Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice

Abstract

We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92-95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations.

Keywords: ABCA1; Cholesterol; Intestine; Lipid Absorption; Lipid Metabolism; Lipids; Lipoprotein Secretion; Lipoprotein Structure; MTP; Triglyceride.

FIGURE 1.

Time-dependent changes in intestinal and plasma lipids with MTP gene deletion. A, schematic diagram showing the time points of tamoxifen injection (0.5 mg/mouse) and specimen collection from 12-week-old chow-fed, male, wild-type (WT, Mttp^f/f) and ER^T2-Villin-Cre;Mttp^f/f (I-Mttp^−/−) mice. At each time point, three mice from each group were sacrificed. Plasma and intestines were collected as described under “Materials and Methods.” Intestines were used to measure MTP activity (B), triglycerides (C), and cholesterol (D). Plasma was used to measure triglycerides (E) and cholesterol (F). Values are mean ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT. The data are representative of three independent experiments.

FIGURE 2.

Intestine-specific ablation of MTP and ABCA1 decreases plasma lipids. Total RNA isolated from the intestine of 12-week-old WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO (n = 5) male mice fed a chow diet was used to quantify mRNA levels of MTP (A) and ABCA1 (B). Oil Red O staining of proximal intestine (C) and liver (D) sections showing lipid staining in these tissues. Plasma was separated by gel filtration to determine mass of triglycerides (E) and cholesterol (F) in different lipoproteins. Values are mean ± S.D. ***, p < 0.001 compared with WT.

FIGURE 3.

Chow diet-fed MTP and ABCA1 gene ablated mice absorb less lipids. 12-Week-old WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO male mice (n = 3) were fasted overnight and injected intraperitoneally with poloxamer 407 (30 mg/mouse). After 1 h, mice were gavaged with 0.5 μCi of [¹⁴C]triolein or [³H]cholesterol and 0.2 mg of cholesterol in 15 μl of olive oil. Plasma was collected 2 h after the gavage of radiolabeled lipids and used to measure triglycerides (A) and cholesterol (B) concentrations. Total plasma was also used to measure radioactivity to determine the absorption of [¹⁴C]triolein (C) and [³H]cholesterol (D). Plasma was precipitated as described under “Materials and Methods” to determine radioactivity cholesterol counts in non-HDL (E) and HDL (F) lipoproteins. Values are mean ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT.

FIGURE 4.

Intestine-specific MTP and ABCA1 gene deletion decrease triglyceride secretion by enterocytes. To study lipid uptake, enterocytes were isolated from 12-week-old chow diet-fed, overnight-fasted mice and radiolabeled for 1 h with 0.5 μCi/ml of [³H]oleate. After 1 h, enterocytes were washed, and lipids were isolated to determine uptake of radiolabeled fatty acid (A). For characterization of secreted lipoproteins, after 1 h of uptake, enterocytes were washed and incubated with fresh media containing 1.4 mm oleic acid containing micelles for 2 h. Isolated lipids from the cells (B) and media (C) were counted to determine total fatty acid-derived radioactivity. Media from B were lipid-extracted, and lipids were separated by thin layer chromatography to determine radioactivity in triglycerides (D), phospholipids (E), and cholesteryl esters (F). Each measurement was done in triplicate with three mice per group. Mean ± S.D. *, p < 0.05; **, p < 0.01 and ***, p < 0.001 compared with WT.

FIGURE 5.

Intestine-specific MTP and ABCA1 gene deletion decrease secretion of cholesterol by enterocytes. A, to study cholesterol uptake, enterocytes were isolated from 12-week-old chow diet-fed, overnight-fasted mice and radiolabeled for 1 h with 0.5 μCi/ml [³H]cholesterol. After 1 h, enterocytes were washed, and lipids were isolated to determine uptake of radiolabeled cholesterol. B, in a separate experiment, total RNA isolated from the intestine of WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO (n = 5) male mice fed a chow diet was used to quantify mRNA levels of different genes. Mean ± S.D., n = 3. ^a, p < 0.05; ^b, p < 0.01, and ^c, p < 0.001 compared with WT. C–E, for characterization of secreted lipoproteins in an experiment described in A, after 1 h of uptake, enterocytes were washed and incubated with fresh media containing 1.4 mm oleic acid containing micelles for 2 h. Isolated lipids from the media (C) were counted to determine total cholesterol radioactivity. [³H]Cholesterol-radiolabeled media were used for separating lipoproteins by density gradient ultracentrifugation, and radioactivity was determined in each fraction (D). Fractions 1–3 and 8–10 represent chylomicrons (CM) and HDL, respectively. For better representation of HDL, fraction 10 was plotted separately (E). Each measurement was done in triplicate with three mice per group. Mean ± S.D., *, p < 0.05; **, p < 0.01 and ***, p < 0.001 compared with WT.

FIGURE 6.

Intestinal MTP deletion increases the expression of intestinal fatty acid oxidation genes. A, total RNA isolated from the intestine of 12-week-old WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO (n = 5) male mice fed a chow diet was used to quantify mRNA levels of different genes. Mean ± S.D., ^a, p < 0.05; ^b, p < 0.01, and ^c, p < 0.001 compared with WT. B, in a separate experiment, 12-week-old WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO male mice (n = 3) were fasted overnight and sacrificed. For fatty acid oxidation, 50-mg aliquots of intestine were incubated with [¹⁴C]oleic acid (0.3 μCi) for 2 h, and the radiolabeled CO₂ was captured and quantified in a scintillation counter. Mean ± S.D. **, p < 0.01 and ***, p < 0.001 compared with WT.

FIGURE 7.

Relative mRNA levels of liver lipid metabolism genes. Total RNA isolated from liver of 12-week-old WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO (n = 5) male mice fed a chow diet was used to quantify mRNA levels of fatty acid and triglyceride biosynthesis genes (A), cholesterol metabolism genes (B), and fatty acid oxidation and triglyceride secretion genes (C). Mean ± S.D. ^a, p < 0.05; ^b, p < 0.01, and ^c, p < 0.001 compared with WT. D, in a separate experiment, 12-week-old WT, I-Mttp^−/−, I-Abca1^−/−, and I-DKO male mice (n = 3) were fasted overnight and sacrificed. For fatty acid oxidation, 50-mg aliquots of liver were incubated with [¹⁴C]oleic acid (0.3 μCi) for 2 h, and the radiolabeled CO₂ was captured and quantified in a scintillation counter. Mean ± S.D. ***, p < 0.001 compared with WT.