Ezetimibe markedly attenuates hepatic cholesterol accumulation and improves liver function in the lysosomal acid lipase-deficient mouse, a model for cholesteryl ester storage disease

Abstract

Lysosomal acid lipase (LAL) plays a critical role in the intracellular handling of lipids by hydrolyzing cholesteryl esters (CE) and triacylglycerols (TAG) contained in newly internalized lipoproteins. In humans, mutations in the LAL gene result in cholesteryl ester storage disease (CESD), or in Wolman disease (WD) when the mutations cause complete loss of LAL activity. A rat model for WD and a mouse model for CESD have been described. In these studies we used LAL-deficient mice to investigate how modulating the amount of intestinally-derived cholesterol reaching the liver might impact its mass, cholesterol content, and function in this model. The main experiment tested if ezetimibe, a potent cholesterol absorption inhibitor, had any effect on CE accumulation in mice lacking LAL. In male Lal(-/-) mice given ezetimibe in their diet (20 mg/day/kg bw) for 4 weeks starting at 21 days of age, both liver mass and hepatic cholesterol concentration (mg/g) were reduced to the extent that whole-liver cholesterol content (mg/organ) in the treated mice (74.3±3.4) was only 56% of that in those not given ezetimibe (133.5±6.7). There was also a marked improvement in plasma alanine aminotransferase (ALT) activity. Thus, minimizing cholesterol absorption has a favorable impact on the liver in CESD.

Keywords: ALT; CE; Cholesterol absorption; Fatty liver; Hepatomegaly; Intrahepatic; LAL; LIPA; NPC1L1; Niemann-Pick C1-Like1; SI; Small intestine; TAG; Triacylglycerol; UC; alanine aminotransferase; cholesteryl ester; gene that encodes LAL; lysosomal acid lipase; small intestine; triacylglycerol; unesterified cholesterol.

Fig 1

Lal^−/− mice manifest marked hepatomegaly and elevations in hepatic cholesterol and triacylglycerol content, and in plasma ALT activity. Absolute (A) and relative (B) liver weights, liver total cholesterol concentration (C) and total cholesterol content (D) in 10-week-old male Lal^−/− and Lal^+/+ mice fed a basal rodent chow diet. Most of the additional cholesterol in the livers of Lal^−/− mice was esterified (E), and this, together with an increased triacylglycerol content, markedly raised hepatic lipid content (F). Plasma ALT activity in the Lal^−/− mice increased dramatically with age (G). The data in E and F are from 10-week-old female and male mice, respectively. In G, all values are for male mice at 7, 13 and 16 weeks. In A to F all values are the mean ± SEM of data from 4 to 6 animals in each group. For the ALT activity data, values for the Lal^+/+ mice are the mean ± SEM of data from 4 to 6 mice at each age, whereas for the Lal^−/− mice the data represent values in individual animals. * p<0.05.

Fig 2

Modulation of liver weight, cholesterol content and function in Lal^−/− mice by manipulation of the amount of cholesterol absorbed from the small intestine. Female Lal^−/− and matching Lal^+/+ mice were fed a rodent chow diet alone or containing either added cholesterol (0.5% w/w) or surfomer (2% w/w) for 28 days starting at 21 days of age. All data are for female mice. Values are the mean ± SEM of data from 5 or 6 mice for every group except the Lal^−/− mice fed the high cholesterol diet where there were 4 mice. Different letters (a-d) denote statistically different values (p<0.05) as determined by 2-way ANOVA with genotype and type of dietary addition as variables.

Fig 3

Ezetimibe, a sterol absorption inhibitor, significantly reduced liver mass and cholesterol content and improved plasma alanine aminotransferase levels in Lal^−/− mice. Male Lal^−/− and matching Lal^+/+ control mice were fed a rodent chow diet either alone or containing ezetimibe (20 mg/day/kg bw) for 28 days starting at 21 days of age. The data for whole liver cholesterol content (E) were obtained by multiplying the liver total cholesterol concentration (D) in each mouse by the whole liver weight for that same animal (A). Values are the mean ± SEM of data from 6 mice in each group. Different letters (a-c) denote statistically significant values (p<0.05) as determined by 2-way ANOVA with genotype and type of dietary addition as variables.