Apolipoprotein E regulates dietary cholesterol absorption and biliary cholesterol excretion: studies in C57BL/6 apolipoprotein E knockout mice

Abstract

The present study examined the role of apolipoprotein E (apoE) in the regulation of dietary cholesterol absorption and biliary cholesterol excretion. Increasing dietary cholesterol from 0.02% to 0.5% in C57BL/6 wild-type mice decreased the percentage of dietary cholesterol that is absorbed by 25%, and this decrease was associated with a 2-fold increase in gallbladder biliary cholesterol concentration. In contrast, increasing dietary cholesterol from 0. 02% to 0.5% in C57BL/6 apoE knockout mice produced no significant suppression of the percentage dietary cholesterol absorption and increased gallbladder biliary cholesterol concentration only 16%. Whereas in wild-type mice, the increase in dietary cholesterol increased the hepatic excretion of biliary cholesterol 4-fold, there was only a 2-fold increase in apoE knockout mice. On both the low- and the high-cholesterol diets, whole liver and isolated hepatocyte cholesterol content was higher in the apoE knockout mice. These results suggest that, in response to dietary cholesterol, apoE may play a critical role in decreasing the percentage absorption of dietary cholesterol and increasing biliary cholesterol excretion. These observations suggest a mechanism whereby the absence of apoE contributes to the propensity for tissue cholesterol deposition and accelerated atherogenesis in apoE knockout mice.

Figure 1

Effect of dietary cholesterol on cholesterol absorption in WT and apoE knockout mice. WT (E+/+) and apoE knockout (E−/−) C57BL/6 males were fed for 3 wk with either dietary 0.02% (open bars) or 0.5% wt/wt cholesterol (filled bars). Mice received a gastric bolus of 100 μl of olive oil containing 1.67 μCi [¹⁴C]cholesterol and 0.67 μCi [³H]β-sitostanol and were placed in metabolic cages, and feces were collected for 24 h. The ratio of [¹⁴C]/[³H] labels in the fecal lipid extracts was determined and % absorption was calculated as detailed in Experimental Procedures. Data presented are the mean ± SD of five animals in each group. *, P < 0.001 vs. E+/+ 0.02% group; **, P < 0.008 vs. E+/+ 0.5% group.

Figure 2

Effect of dietary cholesterol on gallbladder bile concentrations (A) and percent saturation (B) of cholesterol in WT and apoE knockout mice: WT (E+/+) and apoE knockout (E−/−) mice were fed for 3 wk with 0.02% (open bars) and 0.5% cholesterol (filled bars). After 3 wk, the animals were fasted, the peritoneal cavity was exposed, and gallbladder bile was aspirated. Biliary cholesterol concentrations of cholesterol were measured and percent saturation was calculated as described in Experimental Procedures. Data displayed are the mean ± SD of five animals in each group.

Figure 3

Effect of dietary cholesterol on liver (A) and hepatocytes (B) cholesterol content in WT (E+/+) and apoE knockout (E−/−) mice. Animals were fed for 3 wk with either 0.02% or 0.5% cholesterol and fasted for 4–6 h. (A) For liver cholesterol measurements, livers were perfused with PBS and harvested and, (B) for hepatocyte cholesterol measurements, livers were perfused with collagenase and hepatocytes were isolated, as described in Experimental Procedures. Liver and hepatocytes lipids were extracted, and total cholesterol (open bars), free cholesterol (filled bars), and cholesterol ester (hatched bars) contents were measured as described in Experimental Procedures. Data displayed are the mean ± SD of five animals in each group.

Figure 4

Effect of dietary cholesterol on liver HMGR and cholesterol 7α hydroxylase activities in WT and apoE knockout mice: WT (E+/+) and apoE knockout (E−/−) animals were fed with either 0.02% (open bars) or 0.5% cholesterol (filled bars), livers were harvested, and HMGR and cholesterol 7α hydroxylase activities were measured as described in Experimental Procedures. Data displayed are the mean ± SD of five animals in each group.