A new mechanism of thyroid hormone receptor β agonists ameliorating nonalcoholic steatohepatitis by inhibiting intestinal lipid absorption via remodeling bile acid profiles

Abstract

Excessive dietary calories lead to systemic metabolic disorders, disturb hepatic lipid metabolism, and aggravate nonalcoholic steatohepatitis (NASH). Bile acids (BAs) play key roles in regulating nutrition absorption and systemic energy homeostasis. Resmetirom is a selective thyroid hormone receptor β (THRβ) agonist and the first approved drug for NASH treatment. It is well known that the THRβ activation could promote intrahepatic lipid catabolism and improve mitochondrial function, however, its effects on intestinal lipid absorption and BA compositions remain unknown. In the present study, the choline-deficient, L-amino acid defined, high-fat diet (CDAHFD) and high-fat diet plus CCl₄ (HFD+CCl₄)-induced NASH mice were used to evaluate the effects of resmetirom on lipid and BA composition. We showed that resmetirom administration (10 mg·kg^-1·d^-1, i.g.) significantly altered hepatic lipid composition, especially reduced the C18:2 fatty acyl chain-containing triglyceride (TG) and phosphatidylcholine (PC) in the two NASH mouse models, suggesting that THRβ activation inhibited intestinal lipid absorption since C18:2 fatty acid could be obtained only from diet. Targeted analysis of BAs showed that resmetirom treatment markedly reduced the hepatic and intestinal 12-OH to non-12-OH BAs ratio by suppressing cytochrome P450 8B1 (CYP8B1) expression in both NASH mouse models. The direct inhibition by resmetirom on intestinal lipid absorption was further verified by the BODIPY gavage and the oral fat tolerance test. In addition, disturbance of the altered BA profiles by exogenous cholic acid (CA) supplementation abolished the inhibitory effects of resmetirom on intestinal lipid absorption in both normal and CDAHFD-fed mice, suggesting that resmetirom inhibited intestinal lipid absorption by reducing 12-OH BAs content. In conclusion, we discovered a novel mechanism of THRβ agonists on NASH treatment by inhibiting intestinal lipid absorption through remodeling BAs composition, which highlights the multiple regulation of THRβ activation on lipid metabolism and extends the current knowledge on the action mechanisms of THRβ agonists in NASH treatment.

Keywords: CYP8B1; THRβ agonists; bile acids composition; intestinal lipid absorption; nonalcoholic steatohepatitis; resmetirom.

Fig. 1. Resmetirom treatment ameliorated NASH phenotype in CDAHFD-fed mice.

a Schematic representation of the experimental design. b, c Body weight and average food intake. d–h Serum biochemistry. i Representative images of H&E staining (scale bar: 100 μm) and Sirius Red staining (scale bar: 1.5 mm). j NAFLD activity score. k Steatosis score. l Liver TG content. m Quantification of Sirius red staining. n, o Gene expression of α-SMA and Col1-α1. Data were shown as mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001 compared as indicated.

Fig. 2. Resmetirom treatment shaped hepatic lipid composition in CDAHFD-fed mice.

a Principal component analysis of hepatic lipidomic. b Lipid composition in vehicle-and resmetirom-treated mice. c Schematic representation of hepatic FFA metabolic pathway. d Heatmap of TG and PC species containing C18:2 fatty acyl chain in vehicle-and resmetirom-treated CDAHFD-fed mice. e Concentrations of TG (18:2_13:0_22:6), TG (15:0_18:2_20:5), TG (18:0_18:1_18:2), TG (18:2_17:1_18:2) in the liver. f Concentrations of PC (18:2_18:2), PC (18:2_22:1), PC (15:0_18:2), PC (18:2_22:6) in the liver. Data were shown as mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01 compared as indicated.

Fig. 3. Resmetirom treatment modulated hepatic and intestinal bile acid composition in CDAHFD-fed mice.

a Total bile acids concentration in liver. b Relative percentage and concentration of different bile acids in liver. c Concentration of 12-OH and non-12-OH bile acids in liver. d The ratio of 12-OH bile acids to non-12-OH bile acids in liver. e Mean percentage of 12-OH bile acids and non-12-OH bile acids in the liver among three groups. f Total bile acids concentration in intestine. g Relative percentage and concentration of different bile acids in intestine. h Concentration of 12-OH and non-12-OH bile acids in intestine. i The ratio of 12-OH bile acids to non-12-OH bile acids in intestine. j Mean percentage of 12-OH bile acids and non-12-OH bile acids in the intestine among three groups. Data were shown as mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001 compared as indicated.

Fig. 4. Resmetirom treatment altered gene expression involved in bile acid metabolism.

a Gene expression of hepatic bile acids synthesis in chow feeding mice, vehicle-treated and resmetirom-treated CDAHFD feeding mice. b Gene expression of hepatic bile acids conjugation in chow feeding mice, vehicle-treated and resmetirom-treated CDAHFD feeding mice. c Gene expression of hepatic bile acids export in chow feeding mice, vehicle-treated and resmetirom-treated CDAHFD feeding mice. d Gene expression of hepatic bile acids reabsorption in chow feeding mice, vehicle-treated and resmetirom-treated CDAHFD feeding mice. e Gene expression of ileum bile acids transport in chow feeding mice, vehicle-treated and resmetirom-treated CDAHFD feeding mice. Data were shown as mean ± SEM (n = 5–8). *P < 0.05, **P < 0.01 compared as indicated.

Fig. 5. Resmetirom treatment alleviated NASH phenotype and modulated bile acid metabolism in HFD + CCl₄ induced NASH model.

a Schematic representation of the experimental design. b Body weight and average food intake. c Representative images of H&E staining (scale bar: 100 μm) and Sirius Red staining (scale bar: 1.5 mm). d NAFLD activity score and steatosis score. e Liver TG content. f Quantification of Sirius red staining. g Gene expression of α-SMA and Col1-α1. h Heatmap of TG and PC species containing C18:2 fatty acyl chain in vehicle-and resmetirom-treated HFD+CCl₄-treated mice. i Concentration of 12-OH and non-12-OH bile acids in liver. j Mean percentage of 12-OH bile acids and non-12-OH bile acids in the liver among three groups. k Hepatic gene expression of key enzymes involved in bile acid synthesis. Data were shown as mean ± SEM (n = 7–8). *P < 0.05, **P < 0.01, ***P < 0.001 compared as indicated.

Fig. 6. Resmetirom treatment reduced intestinal lipid absorption.

a Representative images of fluorescence from intestinal sections of mice treated with vehicle and resmetirom (scale bar: 100 μm). Intestinal (b), serum (c), and fecal (d) BODIPY concentration in vehicle and resmetirom-treated mice after gavage with BODIPY fatty acid analog. Plasma TG after gavage with olive oil and intraperitoneal injection of tyloxapol (lipid absorption) (e) or intraperitoneal injection of tyloxapol alone (VLDL secretion) (f) in vehicle and resmetirom-treated mice. g Hepatic gene expression involved in bile acids synthesis of vehicle and resmetirom-treated mice. h Different bile acids concentrations in the liver of vehicle and resmetirom-treated mice. i The concentration of 12-OH bile acids in the liver. j Mean percentage of 12-OH bile acids and non-12-OH bile acids in the liver in vehicle and resmetirom-treated mice. k Different bile acids concentrations in the intestinal of vehicle and resmetirom-treated mice. l The concentration of 12-OH bile acids in the intestine. m Mean percentage of 12-OH bile acids and non-12-OH bile acids in the intestine in vehicle and resmetirom-treated mice. Data were shown as mean ± SEM (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 compared as indicated.

Fig. 7. Exogenous CA supplementation abolished the inhibitory effects of resmetirom on intestinal lipid absorption.

a Schematic representation of the CA intervention in normal mice. b Representative images of fluorescence from intestinal sections of normal mice treated with vehicle and resmetirom (scale bar: 100 μm). c Intestinal, serum and fecal BODIPY concentration in vehicle and Resmetirom-treated normal mice after gavage with BODIPY fatty acid analog. d Plasma TG after gavage with olive oil and intraperitoneal injection of tyloxapol (lipid absorption) in vehicle, CA, resmetirom and CA plus resmetirom-treated normal mice. e Schematic representation of the CA intervention in CDAHFD-fed mice. f Representative images of fluorescence from intestinal sections of CDAHFD-fed mice treated with vehicle and resmetirom (scale bar: 100 μm). g Serum, intestinal, and fecal BODIPY concentration in vehicle and resmetirom-treated CDAHFD-fed mice after gavage with BODIPY fatty acid analog. h Plasma TG after gavage with olive oil and intraperitoneal injection of tyloxapol (lipid absorption) in vehicle, CA, resmetirom and CA plus resmetirom-treated CDAHFD-fed mice. Data were shown as mean ± SEM (n = 6). **P < 0.01, ***P < 0.001 compared as indicated.

Fig. 8. T3 treatment reduced intestinal lipid absorption.

a Representative images of fluorescence from intestinal sections of mice treated with vehicle and T3 (scale bar: 100 μm). Intestinal (b), serum (c), and fecal (d) BODIPY concentration in vehicle and T3-treated mice after gavage with BODIPY fatty acid analog. e, f Plasma TG after gavage with olive oil and intraperitoneal injection of tyloxapol (lipid absorption) or intraperitoneal injection of tyloxapol alone (VLDL secretion) in vehicle and T3-treated mice. g Hepatic gene expression involved in bile acid synthesis. Data were shown as mean ± SEM (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 compared as indicated.

Fig. 9. A new mechanism of THRβ agonists ameliorating NASH by inhibiting intestinal lipid absorption via remodeling bile acid profiles.

THRβ agonists suppressed hepatic CYP8B1 expression, thereby leading to reduced production of 12-OH bile acids, and subsequently decreased intestinal lipid absorption, which may contribute to the effects of THRβ activation on NASH improvement.