Role of Esrrg in the Fibrate-Mediated Regulation of Lipid Metabolism Genes in Human ApoA-I Transgenic Mice

We have used a new ApoA-I transgenic mouse model to identify by global gene expression profiling, candidate genes that affect lipid and lipoprotein metabolism in response to fenofibrate treatment. Multilevel bioinformatical analysis and stringent selection criteria (2-fold change, 0% false discovery rate) identified 267 significantly changed genes involved in several molecular pathways. The fenofibrate-treated group did not have significantly altered levels of hepatic human APOA-I mRNA and plasma ApoA-I compared with the control group. However, the treatment increased cholesterol levels to 1.95-fold mainly due to the increase in high-density lipoprotein (HDL) cholesterol. The observed changes in HDL are associated with the upregulation of genes involved in phospholipid biosynthesis and lipid hydrolysis, as well as phospholipid transfer protein. Significant upregulation was observed in genes involved in fatty acid transport and β-oxidation, but not in those of fatty acid and cholesterol biosynthesis, Krebs cycle and gluconeogenesis. Fenofibrate changed significantly the expression of seven transcription factors. The estrogen receptor-related gamma gene was upregulated 2.36-fold and had a significant positive correlation with genes of lipid and lipoprotein metabolism and mitochondrial functions, indicating an important role of this orphan receptor in mediating the fenofibrate-induced activation of a specific subset of its target genes.National Institutes of Health (HL48739 and HL68216); European Union (LSHM-CT-2006-0376331, LSHG-CT-2006-037277); the Biomedical Research Foundation of the Academy of Athens; the Hellenic Cardiological Society; the John F Kostopoulos Foundatio

Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor

In view of the evidence linking plasma high density lipoprotein (HDL)-cholesterol levels to a protective effect against coronary artery disease and the widespread use of fibrates in the treatment of hyperlipidemia, the goal of this study was to analyze the influence of fibrates on the expression of apolipoprotein (apo) A-II, a major protein constituent of HDL. Administration of fenofibrate (300 mg/d) to 16 patients with coronary artery disease resulted in a marked increase in plasma apo A-II concentrations (0.34 +/- 0.11 to 0.45 +/- 0.17 grams/liter; P < 0.01). This increase in plasma apo A-II was due to a direct effect on hepatic apo A-II production, since fenofibric acid induced apo A-II mRNA levels to 450 and 250% of control levels in primary cultures of human hepatocytes and in human hepatoblastoma HepG2 cells respectively. The induction in apo A-II mRNA levels was followed by an increase in apo A-II secretion in both cell culture systems. Transient transfection experiments of a reporter construct driven by the human apo A-II gene promoter indicated that fenofibrate induced apo A-II gene expression at the transcriptional level. Furthermore, several other peroxisome proliferators, such as the fibrate, Wy-14643, and the fatty acid, eicosatetraynoic acid (ETYA), also induced apo A-II gene transcription. Unilateral deletions and site-directed mutagenesis identified a sequence element located in the J-site of the apo A-II promoter mediating the responsiveness to fibrates and fatty acids. This element contains two imperfect half sites spaced by 1 oligonucleotide similar to a peroxisome proliferator responsive element (PPRE). Cotransfection assays showed that the peroxisome proliferator activated receptor (PPAR) transactivates the apo A-II promoter through this AII-PPRE. Gel retardation assays demonstrated that PPAR binds to the AII-PPRE with an affinity comparable to its binding affinity to the acyl coA oxidase (ACO)-PPRE. In conclusion, in humans fibrates increase plasma apo A-II concentrations by inducing hepatic apo A-II production. Apo A-II expression is regulated at the transcriptional level by fibrates and fatty acids via the interaction of PPAR with the AII-PPRE, thereby demonstrating the pivotal role of PPAR in controlling human lipoprotein metabolism

Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates.

Epidemiological and transgenic animal studies have implicated apo C-III as a major determinant of plasma triglyceride metabolism. Since fibrates are very efficient in lowering triglycerides, it was investigated whether fibrates regulate apo C-III gene expression. Different fibrates lowered rat liver apo C-III mRNA levels up to 90% in a dose- and time-dependent manner, whereas intestinal apo C-III mRNA remained constant. This decrease in liver apo C-III mRNA was rapid (1 d) and reversible, since it was restored to control levels within 1 wk after cessation of treatment. In addition, fenofibrate treatment abolished the developmental rise of hepatic apo C-III mRNA observed during the suckling-weaning period. Administration of fibrates to rats induced liver and intestinal expression of the acyl CoA oxidase gene, the rate-limiting enzyme for peroxisomal beta-oxidation of fatty acids. In primary cultures of rat and human hepatocytes, fenofibric acid lowered apo C-III mRNA in a time- and dose-dependent manner. This reduction in apo C-III mRNA levels was accompanied by a decreased secretion of apo C-III in the culture medium of human hepatocytes. In rat hepatocytes fenofibric acid induced acyl CoA oxidase gene expression, whereas acyl CoA oxidase mRNA remained unchanged in human hepatocytes. Nuclear run-on and transient transfection experiments of a reporter construct driven by the human apo C-III gene promoter indicated that fibrates downregulate apo C-III gene expression at the transcriptional level. In conclusion, these studies demonstrate that fibrates decrease rat and human liver apo C-III gene expression. In humans the mechanisms appears to be independent of the induction of peroxisomal enzymes. This downregulation of liver apo C-III gene expression by fibrates may contribute to the hypotriglyceridemic action of these drugs

FADS1 FADS2 Gene Cluster, PUFA Intake and Blood Lipids in Children: Results from the GINIplus and LISAplus Studies

BACKGROUND: Elevated cholesterol levels in children can be a risk factor for cardiovascular diseases in later life. In adults, it has been shown that blood lipid levels are strongly influenced by polymorphisms in the fatty acid desaturase (FADS) gene cluster in addition to nutritional and other exogenous and endogenous determinants. Our aim was to investigate whether lipid levels are determined by the FADS genotype already in children and whether this association interacts with dietary intake of n-3 fatty acids. METHODS: The analysis was based on data of 2006 children from two German prospective birth cohort studies. Total cholesterol, HDL, LDL and triglycerides were measured at 10 years of age. Six single nucleotide polymorphisms (SNPs) of the FADS gene cluster were genotyped. Dietary n-3 fatty acid intake was assessed by food frequency questionnaire. Linear regression modeling was used to assess the association between lipid levels, n-3 fatty acid intake and FADS genotype. RESULTS: Individuals carrying the homozygous minor allele had lower levels of total cholesterol [means ratio (MR) ranging from 0.96 (p = 0.0093) to 0.98 (p = 0.2949), depending on SNPs] and LDL [MR between 0.94 (p = 0.0179) and 0.97 (p = 0.2963)] compared to homozygous major allele carriers. Carriers of the heterozygous allele showed lower HDL levels [β between -0.04 (p = 0.0074) to -0.01 (p = 0.3318)] and higher triglyceride levels [MR ranging from 1.06 (p = 0.0065) to 1.07 (p = 0.0028)] compared to homozygous major allele carriers. A higher n-3 PUFA intake was associated with higher concentrations of total cholesterol, LDL, HDL and lower triglyceride levels, but these associations did not interact with the FADS1 FADS2 genotype. CONCLUSION: Total cholesterol, HDL, LDL and triglyceride concentrations may be influenced by the FADS1 FADS2 genotype already in 10 year old children. Genetically determined blood lipid levels during childhood might differentially predispose individuals to the development of cardiovascular diseases later in life

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Studies in mice have shown that PPARalpha is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARalpha in human liver. Here we set out to compare the function of PPARalpha in mouse and human hepatocytes via analysis of target gene regulation

Genome-Wide Association Study of Plasma Polyunsaturated Fatty Acids in the InCHIANTI Study

Polyunsaturated fatty acids (PUFA) have a role in many physiological processes, including energy production, modulation of inflammation, and maintenance of cell membrane integrity. High plasma PUFA concentrations have been shown to have beneficial effects on cardiovascular disease and mortality. To identify genetic contributors of plasma PUFA concentrations, we conducted a genome-wide association study of plasma levels of six omega-3 and omega-6 fatty acids in 1,075 participants in the InCHIANTI study on aging. The strongest evidence for association was observed in a region of chromosome 11 that encodes three fatty acid desaturases (FADS1, FADS2, FADS3). The SNP with the most significant association was rs174537 near FADS1 in the analysis of arachidonic acid (AA; p = 5.95×10−46). Minor allele homozygotes had lower AA compared to the major allele homozygotes and rs174537 accounted for 18.6% of the additive variance in AA concentrations. This SNP was also associated with levels of eicosadienoic acid (EDA; p = 6.78×10−9) and eicosapentanoic acid (EPA; p = 1.07×10−14). Participants carrying the allele associated with higher AA, EDA, and EPA also had higher low-density lipoprotein (LDL-C) and total cholesterol levels. Outside the FADS gene cluster, the strongest region of association mapped to chromosome 6 in the region encoding an elongase of very long fatty acids 2 (ELOVL2). In this region, association was observed with EPA (rs953413; p = 1.1×10−6). The effects of rs174537 were confirmed in an independent sample of 1,076 subjects participating in the GOLDN study. The ELOVL2 SNP was associated with docosapentanoic and DHA but not with EPA in GOLDN. These findings show that polymorphisms of genes encoding enzymes in the metabolism of PUFA contribute to plasma concentrations of fatty acids

Homozygous staggerer (sg/sg) mice display improved insulin sensitivity and enhanced glucose uptake in skeletal muscle

Homozygous staggerer (sg/sg) mice, which have decreased and dysfunctional Ror alpha (also known as Rora) expression in all tissues, display a lean and dyslipidaemic phenotype. They are also resistant to (high fat) diet-induced obesity. We explored whether retinoic acid receptor-related orphan receptor (ROR) alpha action in skeletal muscle was involved in the regulation of glucose metabolism

Identification and validation of the pathways and functions regulated by the orphan nuclear receptor, ROR alpha1, in skeletal muscle

The retinoic acid receptor-related orphan receptor (ROR) alpha has been demonstrated to regulate lipid metabolism. We were interested in the RORα1 dependent physiological functions in skeletal muscle. This major mass organ accounts for ∼40% of the total body mass and significant levels of lipid catabolism, glucose disposal and energy expenditure. We utilized the strategy of targeted muscle-specific expression of a truncated (dominant negative) RORα1ΔDE in transgenic mice to investigate RORα1 signaling in this tissue. Expression profiling and pathway analysis indicated that RORα influenced genes involved in: (i) lipid and carbohydrate metabolism, cardiovascular and metabolic disease; (ii) LXR nuclear receptor signaling and (iii) Akt and AMPK signaling. This analysis was validated by quantitative PCR analysis using TaqMan low-density arrays, coupled to statistical analysis (with Empirical Bayes and Benjamini–Hochberg). Moreover, westerns and metabolic profiling were utilized to validate the genes, proteins and pathways (lipogenic, Akt, AMPK and fatty acid oxidation) involved in the regulation of metabolism by RORα1. The identified genes and pathways were in concordance with the demonstration of hyperglycemia, glucose intolerance, attenuated insulin-stimulated phosphorylation of Akt and impaired glucose uptake in the transgenic heterozygous Tg-RORα1ΔDE animals. In conclusion, we propose that RORα1 is involved in regulating the Akt2-AMPK signaling pathways in the context of lipid homeostasis in skeletal muscle

Interaction Between Marginal Zinc and High Fat Supply on Lipid Metabolism and Growth of Weanling Rats

The impact of a moderate Zn deficiency on growth and plasma and liver lipids was investigated in two 4-week experiments with male weanling rats fed fat-enriched diets. Semisynthetic, approximately isocaloric diets containing 3% soybean oil were supplemented with either 7 or 100 mg Zn/kg diet and with 22% beef tallow (BT) or sunflower oil (SF). In Experiment 1, which compared the dietary fat level and the fat source in a factorial design of treatments, all diets were fed ad libitum to 6 × 8 animals, whereas intake of the high-Zn BT and SF diets was restricted in Experiment 2 (5 × 6 rats) to the level of intake of the respective low-Zn diets. The low-Zn SF diet consistently depressed food intake and final live weights of the animals to a greater extent than the other low-Zn diets, while intake and growth were comparable among the animals fed the high-Zn diets. The marginal Zn deficit per se did not alter plasma triglyceride and cholesterol concentrations nor hepatic concentrations of triglyceride, cholesterol and phospholipids. The fatty acid pattern of liver phospholipids did not indicate that chain elongation and desaturation of fatty acids was impaired by a lack of zinc. It was concluded that dietary energy and fat intake, and fat source have a greater effect on plasma and liver lipids than a moderate Zn deficiency. Marginally Zn-deficient diets enriched with sunflower oil as a major energy source cause a greater growth retardation than diets rich in carbohydrates or beef tallow