Abstract 172: Characterization and Comparison of High-Density Lipoprotein Subclasses Among Inbred Strains of Mice

Abstract

Aim: Measurement of HDL subclasses provides insight into lipoprotein metabolism and helps predict risk for cardiovascular disease (CVD). HDL subclasses have been studied using biochemical tools in humans, but extensive studies on the role of HDL subclasses and their protein compositions in HDL metabolism have not been conducted using an animal model. In this study, we established a method to identify HDL subclasses in mice and compared HDL subclasses and the protein cargoes associated with them in three inbred strains of mice. Methods: We used a chemical precipitation method described by Warnick et al. in 1982, to identify HDL subclasses. In this method, LDL and VLDL are precipitated out from the serum using dextran sulfate and magnesium chloride solutions. Subsequently, HDL subclass distribution on gels was compared among male mice from 3 inbred strains with low (CAST/EiJ), normal (C57BL/6J) and high (NZB/BINJ) levels of HDL cholesterol (HDL-C). HDL subclasses were visualised by Coomassie Staining and Mass Spectrometry (MS) was done to confirm the presence of ApoA1, a constituent protein of HDL. Results: We observed that CAST/EiJ and NZB/BINJ had significantly different patterns of distribution of HDL subclasses compared to C57BL/6J mice. Furthermore, HDL subclasses from C57BL/6J and NZB/BINJ mice were subjected to quantitative MS. 106 proteins were associated with HDL particles in both strains, 47 proteins were unique to C57BL/6J HDL particles and 7 were unique to NZB/BINJ HDL particles. Using the Ingenuity Pathways Analysis we found that 30 molecules (27%) in NZB/BINJ and 33 (22%) in C57BL/6J mice had well characterized roles in lipid metabolism. 22 proteins that were previously identified as HDL associated in humans were identified in mice as well and 38 proteins were identified to be associated exclusively with mouse HDL in this study. Conclusion: Protein cargoes associated with HDL define its biological function. Thus, differences in HDL subclasses and HDL-associated proteins could potentially affect HDL function in these mouse-inbred strains, which ultimately could influence the development of CVD. Figure: HDL subclasses present in 10-week old C57BL/6J mice (gel was stained with Coomassie Dye to visualize the subclasses; MS was done to confirm presence of ApoA1, a constituent protein of HDL)