ATP citrate lyase inhibitor Bempedoic Acid alleviate long term HFD induced NASH through improvement in glycemic control, reduction of hepatic triglycerides & total cholesterol, modulation of inflammatory & fibrotic genes and improvement in NAS score

Abstract

Non-alcoholic fatty liver disease (NAFLD) and Non-alcoholic steatohepatitis (NASH) are chronic liver disorders, the prevalence of which is increasing worldwide. Long term High Fat Diet (HFD) induced NASH animal models closely mimic the characteristics of human NASH and hence used by investigators as a model system for studying the mechanism of action of new drugs. Bempedoic acid (ETC-1002), a ATP citrate lyase (ACLY) inhibitor that lowers the LDL cholesterol was recently approved by US FDA for the treatment of heterozygous familial hypercholesterolemia (HeFH) and established atherosclerotic cardiovascular disease (ASCVD). ACLY is one of the genes modulated in NASH patients and hence we studied the effect of ACLY inhibitor Bempedoic acid in long term HFD induced NASH animal model to understand the pharmacological benefits and the associated mechanism of action of this newly approved drug in NASH. Mice fed with 60% Kcal High Fat Diet for 32 weeks were used for the study and the animals were given Bempedoic acid for 5 weeks at doses of 10 ​mg ​kg^-1, po, qd, and 30 ​mg ​kg^-1, po, qd. Bempedoic acid treatment resulted in inhibition of body weight gain and improved the glycemic control. Bempedoic acid treated group showed statistically significant reduction in plasma ALT, AST, hepatic triglycerides (TG) and total cholesterol (TC), along with statistically significant reduction in steatosis score by histological analysis. Hepatic gene expression analysis showed significant reduction in inflammatory and fibrotic genes such as Mcp-1/Ccl2, Timp-1 & Col1α1. Histological analysis showed significant improvement in NAS score. Overall, Bempedoic acid alleviated HFD induced Non-Alcoholic Steatohepatitis through inhibition of body weight gain, improvement in glycemic control, reduction of hepatic triglycerides & total cholesterol, modulation of inflammatory & fibrotic genes, and improvement in NAS score. Hence, Bempedoic acid can be a potential therapeutic option for metabolic syndrome and NASH.

Keywords: ATP citrate Lyase; Bempedoic acid; Fibrosis; High fat diet; Inflammation; Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis.

Graphical abstract

Fig. 1

Study Design- 7 weeks old C57BL6/N mice were given either the rodent chow diet (Teklad; Harlan) or HFD (60% Kcal; Research diet, D12492). The animals that were given the rodent chow diet constituted the lean control group and received the chow diet for the duration of the study (37 weeks). These control animals received the vehicle (0.5% w/v methyl cellulose and 0.5% v/v Tween 80) from week 32 till week 37. The animals that were given the HFD were randomized based on body weight & random blood glucose levels after 32 weeks and segregated into three groups. The animals received either the vehicle (0.5% w/v methyl cellulose and 0.5% v/v Tween 80) or Bempedoic acid at 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd for five weeks (till 37 weeks). Oral glucose tolerance test (OGTT) was performed in week 36 (day 31 of treatment). The study was terminated on day 35 of the treatment period and the biochemical, histological and biomarker analysis were performed.

Fig. 2

Effect of Bempedoic acid on body weight gain in long term HFD induced NASH model – The body weight and food intake were measured during the course of the treatment period and represented. (a) Body weight in grams (b) % body weight change (c) Daily energy intake (d) Cumulative energy intake. ∗∗∗∗p<0.0001 when HFD control was compared with Lean control and $ p<0.05, $$ p<0.01, $$$ p<0.001 and $$$$ p<0.0001 when HFD ​+ ​Bempedoic acid, 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd group was compared with HFD control (Two-way ANOVA followed by Bonferroni's post-hoc test). Data shown as Mean ​± ​SEM. n ​= ​10. Bempedoic acid at doses of both 10 ​mg ​kg⁻¹, po, qd, and 30 ​mg ​kg⁻¹, po, qd. inhibited body weight gain starting day 8 of the treatment till end of the study as compared to the HFD control (a & b). There was no significant change in energy intake of all groups (c & d). OGTT was performed on day 31.

Fig. 3

Effect of Bempedoic acid on OGTT profile in long term HFD induced NASH model. (a) Blood glucose curve during OGTT (b) AUC glucose (c) 0 ​min blood glucose levels from fasted (6h) animal samples. $$ p<0.01, $$$ p<0.001 when HFD ​+ ​Bempedoic acid, 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd group were compared with HFD control (Two-way ANOVA followed by Bonferroni's post-hoc test) ∗p<0.05 and ∗∗∗p<0.001 when HFD control was compared with Lean control and $ p<0.05 and $$ p<0.01 when HFD ​+ ​Bempedoic acid, 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd group were compared with HFD control (One-way ANOVA followed by Bonferroni's post-hoc test). Data shown as Mean ​± ​SEM. n ​= ​9–10. Bempedoic acid at both doses has shown statistically significant increase in glucose tolerance as seen by decrease in glucose levels at 15 & 30 ​min after glucose challenge (a), and reduction in AUC blood glucose levels during OGTT (b). Marked decrease in glucose levels were also observed in the blood samples of 6h fasted animals in the Bempedoic acid treatment group tested at 0 ​min during the OGTT (c).

Fig. 4

Effect of Bempedoic acid on lipid profile in long term HFD induced NASH model. The plasma and liver samples were analyzed for ALT, AST, TG and total Cholesterol. (a) Liver Triglyceride levels at the end of the study (b) Liver total cholesterol levels at the end of the study (c) Plasma ALT levels at the end of the study (d) Plasma AST levels at the end of the study. (e) Plasma ALT levels at day 0 (before the start of Bempedoic acid treatment) and on day 35 (after 5 weeks of Bempedoic acid treatment). (f) Plasma AST levels at day 0 (before the start of Bempedoic acid treatment) and on day 35 (after 5 weeks of Bempedoic acid treatment) (g) Plasma Triglycerides at the end of the study (h) Plasma total cholesterol levels at the end of the study. ∗∗p<0.01 and ∗∗∗∗p<0.0001 when HFD control was compared with Lean control and $$ p<0.01, $$$ p<0.001 and $$$$ p<0.0001 when HFD ​+ ​Bempedoic acid, 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd group was compared with HFD control (One-way ANOVA followed by Bonferroni's post-hoc test). Data Shown as Mean ​± ​SEM. n ​= ​9–10. The liver triglycerides (a) and total cholesterol levels (b) were significantly reduced upon treatment with Bempedoic acid. Plasma ALT & AST levels were significantly reduced by both doses of Bempedoic acid (c–f). Plasma Triglycerides and plasma total cholesterol were increased in HFD control group (g, h), however, treatment with Bempedoic acid did not show any modulation of plasma triglycerides and plasma total cholesterol.

Fig. 5

Effect of Bempedoic acid on the liver fibrotic and inflammation gene/protein expression profile in long term HFD induced NASH model. Expression profile of liver inflammation and fibrotic genes/proteins were analyzed at the end of Bempedoic acid treatment. (a–c) mRNA analysis, (a) Col1α1 (b) Timp-1 (c) Mcp-1. (d) MCP-1 protein analysis using ELISA. ∗∗p<0.01 and ∗∗∗p<0.001 when HFD control was compared with Lean control and $ p<0.05 and $$$ p<0.001 when HFD ​+ ​Bempedoic acid, 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd group were compared with HFD control (One-way ANOVA followed by Bonferroni's post-hoc test). Data Shown as Mean ​± ​SEM. n ​= ​9–10. Treatment with Bempedoic acid significantly down regulated the mRNA expression of Col1α1 (a), Timp-1 (b) and protein levels of MCP-1 (d). Modulation of Mcp-1 mRNA levels upon Bempedoic acid was moderate and was observed only in animals treated with Bempedoic acid at 10 ​mg kg-1 dose (c).

Fig. 6

Effect of Bempedoic acid on the NAS score in long term HFD induced NASH model. Histological assessment of liver was performed at the end of Bempedoic acid treatment and the data is provided. (a) Steatosis score (b) Lobular inflammation score (c) NAS score. Representative H&E stained liver microscopic images are provided. (d) Lean control (10X), (e) HFD control (10X), (f) HFD ​+ ​Bempedoic acid 10 ​mg ​kg⁻¹, po, qd (10X), (g) HFD ​+ ​Bempedoic acid 30 ​mg ​kg⁻¹, po, qd (10X). For better visualization, 20X images are provided for lean control and HFD control. (h) Lean control (20X) and (i) HFD control (20X). Green arrow shows steatosis characterized by large vacuoles that occupy the whole cytoplasm and push the nucleus to one side of the cell, and yellow arrows shows hepatocellular ballooning degeneration characterized by cells with swollen and rarefied cytoplasm. ∗∗∗∗p<0.0001 when HFD control is compared with Lean control and $$$$ p<0.0001 when HFD ​+ ​Bempedoic acid, 10 ​mg ​kg⁻¹, po, qd or 30 ​mg ​kg⁻¹, po, qd group is compared with HFD Control (One-way ANOVA followed by Bonferroni's post-hoc test). Data Shown as Mean ​± ​SEM. n ​= ​10. Treatment with Bempedoic acid showed significant reduction of lobular inflammation, hepatocellular ballooning degeneration and NAS scores as compared to the untreated HFD controls. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Effect of Bempedoic acid on collagen deposition and fibrosis score as measured by Sirius red staining in long term HFD induced NASH model. Histological assessment of liver was performed at the end of Bempedoic acid treatment to study the collagen deposition and measure the fibrosis score. Representative Sirius red stained liver microscopic images are provided. (a) Lean control (20X), (b) HFD control (20X), (c) HFD ​+ ​Bempedoic acid 10 ​mg ​kg⁻¹, po, qd (20X), (d) HFD ​+ ​Bempedoic acid 30 ​mg ​kg⁻¹, po, qd (20X). Yellow arrow shows fibrosis (a, b, c, d). Steatosis characterized by large vacuoles that occupy the whole cytoplasm was markedly seen in the sections from HFD control group (b). The scoring for collagen deposition was carried out using Sirius red staining of the liver sections as described in the methods section and represented in a graph (e). Moderate increase in Sirius red staining was observed in HFD controls as compared to the lean controls and this increase was abrogated in the Bempedoic acid treated groups (e). Data was analyzed by One-way ANOVA followed by Bonferroni's post-hoc test. Data Shown as Mean ​± ​SEM. n ​= ​10. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)