A novel role for ABCA1-generated large pre-beta migrating nascent HDL in the regulation of hepatic VLDL triglyceride secretion

Abstract

In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function.

Fig. 1.

siRNA-induced silencing of ABCA1 increases VLDL TG secretion in rat hepatoma cells. A: McA rat hepatoma cells were transfected with either ABCA1 siRNA (0–100 nM) or two control siRNAs for 72 h. Silencing efficiency was assessed by Western blot analysis for ABCA1 expression. All subsequent experiments used 25 nM siRNA to silence ABCA1. B: Control and ABCA1 siRNA transfected McA cells were incubated with radiolabeling medium (10% FBS, 5 μCi/ml ³H-oleate + 0.8 mM oleate) for 24 h. Lipids were extracted from medium and cells, fractionated by TLC, and radiolabeled TG quantified by liquid scintillation spectroscopy. Cellular protein was quantified and radiolabeled TG content was expressed as ³H-TG dpm/μg cell protein (n = 3). C: Conditioned medium (500 µl; pooled, n = 3) from the experiment in B was fractionated by fast protein liquid chromatography and radiolabel in each fraction was determined. VLDL elution position (fractions 30–35) is denoted. D: Control and ABCA1 siRNA transfected McA cells were radiolabeled with [¹⁴C]glycerol (1μCi/ml) and [³H]acetate (5μCi/ml) in the presence of 0.8 mM oleate and cell and medium was harvested at 0, 1, 3, 6, 12, and 24 h. Lipids were extracted from cells and medium and separated into individual lipid classes by TLC, followed by radiolabel quantification in individual lipid classes by liquid scintillation spectroscopy. FC, free cholesterol; TG, triglyceride. ABCA1 siRNA, closed circle and dashed line; control siRNA, open circle and solid line. Results are expressed as mean ± SEM of triplicate wells. * (P < 0.05) and ** (P < 0.01) by Student's t-test. ns, not significant.

Fig. 2.

Silencing of ABCA1 promotes secretion of larger VLDL (i.e., VLDL1) with minimal increase in total apoB secretion. Control and ABCA1 siRNA-transfected McA cells were metabolically radiolabeled with [³⁵S]Met (50μCi/ml) for 4 h in the presence of 0.8 mM oleate. A: Conditioned medium was immunoprecipitated with antibody to apoB. The immunoprecipitated proteins were separated by 4–8% SDS-PAGE and visualized with a phosphorimager. Relative intensities (mean ± range) of apoB100 and apoB48 were quantified and are shown under the image. AU, arbitrary units. B: VLDL from conditioned medium was floated by ultracentrifugation at d = 1.006g/ml and particle size was measured by dynamic laser light scatter. Results are expressed as mean ± SEM of triplicate analyses. ** (P < 0.01). C: [³⁵S]Met radiolabeled conditioned medium was subjected to density gradient ultracentifugation to fractionate the indicated lipoprotein species. ApoB was immunoprecipitated from one ml of individual or pooled fractions, the immunoprecipitated proteins were separated by SDS-PAGE and radiolabel in protein bands was visualized using a phosphorimager. Because fractions 4–6, 7–9, and 10–12 were pooled (i.e., 3 ml total), the volume equivalent for each lane on the gel is indicated below the fraction number. Positions of lipoprotein classes in the gradient are denoted at the top of the gel and migration positions of apoB100 and apoB48 are shown on the right side of the gel image. VLDL1 = S_f 100–400, VLDL2 = S_f 20–100. Images were quantified using a phosphorimager and relative intensities of apoB (B100 + B48) in individual or pooled fractions are presented as a percentage distribution in the gradient. Intensities for the pooled fractions were multiplied by three to correct for the difference in total volume between individual (1 ml) and pooled fractions (3 ml).

Fig. 3.

ABCA1 silencing is associated with decreased activation of PI3 kinase and increased TG secretion. Control and ABCA1 siRNA transfected McA cells were incubated with 0.8 mM oleate for 24 h. A: PI3 kinase activation was determined by Western blot analysis of p-PI3 kinase p85/p55. Data represent mean ± SEM for three samples obtained in two separate experiments. B: Cells were harvested and Western blotted for ABCA1, p-PI3 kinase p85/p55, p-AKT, p-mTOR, MTP, and β-actin (loading control) as indicated. C: Control or ABCA1 siRNA transfected McA cells were incubated with vehicle (DMSO), LY294002 (10 μM), or wortmannin (100 nM) as indicated for 1 h before addition of radiolabeling medium ([³H]oleate (5 μCi/ml) + 0.8 mM oleate). After additional 12 h incubation, lipids were extracted from medium and radiolabel incorporation into TG was quantified by TLC. Data (mean ± SEM, n = 3–4) were normalized to cellular protein content. ** (P < 0.01) by Student's t-test; ns, not significant.

Fig. 4.

Silencing of ABCA1 results in decreased formation of large pre-β HDL concomitant with increased TG secretion. Control and ABCA1 siRNA transfected McA cells were incubated with [³H]oleate (5 µCi/ml) and [¹²⁵I]apoA-I (10 µg/ml; 10⁵ cpm/µg) in the presence of 0.4 mM oleate in serum-free medium for 24 h. A: ABCA1 expression levels were assessed by Western blot analysis (top). Aliquots of conditioned medium were lipid extracted, lipids (PL, TG, and CE) were separated by TLC, and [³H]oleate incorporated into the lipid fractions was determined (mean ± SEM, n = 4). B: Conditioned media and lipid-free [¹²⁵I]apoA-I were separated on agarose gels and radioactivity was visualized using a phosphorimager. Human HDL and LDL were used as markers of α and β migration. C: Aliquots (15µl) of conditioned medium were fractionated by NDGGE and nascent HDL particles were visualized using a phosphorimager. Nascent pre-β migrating HDL formation was quantified by phosphorimager analysis and normalized to control siRNA transfected McA cells for each pre-β HDL fraction. Results are expressed as mean ± SEM of triplicate gels. Image is representative data from three independent experiments. * (P < 0.05), ** (P < 0.01), and *** (P < 0.001) by Student's t-test.

Fig. 5.

Increased TG secretion in ABCA1 silenced McA cells is attenuated by nascent HDL-containing conditioned medium. Conditioned McA medium (+ McA CM) was prepared by incubating 10 μg/ml of apoA-I with McA cells for 24 h; nonconditioned medium (− McA CM) was prepared by incubating the same concentration of apoAI in empty dishes. Conditioned or nonconditioned medium was then transferred to McA cells that had been previously transfected with control or ABCA1 siRNA and cells were then stimulated with 0.4 mM oleate in serum-free radiolabeling medium (containing 5 μCi/ml [³H]oleate) for an additional 12 h. Cells were harvested for Western blot analysis (A) and the medium was harvested to determine the extent of [³H]oleate incorporation into secreted TG (B). A: ABCA1 and p-PI3K p85/p55 expression by Western blot analysis. NS, non-specific band used as load control. B: [³H]TG secretion from control or ABCA1 siRNA-treated McA cells after incubation with conditioned medium from McA cells or nonconditioned medium for 12 h. Results are representative of two separate experiments and are expressed as mean ± SEM of triplicate analyses. * (P < 0.05), ** (P < 0.01), *** (P < 0.001) by Student's t-test.

Fig. 6.

Large nascent HDLs attenuate the increased TG secretion induced by ABCA1 silencing. A: Conditioned medium was prepared by incubating 10 μg apoA-I /ml with control and ABCA1-expressing HEK 293 cells for 24 h. Nonconditioned medium (−CM) was prepared by incubating 10 µg/ml of apoA-I with empty dishes for 24 h. The conditioned or nonconditioned medium along with [³H]oleate + 0.4 mM oleate was then added to McA cells that had been previously transfected with control or ABCA1 siRNA (25 nM for 48 h). After additional 12 h incubation, [³H]TG secretion into the medium was quantified as described in Fig. 1 legend. B: Conditioned medium from ABCA1-expressing HEK293 cells was prepared as described in A and fractionated by high resolution FPLC into individual subfractions of nascent HDL as described previously (8). McA cells that had been previously transfected with control or ABCA1 siRNA (25 nM for 48 h) were incubated with individual nascent HDL subfractions (2 µg protein/ml) in the presence of [³H]oleate + 0.8 mM oleate for an additional 12 h and the amount of newly synthesized TG secreted into the medium was quantified. Results are normalized to control siRNA transfected cells and are expressed as mean ± SEM of triplicate analyses. ** (P < 0.01), *** (P < 0.001); ns, not significant, by one-way ANOVA (Tukey's multiple comparison test).

Fig. 7.

Large rHDL, but not hHDL, attenuate TG secretion in ABCA1-silenced McA cells. A: Small (rHDL_sm) and large (rHDL_lg) recombinant HDL and human HDL were characterized for size by NDGGE (left) and electrophoretic mobility by agarose gel electrophoresis (right). Proteins were visualized by Coomassie Brilliant Blue G-250 staining. B: Control or ABCA1 siRNA transfected (25 nM for 48 h) McA cells were incubated with 10µg protein/ml of either lipid-free apoA-I (LF-AI), rHDL, or hHDL in the presence of [³H]oleate + 0.8 mM oleate for 12 h and the amount of newly synthesized TG secreted into the medium was quantified. C: Cellular protein from the experiment in B was harvested and ABCA1 and p-PI3K p85/p55 expression was examined by Western blot analysis. Western blot data for control siRNA rHDL incubations were similar to that for the lipid-free apoA-I incubation and are not shown. ** (P < 0.01), *** (P < 0.001); ns, not significant, by one-way ANOVA (Tukey's multiple comparison test).

Fig. 8.

Summary diagram of experimental results. In the presence of active ABCA1 (left), lipid free apoA-I is secreted by hepatocytes and lipidated by hepatocyte ABCA1, forming nascent HDL particles. Large nascent HDLs (>10 nm diameter) bind to a putative receptor (Topβ, target of pre-β), which in turn stimulates PI3 kinase activation and results in reduced lipid mobilization and secretion of normal-sized VLDL particles (i.e., VLDL2). In contrast, in the absence of hepatic ABCA1 or diminished ABCA1 activity (right), newly secreted lipid-free apoA-I fails to form large nascent HDL particles. The absence of large nascent HDL particle formation leads to diminished signaling through Topβ, resulting in reduced PI3 kinase activation, increased lipid mobilization, and increased secretion of larger TG-enriched VLDL particles (VLDL1).