Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine

Abstract

The importance of Niemann-Pick C1 Like-1 (NPC1L1) protein in intestinal absorption of dietary sterols, including both cholesterol and phytosterols, is well documented. However, the exact mechanism by which NPC1L1 facilitates cholesterol transport remains controversial. This study administered 22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol) and [(3)H]cholesterol to Npc1l1(+/+) and Npc1l1(-/-) mice to determine whether NPC1L1 facilitates dietary sterol uptake by enterocytes and/or participates in intracellular sterol delivery to the endoplasmic reticulum (ER) for lipoprotein assembly before secretion into plasma circulation. Results showed that [(3)H]cholesterol absorption was reduced but not abolished in Npc1l1(-/-) mice compared with Npc1l1(+/+) mice. In the presence of Pluronic L-81 to block pre-chylomicron exit from the ER, significant amounts of [(3)H]cholesterol were found to be associated with lipid droplets in the intestinal mucosa of both Npc1l1(+/+) and Npc1l1(-/-) mice, and the intracellular [(3)H]cholesterol can be esterified to cholesteryl esters. These results provided evidence indicating that the main function of NPC1L1 is to promote cholesterol uptake from the intestinal lumen but that it is not necessary for intracellular cholesterol transport to the ER. Surprisingly, NBD-cholesterol was taken up by intestinal mucosa, esterified to NBD-cholesteryl esters, and transported to plasma circulation to similar extent between Npc1l1(+/+) and Npc1l1(-/-) mice. Ezetimibe treatment also had no impact on NBD-cholesterol absorption by Npc1l1(+/+) mice. Thus, NBD-cholesterol absorption proceeds through an NPC1L1-independent and ezetimibe-insensitive sterol absorption mechanism. Taken together, these results indicate that NBD-cholesterol can be used to trace the alternative cholesterol absorption pathway but is not suitable for tracking NPC1L1-mediated cholesterol absorption.

Fig. 1.

Uptake of 22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol) into the enterocytes of Niemann-Pick C1 Like-1 (NPC1L1) protein-null (Npc1l1^−/−) and Npc1l1^+/+ mice. Representative images are shown of 20 intestinal sections from Npc1l1^+/+ (left) and Npc1l1^−/− (right) mice (n = 10 mice per group) 3 h after gavaged with 0.4 mg of NBD-cholesterol in 100 μl olive oil. The intestine was fixed with 0.4% paraformaldehyde and embedded in optimal cutting temperature (OCT) medium before fluorescent microscopy. NBD-cholesterol appears to be localized to the lamina propria in Npc1l1^+/+ mice but is not visible in Npc1l1^−/− mice.

Fig. 2.

Uptake of NBD-cholesterol into the enterocytes of Npc1l1^+/+ and Npc1l1^−/− mice in the presence of Pluronic L-81. Representative images are shown of 20 intestinal sections from the proximal (top), middle (middle), and distal (bottom) intestines of Npc1l1^+/+ (left) and Npc1l1^−/− (right) mice (n = 10 mice per group) 3 h after gavaged with 3.45 mg Pluronic L-81 followed by 0.4 mg of NBD-cholesterol in 100 μl olive oil. The intestine was fixed with 0.4% paraformaldehyde and embedded in OCT medium before fluorescent microscopy. NBD-cholesterol is contained within intestinal lipid droplets of both Npc1l1^+/+ and Npc1l1^−/− mice.

Fig. 3.

Cholesterol is esterified in Npc1l1^+/+ and Npc1l1^−/− mice. A: the Npc1l1^+/+ (solid bars; n = 6) and Npc1l1^−/− (open bars; n = 7) mice were fed a diet of 1 μCi [³H]cholesterol in 100 μl olive oil, and intestine was collected after 3 h. Lipids were extracted, and the amount of cholesterol esterification was determined by thin-layer chromatography. Percent cholesterol esterification was determined by [CE counts/(FC counts + CE counts)] × 100. B: the Npc1l1^+/+ (solid bars; n = 4) and Npc1l1^−/− (open bars; n = 5) mice were fed 0.4 mg NBD-cholesterol in 100 μl olive oil, and blood was collected after 4 h. Plasma lipids were extracted for thin-layer chromatography separation of cholesterol and cholesteryl esters. The amount of NBD-cholesterol present in each band was determined by measuring fluorescent intensity at 488 nm. Percent esterification was determined as [CE fluorescence/(FC fluorescence + CE fluorescence)] × 100.

Fig. 4.

Uptake of [³H]cholesterol into the intestine of Npc1l1^+/+ and Npc1l1^−/− mice. The Npc1l1^+/+ (solid bars) and Npc1l1^−/− (open bars) mice were given a dose of 3.45 mg Pluronic L-81 followed by an oral gavage of 1 μCi [³H]cholesterol in 100 μl olive oil. The intestine was removed, cut into 100-mg sections, and dissolved in tissue solubilizer before radioactivity determinations. Data represent means ± SE from 13 Npc1l1^+/+ and 10 Npc1l1^−/− mice. *P < 0.05, **P < 0.01, statistically significant differences from the Npc1l1^+/+ mice.

Fig. 5.

Effect of Pluronic L-81 on intestinal uptake of [³H]cholesterol ether and [¹⁴C]cholesterol. A: the Npc1l1^+/+ mice were given a dose of 3.45 mg Pluronic L-81 followed by an oral gavage of 1 μCi [¹⁴C]cholesterol (solid bars) and [³H]cholesterol ether (open bars). The intestine was collected, cut into 100-mg sections, and dissolved in tissue solubilizer before radioactivity determinations. B: the Npc1l1^+/+ mice were given a dose of saline (hatched bars) or 3.45 mg Pluronic L-81 (open bars) followed by an oral gavage of 1 μCi [³H]cholesterol ether. The intestine was collected, cut into 100-mg sections, and dissolved in tissue solubilizer before radioactivity determinations. Data represent means ± SE from 3 mice in each group. *P < 0.05, **P < 0.01, statistically significant differences from [³H]cholesterol absorption data.

Fig. 6.

Absorption of NBD-cholesterol and [¹⁴C]cholesterol in Npc1l1^+/+, Npc1l1^−/−, and ezetimibe-treated Npc1l1^+/+ mice. Npc1l1^+/+ (solid bars; n = 5), Npc1l1^−/− (open bars; n = 3), and ezetimibe treated Npc1l1^+/+ (hatched bars; n = 4) mice were given an oral gavage of 0.4 mg NBD-cholesterol and 1 μCi [¹⁴C]cholesterol in olive oil. Blood was collected after 4 h via cardiac puncture. Plasma was extracted with hexane, and fluorescence was measured to quantify NBD-cholesterol absorption (A and B). A 10-μl sample of plasma was counted for radioactivity determinations (C and D). Data represent means ± SE of 5 Npc1l1^+/+, 3 Npc1l1^−/−, and 4 ezetimibe-treated Npc1l1^+/+ mice. *P < 0.05, statistically significant difference from Npc1l1^+/+ group.