Apical sodium-dependent bile acid transporter inhibition with volixibat improves metabolic aspects and components of non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice

Abstract

Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis (NASH), and improve the metabolic aspects of the disease. Potential disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased the total amount of bile acid in feces. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition. Non-alcoholic fatty liver disease activity score (NAS) was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1β, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of NASH pathophysiology.

Fig 1. Bile acid synthesis and recirculation.

Primary bile acids are synthesized from cholesterol in the liver, via either the classical pathway, which produces CA, or the alternative pathway, which produces CDCA, or, in mice only, α- or β-MCA.[27, 30] Bile acids are conjugated with glycine or taurine (predominantly taurine in mice) before being released into the bile.[30] In the gut, bile acids are deconjugated and metabolized into secondary bile acids [27, 30, 31] (UDCA is considered a secondary bile acid in humans [32] but a primary bile acid in mice).[26] About 95% of bile acids are reabsorbed from the gut and transported back to the liver via the hepatic portal vein, and the remainder are excreted.[27, 31, 33] The hydrophilicity of the common free and conjugated bile salts decreases in the order UDCA > CA > CDCA > DCA) > LCA, and taurine-conjugated > glycine-conjugated > free species.[34] ASBT inhibition with volixibat blocks the reabsorption of bile acids and increases their excretion, stimulating the liver to synthesize more bile acids from cholesterol.[–37] ASBT: apical sodium-dependent bile acid transporter, CA: cholic acid, CDCA: chenodeoxycholic acid, Cyp: cytochrome P450 family, DCA: deoxycholic acid, G: glycine, HDCA: hyodeoxycholic acid, LCA: lithocholic acid, MCA: muricholic acid, T: taurine, UDCA: ursodeoxycholic acid.

Fig 2. Study design and schedule of assessments during the study.

An oral glucose tolerance test was performed in week 18. HFD: high-fat diet.

Fig 3. Body weight and food intake throughout the study.

(A) body weight and (B) food intake. Error bars show standard error of the mean. *P < 0.05; ***P < 0.001 versus the HFD control group. Chow group, n = 10; HFD control group, n = 20; HFD + volixibat 5 mg/kg, n = 15; HFD + volixibat 15 mg/kg, n = 15; HFD + volixibat, n = 15.

Fig 4. Mean fecal bile acid content at baseline, 12 weeks, and 22 weeks.

(A) total bile acids, (B) LCA, (C) DCA, (D) α-MCA, (E) ω-MCA, (F) β-MCA, (G) HDCA/UDCA and (H) CA. Horizontal lines indicate mean values. Error bars show standard deviation. Fecal bile acid content was measured for mice in each cage over a 2-day period at 0, 12, and 22 weeks. *P < 0.05; **P < 0.01; ***P < 0.001 versus the HFD control group. CA: cholic acid, DCA: deoxycholic acid, HDCA/UDCA: hyodeoxycholic acid/ursodeoxycholic acid, HFD: high-fat diet, LCA: lithocholic acid, MCA: muricholic acid. All data points shown.

Fig 5. Mean plasma bile acid levels at 22 weeks.

(A) total bile acids, (B) CA, (C) TCA, (D) β-MCA, (E) UDCA, (F) DCA, (G) TDCA, (H) HDCA, (I) CDCA, and (J) TCDCA. Dose groups indicate HFD + volixibat dose. Horizontal lines indicate mean values. Error bars show standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001 versus the HFD control group. CA: cholic acid, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, HDCA: hyodeoxycholic acid, HFD: high-fat diet, MCA: muricholic acid, TCA: taurine-conjugated cholic acid, TCDCA: taurine-conjugated chenodeoxycholic acid, TDCA: taurine-conjugated deoxycholic acid, UDCA: ursodeoxycholic acid. All data points shown.

Fig 6. Effects of volixibat treatment on plasma lipids, insulin and liver enzymes throughout the study.

(A) cholesterol, (B) lipoprotein profiles, (C) triglycerides, (D) insulin, (E) ALT and (F) AST. Error bars show standard error of the mean. Lipoprotein analysis was performed using plasma pooled from all mice in each group (at week 16). Data are absolute values and represent cholesterol concentrations in eluted fractions. *P < 0.05; **P < 0.01; ***P < 0.001 versus the HFD control group. ALT: alanine aminotransferase, AST: aspartate aminotransferase, HDL-C: high-density lipoprotein cholesterol, HFD: high-fat diet, LDL-C: low-density lipoprotein cholesterol, VLDL-C: very-low-density lipoprotein cholesterol. Chow group, n = 10; HFD control group, n = 20; HFD + volixibat 5 mg/kg, n = 15; HFD + volixibat 15 mg/kg, n = 15; HFD + volixibat, n = 15.

Fig 7. Effects of volixibat on liver pathology at the end of the study.

Representative hematoxylin and eosin photomicrographs of (i) chow, (ii) HFD, (iii) 5 mg/kg volixibat, (iv) 15 mg/kg volixibat (v) 30 mg/kg volixibat. (A) hepatic triglycerides, (B) hepatic cholesteryl esters, (C) hepatic free cholesterol, (D) steatosis, (E) hepatocyte hypertrophy, (F) inflammatory aggregates and (G) NAFLD activity score. Dose groups indicate HFD + volixibat dose. Horizontal lines indicate mean values. Error bars show standard deviation. Liver histology and liver lipids were assessed after sacrifice of mice at the end of the study. *P < 0.05; **P < 0.01; ***P < 0.001 versus the HFD control group. HFD: high-fat diet, NAFLD: non-alcoholic fatty liver disease. All data points shown.

Fig 8. Effects of volixibat at the end of the study on mesenteric white adipose tissue weight mass.

Horizontal lines indicate mean values. Error bars show standard deviation. Mesenteric white adipose tissue weight was measured after sacrifice of mice at the end of the study. *P < 0.05; ***P < 0.001 versus the HFD control group. HFD: high-fat diet. All data points shown.

Fig 9

(A) Number of differentially expressed genes by treatment; (B) differential expression of selected upstream regulators. (A) The purple shaded boxes show the numbers of differentially expressed genes in the comparisons of chow versus HFD control groups and HFD control versus HFD + volixibat groups (each dose group). The white boxes indicate the numbers of differentially expressed genes shared between corresponding diagonal boxes (purple boxes). (B) NC indicates no consistent directional effect. The HFD control group was compared with the chow group, and each volixibat dose group was compared with the HFD control group. P values are shown as the negative log P value. The Z-score indicates the predicted direction of the effect on a transcription factor: a Z-score below –2 indicates inhibition (blue); a Z-score above +2.0 indicates activation (orange). ESRR-α: estrogen-related receptor α, HFD: high-fat diet, INS-R: insulin receptor, PGC-1β: peroxisome proliferator-activated receptor-γ coactivator-1β, SREBF-2: sterol regulatory element-binding transcription factor 2. Gene expression analysis was performed on eight mice per group.