Influence of apolipoprotein A-I and apolipoprotein A-II availability on nascent HDL heterogeneity

Abstract

It is important to understand HDL heterogeneity because various subspecies possess different functionalities. To understand the origins of HDL heterogeneity arising from the existence of particles containing only apoA-I (LpA-I) and particles containing both apoA-I and apoA-II (LpA-I+A-II), we compared the abilities of both proteins to promote ABCA1-mediated efflux of cholesterol from HepG2 cells and form nascent HDL particles. When added separately, exogenous apoA-I and apoA-II were equally effective in promoting cholesterol efflux, although the resultant LpA-I and LpA-II particles had different sizes. When apoA-I and apoA-II were mixed together at initial molar ratios ranging from 1:1 to 16:1 to generate nascent LpA-I+A-II HDL particles, the particle size distribution altered, and the two proteins were incorporated into the nascent HDL in proportion to their initial ratio. Both proteins formed nascent HDL particles with equal efficiency, and the relative amounts of apoA-I and apoA-II incorporation were driven by mass action. The ratio of lipid-free apoA-I and apoA-II available at the surface of ABCA1-expressing cells is a major factor in determining the contents of these proteins in nascent HDL. Manipulation of this ratio provides a means of altering the relative distribution of LpA-I and LpA-I+A-II HDL particles.

Keywords: ATP binding cassette A1; cholesterol; lipoprotein; phospholipid.

Fig. 1.

Production of nascent HDL by HepG2 cells. HepG2 cells were grown to near confluence, labeled with ³H-cholesterol, and then ABCA1 expression was upregulated. Fresh media with no exogenous apolipoproteins was added and cholesterol efflux was allowed to continue for 24 h. Efflux media was collected, filtered, concentrated, and then run over a Superdex 200 column. The radioactivity in 1 ml fractions of the elution buffer was determined.

Fig. 2.

Time and protein concentration dependence of ABCA1-mediated cholesterol efflux from HepG2 cells induced by apoA-I and apoA-II. HepG2 cells were grown to near confluence, labeled with ³H-cholesterol, and then ABCA1 expression upregulated. (A) Cells were incubated with 2.5 µg/ml of apoA-I (filled squares) or apoA-II (open circles) for the indicated times. (B) Cells were incubated with indicated concentrations of apoA-I (filled squares) or apoA-II (open circles) for 4 h, and the resulting data were fitted to the Michaelis-Menten equation.

Fig. 3.

Profile of nascent HDL containing apoA-I (LpA-I). HepG2 cells were grown to near confluence, labeled with ³H-cholesterol, and treated with 9-cis-retinoic acid and 22-hydroxycholesterol. ABCA1-mediated cholesterol efflux was induced by adding 10 µg/ml of apoA-I. Efflux media was collected after 6 h, filtered, concentrated, and then run over a Superdex 200 column. The radioactivity in 1 ml fractions of the elution buffer was determined. ApoA-I (open squares) cholesterol (filled diamonds).

Fig. 4.

Profile of nascent HDL containing apoA-II (LpA-II). HepG2 cells were prepared as described in Fig. 3. ABCA1-mediated cholesterol efflux was induced by adding 10 µg/ml of apoA-II, and the conditioned medium was analyzed as described in Fig. 3. ApoA-II (open circles) cholesterol (filled diamonds).

Fig. 5.

Profile of nascent HDL containing a mixture of apoA-I and apoA-I (LpA-I+A-II). HepG2 cells were prepared as described in Fig. 3, except that some cells were not labeled with ³H-cholesterol. ABCA1-mediated cholesterol efflux was induced by adding a mixture containing 5 µg/ml of apoA-I and 5 µg/ml of apoA-II. The conditioned medium was analyzed as described in Fig. 3. (A) A mixture of 5 µg/ml of ¹⁴C-apoA-I and 5 µg/ml ³H-apoA-II was incubated with cells containing unlabeled cholesterol. (B) A mixture containing 5 µg/ml each of unlabeled apoA-I and apoA-II was incubated with ³H-cholesterol-labeled cells. ApoA-I, filled squares; ApoA-II, open circles; cholesterol, filled diamonds.

Fig. 6.

Profile of nascent HDL generated at different initial molar ratios of apoA-I and apoA-II. HepG2 cells were prepared as described in Fig. 3, except that the cells were not labeled with ³H-cholesterol. ABCA1-mediated cholesterol efflux was induced by adding apoA-I and apoA-II. The conditioned medium was analyzed as described in Fig. 3. (A) 10 µg/ml of ¹⁴C-apoA-I. (B) 10 µg/ml ³H-apoA-II. (C) 1:1 mol:mol mixture of ¹⁴C-apoA-I and ³H-apoA-II. (D) 4:1 mol:mol mixture of ¹⁴C-apoA-I and ³H-apoA-II. For clarity, only the ³H-apoA-II trace is shown in (C) and (D). The solid vertical line at an elution volume of 66 ml corresponds to the LpA-I peak in (A), and the dashed line V_e = 71 ml corresponds to the maximum for LpA-II in (B).