ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

Abstract

HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

Figure 1

Amplification and identification of Ad-ATPase-B1 in HEK293A cells. Ad-GFP: adenovirus GFP; Ad-ATPase-B1: recombinant adenovirus ATP synthase β-chain. (a) Exogenous ATPase-B1 mRNA level in HEK293A cells with no vector (BLANK), pretreated with Ad-GFP and pretreated with Ad-ATPase-B1 and (b) ATPase-B1 protein detected with anti-V5-tag antibody (1 : 2000), GAPDH (1 : 1000).

Figure 2

Immunofluorescent confocal microscopic analysis of ATPase-B1 expression in primary hepatocytes. Freshly isolated primary hepatocytes grown on cover slips were pretreated with Ad-ATPase-B1 (b) and Ad-GFP (c) at 30 MOIs for 48 h. After washing with PBS, the cells were immunostained with antibody against V5-tag and AlexaFluor 596-conjugated goat anti-mouse IgG and analyzed by confocal microscope.

Figure 3

Multiplicity of infection (MOI) test in HepG2 cells and primary hepatocytes. HepG2 cells (a) and the isolated primary hepatocytes were pretreated with Ad-ATPase-B1 and Ad-GFP with different MOIs, and ATPase-B1 mRNA (b) and protein ((c) A, ATPase-B1 expression in WT primary hepatocytes; B, ATPase-B1 expression in SR-BI−/− primary hepatocytes; C, the internal control of GADPH in WT and SR-BI−/− primary hepatocytes) levels were measured. In HepG2 cells and WT hepatocytes, mRNA expression increased with increased MOI, while mRNA expression peaked at 30 MOIs in SR-BI−/− mouse.

Figure 4

Analysis of DiI-labeled HDL uptake in freshly isolated primary hepatocytes (200x). Primary hepatocytes were pretreated with Ad-ATPase-B1 and Ad-GFP at 30 MOI for 48 h and incubated with 25 μg DiI-HDL for 2 h. (a) Concentration of DiI-HDL and HDL was determined by the dying method with Coomassie brilliant blue. (b) DiI-HDL was detected by fluorescence microscopy (200x) and cellular fluorescent intensities were quantified.

Figure 5

Verification of ATP-B1 expression in the livers of WT and SR-BI−/− mice by RT-PCR (a) and Western blotting ((b) A, ATPase-B1 expression in WT mouse liver; B, ATPase-B1 expression in SR-BI−/− mouse livers; C and D, the internal control of GADPH) after 7 days injection of Ad-ATPase-B1 and Ad-GFP. (**P < 0.001; n = 4, 10).

Figure 6

Plasma lipid levels in WT and SR-BI−/− mice. Before injection, plasma was collected after a 4 h fast. Plasma triglyceride (a), total cholesterol (b), and HDL-C (c) levels were detected.

Figure 7

Plasma triglyceride (a), total cholesterol (b), and HDL-C (c) levels in WT and SR-BI−/− mice 7 days after administration of Ad-ATPase-B1 and Ad-GFP.

Figure 8

Plasma lipoprotein profiles. Seven days after Ad-ATPase-B1 and Ad-GFP administration, mice were fasted for 4 h and euthanized. Aliquots (250 μL) of plasma pooled from each group of mice were fractionated by column gel filtration chromatography. Fractions (500 μL) were eluted and assayed for TG (a) and cholesterol (b) levels.