 min) were higher in the BD-15 and BD-30 rats compared with SH group. N: SH = 5; BD-15 = 7; BD-30 = 8 (ANOVA, post hoc Tukey's: *, P < .05 for BD-15 and BD-30 vs SH).

Figure 2.

A, Body weight change after surgery. Rats in BD had catheter inserted 15 cm distal to ligament of Treitz. Rats in the BD surgery group lost more body weight compared with rats in the SH-PF surgery and Naïve HF surgery Naïve groups. At time of death there was no difference in body weight between Naïve-Chow-fed and BD rats. N: Naïve-HF = 8; SH = 9; SH-PF = 10; BD = 12; Naïve-Chow = 8. B, Food intake after surgery. There was no significant difference between cumulative food intake after surgery from days 14–35 between the 3 ad libitum HF-fed groups; Naïve-HF, SH, and BD groups. N for groups: Naïve-HF = 8; SH = 9; BD = 12. C, Body composition estimation of lean mass change by magnetic resonance. BD and Naïve Chow-fed rats had significantly higher gain in lean mass as a percentage of weight gained after surgery compared with Naïve-HF-fed and SH-PF rats. N for groups: Naïve-HF = 8; SH = 9; SH-PF = 10; BD = 11; Naïve-Chow = 8 (ANOVA, post hoc Tukey's: ***, P < .001). D, Body composition estimation of fat mass change by magnetic resonance. BD and Naïve-Chow-fed rats had significantly lower gain in fat mass as a percentage of weight gained after surgery compared with Naïve-HF-fed and SH-PF rats. N for groups: Naïve-HF = 8; SH = 9; HF-PF = 10; BD = 11; Naïve-Chow = 8 (ANOVA, post hoc Tukey's: ***, P < .001). E, Glucose tolerance after BD. BD rats had an improvement in their oral glucose tolerance compared with Naïve-HF and SH-PF rats and F, glucose tolerance test (GTT) AUC. AUC for 120 minutes was significantly less after BD surgery. N for groups: Naïve-HF = 8; SH = 9; SH-PF = 9; BD = 12; Naïve-Chow = 8 (ANOVA, post hoc Tukey's: *, P < .05; **, P < .01).

Figure 3.

A, Negative ion ESI-Liquid chromatography-mass spectroscopy summed ion chromatograms. Mass size 514, 498, 448, 464, 407, 391 specific to the major conjugated bile acids in extracts of bile of rats after BD (top chromatogram) or SH operated (bottom chromatogram). The ion chromatogram for m/z 498 is separately depicted to indicate the presence of taurine-conjugated dihydroxy-cholanoic acids, including TUDCA. The following peaks are indicated: taurocholate (TCA), taurodeoxycholate (TDCA), tauro-β-muricholic (TβMCA), tauro-chenodeoxycholate (TCDCA), glycocholate (GCA), glycoursodeoxycholate (GUDCA), uncnjugtaed hyodeoxycholate (HDCA), unconjugated UDCA, and unconjugated deoxycholate (DCA). These chromatograms are normalized to the most intense ion in the profile and note the difference (27-fold) in relative intensity of the ions between the BD and SH-operated bile samples. B, Serum bile acid levels after surgery. Postprandial total serum bile acid levels were higher in BD rats compared with all other groups including weight-matched Chow Naïve and food intake-matched SH-PF. N for groups: Naïve-HF = 8; SH = 9; SH-PF = 10; BD = 11; Naïve-Chow = 7 (ANOVA, post hoc Tukey's: *, P < .05). C, Serum bile acid composition analysis. Bile acid composition analyzed by liquid chromatography-mass spectroscopy in serum is shown with a significant proportion being conjugated (glycine or taurine) bile acids. There is a significant increase seen in the taurine-conjugated bile acids from a mean of 75% to that of 82% between these 2 rat groups. The following unconjugated bile acids are also indicated: cholic acid (CA), deoxycholate (DCA), α-muricholic acid (α-MCA) β-muricholic acid (β-MCA), chenodeoxycholate (CDCA), and UDCA. D, Taurine and unconjugated UDCA serum levels after surgery. Postprandial serum TUDCA levels were higher in BD rats compared with all other groups including weight-matched Chow Naïve and food-intake-matched SH-PF whereas no difference was observed in serum UDCA (E) levels between groups. N for groups: Naïve-HF = 8; SH = 9; SH-PF = 10; BD = 11; Naïve-Chow = 7 (ANOVA, post hoc Tukey's: *, P < .05].

Figure 4.

A, Ileal histology after surgery. Rats humanely destroyed 5 weeks after surgery from the BD group showed histologic adaptation of the ileum. Hematoxylin and eosin-stained sections) (magnification, times]10)of the BD rat's ileum at time of death shows marked increase in length and number of villi compared with the SH-PF (B) animals ileal histology, which has remained typical of a rat ileal with short villi length. C, Response of ileal epithelial bile acid-responsive gene FXR. mRNA levels of bile acid-responsive nuclear receptor gene FXR were measured by RT-PCR and expressed in relative expression units. FXR mRNA was decreased in BD rats compared with SH-PF. N for groups: BD = 7; SH-PF = 5; Chow Naive = 5 (ANOVA, Post hoc Tukey's: ***, P < .001). D, Fecal bile acid quantification. Using a 3-day fecal collection, total fecal bile acid levels were measured and found to be similar in BD rats compared with all other groups including weight-matched Chow Naïve and food-intake-matched SH-PF. E, Hepatic bile acid production. mRNA levels of the gene coding for the rate-limiting bile acid production enzyme 7α-hydroxylase (CYP7A1) were measured by RT-PCR and expressed in relative expression units. CYP7A1 mRNA was decreased in BD rats compared with SH-PF. N for groups: BD = 6; SH-PF = 4; Naïve-Chow = 6 (ANOVA, post hoc Tukey's: *, P < .05; **, P < .01). F, Hepatic bile acid export. mRNA levels of the gene coding for the major BSEP and OATPs were measured by RT-PCR and expressed in relative expression units. BSEP and OATPs mRNA were decreased in BD rats compared with SH-PF. BD = 6, SH-PF = 4, Naïve-Chow = 6 (ANOVA, post hoc Tukey's: **, P < .01; ***, P < .001).

Figure 5.

A, Hepatic histology postsurgery. Rats humanely destroyed at 5 weeks after surgery from the BD group showed decreased steatosis by oil-red O staining on frozen liver sections. Sections (×10) of the BD rat's liver at time of death show marked decrease in number and size of steatosis compared with the SH-PF (B) animals, which has remained typical of a HF-fed obese rat. C, Hepatic TG content. Liver TG levels were lower in BD rats compared with SH-PF and Naïve-HF groups. N for groups: BD = 9; SH-PF = 6; Chow Naive = 8 (ANOVA, Post hoc Tukey's: **, P < .01; ***, P < .001). D, Hepatic ER Stress gene expression. mRNA levels of the gene coding for the ER stress component CHOP were measured by RT-PCR and expressed in relative expression units. CHOP mRNA was decreased in BD rats compared witho SH-PF. N for groups: BD = 9; SH-PF = 6; Chow Naive = 8 (ANOVA, post hoc Tukey's: *, P < .05). E, Hepatic ER Stress-protein expression. pJNK2 by Western blot was decreased in BD liver tissue compared with SH-PF and Naïve-HF. Equal protein loading confirmed by β-actin levels. Representative blots shown. Representative blots shown. Quantification done by densitometry ratios of pJNK to total JNK. N for groups: BD = 9; SH-PF = 6; Chow Naive = 8 (ANOVA, post hoc Tukey's: *, P < .05).

Figure 6.

A, Serum bile acid levels after gavage for 3 weeks. Postprandial total serum bile acid levels were higher in UDCA and TUDCA gavage rats compared with saline gavage rats. N for groups: saline = 4; UDCA = 5; TUDCA = 5 (ANOVA, post hoc Tukey's: *, P < .05). B, Serum bile acid composition analysis. Serum bile acid composition analyzed by liquid chromatography-mass spectroscopy shows increased level of TUDCA in both UDCA- and TUDCA-gavaged rats after 3 weeks. N for groups: Saline = 4; UDCA = 5; TUDCA = 5 (ANOVA, post hoc Tukey's: *, P < .05). C, Hepatic histology after gavage for 3 weeks. Diet-induced obese rats gavaged for 3 weekd showed decreased steatosis by oil-red O staining on frozen liver sections for TUDCA- and UDCA-gavaged rats. Sections (×10)of the saline gavaged rat's liver at time of death show marked steatosis. D, Hepatic TG content. Liver TG levels were lower in UDCA- and TUDCA-gavaged rats compared with saline-gavaged group. N for groups: saline = 4; UDCA = 5; TUDCA = 5 (ANOVA, post hoc Tukey's: *, P < .05). E, Postgavage hepatic ER stress-gene expression. mRNA levels of the gene coding for the ER stress component CHOP were measured by RT-PCR and expressed in relative expression units. CHOP mRNA was decreased in UDCA- and TUDCA-gavaged rats compared with saline-gavaged group. N for groups: saline = 4; UDCA = 5; TUDCA = 5 (ANOVA, post hoc Tukey's: *, P < .05).