Colesevelam suppresses hepatic glycogenolysis by TGR5-mediated induction of GLP-1 action in DIO mice

Abstract

Bile acid sequestrants are nonabsorbable resins designed to treat hypercholesterolemia by preventing ileal uptake of bile acids, thus increasing catabolism of cholesterol into bile acids. However, sequestrants also improve hyperglycemia and hyperinsulinemia through less characterized metabolic and molecular mechanisms. Here, we demonstrate that the bile acid sequestrant, colesevelam, significantly reduced hepatic glucose production by suppressing hepatic glycogenolysis in diet-induced obese mice and that this was partially mediated by activation of the G protein-coupled bile acid receptor TGR5 and glucagon-like peptide-1 (GLP-1) release. A GLP-1 receptor antagonist blocked suppression of hepatic glycogenolysis and blunted but did not eliminate the effect of colesevelam on glycemia. The ability of colesevelam to induce GLP-1, lower glycemia, and spare hepatic glycogen content was compromised in mice lacking TGR5. In vitro assays revealed that bile acid activation of TGR5 initiates a prolonged cAMP signaling cascade and that this signaling was maintained even when the bile acid was complexed to colesevelam. Intestinal TGR5 was most abundantly expressed in the colon, and rectal administration of a colesevelam/bile acid complex was sufficient to induce portal GLP-1 concentration but did not activate the nuclear bile acid receptor farnesoid X receptor (FXR). The beneficial effects of colesevelam on cholesterol metabolism were mediated by FXR and were independent of TGR5/GLP-1. We conclude that colesevelam administration functions through a dual mechanism, which includes TGR5/GLP-1-dependent suppression of hepatic glycogenolysis and FXR-dependent cholesterol reduction.

Fig. 1.

Colesevelam modulates hepatic glucose and lipid metabolism. A–M: wild-type (WT) mice were fed a chow diet or a high-fat diet (HFD) for 15 wk and treated with or without colesevelam HCl (HFD + Col) for 7 days. A: body weight. B: body mass composition. C: food intake. D: hepatic cholesterol. E: plasma glucose. F: intraperitoneal glucose tolerance test (2 g/kg). G: plasma insulin. H: plasma glucagon. I: in vivo endogenous glucose production in awake and unrestrained HFD or HFD + Col mice following a brief 4-h morning fast was determined by GC-MS analysis of tracer dilution after steady-state infusion of [U-¹³C]glucose. J–L: sources of glucose production were determined by the deuterated water method and ²H nuclear magnetic resonance (NMR) in isolated perfused livers from fed WT DIO mice on a HFD for 15 wk treated with and without HFD + Col for 7 days. M: hepatic glycogen content in fed and 12-h fasted (9 PM to 9 AM) HFD and HFD + Col mice. Data are presented as means ± SE (n = 6–7). The presence of different lowercase letters indicates statistical significance (^aP < 0.05; ^bP < 0.01; ^cP < 0.005; and ^dP < 0.001 vs. control).

Fig. 2.

TGR5-mediated induction of glucagon-like peptide (GLP)-1 is required for Col-mediated suppression of hepatic glycogenolysis. A: GLP-1 measured in portal blood of fed WT DIO mice on a chow or HFD for 15 wk and treated with or without colesevelam HCl (HFD + Col) for 7 days (n = 6/group). B–F: WT DIO mice on HFD for 15 wk were administered either saline, the GLP-1R agonist exendin-4 (Ex-4), the GLP-1R antagonist exendin-(9–39) (Ex-9), or colesevelam (Col) with or without Ex-9 for 7 days (n = 5–6/group). B: livers from fed mice were perfused with media containing ²H₂O, and the deuterium enrichment of glucose was measured by NMR, where higher H5/H2 enrichment indicates less glycogenolysis. C: hepatic glycogenolysis. D: hepatic glycogen from livers in B (n = 5–6/group). Portal GLP-1 (E) and hepatic glycogen (F) levels analyzed from WT or TGR5 knockout (KO) mice fed 15 wk with chow or a HFD and then treated with or without colesevelam HCl (HFD + Col) for 7 days (n = 5–6/group). Data are presented as means ± SE. Lowercase letters indicate statistical significance (^aP < 0.05; ^bP < 0.01; ^cP < 0.005; and ^dP < 0.001 vs. control; n/s = not significant).

Fig. 3.

Bile acid-mediated activation of TGR5 initiates a novel cAMP signaling cascade. Real-time analysis of TGR5 activation (cAMP signaling) in TGR5-bioluminescence resonance energy transfer (BRET) cells after treatment with the indicated concentrations of bile acid (BA) (A) or isoproterenol (B). C: Real-time analysis of TGR5 activation (cAMP signaling) in TGR5-BRET cells after treatment with bile acid [3, 10, and 30 μM taurocholic acid (TCA)], Colesevelam (Col) alone, oleic acid alone, or bound Col/TCA equivalent to 55 μM (see materials and methods). Arrow indicates when factors were added to cells. GCA, glycocholic acid; TCDCA, taurine chenoxycholic acid; GCDCA, glycine chenoxycholic acid.

Fig. 4.

Col-mediated activation of TGR5 in the colon induces GLP-1. A: WT C57Bl/6 mice fed a chow diet or induced to obesity by a 60% HFD for 15 wk were supplemented with and without Colesevelam (Col) for 7 days. Tgr5 mRNA expression in ileum, mid-colon (M. Colon), and distal colon (D. Colon) was analyzed by quantitative real-time PCR (n = 6/group). B: portal GLP-1 levels in WT mice 40 min after rectal administration of vehicle, INT-777, TCA, Col alone, or Col preincubated with TCA (Col + TCA) (n = 4–5/group). C: ileum and distal colon (D. Colon) gene expression from mice in B. Data are presented as means ± SE. The presence of different lowercase letters indicates statistical significance relative to ileal gene expression from chow fed mice (^aP < 0.05; ^bP < 0.01; and ^dP < 0.001 vs. control; n.d. = not detected).

Fig. 5.

TGR5-mediated induction of GLP-1 contributes to Col-mediated suppression of hyperglycemia but not hyperinsulinemia in DIO mice. Plasma glucose (A) and insulin levels (B) from WT DIO mice on HFD for 15 wk were administered either saline, the GLP-1R agonist exendin-4 (Ex-4), the GLP-1R antagonist exendin-(9–39) (Ex-9), or colesevelam (Col) with or without Ex-9 for 7 days (n = 5–6/group). Plasma glucose (C) and (D) insulin levels from WT or TGR5 KO mice fed 15 wk a HFD and then treated with or without colesevelam HCl (HFD + Col) for 7 days (n = 5–6/group). Data are presented as means ± SE. Lowercase letters indicate statistical significance (^aP < 0.05; ^bP < 0.01; ^cP < 0.005; and ^dP < 0.001 vs. control; n/s = not significant).

Fig. 6.

Farnesoid X receptor (FXR), but not TGR5/GLP-1, is required for Col-mediated remediation of hepatic cholesterol levels. A: hepatic cholesterol levels from WT DIO mice on HFD for 15 wk were administered either saline, the GLP-1R agonist exendin-4 (Ex-4), the GLP-1R antagonist exendin-(9–39) (Ex-9), or colesevelam (Col) with or without Ex-9 for 7 days (n = 5–6/group). B: hepatic cholesterol levels from WT or TGR5 KO mice fed 15 wk a HFD and then treated with or without colesevelam HCl (HFD + Col) for 7 days (n = 5–6/group). Ileum (C) or hepatic gene (D) expression from WT chow, HFD, and HFD + Col fed mice. E: measurement of de novo cholesterol synthesis in HFD and HFD + Col mice. Plasma insulin (F), plasma glucose (G), and hepatic cholesterol (H) levels from WT or FXR KO mice fed HFD for 15 wk and then treated with or without colesevelam HCl (HFD + Col) for 7 days (n = 5–6/group). Data are presented as means ± SE. Lowercase letters indicate statistical significance (^aP < 0.05; ^bP < 0.01; ^cP < 0.005; and ^dP < 0.001 vs. control; n/s = not significant).