Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats

Abstract

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of β-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and β-cell lines with cholic acid decreased ER stress. These results suggest that postoperative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress.

Fig. 1.

IT surgery does not affect food intake or body weight, but increases energy expenditure. Energy intake (A), body weight (B), energy expenditure (C), energy expenditure AUC (D), total physical activity (E) and sum of total physical activity (F) in control (n=16), sham (n=16) and IT-operated (n=16) animals. *P<0.05 compared with control and sham by Student's t-test.

Fig. 2.

IT surgery improves glucose tolerance, islet function and nutrient-stimulated GLP-1 secretion. (A-C) Circulating glucose concentrations during OGTTs at 1 (A), 3 (B) and 4 (C) months after surgery. (D-F) Circulating insulin concentrations during OGTTs at 1 (D), 3 (E) and 4 (F) months after surgery. (G,H) Circulating GLP-1 concentrations during OGTTs at 1 (G) and 4 (H) months after surgery. (I) Circulating GIP concentrations during the OGTT at 3 months after surgery. (J,K) Circulating PYY (J) and TG concentrations (K) during the OFTT at 2 months after surgery. During the first month OGTT, n=28 per group. During the third and fourth month OGTTs and second month OFTT, n= 16 per group. **P<0.01, ***P<0.001 for IT compared with control and sham by Student's t-test of the AUC. *P<0.05 for IT compared with control by Student's t-test of the AUC. ⁺⁺P<0.01, ⁺⁺⁺P<0.001 for IT compared with control and sham by Student's t-test of the percentage change from fasting to peak insulin values.

Fig. 3.

IT surgery improves insulin signaling in liver, skeletal muscle, mesenteric adipose and pancreas. (A) Representative immunoblots for pAkt (Ser473), total Akt, pMAPK(Thr202/Tyr204), total MAPK, pJNK(Thr183/Tyr185) and total JNK in liver, adipose, skeletal muscle and pancreas at 1.5 and 4.5 months after surgery. (B-E) Results were quantified in densitromic units and expressed relative to the total protein of interest in liver (B), skeletal muscle (C), mesenteric adipose (D) and pancreas (E). *P<0.05, **P<0.01, ***P<0.001 for IT compared with control and sham by Student's t-test; n=12 per group at 1.5 months and n=16 per group at 4.5 months.

Fig. 4.

IT surgery preserves β-cell mass. (A-F) Representative images of pancreas sections immunostained for insulin from pre-diabetic control (A), sham (B) and IT-operated animals (C) at 1.5 months after surgery and control (D), sham (E) and IT-operated animals (F) at 4.5 months after surgery. (G) β-cell quantification in a subset of samples from control (n=6), sham (n=6) and IT-operated animals (n=6) at 1.5 months after surgery and control (n=6), sham (n=7) and IT-operated animals (n=8) at 4.5 months after surgery. *P<0.05, **P<0.01 for IT compared with control and sham by Student's t-test.

Fig. 5.

IT surgery preferentially increases nonconjugated primary bile acids. (A-C) Fasting plasma bile acid profiles in control (A), sham (B) and IT-operated animals (C) at 2 months after surgery. (D) Fasting plasma bile acid concentrations. (E) Fasting plasma non-conjugated bile acid, non-conjugated cholic acid, primary bile acid and secondary bile acid concentrations. (F) Hepatic bile acid profiles. (G) Bile acid profiles in cecal contents. (H) Fasting plasma glycine-conjugated bile acid concentrations. (I) Fasting plasma glycine-conjugated bile acid concentrations expressed as a percentage of conjugated bile acids. (J) Hepatic glycine-conjugated bile acid concentrations expressed as an absolute value and as a percentage of the total hepatic bile acid pool. (K) Hepatic mRNA expression of genes involved in bile acid metabolism and the FXR pathway relative to ARBP. All plasma values were measured at 2 months after surgery. Liver and cecal contents values were measured at 4.5 months after surgery; n=16 per group. *P<0.05, **P<0.01, ***P<0.001 for IT compared with control and sham, ⁺P<0.05 compared with sham by Student's t-test. C, cholate; CDC, chenodeoxycholate; DCA, deoxycholate; GC, glycocholate; GCDC, glycochenodeoxycholate; GDC, glycodeoxycholate; GHDC, glycohyodeoxycholate; HDC, hyodeoxycholate; T-a-MC, tauro-α-muricholate; T-b-MC, tauro-β-muricholate; TC, taurocholate; TCDC, taurochenodeoxycholate; TDC, taurodeoxycholate; THDC, taurohyodeoxycholate; TTHC, taurotetrahyodeoxycholate; TUDC, tauroursodeoxycholate.

Fig. 6.

IT surgery increases the relative expression of Gammaproteobacteria in cecal contents. (A) Average phylum-level composition of cecal contents. (B) Average class-level composition of cecal contents. (C) Average genus-level composition of cecal contents. *P<0.05 for IT compared with control and sham by Student's t-test; n=10-14 per group.

Fig. 7.

IT surgery decreases markers of ER stress in liver, adipose, muscle and pancreas. (A) Representative immunoblots for pPERK(Thr980) and total PERK, peIF-2α(Ser51) and total eIF-2α, pIRE1(Ser724) and total IRE1, sXBP1, BiP and tubulin in liver, skeletal muscle, mesenteric adipose and pancreas at 1.5 and 4.5 months after surgery. All blots were scanned and quantified using FluorChem 9900. (B-E) Results were quantified in densitromic units and expressed relative to the total protein of interest or relative to tubulin for BiP, ATF6 and sXBP1 in liver (B), skeletal muscle (C), mesenteric adipose (D) and pancreas (E). *P<0.05, **P<0.01, ***P<0.001 for IT compared with control and sham by Student's t-test; n=12 per group at 1.5 months and n=16 per group at 4.5 months.

Fig. 8.

Preincubation of hepatocytes, adipocytes and β-cells with cholate protects cells from the development of ER stress. Cells were incubated with 100 μM cholic acid or DMSO for 24 hours then exposed to 2 μM thapsigargin for 2 hours. (A) Representative immunoblots for pPERK(Thr980) and total PERK, peIF-2α(Ser51) and total eIF2α, pIRE1(Ser724) and total IRE1, sXBP1, pJNK(Thr183/Tyr185) and total JNK and BiP. Experiments were performed in HepG2, differentiated 3T3 L1 adipocytes and β-TC6 cells. (B-D) Control cells (Con) were not exposed to cholic acid or thapsigargin. Other cells were exposed to chloic acid but not to thapsigargin (CA), to thapsigargin but not cholic acid (Th), or preincubated with cholic acid and then exposed to thapsigargin (Th + CA). Results were quantified in densitromic units and expressed relative to the total protein of interest or relative to tubulin for BiP, ATF6 and sXBP1 in hepatocytes (B), adipocytes (C) and β-cells (D). *P<0.05, **P<0.01 for IT compared with Th by Student's t-test. ⁺P<0.05, ⁺⁺P<0.01 compared with Con and CA by Student's t-test; n=3 per group.