FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption

Abstract

FXR agonists are used to treat non-alcoholic fatty liver disease (NAFLD), in part because they reduce hepatic lipids. Here, we show that FXR activation with the FXR agonist GSK2324 controls hepatic lipids via reduced absorption and selective decreases in fatty acid synthesis. Using comprehensive lipidomic analyses, we show that FXR activation in mice or humans specifically reduces hepatic levels of mono- and polyunsaturated fatty acids (MUFA and PUFA). Decreases in MUFA are due to FXR-dependent repression of Scd1, Dgat2, and Lpin1 expression, which is independent of SHP and SREBP1c. FXR-dependent decreases in PUFAs are mediated by decreases in lipid absorption. Replenishing bile acids in the diet prevented decreased lipid absorption in GSK2324-treated mice, suggesting that FXR reduces absorption via decreased bile acids. We used tissue-specific FXR KO mice to show that hepatic FXR controls lipogenic genes, whereas intestinal FXR controls lipid absorption. Together, our studies establish two distinct pathways by which FXR regulates hepatic lipids.

Keywords: FXR; NAFLD; bile acids; intestinal lipid absorption.

Figure 1.. Humans and mice treated with FXR agonists have selective reductions in hepatic TAG containing MUFAs and PUFAs

(A) Experiment schematic of double-blinded placebo-controlled randomized trial for obeticholic acid (OCA) in human patients. (B and C) Lipidomic analyses of (B) hepatic lipid classes (nmol/mg liver) and (C) hepatic TAG species (nmol/mg liver) in human patients treated as in (A). (D) Experiment schematic: wild-type or Fxr^−/− mice (n = 11–12/group) fed a standard rodent diet were treated for 3 days with vehicle or FXR agonist GSK2324. (E) Hepatic mRNA expression of FXR target genes Shp and MafG in mice from (D). (F) Total hepatic TAG in mice from D. (G and H) Lipidomic analyses of (G) hepatic lipid classes (nmol/mg liver) and (H) hepatic TAG species (nmol/mg liver) in mice from (D). (I and J) Bar graphs of individual TAG species containing (I) monounsaturated and (J) polyunsaturated fatty acids in livers of mice from (D). Data are represented as mean ± SEM with individual animals noted as dots. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1.

Figure 2.. Treatment with GSK2324 selectively reduces the expression of fatty acid and triglyceride synthesis genes

(A) Schematic of the hepatic de novo lipogenesis pathway, where genes are represented in yellow and metabolites in green. (B–H) Hepatic mRNA expression for 24 genes encoding major enzymes in hepatic lipogenesis from livers of wild-type or Fxr^−/− mice fed a standard rodent diet and treated with vehicle or GSK2324 with most significantly changing genes denoted with red asterisks in (A). (I) Experimental schematic outlining de novo lipogenesis measurements using deuterium oxide (D₂O) in mice treated for 3 days with FXR agonist GSK2324 (n = 8/group). (J) Fatty acid methyl ester analysis for newly synthesized neutral lipids in livers of mice from (I). Data are represented as mean ± SEM with individual animals noted as dots. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2.

Figure 3.. FXR activation reduces hepatic TAG levels independently of SHP and SREBP1c

(A and H) Experiment schematics: wild-type mice and (A) Shp^−/− or (H) Srebp1c^−/− mice (n = 9–12/group) fed a standard rodent diet were treated for 3 days with vehicle or FXR agonist GSK2324. (B and I) Hepatic mRNA expression of FXR target genes Shp and MafG in wild-type and (B) Shp^−/− or (H) Srebp1c^−/− mice. (C and J) Hepatic mRNA expression of select lipogenic genes in wild-type and (C) Shp^−/− or (J) Srebp1c^−/− mice. (D and K) Hepatic lipid classes (nmol/mg liver) in wild-type and (D) Shp^−/− or (K) Srebp1c^−/− mice. (E and L) Hepatic TAG species (nmol/mg liver) in wild-type and (E) Shp^−/− or (L) Srebp1c^−/− mice. (F, G, M, and N) Individual TAG species containing MUFA and PUFA acyl tails in wild-type and (F and G) Shp^−/− or (M and N) Srebp1c^−/− mice. Data are represented as mean ± SEM with individual animals noted as dots. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.

Figure 4.. FXR activation decreases intestinal lipid absorption

(A) Total hepatic C18:2 in wild-type mice treated with vehicle or GSK2324. (B, E, and H) Experiment schematics for three different lipid absorption methods. Wild-type mice fed a standard rodent diet were treated with either vehicle or GSK2324 and (B) gavaged with ¹⁴C triolein, (E) gavaged with fatty acid conjugated to BODIPY fluorophore, or (H) fed a diet containing non-absorbable lipid standard sucrose polybehenate (C22:0, n = 10/group). (C and D) Total plasma TAG (C) and plasma radioactive counts (D) from ¹⁴C triolein from mice as in (B). (F and G) (F) Representative images and (G) fluorescence quantification from intestinal sections of mice treated as in (E). (I) Fatty acid methyl ester analysis of fecal lipids normalized to C22:0 from mice treated as in (H). Data are represented as mean ± SEM with individual animals noted as dots. **p < 0.01, ***p < 0.001. See also Figure S4.

Figure 5.. Decreased intestinal lipid absorption requires FXR-dependent changes in bile acids

(A) Experiment schematic: wild-type or Fxr^−/− mice (n = 11–12/group) fed a standard rodent diet were treated for 3 days with vehicle or FXR agonist GSK2324. (B) Fecal fatty acids in mice from (A). (C and D) Total biliary bile acid concentration (C) and biliary bile acid composition (D) in mice from (A). (E) Experiment schematic: wild-type mice fed either a standard rodent diet or a diet supplemented with cholic acid were treated with vehicle or GSK2324 for3 days (n = 10/group). (F) Hepatic mRNA expression of Shp and MafG in mice from (E). (G–J) Total biliary bile acid concentration (G) and biliary bile acid composition (H) in mice from (E). Lipidomic analyses of (I) total hepatic TAG and (J) individual hepatic TAG species (nmol/mg liver) in mice from (E). (K) Fecal fatty acids in mice from (E). Data are represented as mean ± SEM with individual animals noted as dots. *p < 0.05, ***p < 0.001. See also Figure S5.

Figure 6.. In an NAFLD model, FXR activation dramatically decreases hepatic TAG and intestinal lipid absorption

(A) Experiment schematic of diet-induced NAFLD model. Wild-type or Fxr^−/− mice fed WD for 8 weeks were treated for 3 days with vehicle or FXR agonist GSK2324 (n = 8–10/group). (B) Hepatic mRNA expression of FXR target genes Shp and MafG in mice from (A). (C and D) Lipidomic analyses of (C) hepatic lipid classes (nmol/mg liver) and (D) hepatic TAG species (nmol/mg liver) in mice from (A). (E) Total bile acid pool amount from mice in (A). (F and J) Experiment schematic for absorption methods. WD-fed wild-type mice were treated with either vehicle or GSK2324 and (F) gavaged with ¹⁴C triolein or with fatty acid conjugated to BODIPY fluorophore, or (J) fed a diet containing non-absorbable lipid standard sucrose polybehenate (C22:0, n = 10/group). (G and H) Representative images (G) and fluorescence quantification (H) from intestinal sections of BODIPY-gavaged mice treated as in (F). (I) Liver radioactive counts from ¹⁴C triolein-gavaged mice as in (F). (K) Fecal fatty acids after treatment with either vehicle or GSK2324 in mice as in (J). Data are represented as mean ± SEM with individual animals noted as dots. **p < 0.01, ***p < 0.001. See also Figure S6.

Figure 7.. Intestinal FXR mediates changes in lipid absorption, whereas hepatic FXR controls lipid synthesis

(A, C, and E) Experiment schematics: wild-type or (A) Shp^−/− mice, (C) Srebp1c^−/− mice, or (E) Fxr^fl/fl, Fxr^Liv-KO, and Fxr^Int-KO mice were fed a Western diet for 8 weeks and treated with either vehicle or GSK2324. (B and D) Lipidomic analysis of hepatic lipid classes in WD-fed (B) Shp^−/− mice and (D) Srebp1c^−/− mice. (F) Hepatic mRNA expression of FXR target gene Shp in mice from (E). (G) Ileal mRNA expression of FXR target gene FGF15 in mice from (E). (H) Lipidomic analysis of hepatic lipid classes in mice from (E). (I) Hepatic mRNA expression of select lipogenic genes in mice from (E). (J) Fecal fatty acids after treatment with either vehicle or GSK2324 in mice as in (E). (K) Total bile acid pool amount in mice from (E). Data are represented as mean ± SEM with individual animals noted as dots. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.