Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL

Abstract

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.

Fig. 1.

Analysis of ABCA1 expression and nascent pre-β HDL size distribution from different cell types. A: ABCA1 protein expression by Western blot. Twenty-five micrograms of cell lysate protein isolated from HEK293-FlpIn cells (non-transfected control), HEK293 cells stably transfected with ABCA1, rat McArdle-RH7777 hepatoma cells, and elicited mouse peritoneal macrophages (MPMs) were separated by 4–16% SDS-PAGE. Proteins were then transferred to a nitrocellulose membrane, blotted with rabbit anti-mouse ABCA1 antisera or anti-GAPDH monoclonal antibody, and developed by chemiluminescence using an LAS-3000 imaging system (FujiFilm). Images were quantified using Multi Gauge software and normalized to GAPDH expression. For comparison among cell types, the relative expression of ABCA1 was normalized to McArdle cells. B: Nascent pre-β HDL size distribution determined by 4–30% nondenaturing gradient gel electrophoresis (NDGGE). Nontransfected (HEK293) and stably transfected (HEK293-ABCA1) cells, McArdle-RH7777 cells, and elicited MPMs were incubated with 10 μg/ml of [¹²⁵I]tyramine cellobiose (TC)-apolipoprotein A-I (apoA-I) for 24 h. Aliquots of conditioned media were subfractionated by NDGGE, and nascent HDLs were visualized by phosphorimager analysis. Numbers shown on left side are nm diameter. C: Nascent HDL fractionation by high-resolution fast-protein liquid chromatography (FPLC). [¹²⁵I]TC-apoA-I (10 μg/ml) was incubated for 24 h with the indicated cells, after which the conditioned medium was fractionated by FPLC. Radiolabel in fractions from the FPLC separation was quantified by γ counting and represented a percentage of total count recovered from the column. Vertical dashed lines denote fractions corresponding to pre-β1 to -5 HDL tracers. D: NDGGE and phosphorimager analysis of an aliquot of FPLC-fractionated pre-β1 to -5 HDL tracers generated by incubation of [¹²⁵I]TC-apoA-I with HEK293-ABCA1 cells for 24 h. Numbers above each lane denote pre-β HDL fraction. HMW, high-molecular-weight standard. Numbers shown on left side are nm diameter.

Fig. 2.

Whole-plasma decay, plasma fractional catabolic rate (FCR) and tissue uptake of pre-β HDL tracers in human apolipoprotein A-I transgenic (hA-I ^Tg) mice. [¹²⁵I]TC-pre-β1, -2, -3, and -4 HDLs were injected into hA-I ^Tg mice, and plasma samples were drawn over 24 h, after which animals were euthanized and tissues were harvested and digested to quantify radiolabel uptake. Data represent mean ± SD (n = 4 for all tracers). A: Whole-plasma decay of pre-β HDL tracers in hA-I ^Tg mice. Radioactivity of plasma samples at each time point was quantified and percent of injected radioactivity remaining in the plasma after dose injection was plotted versus time. B: Plasma FCR of pre-β HDL tracers in hA-I ^Tg mice. FCR is expressed as pools/day. Values were calculated using SAAM software and a two-pool model with rate constants from the plasma pool to the liver and kidney, as described in the Methods section. C: Percentage of injected dose recovered in liver and kidney. Liver and kidney were harvested at the end of the study and digested overnight with 1 N NaOH at 60°C. Digested tissues were quantified for ¹²⁵I radioactivity and expressed as percentage of initial dose. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test to ascertain individual differences within a tissue. Bars with different letters are significantly different from one another at P < 0.05.

Fig. 3.

Whole-plasma decay, plasma FCR, and tissue uptake of apoA-I and pre-β1 HDL tracers in hA-I ^Tg mice. [¹²⁵I]TC-apoA-I and pre-β1 HDL tracers were injected into hA-I ^Tg mice (see Fig. 2 legend). A: Whole-plasma decay of apoA-I and pre-β1 HDL tracers in hA-I ^Tg mice. B: Plasma FCR of apoA-I and pre-β HDL tracers in hA-I ^Tg mice. C: Percentage of injected dose recovered in liver and kidney of apoA-I and pre-β1 HDL tracers in hA-I ^Tg mice. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test to ascertain individual differences. Bars with different letters are significantly different from one another at P < 0.05. Error bars indicate SEM.

Fig. 4.

Size distribution of pre-β HDL tracers after intravenous injection into hA-I ^Tg-recipient mice. Plasma samples were harvested at the indicated times after [¹²⁵I]TC-apoA-I and pre-β1, -2, -3, and -4 HDL tracer injection. Plasma samples (15 μl aliquot) were fractionated on 4–30% NDGGE, and radiolabel distribution was visualized using a phosphorimager. A phosphorimager result of one representative recipient mouse for each tracer is shown. HMW protein standard (lane 1) and dose (lane 2) are shown for reference. Lanes 3–9 contain plasma samples taken at 5 min, 30 min, and 1, 2, 3, 5, and 24 h, respectively.

Fig. 5.

In vitro remodeling of pre-β HDL tracers in the presence or absence of LCAT or PLTP. [¹²⁵I]TC-radiolabeled pre-β1 to -4 HDLs were incubated with plasma with or without LCAT (top panel) or PLTP (lower panel). C57Bl/6 mouse plasma was incubated with anti-mouse LCAT antiserum to immunodeplete LCAT or preimmune serum (control) (see Methods section). LCAT immunodepletion was >99% compared with preimmune incubated plasma. Plasma from wild-type (WT) or PLTP^−/− plasma was used to investigate pre-β HDL remodeling in the presence or absence of PLTP. Incubations were performed for 1 h at 4°C or for 5 min and 1 h at 37°C. Aliquots of the incubation mixtures were subjected to 4–30% NDGGE and developed with a phosphorimager. Top panel, incubations ± LCAT; bottom panel, incubations ± PLTP. 1: HMW marker; hydrodynamic size of standard proteins is shown on the left; 2: WT, 4°C, 1 h; 3: LCAT-depleted (−LCAT) or PLTP^−/−, 4°C, 1 h; 4: WT, 37°C, 5 min; 5: −LCAT or PLTP^−/−, 37°C, 5 min; 6: WT, 37°C, 1 h; 7: −LCAT or PLTP^−/−, 37°C, 1 h.

Fig. 6.

In vitro reactivity of pre-β HDL with human LCAT. Individual pre-β HDLs were assembled and isolated (see Fig. 1 legend). An equal amount of pre-β HDL cholesterol (10⁴ dpm) was incubated with 50 ng of purified human recombinant LCAT at 37°C for 1 h (see Methods). The incubation mixtures were then lipid extracted and the percentage of radiolabeled free cholesterol converted to cholesteryl ester was determined. The percentage cholesterol esterification in the absence of added LCAT enzyme (i.e., blank) was measured and subtracted from that measured in the presence of LCAT for each pre-β HDL particle. Results present duplicate incubations, with the horizontal line denoting the average value of the duplicates.