Liver-specific induction of Abcg5 and Abcg8 stimulates reverse cholesterol transport in response to ezetimibe treatment.

OBJECTIVE: Previous studies have shown ezetimibe treatment results in a 2-6-fold increase in reverse cholesterol transport (RCT). However, recent sterol balance studies question the role of biliary sterol secretion in RCT, and challenge the hypothesis that ezetimibe increases RCT through decreased absorption of biliary cholesterol in the intestine. We set out to determine whether ezetimibe may increase RCT by mechanisms that are independent of its well-established inhibition of intestinal cholesterol absorption. METHODS: C57BL/6J, Npc1l1-KO, and/or Abcg8-KO mice were fed a chow diet with or without ezetimibe and fecal [(14)C]-neutral and [(14)C]-acidic sterols were measured to examine macrophage-to-feces RCT. We measured the expression of RCT related genes in the liver and jejunum in these mice. To confirm our significant gene expression findings, we utilized primary human hepatocytes cultured with or without a glucuronated metabolite of ezetimibe. RESULTS: Our studies revealed that treatment with ezetimibe was associated with increased expression of hepatic Abcg5 and Abcg8. Ezetimibe did not directly affect expression in the liver, but this expression was due to the inhibition of intestinal cholesterol absorption. This conclusion was supported by the absence of an ABCG5/ABCG8 expression response to treatment with an ezetimibe metabolite in primary human hepatocytes. Finally, we found that the ezetimibe-dependent stimulation of RCT was attenuated in the absence of Abcg8. CONCLUSIONS: Our study is the first to demonstrate ezetimibe treatment cooperatively stimulated macrophage-to-feces RCT by indirectly increasing liver Abcg5/Abcg8 expression in addition to its known suppression of intestinal cholesterol absorption.

Maternal nutrition induces pervasive gene expression changes but no detectable DNA methylation differences in the liver of adult offspring.

AIMS: Epidemiological and animal studies have shown that maternal diet can influence metabolism in adult offspring. However, the molecular mechanisms underlying these changes remain poorly understood. Here, we characterize the phenotypes induced by maternal obesity in a mouse model and examine gene expression and epigenetic changes induced by maternal diet in adult offspring. METHODS: We analyzed genetically identical male mice born from dams fed a high- or low-fat diet throughout pregnancy and until day 21 postpartum. After weaning, half of the males of each group were fed a high-fat diet, the other half a low-fat diet. We first characterized the genome-wide gene expression patterns of six tissues of adult offspring - liver, pancreas, white adipose, brain, muscle and heart. We then measured DNA methylation patterns in liver at selected loci and throughout the genome. RESULTS: Maternal diet had a significant effect on the body weight of the offspring when they were fed an obesogenic diet after weaning. Our analyses showed that maternal diet had a pervasive effect on gene expression, with a pronounced effect in liver where it affected many genes involved in inflammation, cholesterol synthesis and RXR activation. We did not detect any effect of the maternal diet on DNA methylation in the liver. CONCLUSIONS: Overall, our findings highlighted the persistent influence of maternal diet on adult tissue regulation and suggested that the transcriptional changes were unlikely to be caused by DNA methylation differences in adult liver.

Targeting of Acyl-CoA synthetase 5 decreases jejunal fatty acid activation with no effect on dietary long-chain fatty acid absorption.

BACKGROUND: The absorption of dietary long chain fatty acids (LCFA) largely occurs in the jejunum. LCFA are activated via conjugation with Coenzyme A (CoA), a reaction catalyzed by Acyl-CoA synthetases (ACS). Acyl-CoA sythesis is critical for dietary LCFA absorption; yet, the jejunal ACS enzymes that catalyze the reaction are largely unknown. FINDINGS: High throughput mRNA sequencing of the mouse jejunum revealed that the expression of acyl-CoA synthetase 5 (Acsl5) and fatty-acid transport protein 4 (Fatp4) largely exceeded all other annotated ACS genes that activate LCFA. Interestingly, Acsl5 knockout (KO) mice displayed a decrease of 60% in jejunal total long chain acyl-CoA synthesis rate. Nevertheless, and despite of this decrease, dietary LCFA absorption and body-weight gain in response to high fat diet remained unaffected. CONCLUSION: Acsl5 is a major activator of dietary LCFA, yet in Acsl5 KO mice residual ACS activity is sufficient for maintaining a normal LCFA absorption. Our findings provide further evidence for a robust small intestine LCFA absorption capacity.

Dysregulation of cholesterol homeostasis in human prostate cancer through loss of ABCA1.

Recent epidemiologic data show that low serum cholesterol level as well as statin use is associated with a decreased risk of developing aggressive or advanced prostate cancer, suggesting a role for cholesterol in aggressive prostate cancer development. Intracellular cholesterol promotes prostate cancer progression as a substrate for de novo androgen synthesis and through regulation of AKT signaling. By conducting next-generation sequencing-based DNA methylome analysis, we have discovered marked hypermethylation at the promoter of the major cellular cholesterol efflux transporter, ABCA1, in LNCaP prostate cancer cells. ABCA1 promoter hypermethylation renders the promoter unresponsive to transactivation and leads to elevated cholesterol levels in LNCaP. ABCA1 promoter hypermethylation is enriched in intermediate- to high-grade prostate cancers and not detectable in benign prostate. Remarkably, ABCA1 downregulation is evident in all prostate cancers examined, and expression levels are inversely correlated with Gleason grade. Our results suggest that cancer-specific ABCA1 hypermethylation and loss of protein expression direct high intracellular cholesterol levels and hence contribute to an environment conducive to tumor progression.

Cathepsin B is a novel gender-dependent determinant of cholesterol absorption from the intestine.

We used a mouse C57BL/6J×CASA/Rk intercross to map a locus on chromosome 14 that displayed a gender-dependent effect on cholesterol absorption from the intestine. Studies in congenic animals revealed a complex locus with multiple operating genetic determinants resulting in alternating gender-dependent phenotypic effects. Fine-mapping narrowed the locus to a critical 6.3 Mb interval. Female subcongenics, but not males, of the critical interval displayed a decrease of 33% in cholesterol absorption. RNA-Seq analysis of female subcongenic jejunum revealed that cysteine protease cathepsin B (Ctsb) is a candidate to explain the interval effect. Consistent with the phenotype in critical interval subcongenics, female Ctsb knockout mice, but not males, displayed a decrease of 31% in cholesterol absorption. Although studies in Ctsb knockouts revealed a gender-dependent effect on cholesterol absorption, further fine-mapping dismissed a role for Ctsb in determining the effect of the critical 6.3 Mb interval on cholesterol absorption.

Systematic haplotype analysis resolves a complex plasma plant sterol locus on the Micronesian Island of Kosrae.

Pinpointing culprit causal variants along signal peaks of genome-wide association studies (GWAS) is challenging. To overcome confounding effects of multiple independent variants at such a locus and narrow the interval for causal allele capture, we developed an approach that maps local shared haplotypes harboring a putative causal variant. We demonstrate our method in an extreme isolate founder population, the pacific Island of Kosrae. We analyzed plasma plant sterol (PPS) levels, a surrogate measure of cholesterol absorption from the intestine, where previous studies have implicated 2p21 mutations in the ATP binding cassette subfamily G members 5 or 8 (ABCG5 or ABCG8) genes. We have previously reported that 11.1% of the islanders are carriers of a frameshift ABCG8 mutation increasing PPS levels in carriers by 50%. GWAS adjusted for this mutation revealed genomewide significant signals along 11 Mb around it. To fine-map this signal, we detected pairwise identity-by-descent haplotypes using our tool GERMLINE and implemented a clustering algorithm to identify haplotypes shared across multiple samples with their unique shared boundaries. A single 526-kb haplotype mapped strongly to PPS levels, dramatically refining the mapped interval. This haplotype spans the ABCG5/ABCG8 genes, is carried by 1.8% of the islanders, and results in a striking 100% increase of PPS in carriers. Resequencing of ABCG5 in these carriers found a D450H missense mutation along the associated haplotype. These findings exemplify the power of haplotype analysis for mapping mutations in isolated populations and specifically for dissecting effects of multiple variants of the same locus.

Moderately decreased cholesterol absorption rates are associated with a large atheroprotective effect.

OBJECTIVE: Human treatment with ezetimibe results in a moderate 50% to 54% decrease in cholesterol absorption and a 15% to 20% decrease in plasma LDL-cholesterol levels; nevertheless, the efficacy of ezetimibe therapy has been recently challenged by the ENHANCE trial. We examined the efficacy of a moderate decrease in cholesterol absorption in preventing atherosclerosis formation in the mouse. METHODS AND RESULTS: Congenic 14DKK animals, consisting of a castaneus (CASA/Rk) chromosome 14 interval introgressed onto the C57BL/6J background, displayed a moderate decrease in cholesterol absorption rates. The effect of moderately decreased absorption on atherosclerosis formation was determined in 14DKK apolipoprotein E knockouts (14DKK-apoEKO). When compared to chow diet-fed control apoEKO mice, congenic 14DKK-apoEKO displayed a moderate 41% decrease in cholesterol absorption rates, 30% to 37% decrease in plasma cholesterol levels, and a 70% decrease in atherosclerosis formation. Studies on cholesterol efflux and reverse cholesterol transport (RCT) from 14DKK bone marrow-derived macrophages rejected a 14DKK interval-dependent atheroprotective effects that operate in macrophages. In contrast, 14DKK-apoEKO congenics were characterized by a 60% increase in RCT from peripheral tissue macrophages. CONCLUSIONS: These studies strongly suggest that moderately decreased cholesterol absorption rates result in a large atheroprotective effect attributable to a decrease in plasma cholesterol levels and an increase in RCT from peripheral tissue macrophages.

Cholesterol absorption from the intestine is a major determinant of reverse cholesterol transport from peripheral tissue macrophages.

OBJECTIVE: We examined the effect of ezetimibe, a cholesterol absorption (CA) inhibitor, and genetic determinants of CA on reverse cholesterol transport (RCT) from subcutaneously injected macrophages using a new dual isotope label technique. METHODS AND RESULTS: Treatment of C57BL/6J mice with ezetimibe decreased dietary CA by 86% and increased RCT from peripheral tissue macrophages (PTM) by 6-fold (P<0.0001). Moreover, congenic 14DKK mice with a modest 41% decrease in dietary CA displayed a 67% increase in RCT from PTM (P<0.007). CONCLUSIONS: These findings indicate that pharmacological and genetic modifiers of cholesterol absorption are major determinants of reverse cholesterol transport from peripheral tissue macrophages.

Identification of quantitative trait loci affecting body composition in a mouse intercross.

Gravimetric analysis and dual energy x-ray absorptiometry densitometry were used to determine lean, fat, and bone tissue traits in a F(2) mouse population from a C57BL/6J and CASA/Rk intercross (B6CASAF2). These traits were used in a linkage analysis to identify quantitative trait loci that affect body composition. Linkage mapping showed that body weight (BW) loci on proximal chromosome 2 occurred in the same region as body length, lean tissue mass, and bone mineral content and on chromosome 13 in the same region as lean tissue mass, bone mineral density, and bone mineral content. Fat-related loci occurring on mid-chromosome 2 near 60 cM, proximal chromosome 6, and mid-chromosome 10 were distinct from BW, lean tissue, and bone tissue loci. In B6CASAF2 females, heterozygotes and CASA/Rk homozygotes at the chromosome 6 locus marker had higher body fat percentages, and this locus was responsible for 11% of the variance for body fat percentage. Female heterozygotes and C57BL/6J homozygotes at the chromosome 15 locus marker had higher bone mineral densities, and this locus could explain 8% of that trait's variance. A survey of the literature did not reveal any previous reports of fat-specific loci in the chromosomal 10 region near 42 cM reported in this study. The results of this study indicate that BW and BMI have limited usefulness as phenotypes in linkage or association studies when used as obesity phenotypes.

A complex plasma plant sterol locus on mouse chromosome 14 has at least two genes regulating intestinal sterol absorption.

We previously identified two inbred mouse strains, C57BL/6J and CASA/Rk, with different plasma plant sterol levels. An intercross between these strains revealed a broad plasma plant sterol locus on chromosome 14, which peaked at 17 centimorgan (cM) with a maximum logarithm of the odds score of 9.9. Studies in a chromosome 14 congenic strain, 14KK, with a 4-60 cM CASA/Rk interval on the C57BL/6J background revealed that males, but not females, had decreased plasma plant sterol levels and intestinal cholesterol absorption. In two subcongenic strains, 14PKK and 14DKK, with 4-19.5 and 19.5-60 cM CASA/Rk intervals, respectively, both males and females had decreased plasma plant sterol levels and decreased intestinal cholesterol absorption. Compatible with the decreased plasma plant sterol phenotype, 14PKK mice had increased biliary plant sterol excretion, whereas 14DKK mice did not. Therefore, gender-dependent interactions of genes at the 14PKK and 14DKK intervals are likely to underlie the 14KK interval effect on plasma plant sterol levels and sterol absorption from the intestine. These studies confirm the plasma plant sterol locus on mouse chromosome 14 and provide evidence that there are at least two sets of genes operating: one set affecting intestinal sterol absorption and biliary excretion, and the other set mainly affecting intestinal sterol absorption.

Two loci on chromosome 9 control bile acid composition: evidence that a strong candidate gene, Cyp8b1, is not the culprit.

An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower beta-muricholic acid (betaMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %betaMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38-73 cM (9KK) and 68-73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %betaMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %betaMC was decreased. Sterol 12alpha-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or high-low biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68-73 cM) major determinant in males, and a more proximal (38-68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible.

Bioinformatics tools enabling u-statistics for microarrays.

It is rare that a single gene is sufficient to represent all aspects of genomic activity. Similarly, most common diseases cannot be explained by a mutations at a single locus. Since complex systems tend to be neither linear nor hierarchical in nature, but to have correlated components of unknown relative importance, the assumptions of traditional (parametric) multivariate statistical methods can rarely be justified on theoretical grounds. Empirical "validation" is not only problematic, but also time consuming. Here we demonstrates how bioinformatics tools, ranging from spreadsheets to grids, can enable u-statistics as a non-parametric alternative for scoring multivariate ordinal data. Applications are shown to improve assessment of genetic risk factors, quality control of microarrays and signal value estimation, scoring genomic profiles that best correlated with complex risk factors (cardiovascular diseases), and complex responses to an intervention (treatment of psoriasis).

Phytosterolemia on the island of Kosrae: founder effect for a novel ABCG8 mutation results in high carrier rate and increased plasma plant sterol levels.

Screening of 932 adults on the Pacific island of Kosrae for plasma plant sterol levels disclosed three subjects, two of them asymptomatic, with phytosterolemia. Sequencing the ATP binding cassette subfamily G member 8 (ABCG8) gene revealed a novel exon 2 mutation that causes a change in codon 24 from glutamine to histidine and a frame shift followed by a premature stop codon, precluding the formation of a functional ABCG8 protein. Genotyping of 1,090 Kosraens revealed 150 as carriers, a 13.8% carrier rate. DNA sequencing of 67 carriers revealed the same mutation as in the probands. In carriers, plasma campesterol and sitosterol levels were 55% and 30% higher, respectively, than in noncarriers. Moreover, compared with noncarriers, carriers showed 21% lower plasma levels of lathosterol, a surrogate marker for cholesterol biosynthesis. There was no difference between the groups in plasma total cholesterol, triglycerides, apolipoprotein B, or apolipoprotein A-I levels. In summary, on the island of Kosrae, a strong founder effect of a mutant ABCG8 allele results in a large number of carriers with increased plasma plant sterol levels and decreased lathosterol levels. The latter finding suggests that heterozygosity for a mutated ABCG8 allele results in a modest increase in dietary cholesterol absorption and a decrease in cholesterol biosynthesis.

Cholesterol feeding of mice expressing cholesterol 7alpha-hydroxylase increases bile acid pool size despite decreased enzyme activity.

Dietary cholesterol regulation of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classical pathway of bile acid synthesis, has been implicated in plasma cholesterol responsiveness. In the current study, the effects of 0.0% and 0.5% cholesterol diets were examined in Cyp7a1 knockout (KO), heterozygous Cyp7a1 KO (Het), and human Cyp7a1 transgenic mice on the mouse Cyp7a1 KO background (Tg+KO). We confirmed previous findings that dietary cholesterol increased mouse Cyp7a1 activity in Het mice but decreased human Cyp7a1 activity in Tg+KO mice. However, in both Het and Tg+KO mice, dietary cholesterol increased bile acid pool size (36% and 72%, respectively) and fecal bile acid excretion (2.2- and 3.6-fold, respectively). The expression of cholesterol 27-hydroxylase (Cyp27), the major enzyme of the alternative pathway of bile acid synthesis, was not significantly different in cholesterol-fed KO, Het, or Tg+KO mice. Furthermore, dietary cholesterol had comparable effects on total plasma cholesterol and non-high-density lipoprotein cholesterol in KO, Het, and Tg+KO mice. Thus, in Tg+KO mice, dietary cholesterol regulates bile acid pool size, fecal bile acid excretion, and plasma cholesterol independently of Cyp7a1 activity. These results challenge the notion that dietary cholesterol regulation of Cyp7a1 is a major determinant of plasma cholesterol responsiveness.

Effects of streptozotocin-induced diabetes in apolipoprotein AI deficient mice.

During the past decade a number of investigators have attempted to develop mouse models of diabetic macrovascular disease. Hyperglycemia might increase vascular damage because it increases oxidant stress. For this reason we studied animals that were deficient in HDL; HDL is widely believed to protect against oxidant stress. An inbred line of mice doubly deficient in LDL receptor and apoAI was made diabetic with streptozotocin (STZ); control mice had an average glucose of 7.2+/-2mmol/l and STZ-treated mice had an average glucose of 19.4+/-6.5mmol/l. The animals were fed a high cholesterol but low fat diet leading to plasma cholesterol levels of 9.4+/-1.6mmol/l in control animals and 10.1+/-1.8mmol/l in STZ-treated mice. The control and STZ-treated animals had similar plasma lipoprotein profiles. Atherosclerosis assessed at 23 weeks averaged 38154microm(2) in control and 32962microm(2) in STZ-treated mice. Therefore STZ-induced diabetes does not alter plasma lipoproteins or atherosclerosis in HDL deficient mice.

Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice.

High-cholesterol diets elicit changes in gene expression via such transcription factors as sterol-regulatory element binding proteins (SREBPs) and liver X receptors (LXRs). We used Affymetrix microarrays to identify genes in mouse liver regulated by dietary cholesterol (0.0% vs. 0.5% cholesterol wt/wt). Three independent experiments were performed, and data were analyzed with Affymetrix Microarray Suite and ANOVA statistical software. There were 69 unique Unigene clusters consistently regulated by dietary cholesterol (37 downregulated and 32 upregulated). The array results were confirmed by quantitative RT-PCR (Q-PCR) for seven of nine downregulated genes and five of six upregulated genes. A time course of dietary cholesterol feeding over 1 week revealed different temporal patterns of gene regulation for these confirmed genes. Six downregulated genes were examined in transgenic mice overexpressing truncated nuclear forms of SREBP-1a and SREBP-2, and all were induced in these mice. A second microarray analysis of mice treated with the LXR agonist TO901317 confirmed that 13 of the 32 cholesterol upregulated genes were also LXR-activated. This array result was confirmed by Q-PCR for three of three genes. In summary, these studies identified and confirmed six novel dietary cholesterol-regulated genes, three putative SREBP target genes (calcium/calmodulin-dependent protein kinase 1D, fatty acid binding protein 5, and proprotein convertase subtilisin/kexin 9), and three putative LXR target genes (a disintegrin and metalloprotease domain 11, apoptosis-inhibitory 6, and F-box-only protein 3).

Genetic regulation of cholesterol absorption and plasma plant sterol levels: commonalities and differences.

The molecular basis of the processes that control two closely related traits, the absorption of cholesterol from the intestines and plasma plant sterol levels, are only partially understood. The discovery that mutations in two novel hemitransporters, ATP binding cassette transporter G5 (ABCG5) and ABCG8, underlie a rare inborn error in plant sterol metabolism, beta-sitosterolemia, represents a major breakthrough in this field. More recently, genetic studies in the mouse that mapped loci in linkage with cholesterol absorption and plasma plant sterol levels and studies in humans that examined the relationship of plasma plant sterol levels to sequence variation in the ABCG5/ABCG8 locus suggested the involvement of other genes. Moreover, studies in beta-sitosterolemic patients, in ABCG5/ABCG8-targeted animals, and on a newly developed cholesterol absorption inhibitor, ezetimibe, suggest commonalities and differences in the regulation of the two traits. This review summarizes the evidence for genetic control of cholesterol absorption and plasma plant sterol levels, presents the evidence for commonalities and differences between the two traits, and discusses recent developments and future perspectives in this field.

Localization of the PE methylation pathway and SR-BI to the canalicular membrane: evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol.

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.