ABCA1-Derived Nascent High-Density Lipoprotein-Apolipoprotein AI and Lipids Metabolically Segregate

Abstract

Objective: Reverse cholesterol transport comprises cholesterol efflux from ABCA1-expressing macrophages to apolipoprotein (apo) AI, giving nascent high-density lipoprotein (nHDL), esterification of nHDL-free cholesterol (FC), selective hepatic extraction of HDL lipids, and hepatic conversion of HDL cholesterol to bile salts, which are excreted. We tested this model by identifying the fates of nHDL-[³H]FC, [¹⁴C] phospholipid (PL), and [¹²⁵I]apo AI in serum in vitro and in vivo.

Approach and results: During in vitro incubation of human serum, nHDL-[³H]FC and [¹⁴C]PL rapidly transfer to HDL and low-density lipoproteins (t_1/2=2-7 minutes), whereas nHDL-[¹²⁵I]apo AI transfers solely to HDL (t_1/2<10 minutes) and to the lipid-free form (t_1/2>480 minutes). After injection into mice, nHDL-[³H]FC and [¹⁴C]PL rapidly transfer to liver (t_1/2=≈2-3 minutes), whereas apo AI clears with t_1/2=≈460 minutes. The plasma nHDL-[³H]FC esterification rate is slow (0.46%/h) compared with hepatic uptake. PL transfer protein enhances nHDL-[¹⁴C]PL but not nHDL-[³H]FC transfer to cultured Huh7 hepatocytes.

Conclusions: nHDL-FC, PL, and apo AI enter different pathways in vivo. Most nHDL-[³H]FC and [¹⁴C]PL are rapidly extracted by the liver via SR-B1 (scavenger receptor class B member 1) and spontaneous transfer; hepatic PL uptake is promoted by PL transfer protein. nHDL-[¹²⁵I]apo AI transfers to HDL and to the lipid-free form that can be recycled to nHDL formation. Cholesterol esterification by lecithin:cholesterol acyltransferase is a minor process in nHDL metabolism. These findings could guide the design of therapies that better mobilize peripheral tissue-FC to hepatic disposal.

Keywords: apolipoprotein AI; atherogenesis; cholesterol; lipids; lipoprotein.

Figure 1

SEC Isolation of nHDL. nHDL was collected from the media after apo AI incubation of BHK cells over expressing ABCA1 with no FC-loading (A, 0 CDX) and with FC-loading with 2.5 mM (B) and 5.0 mM CDX (C) and analyzed by SEC. Grey-filled plots are the A280 SEC of the media; red bars denote the fractions from media that were collected and pooled as large (L), medium (M) and small (S) nHDL. Analytical A280 SEC of the pooled fractions L, M and S are shown in the chromatograms labeled a, b and c respectively. Non-denaturing PAGE (D) and SDS-PAGE (E) of the L, M and S pooled fractions from each of the three cell cholesterol loading conditions as labeled. Molecular weight standards are in the left lanes, and apo AI and BSA are as labelled.

Figure 2

Transfer of nHDL-[3H]FC to Serum Lipoproteins. nHDL-[3H]FC was prepared from BHK-ABCA1 cells labeled with [3H]FC as in Figure 1. nHDL-[3H]FC were tested under several conditions and analyzed by SEC, which was monitored by β-counting of the collected fractions. A) nHDL-[3H]FC; B) nHDL-[3H]FC incubated with HDL at 4 °C and immediately analyzed by SEC (0 min). C) nHDL-[3H]FC incubated with serum at 4 °C or 37oC for various times as labeled and analyzed by SEC. D) Kinetics of [3H]FC accumulation in LDL (fractions encompassed by grey box in Panel C). E) The kinetics of nHDL-[3H]FC transfer to isolated LDL determined by heparin-Mn+2 precipitation of LDL.

Figure 3

Transfer of nHDL-[14C]PL to Serum Lipoproteins: A) Control nHDL-[14C]PL, no serum (O). B) nHDL-[14C]PL was incubated with serum at 37 °C for various times (as labeled) and separated by SEC after which collected aliquots were β-counted (●). The grey rectangle circumscribes the regions of LDL-[14C]PL that were summed to calculate kinetics of nHDL-[14C]PL transfer to LDL, which is shown in Panel C.

Figure 4

Transfer of nHDL-[125I]Apo AI to Serum Lipoproteins. A) SEC Profile of nHDL-[125I]apo AI (O). B) SEC of [125I]Apo AI(●) and HDLA280 nm (grey fill). C–K) SEC profiles of [125I]apo AI after incubation with serum at various temperatures and times as labeled.

Figure 5

Distributions of nHDL-[3H]FC, [14C]PL, and [125I]apo AI into Serum Lipoproteins and Lipid-Poor Apo AI. Bars represent the percent distribution of each radio-analyte from Figures 2–4 at 2 h. The 24 h values for [125I]apo AI are shown as a red line above the 2 h bar.

Figure 6

nHDL-FC, PL and Apo AI Kinetics for Plasma Clearance and Hepatic Uptake in Mice. Plasma and tissue were collected and analyzed at various times following the injection of nHDL-[3H]FC, -[14C]PL, and -[125I]Apo AI; details are in Material and Methods. A – C) Plasma decay plots for each analyte. Data are plotted as smoothed curves with the ordinates adjusted to the same scale for comparison. For C, data were fitted to a four-parameter bi-exponential equation having the form, Weight-Normalized Plasma DPM = ae-k1t + be-k2t where t is time, k1 and k2 are rate constant, and a and b are their respective pre-exponential constants that reflect injected dose. D – E) Hepatic uptake plots for each analyte. Inserts in D and E show the kinetics for the initial (0 – 30 min) rapid hepatic uptake of [3H]FC and [14C]PL.

Figure 7

Comparison of the rates of Hepatic nHDL- and HDL-[14C]PL Uptake In Vitro. nHDL- and HDL-[14C]PL (20 µg/mL protein) were incubated with Huh7 cells in the presence or absence of PLTP (10 µg/mL) and the cell-associated radioactivity determined at various times as shown. Additional details are provided in the Materials and Methods. Slopes in panel B were different, p = 0.0016. Slopes in the other panels were not significantly different.

Figure 8

Model of nHDL Remodeling and Metabolism In Vivo. Cells extrude FC and PL via the interaction of apo AI with ABCA1 giving nHDL (1). nHDL-FC, PL, and apo AI rapidly transfer to HDL, t1/2 < 2 min (2); concurrently nHDL-FC and PL transfer to LDL (3). Over the same time frame, nHDL- and HDL-FC and PL transfer mainly to the liver (4, 5, 6) while some nHDL-apo AI is recycled to ABCA1 (7). Over time, FC and PL equilibrate among erythrocytes (8), extrahepatic tissues, and lipoproteins, which achieve a steady state concentration that is the target of enzymes, transfer proteins, and hepatic receptors. nHDL- and HDL-FC and PL accretion in the liver occurs via spontaneous transfer and SR-B1 (4, 5, 6), with the latter being promoted by PLTP (4). Symbols for FC, PL, and apo AI are labeled as shown in the legend at the top of the figure.