Scavenger receptor class B type I protein as an independent predictor of high-density lipoprotein cholesterol levels in subjects with hyperalphalipoproteinemia

Abstract

Context: In mice, scavenger receptor class B, type I (SR-BI) receptor protein deficiency is associated with elevated high-density lipoprotein (HDL)-cholesterol (HDL-C) levels.

Objective: Our objective was to determine the relationship between SR-BI protein and HDL-C levels in humans.

Design: This was a prospective study of adults with hyperalphalipoproteinemia. Fasting blood was obtained for lipid and lipoprotein measurement, genomic DNA, and monocyte-derived macrophages. SR-BI protein levels were measured by Western blots, and SR-BI activity was measured by cholesteryl ester (CE) uptake of each donor's radiolabeled HDL with their monocyte-derived macrophages, or by degradation and specific cell association of dual-labeled HDL in vitro.

Setting: The study was performed in a tertiary university teaching hospital.

Results: The mean age was 57.2 +/- 10.9 yr (n = 65). SR-BI protein levels were inversely associated with HDL-C levels (P < 0.002), HDL particle size (P < 0.05), and positively associated with CE uptake (P < 0.004); there was no association with plasma apolipoprotein levels. SR-BI protein levels (P = 0.01) were independent predictors of HDL-C levels. Subjects who were carriers of the A allele for the rs4238001 (glycine to serine at position 2) polymorphism [single nucleotide polymorphism (SNP)] had lower SR-BI protein levels (P = 0.01), whereas carriers of the C allele for the rs2278986 SNP also had lower SR-BI protein levels (P = 0.02). Body mass index (P = 0.05), rs4238001 (P = 0.01), and rs2278986 (P = 0.01) SNPs were independent predictors of SR-BI protein levels. In vitro studies of murine macrophages stably expressing the glycine to serine at position 2 SNP showed less degradation (P < 0.0004) and specific cell association (P < 0.0004) of [(125)I, (3)H]-CE-labeled HDL.

Conclusions: SR-BI protein has an independent effect on HDL-C levels in women with hyperalphalipoproteinemia. Two SNPs were significantly associated with lower SR-BI protein levels.

Figure 1

HDL-C is significantly inversely associated with SR-BI protein levels. SR-BI protein levels were measured by Western blotting of cell lysates isolated from MDMs from each subject (n = 43). P = 0.002.

Figure 2

HDL particle size is inversely associated with SR-BI protein levels. HDL particle size was determined by nuclear magnetic resonance spectroscopy (n = 42). P < 0.05.

Figure 3

CE uptake from HDL is positively associated with SR-BI protein levels. HDL isolated from each study subject was radiolabeled with [1,2-³H]cholesteryl oleyl ether, and then incubated with each donor’s respective MDMs (n = 34). P = 0.004.

Figure 4

The association of macrophage SR-BI protein levels with the rs4238001 and rs2278986 SNPs. A, SR-BI protein levels were significantly lower in carriers of the A allele (AA/AG) for the rs4238001 SNP compared with noncarriers (GG) (P = 0.01; r = 0.40; n = 40). B, COS-7 cells transiently transfected with plasmid expressing the rs4238001 or G2S SNP had approximately 37% lower SR-BI protein expression compared with cells transfected with the wild-type (WT) SR-BI plasmid. The Western blot is representative of two independent experiments. C, SR-BI protein levels were significantly lower in carriers of the C allele (CC/TC) for the rs2278986 SNP compared with noncarriers (TT) (P = 0.02; r = 0.40; n = 36).

Figure 5

The rs4238001 (G2S) variant is associated with alterations in SR-BI protein translation and degradation, but not SR-BI mRNA transcription. A, Transcription of SR-BI RNA was similar in rabbit reticulocytes transfected with plasmids expressing either SR-BI wild-type (WT) or GS2 variant. B, Translation of SR-BI protein was reduced in rabbit reticulocytes transfected with plasmids expressing the G2S variant. C, Stably expressing wild-type and G2S variant murine RAW macrophages were incubated with cycloheximide (140 μg/ml) for varying periods of time (0–7 h). SR-BI turnover was significantly greater in G2S cells by 4 and 7 h compared with wild-type cells (P < 0.0007 and P < 0.04, respectively). *, P < 0.0007 and **, P < 0.04 compared to murine RAW macrophages transfected with wild-type SR-BI.

Figure 6

Degradation and cell association of HDL are reduced in murine RAW macrophages stably expressing the SR-BI G2S variant. A, Degradation of [¹²⁵I, ³H]-labeled HDL was significantly lower in G2S expressing cells (P < 0.0004). B, Specific ¹²⁵I and [³H]CE cell association (panel C) were significantly lower in G2S expressing cells (*, P < 0.0004 compared to wild-type).