Inhibition of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) prevents dietary cholesterol-associated steatosis by enhancing hepatic triglyceride mobilization

Abstract

Acyl-CoA:cholesterol O-acyl transferase 2 (ACAT2) promotes cholesterol absorption by the intestine and the secretion of cholesteryl ester-enriched very low density lipoproteins by the liver. Paradoxically, mice lacking ACAT2 also exhibit mild hypertriglyceridemia. The present study addresses the unexpected role of ACAT2 in regulation of hepatic triglyceride (TG) metabolism. Mouse models of either complete genetic deficiency or pharmacological inhibition of ACAT2 were fed low fat diets containing various amounts of cholesterol to induce hepatic steatosis. Mice genetically lacking ACAT2 in both the intestine and the liver were dramatically protected against hepatic neutral lipid (TG and cholesteryl ester) accumulation, with the greatest differences occurring in situations where dietary cholesterol was elevated. Further studies demonstrated that liver-specific depletion of ACAT2 with antisense oligonucleotides prevents dietary cholesterol-associated hepatic steatosis both in an inbred mouse model of non-alcoholic fatty liver disease (SJL/J) and in a humanized hyperlipidemic mouse model (LDLr(-/-), apoB(100/100)). All mouse models of diminished ACAT2 function showed lowered hepatic triglyceride concentrations and higher plasma triglycerides secondary to increased hepatic secretion of TG into nascent very low density lipoproteins. This work demonstrates that inhibition of hepatic ACAT2 can prevent dietary cholesterol-driven hepatic steatosis in mice. These data provide the first evidence to suggest that ACAT2-specific inhibitors may hold unexpected therapeutic potential to treat both atherosclerosis and non-alcoholic fatty liver disease.

FIGURE 1.

Mice lacking ACAT2 or treated with ACAT2 ASO are protected against dietary cholesterol-driven hepatic steatosis. Liver lipids from female mixed strain mice fed a very low fat (10% of energy as fat) diet containing either low (0.001% w/w) or high (0.2% w/w) cholesterol (Chol) for 6 weeks were compared. Livers from littermates with ACAT2 intact (ACAT2^+/+) (dark cross-hatched bars and gray cross-hatched bars) were compared with ACAT2 knock-out (ACAT2^−/−) mouse livers (open shaded bars and gray shaded bars). Livers from male LDLr^−/−, apoB^100/100 mice fed a low fat (20% of energy as palm oil), moderate cholesterol (0.1% w/w) diet for 8 weeks and treated biweekly with a non-targeting ASO (ASO control (Cont.)) (black bars) or an ASO targeting ACAT2 (ACAT2 ASO) (open bars) were also compared. A, hepatic free (unesterified) cholesterol concentration. PR, protein. B, hepatic cholesteryl ester (Chol Ester) concentration. C, hepatic triglyceride concentration. D, plasma triglyceride concentrations at the end of the treatment periods. Data represent the average (±S.E.) of individual samples (n = 5–8) per group; p values obtained by Student's t test with p ≤ 0.05 being considered statistically significant.

FIGURE 2.

Histological appearance consistent with NAFLD in ACAT2^+/+ mice. Liver sections stained with hematoxylin and eosin from two ACAT2^+/+ mice and two ACAT2^−/− mice fed high cholesterol diets are shown. ACAT2^+/+ livers had many large lipid droplets within the hepatocytes, whereas ACAT2^−/− livers had fewer and smaller lipid droplets (arrows). The black bar in the upper right corner indicates 30 μm. WT, wild type.

FIGURE 3.

ASO-mediated knockdown of ACAT2 in adult inbred SJL mice protects against dietary cholesterol-driven hepatic steatosis. A, hepatic triglyceride concentrations in female (F) and male (M) mice of five different inbred strains purchased from The Jackson Laboratory and fed a low fat (10% of energy as palm oil) diet containing 0.2% (w/w) cholesterol for 6 weeks. liver prot, liver proteins. B–F, ACAT2 and lipid values in livers of SJL/J male mice fed the high cholesterol diet for 6 weeks and treated biweekly with injections (25 mg/kg) of either an ASO targeting the knockdown of ACAT2 (ACAT2 ASO) (open bars) or a non-targeting ASO (ASO control) (black bars). B, ACAT2 protein in three individual mouse liver microsomal preparations by Western blotting. C, ACAT2 activity. D, hepatic triglyceride concentration. Liver PR, liver proteins. E, hepatic cholesteryl ester concentration. F, hepatic phospholipid concentration. Data in panels A (n = 3) and C–F (n = 6) represent the mean ± S.E.; p values obtained by Student's t test with p ≤ 0.05 being considered statistically significant.

FIGURE 4.

Relationship between hepatic cholesteryl ester and triglyceride mass. Hepatic cholesteryl ester and triglyceride concentrations were quantified by group as mean ± S.E. and correlated among several groups of animals with and without ACAT2 fed for at least 6 weeks of low (0.001%), moderate (0.1%), or high (0.2%) cholesterol diets. Several groups of animals were compared, as shown in the legend. ASO treatment was with either ASO control or ACAT2 ASO. Dietary cholesterol is indicated as a percentage (w/w) followed by the number of animals in each group (n = #). All points together describe a regression line with a correlation coefficient of r = 0.87, p < 0.0001). Liver PR, liver proteins.

FIGURE 5.

Liver triglyceride lowering seen with ACAT2 deficiency is linked to elevated VLDL-TG secretion rate. Isolated recirculating liver perfusion was done with livers from female mixed strain ACAT2^+/+ and ACAT2^−/− mice fed a 0.2% cholesterol, low fat (10% of energy) diet for 6 weeks. The rates of accumulation in perfusate of triglycerides (A), cholesteryl esters (B), and free cholesterol (FC) (C) were measured after collection of perfusate samples every 30 min during 3 h of perfusion. The data in D show the VLDL particle size measurements made in VLDL isolated from several perfusate time point samples to help sort the difference between enrichment (enlargement) of particles with triglycerides versus the increase in the number of particles that transport secreted triglycerides. Data represent the mean ± S.E. (n = 5 liver perfusion experiments). p values obtained by Student's t test with p ≤ 0.05 being considered statistically significant.

FIGURE 6.

ACAT2 deficiency is associated with increased mobilization of stored hepatic triglyceride for VLDL secretion. Hepatic triglycerides were isotopically labeled with [³H]oleic acid before and with [¹⁴C]oleic acid during isolated liver perfusion of livers from female C57Bl/6 ACAT2^+/+ and ACAT2^−/− mice fed the 0.2% cholesterol, low fat diet for 6 weeks. [¹⁴C]Oleate on albumin was continuously infused at a constant rate (∼11,000 cpm/min) during recirculating liver perfusion (A and B), and the incorporation into ¹⁴C-labeled hepatic triglyceride was monitored in liver samples collected hourly during perfusion (B), whereas the appearance of ¹⁴C-lipid in perfusate lipoproteins was also monitored (A). The rate of appearance of [³H]oleate in perfusate lipids (mainly in triglycerides (>85%)) was monitored in perfusate samples with collections made every 30 min (C). Data represent the mean ± S.E. from n = 5–6. p values obtained by Student's t test with p ≤ 0.05 being considered statistically significant.