Accelerated lipid absorption in mice overexpressing intestinal SR-BI

Abstract

Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [(14)C]cholesterol and [(3)H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.

Figure 1. Tissue distribution of SR-BI in transgenic mice

Panel A: 50 μg of membrane protein from total small intestine were analyzed by Western blotting with anti-SR-BI antibodies. W, wild type mice; T, SR-BI transgenic mice. Panel B: 100 μg of protein from homogenates of different tissues from transgenic and wild type mice were analyzed by Western blotting with anti-SR-BI antibodies. Panel C: 50 μg of membrane protein from different intestinal segments (duodenum to colon) of wild type and transgenic mice were analyzed by Western blotting with anti-SR-BI antibodies. P, Proximal; M, Medium, D, Distal part of the small intestine; C, Colon. Panel D, enterocytes from wild type and transgenic mice were separated into 8 different fractions along the villus-crypt axis according to Weiser (20) and homogenized. 50 μg of membrane proteins were analyzed by Western blotting with anti-SR-BI antibodies.

Figure 2. Intestinal localization of SR-BI

Sections of small intestine from wild type and transgenic mice were incubated with anti-SR-BI antibodies followed by incubation with biotinylated secondary antibodies and with streptavidin-peroxidase complex. Peroxidase activity was revealed using 3′ diaminobenzidine tetrahydrochloride (DAB). A,D,G: control was obtained with an incubation of wild type sections in only the secondary antibodies; B,E,H: wild type mice; C,F,I: SR-BI transgenic mice. P, Proximal; M, Medium; D, Colon. V, Villosity; C, Crypt.

Figure 3. Effect of intestinal over-expression of SR-BI on plasma cholesterol and triglycerides levels

Plasma was collected from 8 weeks aged mice. Total cholesterol, HDL cholesterol, LDL/VLDL cholesterol and triglycerides were determined as described under “Experimental procedures”. Wild type (closed bar), SR-BI Transgenic mice (open bar). Data are presented as mean ± SEM from 10 mice in each group. Statistical significance between wild type and SR-BI Transgenic mice was determined by Student’s t test. *** p < 0.01. * p < 0.05

Figure 4. Analysis of plasma apolipoprotein content

An aliquot of lipoproteins isolated by sequential density ultracentrifugation from equal amount (400 μl) of pooled plasma from 5 mice were loaded onto reducing 4 – 16 % SDS polyacrylamide gel and stained with Coomassie blue. Proteins were identified with respect to the migration of molecular weight standards. W, wild type mice; T, SR-BI transgenic mice.

Figure 5. Lipid absorption rates in wild type and SR-BI Tg mice

Wild type (square) and SR-BI Tg (triangle) mice were fed by stomach gavage with a 100 μl test meal containing 2 μCi [¹⁴C] cholesterol, 1μCi [³H] triolein and 2 g/l cholesterol suspended in corn oil, without (panels A and B) or after (panels C and D) retro-orbitaly injection of 0.1ml Triton WR 1339 in NaCl 0.9 % (1:6, v/v). Blood samples were collected up to 4 h and radioactivity was measured by liquid scintillation counting. Cholesterol (A, C) and triglycerides (B, D) absorption rates are presented as mean ± SEM from 6 mice in each group (A, B) and mean ± SEM from 3 mice (C, D). * Statistical significance, p < 0.05 between wild type and SR-BI Tg mice as determined by Student’s t test. *** p < 0.01.

Figure 6. Tissue distribution of [¹⁴C] and [³H] radiolabels in wild type and SR-BI Tg mice following gavage

On the same animals as in Figure 5 (A and B), liver and intestinal mucosa (divided in 3 parts) were harvested 6 h after gavage and homogenized as described under “Experimental procedures”. Tissue distribution of [¹⁴C] cholesterol (A) and [³H] radiolabel (B) were determined by scintillation counting. Wild type (closed bar), SR-BI Transgenic mice (open bar). Data are presented as mean ± SEM from 6 mice in each group. Statistical significance between wild type and SR-BI Transgenic was determined by Student’s t test. * p < 0.05

Figure 7. contribution of hepatic SR-BI over-expression to the catabolism of cholesterol

Mice were injected intravenously with 100 μl of intralipid containing 2 μCi [¹⁴C] cholesterol, then biles were collected from 30 min to 120 min (see “Experimental procedures”). [¹⁴C] radiolabel excreted into bile was determined by scintillation counting. Data are presented as the percentage of [¹⁴C] radiolabel in the bile per [¹⁴C] radiolabel in plasma ± SEM from 5–6 mice in each group. Wild type (closed bar), SR-BI Tg (open bar). Statistical significance between wild type and SR-BI Tg was determined by Student’s t test. * p < 0.05