Annabelle Rodriguez

Intermittent Hypoxia Induces Hyperlipidemia in Lean Mice

Obstructive sleep apnea, a syndrome leading to recurrent intermittent hypoxia (IH), has been associated previously with hypercholesterolemia, independent of underlying obesity. We examined the effects of experimentally induced IH on serum lipid levels and pathways of lipid metabolism in the absence and presence of obesity. Lean C57BL/6J mice and leptin-deficient obese C57BL/6J-Lepob mice were exposed to IH for five days to determine changes in serum lipid profile, liver lipid content, and expression of key hepatic genes of lipid metabolism. In lean mice, exposure to IH increased fasting serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, phospholipids (PLs), and triglycerides (TGs), as well as liver TG content. These changes were not observed in obese mice, which had hyperlipidemia and fatty liver at baseline. In lean mice, IH increased sterol regulatory element binding protein 1 (SREBP-1) levels in the liver, increased mRNA and protein levels of stearoyl–coenzyme A desaturase 1 (SCD-1), an important gene of TG and PL biosynthesis controlled by SREBP-1, and increased monounsaturated fatty acid content in serum, which indicated augmented SCD-1 activity. In addition, in lean mice, IH decreased protein levels of scavenger receptor B1, regulating uptake of cholesterol esters and HDL by the liver. We conclude that exposure to IH for five days increases serum cholesterol and PL levels, upregulates pathways of TG and PL biosynthesis, and inhibits pathways of cholesterol uptake in the liver in the lean state but does not exacerbate the pre-existing hyperlipidemia and metabolic disturbances in leptin-deficient obesity.

Variants in scavenger receptor class B type I gene are associated with HDL cholesterol levels in younger women

Objective:Variants within the scavenger receptor class B type I (SCARB1) receptor gene have been previously associated with lipid levels, especially in women, with some studies reporting the association to be stronger in the presence of diabetes or post-menopausal estrogen use. Based on the reported gender-specific association and modification effect of estrogen on lipid levels according to SCARB1 variants, we explored the relationship between SCARBI single nucleotide polymorphisms (SNPs) and lipid levels in an Amish population to assess sex and age differences. Methods: Eight SCARB1 SNPs, identified from public databases, were genotyped in 919 subjects. Results: Rs5888 and rs3782287 were in high linkage disequilibrium (LD), with r2 > 0.8. None of the SNPs were significantly associated with lipid levels in men; however in women, rs5888 (p = 0.04) and rs5891 (p < 0.001) were significantly associated with higher HDL-C levels. Rs5891 had an allele frequency of 3% and predicts a missense mutation (Ile135Val), which may be functional. Moreover, rs3782287 (p = 0.023) and rs5888 (p = 0.003) were significantly associated with higher HDL-C levels in women younger than 50 years but not in women aged 50 years or older (p for interaction between age and rs5888 = 0.045). None of the SNP effects on HDL-C were modified in the presence of diabetes, in either men or women. Conclusions:SCARB1 SNPs influence HDL-C levels in women, particularly in those less than 50 years old. Condensed Abstract: We assessed associations between SCARB1 SNPs and lipid traits in 919 Amish men and women. Two SNPs, rs3782287 and rs5888, were significantly associated with higher HDL-C levels in women younger than 50 years but not in women aged 50 years or older, supporting an interaction between common sequence variants in SCARB1 and estrogen on HDL-C.

Novel Effects of the Acyl-Coenzyme A:Cholesterol Acyltransferase Inhibitor 58-035 on Foam Cell Development in Primary Human Monocyte–Derived Macrophages

We examined the effect of acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors on intracellular cholesterol stores in primary human monocyte-derived macrophages (HMMs) during foam cell formation. HMMs were exposed to acetylated low density lipoprotein (acLDL, 500 microg protein per mL) with or without 58-035 (1 to 10 microg/mL) or CI-976 (2 microg/mL) for 2 to 48 hours. Total cholesterol (TC) and esterified cholesterol (EC) mass was significantly lower while unesterified cholesterol (UC) increased slightly in cells incubated with acLDL plus ACAT inhibitors. Sterol mass was also measured in cells coincubated with acLDL (500 microg protein per mL) with or without 58-035 (2 microg/mL), high density lipoprotein (HDL, 400 microg protein per mL), or HDL+58-035 for 48 hours. TC and EC were 23% and 55% lower, respectively (P<0.0004), while UC was 11% higher (P<0.04) in cells incubated with acLDL plus 58-035. In contrast, coincubation with HDL alone did not significantly affect TC, EC, or UC mass compared with acLDL alone. The effect of 58-035 could not be explained by cytotoxicity, because adenine release, secreted lactate dehydrogenase, glucose utilization, and cell protein were similar in cells exposed to acLDL regardless of the presence of 58-035. We investigated several potential mechanisms for the decreased TC mass, including increased UC efflux and decreased acLDL binding and uptake. Efflux was measured in cells exposed to [1,2-(3)H]cholesteryl oleate-labeled acLDL, unlabeled control acLDL, and native untreated acLDL (500 microg protein per mL) with or without 58-035 (5 microg/mL) for 24 or 48 hours. UC efflux increased in a time-dependent manner from cells exposed to acLDL plus 58-035 compared with cells exposed to acLDL alone (P<0. 04). High-affinity binding was measured in cells exposed to (125)I-acLDL (5 microg protein per mL) with or without excess unlabeled acLDL (100 or 500 microg protein per mL) for 4 hours at 4 degrees C. Specific acLDL binding, uptake, and total degradation were significantly lower when 58-035 was present during cholesterol enrichment compared with cells exposed to acLDL alone (P<0.001). Unlike the effects of ACAT inhibitors on foam cell formation in rodent macrophages, these compounds lowered TC accumulation in HMMs during foam cell formation by limiting the uptake of acLDL and enhancing UC efflux. They may offer promise as drug therapies for atherosclerosis.

Scavenger Receptor Class B Type I Protein as an Independent Predictor of High-Density Lipoprotein Cholesterol Levels in Subjects with Hyperalphalipoproteinemia

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 donors 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.

Clinical impact of scavenger receptor class B type I gene polymorphisms on human female fertility

BACKGROUND The goal of this study was to evaluate the association of SCARB1 single nucleotide polymorphisms (SNPs) and fertility outcomes in women undergoing IVF. METHODS Between November 2007 and March 2010, granulosa cells and follicular fluid were collected from women undergoing IVF. Five SCARB1 SNPs were sequenced and progesterone levels were measured in the follicular fluid. Fertility measurements were defined as the presence of gestational sac(s) and fetal heartbeat(s). RESULTS The study group consisted of 274 women (mean age of 36.4 ± 4.6 years). The racial/ethnic composition was 55% Caucasian (n = 152), 25% African-American (n = 68), 12% Asian (n = 34), 5% Hispanic, (n = 14) and 2% other (n = 6). There was a significant difference in the genotype frequencies of the SCARB1 SNPs across the groups. Subjects who were homozygous for the minor allele in the rs5888 SNP had higher follicular progesterone levels than those who were homozygous for the major allele (P = 0.03). In the Caucasian group, carriers of the minor A allele of the rs4238001 SNP had lower follicular progesterone levels compared with homozygous carriers of the major G allele (P = 0.04). In this group, follicular progesterone levels were highly predictive of the rs4238001 SNP (P = 0.03). In the entire cohort, minor allele carriers of rs4238001 did not have any viable fetuses at Day 42 following embryo transfers (P = 0.04). In the African-American group in particular, there was also an association between rs10846744 and gestational sac(s) (P = 0.006), and fetal heartbeat(s) (P = 0.005). CONCLUSIONS In part, SCARB1 rs4238001 and rs10846744 SNPs may contribute to human female infertility.

Anti-atherogenic effects of the acyl-CoA:cholesterol acyltransferase inhibitor, avasimibe (CI-1011), in cultured primary human macrophages.

Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to reduce atherosclerotic lesions in animals; however, the mechanism(s) for this effect remains unclear. Therefore, we used cultured primary human monocyte-derived macrophages (HMMs) to examine the effect of the ACAT inhibitor, avasimibe (CI-1011), during foam cell formation and during cholesterol efflux from established foam cells. To examine the effect of CI-1011 on foam cell development, HMMs were incubated with aggregated acetylated LDL (ag-acLDL)+/-CI-1011 for 48 h. Total cholesterol (TC) was 29% lower in HMMs incubated with ag-acLDL and CI-1011 compared with ag-acLDL (P<0.05). To determine if TC reduction was due to reduced ag-acLDL uptake by CI-1011, 125I-acLDL binding at 4 degrees C for 4 h to HMMs preincubated with acLDL or ag-acLDL, CI-1011, acLDL+CI-1011, or ag-acLDL+CI-1011 for 48 h was measured. Specific binding was 40% lower in cells preincubated with acLDL+CI-1011, 52% lower in cells preincubated with ag-acLDL+CI-1011 and 49% lower in cells preincubated with CI-1011 compared with cells preincubated with acLDL (P<0.0003). Because CI-1011 appeared to directly affect acLDL binding, 125I-acLDL (3-80 microg protein/ml) binding was done in HMMs preincubated with CI-1011 (0-10 microg/ml) for 48 h. The calculated B(max) decreased in HMMs exposed to increasing concentrations of CI-1011, suggesting that CI-1011 altered scavenger receptor function and/or number. To examine the effects of CI-1011 on cholesterol efflux from established foam cells, we first examined whether CI-1011 was cytotoxic. HMMs were preincubated with ag-acLDL for 24 h, and then radiolabeled with [14C]adenine for 2 h (time zero). The radiolabeled cells were exposed to control RPMI medium or the same medium+HDL, CI-1011, or HDL+CI-1011 for 24 h. The release of [14C]adenine into the medium was not significantly different between cells exposed to RPMI, HDL, CI-1011, or HDL+CI-1011, suggesting that CI-1011 was not cytotoxic. Foam cells exposed to RPMI and CI-1011 (1-10 microg/ml) for 48 h showed time dependent reduction in cellular TC mass, with a corresponding increase in radiolabeled unesterified cholesterol into the medium. We then asked whether CI-1011 enhanced apoE mediated cholesterol efflux. Although cellular apoE increased between 2- and 7-fold in foam cells compared to control macrophages, apoE secreted into the medium was not significantly different between cells exposed to RPMI or CI-1011. Thus, CI-1011 exerted anti-atherogenic effects by reducing TC accumulation, inhibiting acLDL binding, and by limiting lipid storage in HMMs.

Association of SCARB1 Variants With Subclinical Atherosclerosis and Incident Cardiovascular Disease The Multi-Ethnic Study of Atherosclerosis

Objective—We previously reported a statistically significant association of SCARB1 intronic single nucleotide polymorphism (SNP) rs10846744 with common carotid intimal-medial artery thickness in each of the 4 Multi-Ethnic Study of Atherosclerosis racial/ethnic groups (white, Chinese, black, and Hispanic). Methods and Results—Using an expanded sample of 7936 Multi-Ethnic Study of Atherosclerosis participants, phenotyped for measures of subclinical atherosclerosis, incident myocardial infarction, and cardiovascular disease, and genotyped through the SNP Health Association Resource project, we have now examined the genetic association of these phenotypes with 126 genotyped and imputed SCARB1 SNPs. We also performed stratified analyses to examine whether SCARB1 SNP effects differed by sex. Our analysis of the full Multi-Ethnic Study of Atherosclerosis cohort provides strong evidence for the association of rs10846744 with common carotid intimal-medial thickness (P=1.04E-4 in combined analysis of all 4 Multi-Ethnic Study of Atherosclerosis racial/ethnic groups). In sex-stratified analysis, we observed statistically significant association of rs10846744 with incident cardiovascular disease events in males (P=0.01). Examining analytical results from the Myocardial Infarction Genetics Consortium for replication, we observed further support for the association of rs10846744 with myocardial infarction. Conclusion—The SCARB1 SNP, rs10846744, exerts a major effect on subclinical atherosclerosis and incident cardiovascular disease in humans.

Association of scavenger receptor class B type I polymorphisms with subclinical atherosclerosis: The multi-ethnic study of atherosclerosis

Background—Little is known about the association of scavenger receptor class B type I (SCARB1) single-nucleotide polymorphisms (SNPs) and subclinical atherosclerosis, particularly in subjects of different racial/ethnic backgrounds. We examined this relationship in the Multi-Ethnic Study of Atherosclerosis. Methods and Results—Forty-three SCARB1-tagging SNPs were genotyped. Baseline examinations included fasting lipids and subclinical atherosclerosis phenotypes (coronary artery calcification, common carotid intimal-medial artery thickness [CCIMT], and internal carotid intimal-medial artery thickness). Examining SNP associations with different subclinical atherosclerosis phenotypes across multiple racial/ethnic groups with adjustment for multiple covariates, we found that the C allele of SNP rs10846744 was associated with higher CCIMT in African American (P=0.03), Chinese (P=0.02), European American (P=0.05), and Hispanic participants (P=0.03) and was strongly associated in pooled analyses (P=0.0002). The results also showed that the association of this SNP with CCIMT was independent of lipids and other well-established cardiovascular risk factors. Stratifying by sex, there seemed to be a strong association of rs10846744 with CCIMT in women, but no genotype-sex interactions were observed. Conclusions—Variation in SCARB1 at rs10846744 was significantly associated with CCIMT across racial/ethnic groups in Multi-Ethnic Study of Atherosclerosis.

Deficiency of Scavenger Receptor Class B Type I Negatively Affects Progesterone Secretion in Human Granulosa Cells

Our goal was to examine the effect of deficiency of the lipoprotein receptor, scavenger receptor class B type I (SR-BI), on progesterone secretion in human granulosa cells (HGL5). Scrambled or SR-BI small interfering RNA [knockdown (KD)] cells were exposed to dimethylsulfoxide [DMSO, vehicle for forskolin (Fo)], Fo, serum, high-density lipoprotein, low-density lipoprotein (LDL), or Fo plus lipoproteins or serum for 24 h. Progesterone secretion was lower in all of the SR-BI KD cells regardless of treatment. We examined progesterone secretion in SR-BI KD, LDL receptor KD, and double KD cells incubated with DMSO, Fo, LDL, or Fo + LDL for 6-24 h. As compared with scrambled cells, progesterone secretion was lower in SR-BI and double KD cells regardless of treatment; whereas progesterone secretion was only lower in LDL receptor KD cells incubated with LDL and Fo + LDL. We measured phosphorylation of hormone-sensitive lipase (pHSL) expression, intracellular total cholesterol (TC) mass, and progesterone secretion in scrambled and SR-BI KD cells incubated with DMSO or Fo for 2-24 h. The expression of pHSL was similar between the cells and conditions. The mean change in TC mass and progesterone secretion was lower in SR-BI KD cells exposed to DMSO and Fo. Incubating SR-BI KD cells with 22-hydroxy cholesterol did not overcome the reduction in progesterone secretion. At different time points, RNA expression of steroidogenic acute regulatory protein, side-chain cleavage, and 3β-hydroxysteroid dehydrogenase was significantly lower in SR-BI KD cells incubated with Fo. In conclusion, SR-BI protein deficiency, in part, might explain progesterone deficiency in some infertile women.

Expression of Scavenger Receptor-BI and Low-Density Lipoprotein Receptor and Differential Use of Lipoproteins to Support Early Steroidogenesis in Luteinizing Macaque Granulosa Cells

An ovulatory hCG stimulus to rhesus macaques undergoing controlled ovarian stimulation protocols results in a rapid and sustained increase in progesterone synthesis. The use of lipoproteins as a substrate for progesterone synthesis remains unclear, and the expression of lipoprotein receptors [very-low-density lipoprotein receptor (VLDLR), low-density lipoprotein receptor (LDLR), and scavenger receptor-BI (SR-BI)] soon after human chorionic gonadotropin (hCG) (<12 h) has not been characterized. This study investigated lipoprotein receptor expression and lipoprotein (VLDL, LDL, and HDL) support of steroidogenesis during luteinization of macaque granulosa cells. Granulosa cells were aspirated from rhesus monkeys undergoing controlled ovarian stimulation before or up to 24 h after an ovulatory hCG stimulus. The expression of VLDLR decreased within 3 h of hCG, whereas LDLR and SR-BI increased at 3 and 12 h, respectively. Granulosa cells isolated before hCG were cultured for 24 h in the presence of FSH or FSH plus hCG with or without VLDL, LDL, or HDL. Progesterone levels increased in the presence of hCG regardless of lipoprotein addition, although LDL, but not HDL, further augmented hCG-induced progesterone. Other cells were cultured with FSH or FSH plus hCG without an exogenous source of lipoprotein for 24 h, followed by an additional 24 h culture with or without lipoproteins. Cells treated with hCG in the absence of any lipoprotein were unable to maintain progesterone levels through 48 h, whereas LDL (but not HDL) sustained progesterone synthesis. These data suggest that an ovulatory stimulus rapidly mobilizes stored cholesterol esters for use as a progesterone substrate and that as these are depleted, new cholesterol esters are obtained through an LDLR- and/or SR-BI-mediated mechanism.