Abstract 231: ABCG1 Null and SR-BI Null Mice Reduced Plasma HDL Uptake by the Liver and Small Intestine
Aim: To reveal cholesterol transport via non-liver excretion pathway of plasma HDL-cholesterol, we focused on small intestinal and kidney in hyper- and hypo-lipoproteinemia model mice.
Method: Mouse plasma was collected within EDTA coated tubes. Plasma lipoprotein profiles were measured by size exclusion HPLC and plasma preβHDL level was detected by immno-blotting of 2D PAGE by anti-mouse apoA-I peptide polyclonal antibody. Mouse HDL was co-incubated with 3H-cholesteryl oleyl ether (CEt) presence of CETP containing human plasma proteins for 48 hrs. The (100μL) of 3H-CEt-HDL was injected trough tail vein in anesthetized mice. 30μL of blood was collected each time point to chase the radioactivity, Mice organs were harvested after 3 hrs of the injection. Since lack of cellular catabolic pathway, CEt within the cells remained. For the immunohistochemical study, mouse was anesthetized and perfused thoroughly with PBS/5mM EDTA followed by 4% PFA/0.1M PB for the fixation. Leica CM1850 Cryostat was used for sectioning (thickness 16 μm). The sections were incubated in PBS containing 5% skim milk, 0.5% Triton X-100 and 5% inactivated normal horse serum for 3 hrs at room temperature, then incubated with primary antibodies (anti-SR-BI antibody diluted to 1:10,000) for overnight at 4°C. Secondary antibodies (Alexa fluor594) dissolved in the same blocking solution were applied to the sections for 90 min at room temperature. The fluorescence samples were observed by Nikon A1RSi confocal microscopy system.
Results and conclusion: HDL derived CEt in intestinal cells were significantly reduced in ABCG1 null and SR-BI null mice during this experimental period. The other hand, kidney radioactivity showed no difference. Interestingly, preβHDL in SR-BI null mice was reduced by 2D western blotting of anti-mouse apoA-I peptide. Indicates that reduced speed of the plasma apoA-I recycling in SR-BI null mice, it may also cause reduced uptake of HDL-CEt by alternative pathway. Here we show that strong SR-BI-immunoreactivity was also found in the basolateral membrane and even higher intensity in some region. SR-BI located at the basolateral membrane may function as HDL-CE receptor in the small intestine.
Author Disclosures: M. Tsujita: None. N. Akita: None. M. Hossain: None. T. Yokota: None. A.T. Remaley: None. N. Kumamoto: None. T. Ueda: None. S. Ugawa: None. S. Yokoyama: None.
- © 2014 by American Heart Association, Inc.