Multiple mechanisms limit the accumulation of unesterified cholesterol in the small intestine of mice deficient in both ACAT2 and ABCA1

Abstract

Cholesterol homeostasis in the enterocyte is regulated by the interplay of multiple genes that ultimately determines the net amount of cholesterol reaching the circulation from the small intestine. The effect of deleting these genes, particularly acyl CoA:cholesterol acyl transferase 2 (ACAT2), on cholesterol absorption and fecal sterol excretion is well documented. We also know that the intestinal mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1) increases in Acat2(-/-) mice. However, none of these studies has specifically addressed how ACAT2 deficiency impacts the relative proportions of esterified and unesterified cholesterol (UC) in the enterocyte and whether the concurrent loss of ABCA1 might result in a marked buildup of UC. Therefore, the present studies measured the expression of numerous genes and related metabolic parameters in the intestine and liver of ACAT2-deficient mice fed diets containing either added cholesterol or ezetimibe, a selective sterol absorption inhibitor. Cholesterol feeding raised the concentration of UC in the small intestine, and this was accompanied by a significant reduction in the relative mRNA level for Niemann-Pick C1-like 1 (NPC1L1) and an increase in the mRNA level for both ABCA1 and ABCG5/8. All these changes were reversed by ezetimibe. When mice deficient in both ACAT2 and ABCA1 were fed a high-cholesterol diet, the increase in intestinal UC levels was no greater than it was in mice lacking only ACAT2. This resulted from a combination of compensatory mechanisms including diminished NPC1L1-mediated cholesterol uptake, increased cholesterol efflux via ABCG5/8, and possibly rapid cell turnover.

Fig. 1.

Fractional cholesterol absorption and total cholesterol concentration in the small intestine of Acat2^+/+ and Acat2^−/− mice fed diets containing various agents that alter the enterohepatic flux of cholesterol. Values are means ± SE of data from 7–12 mice in A, B, D and E, and from 5–7 mice in C and F. Two-way ANOVA revealed no interaction between genotype and diet (A–C). There was a statistically significant (P < 0.05) effect of diet (as denoted by ‡) but no effect of genotype. One-way ANOVA revealed significant effects of genotype or diet (D–F). Bars designated with different letters denote statistically different values, P < 0.05.

Fig. 2.

Concentrations of total, unesterified, and esterified cholesterol in the intestine (A) and liver (B) of Acat2^+/+ and Acat2^−/− mice fed diets containing cholesterol or ezetimibe, alone or in combination. The height of each bar defines the total cholesterol concentration, which is presented as the means ± SE of data from 6–8 mice in each group. For both organs the portion of each bar with dark shading represents the absolute fraction of the total cholesterol that was esterified. The lighter shaded portion represents the unesterified cholesterol fraction. The respective SE for the mean values for the esterified and unesterified fractions are not shown. Bars designated with different letters denote statistically different values, P < 0.05.

Fig. 3.

Relative change in unesterified cholesterol concentration and relative levels of expression of mRNA for various proteins in the small intestine of Acat2^+/+ and Acat2^−/− mice fed diets containing cholesterol or ezetimibe, alone and in combination. Matching groups of female Acat2^+/+ and Acat2^−/− mice that were fed the same diets as described in the legend to Fig. 2 were used for the measurement of the relative level of expression of mRNA for multiple proteins by real-time PCR. The values in A were calculated by using the absolute concentration data given in Fig. 2A. The concentration of unesterified cholesterol in the small intestine of the Acat2^+/+ fed the control diet was arbitrarily set at 1.0, and the change in the unesterified cholesterol concentration in each of the other groups of mice was expressed relative to that for the Acat2^+/+ control diet group (A). Relative mRNA levels are expressed relative to that obtained for Acat2^+/+ mice fed the control diet. All values are means ± SE of data from 6–8 mice in each group. Two-way ANOVA revealed interaction between diet and genotype (A–E). One-way ANOVA revealed significant effects of genotype or diet. Bars designated with different letters denote statistically different values, P < 0.05.

Fig. 4.

Fractional cholesterol absorption, fecal neutral sterol excretion, total cholesterol concentration, and rate of sterol synthesis in the small intestine of mice deficient in both ABCA1 and ACAT2. The data in A and B are for females and those in C–F are for males. Values are means ± SE of data for a minimum of 9 mice per group except in E where there were 5–8 animals per group. Bars designated with different letters denote statistically significant values, P < 0.05.

Fig. 5.

Relative levels of expression of mRNA for various proteins in the small intestine of mice deficient in both ABCA1 and ACAT2. All the mice were females fed a basal rodent chow diet. Relative mRNA levels were determined by expressing the amount of mRNA found relative to that for the Acat2^+/+/Abca1^+/+ mice that in each case was arbitrarily set at 1.0. In B the open bars for the 2 groups lacking ABCA1 reveal that the PCR primers utilized for this assay detect an aberrant transcript that does not produce functional ABCA1 protein. In all panels values are means ± SE of data from a minimum of 5–7 mice per group. One-way ANOVA revealed significant differences among groups (A–E) and bars designated with different letters denote statistically different values, P < 0.05.

Fig. 6.

Effect of cholesterol feeding on the concentration of total, unesterified, and esterified cholesterol in the small intestine and liver of mice deficient in both ABCA1 and ACAT2. All mice were females fed a basal diet containing added cholesterol at a level of 0.5% wt/wt for 8–10 days. The height of each bar defines the total cholesterol concentration, which is presented as means ± SE of data from 12–17 animals in all groups. For both organs, the portion of each bar with dark shading represents the absolute fraction of the total cholesterol that was esterified. The respective SE for the mean values for the esterified and unesterified fractions are not shown. One-way ANOVA revealed significant effects of genotype on total cholesterol concentrations. Bars designated with different letters denote statistically different values, P < 0.05.