Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

Abstract

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.

Fig. 1.

Expression profile and localization of mouse NPC1L1. A: NPC1L1 is specifically expressed in mouse small intestine. Tissues taken from C57BL/6 mice were immediately homogenized. From each sample, 50 μg of total protein was loaded in SDS-PAGE and immunoblotted with the affinity-purified rabbit anti-NPC1L1 polyclonal antibody. For glycosylation analysis, homogenates of small intestine were subjected to PNGase F digestion. B: NPC1L1 protein mainly distributes in the villi of mouse small intestine. Intestinal sections (4 μm) were deparaffinized and stained with anti-NPC1L1 and anti-Villin (microvilli) antibodies. Scale bar: 50 μm. C: Enlarged view of the villi tips, showing that NPC1L1 localizes on the apical membrane of enterocytes. Small intestinal samples were costained with anti-NPC1L1 and anti-Villin (absorptive enterocytes) or anti-Mucin2 (goblet cells) antibodies. Scale bar: 10 μm. D: Enlarged view of the crypts showing that NPC1L1 also expresses in the transit-amplifying cells. Small intestinal samples were costained with anti-NPC1L1 and anti-Ki67 (proliferating cells) or anti-Lysozyme (Paneth cells) antibodies. The color of the title matches the stain color in the photomicrographs. Nuclear counterstaining with Hoechst is blue. Scale bar: 20 μm.

Fig. 2.

Localization of ACAT2 in mouse small intestine. A, B: Validation of the affinity-purified anti-ACAT2 antibody. Liver, small intestine, and colon samples from ACAT2^+/+ and ACAT2^−/− mice were homogenized and subjected to SDS-PAGE followed by immunoblotting with affinity-purified rabbit anti-ACAT2 polyclonal antibody (A). Deparaffinized 4-μm sections of small intestine sample from ACAT2^+/+ and ACAT2^−/− mice were stained with anti-ACAT2 and anti-Villin antibodies (B). C, D: Enlarged view of the villi tips showing that NPC1L1 and ACAT2 localize to the different regions of intestinal epithelium. Intestinal samples were costained with purified anti-ACAT2 and anti-Villin (C) or anti-NPC1L1 (D) antibodies.

Fig. 3.

Localization of ACAT1 in mouse small intestine. A, B: Validation of purified anti-ACAT1 antibody. Lung, small intestine, and colon samples from ACAT1^+/+ and ACAT1^−/− mice were homogenized and subjected to SDS-PAGE followed by immunoblotting with the affinity-purified rabbit anti-ACAT1 polyclonal antibody (A). Deparaffinized 4-μm sections of small intestine samples from ACAT1^+/+ and ACAT1^−/− mice were stained with anti-ACAT1 and anti-Villin antibodies (B). C: ACAT1 localized to mesenchymal cells of the villi and Paneth cells of the crypts. Paraffin-embedded intestinal sections were costained with anti-ACAT1 and anti-Villin or anti-Lysozyme antibodies, respectively.

Fig. 4.

Distribution of NPC1L1, ACAT1, and ACAT2 in mouse intestine. A: Immunoblot analysis of NPC1L1, ACAT1, and ACAT2 in mouse duodenum, jejunum, ileum, and colon. Immunoblots were performed with tissue homogenates using the purified polyclonal anti-NPC1L1, ACAT1, and ACAT2 antibodies. Three mice were used per segment. B: Quantitative PCR analysis of the gene expression of NPC1L1, ACAT1, and ACAT2 in mouse duodenum, jejunum, ileum, and colon. The expression level of cyclophilin was used as an internal control. The relative amounts of mRNA in different segments of intestine were normalized with relative amounts of mRNA in colon. Values are mean ± SD.

Fig. 5.

Cholesterol induces endocytosis of NPC1L1 from brush border membrane toward the inside in mouse intestine. Male C57BL/6 mice (12 weeks old) were administered 200 μl corn oil or corn oil containing 40 mg/ml cholesterol by oral gavage. Thirty minutes later, proximal segments of small intestine were subjected to immunohistochemistry and filipin staining, respectively. (A) Cholesterol induced endocytosis of NPC1L1. Paraffin-embedded sections were applied to immunohistochemistry staining with anti-NPC1L1, anti-ACAT2, anti-Villin, and anti-Rab11 antibodies. Rab11 is a marker of recycling endosomes. B: Frozen sections were stained with filipin to indicate free cholesterol. C: Intestinal total cholesterol and phospholipids were measured enzymatically. The relative amount of cholesterol was normalized with that of phospholipids. Values are mean ± SD. P values were calculated by ANOVA. **0.001 < P < 0.01.

Fig. 6.

Ezetimibe inhibits the internalization of NPC1L1 and cholesterol in mouse intestine. Male C57BL/6 mice (12 weeks old; n = 6 per group) were pretreated with ezetimibe or vehicle before being gavaged with cholesterol. Thirty minutes later, intestinal samples were excised and subjected to immunohistochemistry and filipin staining. A: Ezetimibe blocked cholesterol-induced endocytosis of NPC1L1. Paraffin-embedded sections were applied to immunohistochemistry staining with anti-NPC1L1 and anti-Villin or anti-Rab11 antibodies. B: Ezetimibe blocked dietary cholesterol from entering enterocytes. Frozen sections of mouse intestine were stained with filipin to indicate free cholesterol. C: Cholesterol and phospholipids were measured enzymatically. The relative amount of cholesterol was normalized to that of phospholipids. D: Male C57BL/6 mice were orally gavaged with vehicle (control) or ezetimibe once daily for 3 days. Then [¹⁴C]cholesterol and [³H]sitostanol in 150 μl corn oil were orally gavaged. Cholesterol absorption was determined by the fecal ratio method. Values are mean ± SD. P values were calculated by ANOVA. **0.001 < P < 0.01.