ATP-binding Cassette G1 Research Articles

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Adenosine Monophosphate Activated Protein Kinase Regulates ABCG1-Mediated Oxysterol Efflux From Endothelial Cells and Protects Against Hypercholesterolemia-Induced Endothelial Dysfunction

Adenosine monophosphate activated protein kinase (AMPK) has been identified as a regulator of vascular function via the preservation of endothelial cell (EC) function. In this study, we examined whether the beneficial effects of AMPK on ECs are dependent on its involvement in cholesterol efflux and its impact on hypercholesterolemia-induced endothelial dysfunction. Using human aortic ECs and bovine aortic ECs, we show that AMPK activation upregulates ATP binding cassette G1 (ABCG1) expression independently of liver X receptor alpha (LXR alpha) transcriptional activity but through a posttranscriptional mechanism that increases mRNA stability. Using a heterologous system and a luciferase reporter, we further identify that the 3'-untranslated region of the ABCG1 mRNA is responsible for the regulatory effects of AMPK activation. 5-Aminoimidazole-4-carboxamide-1-beta-D-riboside treatment promotes endothelial 7-ketocholesterol efflux and prevents 7-ketocholesterol (7-KC)-induced reactive oxygen species production in an ABCG1-dependent manner, thus preserving endothelial nitric oxide synthase activity and nitric oxide bioavailability. Notably, in vivo studies using C57BL/6J mice receiving a high-cholesterol diet revealed that the infusion of 5-aminoimidazole-4-carboxamide-1-beta-d-riboside increases vascular ABCG1 expression and improves vascular reactivity. These effects are abrogated by the AMPK antagonist compound C and by the vascular gene transfer of ABCG1 small interfering RNA. Our current findings uncover a novel mechanism by which AMPK protects against hypercholesterolemia-mediated endothelial dysfunction.

ATP-binding Cassette Transporter G1 Deficiency Dysregulates Host Defense in the Lung

Mice with genetic deletion of the cholesterol efflux transporter, ATP-binding cassette (ABC) G1, have pulmonary lipidosis and chronic pulmonary inflammation. Whether ABCG1 regulates host defense is unknown. To determine whether ABCG1 regulates pulmonary innate immunity and host defense, and to investigate the underlying molecular/cellular mechanisms. Abcg1(+/+) and Abcg1(-/-) mice were challenged with intrapulmonary lipopolysaccharide (LPS) or Klebsiella pneumoniae, intravenous K. pneumoniae, or intraperitoneal LPS. Phenotypic responses were profiled. Bone marrow chimeras and in vitro assays were used to differentiate and characterize the role of hematopoietic versus nonhematopoietic ABCG1 in host defense. Unexposed Abcg1(-/-) mice had normal numbers of circulating neutrophils, but increased neutrophil recruitment to the airspace and lung parenchyma, and increased airspace cytokines and chemokines in the steady state. After intrapulmonary LPS or K. pneumoniae, Abcg1(-/-) mice displayed exaggerated further neutrophil recruitment to and degranulation in the airspace, and elevated airspace cytokine/chemokine induction. Alveolar macrophage ABCG1 was critical, as ABCG1 deficiency in hematopoietic cells was sufficient to enhance responses in vivo, and Abcg1(-/-) alveolar macrophages adopted a "foam cell" phenotype, and were hyperresponsive ex vivo. Pulmonary compartmentalization and clearance of K. pneumoniae were increased in Abcg1(-/-) mice, indicating enhanced host defense. By contrast, Abcg1(+/+) and Abcg1(-/-) mice had equivalent responses to intravenous K. pneumoniae and intraperitoneal LPS, suggesting that ABCG1 regulates innate immunity in a tissue-selective manner. Abcg1(-/-) mice have an enhanced pulmonary host defense response driven predominantly by hematopoietic cells.

Polyphenols and Cholesterol Efflux

Despite strong evidence for an inverse association between high-density lipoprotein cholesterol (HDL-C) levels and cardiovascular risk,1 successful therapeutic strategies to target HDL have remained elusive. A recent Phase III clinical trial failed to show clinical benefit with the cholesteryl ester transfer protein inhibitor torcetrapib despite markedly increased HDL-C levels.2,3 This outcome reinforced a growing consensus that measurement of HDL-C alone may be an incomplete surrogate for the in vivo functionality of HDL and the clinical efficacy of targeting HDL. The careful mechanistic assessment of HDL function has thus emerged as a potential way forward.4 Macrophage reverse cholesterol transport (RCT), the process by which cholesterol is transported from macrophage “foam” cells to the liver for ultimate fecal excretion, has been postulated to play a major role in HDL-mediated atheroprotection.5 Indeed, quantitative measures of macrophage RCT are more strongly associated with atherosclerosis than plasma HDL-C concentrations in mice.6 The first critical step of macrophage RCT involves efflux of cellular cholesterol to circulating HDL particles. Research in recent years has documented a specific role for the macrophage transporters ATP-binding cassette subfamilies A1 and G1 (ABCA1 and ABCG1) in cholesterol efflux (see the Figure). These findings have stimulated efforts to target the macrophage at the cellular level as a means of enhancing overall RCT. For example, pharmacological agonism of the liver X receptor (LXR) upregulates both ABCA1 and ABCG1 expression,7,8 promotes macrophage cholesterol efflux ex vivo and RCT in vivo, …

PPARγ regulates the expression of cholesterol metabolism genes in alveolar macrophages

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear transcription factor involved in lipid metabolism that is constitutively expressed in the alveolar macrophages of healthy individuals. PPARγ has recently been implicated in the catabolism of surfactant by alveolar macrophages, specifically the cholesterol component of surfactant while the mechanism remains unclear. Studies from other tissue macrophages have shown that PPARγ regulates cholesterol influx, efflux, and metabolism. PPARγ promotes cholesterol efflux through the liver X receptor-alpha (LXRα) and ATP-binding cassette G1 (ABCG1). We have recently shown that macrophage-specific PPARγ knockout (PPARγ KO) mice accumulate cholesterol-laden alveolar macrophages that exhibit decreased expression of LXRα and ABCG1 and reduced cholesterol efflux. We hypothesized that in addition to the dysregulation of these cholesterol efflux genes, the expression of genes involved in cholesterol synthesis and influx was also dysregulated and that replacement of PPARγ would restore regulation of these genes. To investigate this hypothesis, we have utilized a Lentivirus expression system (Lenti-PPARγ) to restore PPARγ expression in the alveolar macrophages of PPARγ KO mice. Our results show that the alveolar macrophages of PPARγ KO mice have decreased expression of key cholesterol synthesis genes and increased expression of cholesterol receptors CD36 and scavenger receptor A-I (SRA-I). The replacement of PPARγ (1) induced transcription of LXRα and ABCG1; (2) corrected suppressed expression of cholesterol synthesis genes; and (3) enhanced the expression of scavenger receptors CD36. These results suggest that PPARγ regulates cholesterol metabolism in alveolar macrophages.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Changes in the expression of cholesterol metabolism-associated genes in HCV-infected liver: A novel target for therapy?

Recent investigations indicate that hepatitis C virus (HCV) infection is closely associated with hepatocytic lipid metabolism and induces hepatic steatosis. However, the actual lipid metabolism in HCV-infected liver has not been extensively investigated in humans. In this study, we evaluated the expression of lipid metabolism-associated genes in patients with HCV infection by real-time PCR. Sterol regulatory element-binding protein (SREBP)-2 expression was unchanged and low density lipoprotein receptor expression was markedly reduced by 90% in HCV-infected liver. The expression of apolipoprotein B100, microsomal triglyceride transfer protein and ATP-binding cassette G5 was significantly increased. Up-regulation of cholesterol synthesis-associated genes, including HMG-CoA reductase, HMG-CoA synthase, farnesyl-diphosphate synthase and squalene synthase, confirmed enhanced de novo cholesterol synthesis. The expression of cholesterol 7alpha-hydroxylase and farnesoid X receptor was enhanced, while bile salt export pump expression was unchanged. Fatty acid synthase expression was increased which was accompanied by increased expression of liver X receptor alpha and SREBP-1c. In summary, the regulation of lipid metabolism was impaired and cholesterol and fatty acid synthesis continued to increase without negative feedback in HCV-infected liver. These changes may be beneficial for HCV replication.

CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel highly expressed in epithelial cells of the gastrointestinal tract. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease characterized by pancreatic insufficiency, fat malabsorption, and steatorrhea. Despite the administration of pancreatic enzymes to normalize malabsorption, CF patients still experienced lipid fecal loss, nutritional deficiencies, and abnormalities in serum lipid profile, suggesting the presence of intrinsic defects in the intestinal handling of nutrients. The objective of the present study was to assess the impact of CFTR gene knockdown on intracellular lipid metabolism of the intestinal Caco-2/15 cell line. Partial CFTR gene inactivation led to cellular lipid accretion of phospholipids, triglycerides, and cholesteryl esters. Likewise, secretion of these lipid fractions was significantly increased following CFTR gene manipulation. As expected from these findings, the output of triglyceride-rich lipoproteins showed the same increasing pattern. Investigation of the mechanisms underlying these changes revealed that CFTR knockdown resulted in raised levels of apolipoproteins in cells and media and microsomal transfer protein activity, two important factors for the efficient assembly and secretion of lipoproteins. Similarly, scrutiny of the enzymatic monoacylglycerol acyltransferase and diacylglycerol acyltransferase, which exhibit dynamic function in triacylglycerol resynthesis and chylomicron formation in enterocytes, revealed a significant augmentation in their activity. Conversely, cholesterol uptake mediated by Niemann-Pick C1 like 1, Scavenger Receptor Class B Type I, and ATP-binding cassette G8 remains unaffected by genetic modification of CFTR. Collectively, these results highlight the role played by CFTR in intestinal handling of lipids and may suggest that factors other than defective CFTR are responsible for the abnormal intracellular events leading to fat malabsorption in CF patients.

LXR and ABCA1 control cholesterol homeostasis in the proximal mouse epididymis in a cell-specific manner

Mammalian spermatozoa undergo important plasma membrane maturation steps during epididymal transit. Among these, changes in lipids and cholesterol are of particular interest as they are necessary for fertilization. However, molecular mechanisms regulating these transformations inside the epididymis are still poorly understood. Liver X receptors (LXRs), the nuclear receptors for oxysterols, are of major importance in intracellular cholesterol homeostasis, and LXR(-/-)-deficient male mice have already been shown to have reduced fertility at an age of 5 months and complete sterility for 9-month-old animals. This sterility phenotype is associated with testes and caput epididymides epithelial defects. The research presented here was aimed at investigating how LXRs act in the male caput epididymidis by analyzing key actors in cholesterol homeostasis. We show that accumulation of cholesteryl esters in LXR(-/-) male mice is associated with a specific loss of ABCA1 and an increase in apoptosis of apical cells of the proximal caput epididymidis. ATP-binding cassette G1 (ABCG1) and scavenger receptor B1 (SR-B1), two other cholesterol transporters, show little if any modifications. Our study also revealed that SR-B1 appears to have a peculiar expression pattern along the epididymal duct. These results should help in understanding the functional roles of LXR in cholesterol trafficking processes in caput epididymidis.

Ligand, receptor, and cell type–dependent regulation of ABCA1 and ABCG1 mRNA in prostate cancer epithelial cells

Recent evidence suggests that the liver X receptor (LXR) is a potential anticancer target in prostate carcinoma. There is little characterization, however, of which of the two LXR isoforms, LXRalpha or LXRbeta, regulates the LXR-responsive genes ATP-binding cassette subfamily members A1 (ABCA1) and G1 (ABCG1) in transformed prostatic epithelial cells. In this study, small interfering RNA (siRNA) was used to determine whether LXRalpha or LXRbeta is involved in regulating ABCA1 and ABCG1 mRNA expression in LNCaP and PC-3 cells. Treatment of both cell lines with the synthetic LXR ligand T0901317 and oxysterols: 25-hydroxycholesterol (25HC) and 24(S), 25-epoxycholesterol (24,25EC), resulted in more than a 10-fold increase of ABCA1 and ABCG1 mRNA expression. Transfection of LNCaP cells with siRNA against either LXRbeta or LXRalpha failed to inhibit T0901317 and 25HC-mediated increase of ABCA1 mRNA. siRNA silencing of LXRbeta did, however, inhibit ABCA1 mRNA expression in 24,25EC-treated LNCaP cells. In contrast, LXRbeta siRNA inhibited T0901317, 25HC, and 24,25EC induction of ABCA1 mRNA in PC-3 cells and ABCG1 mRNA in both LNCaP and PC-3 cells. Additional experiments revealed that T0901317 and 25HC induction of ABCA1 mRNA expression was significantly inhibited by the p38 stress kinase antagonist SB202190 and PKA inhibitor H89. Our study is the first to show that LXRbeta, but not LXRalpha, is the major regulatory isoform of ABCG1 mRNA expression in LNCaP and PC-3 cells. Our study also reveals that ABCA1 gene expression is differentially regulated by synthetic and natural LXR ligands, possibly involving kinase mediated signal transduction.

The state of cholesterol metabolism in the liver of patients with primary biliary cirrhosis: the role of MDR3 expression

Because dyslipidemia, such as hypercholesterolemia, is a characteristic of primary biliary cirrhosis (PBC), hepatic lipid metabolism may be disturbed in PBC patients. We examined the expression of lipid metabolism-associated genes in PBC liver. All of the patients examined were in stage I or II PBC and without medication. RNA was isolated from liver specimens by needle biopsies of PBC patients and controls. The expression levels of various genes were measured by real-time RT-PCR. Multidrug resistance 3 (MDR3) expression was examined immunohistochemically. Statistical correlations between the gene expression levels and indices of blood testing were calculated. The expression levels of sterol regulatory element-binding protein (SREBP) 2 and LDL receptor were significantly lower, and those of apolipoprotein B, microsomal triglyceride transfer protein, ATP-binding cassette G5, and liver X receptor α (LXRα) were significantly higher in the PBC liver than in the normal control liver. The expression levels of bile acid synthesis- and excretion-associated genes did not change, and those of farnesoid X receptor, peroxisome proliferator-activated receptor α, and SREBP-1c were similar between the PBC and normal liver. MDR3 gene expression levels in the PBC liver were more than 4-fold higher than those in the control liver. Immunohistochemically, strong canalicular staining for MDR3 was observed in the PBC liver. LXRα expression was positively correlated with MDR3 levels. Serum levels of γ-glutamyl transpeptidase (GGT) and IgM were negatively correlated with MDR3 levels. Hepatocellular cholesterol metabolism was at least partially disturbed, even in the early stage of PBC. The most characteristic finding was a distinct elevation of MDR3 expression, and the MDR3 levels were negatively correlated with GGT and IgM levels.

Altered expression of genes functioning in lipid homeostasis is associated with lipid deposition in NOD mouse lacrimal gland

Functional atrophy and accompanying lymphocytic infiltration and destruction of the lacrimal gland (LG) are characteristics of Sjögren's Syndrome (SjS). The male NOD mouse is an experimental model for the autoimmune exocrinopathy that develops in the LG of SjS patients. Acinar cells in LG of male NOD mice aged 3–4 months were previously shown to accumulate lipid droplets. In the current study, analysis of lipid components revealed that the accumulated lipids were mostly cholesteryl esters (CE). Gene expression microarray analysis followed by real-time RT-PCR revealed alterations in the expression of several genes involved in lipid homeostasis in LG of 12-week-old male NOD mice relative to matched BALB/c controls. A series of upregulated genes including apolipoprotein E, apolipoprotein F, hepatic lipase, phosphomevalonate kinase, ATP-binding cassette D1 and ATP-binding cassette G1 were identified. Comparison of liver mRNAs to LG mRNAs in BALB/c and NOD mice revealed that the differential expressions were LG-specific. Gene expression profiles were also characterized in LGs of female mice, younger mice and immune-incompetent NOD SCID mice. Investigation of the cellular distribution of Apo-E and Apo-F proteins suggested that these proteins normally coordinate to mediate lipid efflux from the acinar cells but that dysfunction of these processes due to missorting of Apo-F may contribute to CE deposition. Finally, the initiation and extent of lipid deposition were correlated with lymphocytic infiltration in the LG of male NOD mice. We propose that impaired lipid efflux contributes to lipid deposition, an event that may contribute to the development and/or progression of dacryoadenitis in the male NOD mouse.

Cognition, learning behaviour and hippocampal synaptic plasticity are not disrupted in mice over-expressing the cholesterol transporter ABCG1

BackgroundCognitive deficits are a hallmark feature of both Down Syndrome (DS) and Alzheimer's Disease (AD). Extra copies of the genes on chromosome 21 may also play an important role in the accelerated onset of AD in DS individuals. Growing evidence suggests an important function for cholesterol in the pathogenesis of AD, particularly in APP metabolism and production of Aβ peptides. The ATP-Binding Cassette-G1 (ABCG1) transporter is located on chromosome 21, and participates in the maintenance of tissue cholesterol homeostasis.ResultsTo assess the role of ABCG1 in DS-related cognition, we evaluated the cognitive performance of mice selectively over-expressing the ABCG1 gene from its endogenous regulatory signals. Both wild-type and ABCG1 transgenic mice performed equivalently on several behavioral tests, including measures of anxiety, as well as on reference and working memory tasks. No deficits in hippocampal CA1 synaptic plasticity as determined with electrophysiological studies were apparent in mice over-expressing ABCG1.ConclusionThese findings indicate that although ABCG1 may play a role in maintaining cellular or tissue cholesterol homeostasis, it is unlikely that excess ABCG1 expression contributes to the cognitive deficits in DS individuals.

Polymorphisms in ABCG5/G8 transporters linked to hypercholesterolemia and gallstone disease

ATP-binding cassette (ABC) transporters function in the homeostasis of lipids. Dysfunction of ABC transporters is frequently associated with disease. This review examines links between polymorphisms of ABC G5 (ABCG5) and G8 (ABCG8) transporter genes to hypercholesterolemia and to gallstone disease risk. Various polymorphisms (A632V, T400K, D19H, M429V, and C54Y) in the ABCG8 and ABCG5 (Q604E) gene have been found to be associated with several facets of cholesterol metabolism, including baseline cholesterol level, cholesterol kinetics, individual responsiveness of plasma cholesterol to dietary and pharmaceutical interventions for hypercholesterolemia, and increased risk of gallstones. Clearly, the ABCG5 and ABCG8 genes play an important role in cholesterol homeostasis. However, more research is needed to establish how specific polymorphisms of these genes confer to higher risk of these diseases.

Corn fiber oil and sitostanol decrease cholesterol absorption independently of intestinal sterol transporters in hamsters

Objective The aim of this study was to investigate the cholesterol-lowering mechanisms of corn fiber oil (CFO), ferulate phytostanyl esters (FPEs) and parent compounds of FPE, including sitostanol and ferulic acid, in hamsters. Method Seventy male Golden Syrian hamsters were randomly assigned to six experimental diets for 4 weeks: (1) cornstarch–casein–sucrose-based control diet (control); and (2) control diet plus 0.1% (wt/wt) cholesterol (cholesterol–control). The remaining four groups were given cholesterol–control diet with: (3) 10% (wt/wt) CFO; (4) 0.5% (wt/wt) sitostanol; (5) 0.23% (wt/wt) ferulic acid; and (6) 0.73% (wt/wt) FPE. At the end of dietary intervention, total plasma cholesterol, high-density lipoprotein cholesterol and triglyceride concentrations were determined. Parameters of cholesterol kinetics, including cholesterol absorption and synthesis, as well as mRNA expression of sterol transporters such as Niemann–Pick C1 like 1 (NPC1L1), ATP-binding cassette G5 (ABCG5) and ABCG8, were assessed. Results Supplementation with CFO decreased ( P<.0001) plasma total cholesterol levels by 29% as compared with the cholesterol–control group, while FPE and sitostanol reduced ( P<.02) cholesterolemia by 15% and 14%, respectively. CFO and sitostanol decreased ( P<.05) cholesterol absorption by 24% compared to the cholesterol–control group. Dietary intervention did not alter the intestinal gene expression of ABCG5, ABCG8 and NPC1L1. Conclusion The present results show that the CFO-induced and sitostanol-induced decrease in cholesterol absorption is independent of intestinal enterocyte sterol transporters such as ABCG5, ABCG8 and NPC1L1 in hamsters.

ATP binding cassette G8 T400K polymorphism may affect the risk of gallstone disease among Chinese males

BackgroundSupersaturation of bile with cholesterol is a primary step in the formation of cholesterol gallstones. ATP binding cassette (ABC) G5 and G8 play an important role in regulating sterol absorption and secretion. To investigate a possible association between transporter gene polymorphism and gallstone formation, we examined five common polymorphisms in the ABCG5 (Q604E) and ABCG8 (D19H, Y54C, T400K, A632V) genes in patients with gallstone disease (GS). MethodsStudy subjects included 287 patients with GS and 219 gallstone free controls (GSF). Polymorphisms were determined using PCR-RFLP analysis or the Taqman MGB assay. Plasma and biliary lipid levels were measured. Results2 SNPs of ABCG8 gene (Y54C and T400K) showed strong linkage disequilibrium (D'=0.824, r2=0.579). Male carriers of the less frequent K allele of ABCG8 T400K had a 2.31-fold elevated risk [95% confidence interval (CI) 1.12∼4.76, P=0.023] for gallstone disease compared to male with the common genotype after the adjustment for age, body mass index. Males with the K allele had lower plasma triglyceride (P=0.044) and biliary phospholipid (P=0.035) levels than TT homozygotes. No such association was found in female or other 4 SNPs. ConclusionsThese findings indicate that the T400K polymorphism in ABCG8 may be associated with the incidence of gallstone disease in males.

A Growth Hormone-Releasing Peptide that Binds Scavenger Receptor CD36 and Ghrelin Receptor Up-Regulates Sterol Transporters and Cholesterol Efflux in Macrophages through a Peroxisome Proliferator-Activated Receptor γ-Dependent Pathway

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

The ABCG5 Polymorphism Contributes to Individual Responses to Dietary Cholesterol and Carotenoids in Eggs

The ATP binding cassette G5 (ABCG5) polymorphisms have been postulated to play a role in the response to dietary cholesterol. The objective of this study was to examine the contribution of the ABCG5 polymorphism on the plasma response to consumption of cholesterol and carotenoids from eggs. For this purpose, genotyping was conducted for 40 men and 51 premenopausal women who were randomly assigned to consume an egg (EGG, 640 mg/d additional dietary cholesterol and 600 microg lutein+ zeaxanthin) or placebo (SUB, 0 mg/d cholesterol, 0 microg lutein + zeaxanthin) diet for 30 d. The two arms of the dietary intervention were separated by a 3-wk washout period. Plasma concentrations of total cholesterol, LDL cholesterol (LDL-C), and HDL cholesterol were determined. Because eggs are an excellent source of lutein and zeaxanthin, the plasma levels of these carotenoids were also measured in a subset of subjects to determine whether the response to carotenoid intake was similar to that seen for dietary cholesterol and to evaluate the contribution of ABCG5 polymorphism to both responses. Individuals possessing the C/C genotype experienced a greater increase in both LDL-C (P < 0.05) and a trend for lutein (P = 0.08) during the EGG period compared with those individuals with the C/G (heterozygote) or G/G genotypes (homozygotes). These results, although obtained from a small number of subjects, suggest that the ABCG5 polymorphism may play a role in the plasma response to dietary cholesterol and carotenoids.

Cholesterol kinetics and intestinal sterol transporter gene expression in response to corn fiber oil and its constituents in hamsters

The objective of this study was to evaluate the cholesterol-lowering mechanisms of corn fiber oil (CFO), ferulate phytostanyl esters (FPE) and parent compounds including sitostanol and ferulic acid in hamsters. Seventy male golden Syrian hamsters were randomly assigned to six experimental diets for 4 weeks. 1) Control diet without cholesterol 2) Control diet plus 0.1% (w/w) cholesterol. The remaining four groups were given 0.1 % cholesterol diet with: 3) 10% (w/w) CFO 4) 0.5% (w/w) sitostanol, 5) 0.23% (w/w) ferulic acid and 6) 0.73% (w/w) FPE. At the end of four weeks of dietary intervention, plasma cholesterol levels, cholesterol absorption and synthesis as well as mRNA expression of sterol transporters were assessed. Supplementation with corn fiber oil decreased (p< 0.0001) plasma total cholesterol levels by 29% as compared with cholesterol-control, while feruloyl sitostanol (p< 0.02) and sitostanol (p< 0.02) reduced cholesterolemia by 15% and 14% respectively. Moreover, CFO (p ≤ 0.02) and sitostanol (p ≤ 0.02) decreased cholesterol absorption by 24% compared to cholesterol-control group. While only moderate changes in mRNA expression of intestinal enterocyte sterol transporters were observed, CFO and sitostanol showed 1.5- to 2- fold up-regulation of mRNA expression of ATP-binding cassette G5 (ABCG5) and ABCG8. Results of present study suggest that CFO and sitostanol-induced decreases in cholesterol absorption may be due to up-regulation of intestinal enterocyte efflux sterol transporters such as ABCG5 and ABCG8 in hamsters. Funded by NSERC.

Polymorphisms of ATP binding cassette G5 and G8 transporters: their effect on cholesterol metabolism after moderate weight loss in overweight and obese hyperlipidemic women

Polymorphisms of ATP binding cassette G5 and G8 transporters: their effect on cholesterol metabolism after moderate weight loss in overweight and obese hyperlipidemic women