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Polymorphisms of the human Delta-5 (FADS1) and Delta-6 (FADS2) desaturase genes have been recently described to be associated with the level of several long-chain n-3 and n-6 polyunsaturated fatty acids (PUFAs) in serum phospholipids. We have genotyped 13 single nucleotide polymorphisms (SNPs) located on the FADS1-FADS2-FADS3 gene cluster (chromosome 11q12-13.1) in 658 Italian adults (78% males; mean age 59.7 +/- 11.1 years) participating in the Verona Heart Project. Polymorphisms and statistically inferred haplotypes showed a strong association with arachidonic acid (C20:4n-6) levels in serum phospholipids and in erythrocyte cell membranes (rs174545 adjusted P value for multiple tests, P < 0.0001 and P < 0.0001, respectively). Other significant associations were observed for linoleic (C18:2n-6), alpha-linolenic (C18:3n-3) and eicosadienoic (C20:2n-6) acids. Minor allele homozygotes and heterozygotes were associated to higher levels of linoleic, alpha-linolenic, eicosadienoic and lower levels of arachidonic acid. No significant association was observed for stearidonic (C18:4n-3), eicosapentaenoic (C20:5n-3) and docosahexaenoic (C22:6n-3) acids levels. The observed strong association of FADS gene polymorphisms with the levels of arachidonic acid, which is a precursor of molecules involved in inflammation and immunity processes, suggests that SNPs of the FADS1 and FADS2 gene region are worth studying in diseases related to inflammatory conditions or alterations in the concentration of PUFAs. Show less

The molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novo synthesis by the mammary gland. Marked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (LPL, CD36), intracellular FA trafficking (FABP3), long-chain (ACSL1) and short-chain (ACSS2) intracellular FA activation, de novo FA synthesis (ACACA, FASN), desaturation (SCD, FADS1), triacylglycerol synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (BTN1A1, XDH), ketone body utilization (BDH1), and transcription regulation (INSIG1, PPARG, PPARGC1A). Change in SREBF1 mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was < or =2-fold in magnitude, while expression of INSIG1, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to PPARGC1A. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of ABCG2 during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured. A network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that SREBF1 is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1. Expression of SCD, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., SCD). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation. Show less

The delta-5 and delta-6 desaturases, encoded by FADS1 and FADS2 genes, are key enzymes in polyunsaturated fatty acid (PUFA) metabolism that catalyze the conversion of linoleic acid (LA) into arachidonic acid (AA) and that of alpha-linolenic acid (ALA) into eicosapentaenoic acid (EPA). Single-nucleotide polymorphisms (SNPs) in FADS1 and FADS2 have been associated with different concentrations of AA and LA, and those associations have possible functional consequences for desaturase activity. We aimed to evaluate the possible association among FADS genotypes, desaturase activity, inflammation, and coronary artery disease (CAD). Thirteen FADS SNPs and the ratio of AA to LA (AA/LA) on red blood cell (RBC) membranes, a marker of desaturase activity, were evaluated in 876 subjects with (n = 610) or without (n = 266) angiographically documented CAD. Both AA/LA and the ratio of EPA to ALA (EPA/ALA) were higher in patients with CAD than in those without CAD, but, in a multiple logistic regression model, only a higher AA/LA resulted an independent risk factor for CAD (odds ratio: 2.55; 95% CI: 1.61, 4.05 for higher compared with lower ratio tertile; P for trend < 0.001). Furthermore, concentrations of high-sensitivity C-reactive protein increased progressively across tertiles of AA/LA. Graded increases in high-sensitivity C-reactive protein concentrations and CAD risk were related to the carriership of FADS haplotypes, including the alleles associated with a higher ratio. In populations following a Western diet, subjects carrying FADS haplotypes that are associated with higher desaturase activity may be prone to a proinflammatory response favoring atherosclerotic vascular damage. Show less

Starvation triggers a complex array of adaptative metabolic responses including energy-metabolic responses, a process which must imply tissue specific alterations in gene expression and in which the liver plays a central role. The present study aimed to describe the evolution of global gene expression profiles in liver of 4-week-old male chickens during a 48 h fasting period using a chicken 20 K oligoarray. A large number of genes were modulated by fasting (3532 genes with a pvalue corrected by Benjamini-Hochberg < 0.01); 2062 showed an amplitude of variation higher than +/- 40% among those, 1162 presented an human ortholog, allowing to collect functional information. Notably more genes were down-regulated than up-regulated, whatever the duration of fasting (16 h or 48 h). The number of genes differentially expressed after 48 h of fasting was 3.5-fold higher than after 16 h of fasting. Four clusters of co-expressed genes were identified by a hierarchical cluster analysis. Gene Ontology, KEGG and Ingenuity databases were then used to identify the metabolic processes associated to each cluster. After 16 h of fasting, genes involved in ketogenesis, gluconeogenesis and mitochondrial or peroxisomal fatty acid beta-oxidation, were up-regulated (cluster-1) whereas genes involved in fatty acid and cholesterol synthesis were down-regulated (cluster-2). For all genes tested, the microarray data was confirmed by quantitative RT-PCR. Most genes were altered by fasting as already reported in mammals. A notable exception was the HMG-CoA synthase 1 gene, which was up-regulated following 16 and 48 h of fasting while the other genes involved in cholesterol metabolism were down-regulated as reported in mammalian studies. We further focused on genes not represented on the microarray and candidates for the regulation of the target genes belonging to cluster-1 and -2 and involved in lipid metabolism. Data are provided concerning PPARa, SREBP1, SREBP2, NR1H3 transcription factors and two desaturases (FADS1, FADS2). This study evidences numerous genes altered by starvation in chickens and suggests a global repression of cellular activity in response to this stressor. The central role of lipid and acetyl-CoA metabolisms and its regulation at transcriptional level are confirmed in chicken liver in response to short-term fasting. Interesting expression modulations were observed for NR1H3, FADS1 and FADS2 genes. Further studies are needed to precise their role in the complex regulatory network controlling lipid metabolism. Show less

Changes in fatty acid metabolism associated with insulin resistance have been described in rats and humans but have not been well characterized in the frequently used mouse model of diet-induced obesity. To analyse the early phase as well as established insulin resistance, C57BL/6 mice were placed for 1 or 16 weeks on a high fat diet (1w-HFD, 16w-HFD). Endocrine and metabolic parameters indicated that 1w-HFD mice showed a moderate but significant induction of insulin resistance while 16w-HFD mice exhibited profound obesity-associated insulin resistance and dyslipidemias. Significant alterations in fatty acid composition were observed in plasma and liver in both groups. Liver phospholipid-associated arachidonate and docosahexaenoate were increased in both 1w-HFD and 16w-HFD mice, possibly due to increased expression of the desaturases Fads1 and Fads2. Unexpectedly, SCD1 activity and gene expression in liver were decreased in the 1w-HFD group accompanied by diminished total hepatic lipid levels, while they were increased in chronically fed mice. Our data indicate that the early phase of HFD-induced insulin resistance is not associated with elevated liver lipid concentration. Furthermore, the early and persistent rise of arachidonate and docosahexaenoate indicates that insulin resistance is not due to insufficient availability (or concentrations) of polyunsaturated fatty acids as postulated previously. Show less

The enzymes encoded by fatty acid desaturase (FADS) 1 and FADS2 are rate-limiting enzymes in the desaturation of linoleic acid [LA; 18:2(n-6)] to arachidonic acid [ARA; 20:4(n-6)], and alpha-linolenic acid [ALA; 18:3(n-3)] to eicosapentaenoic acid [EPA; 20:5(n-3)] and docosahexaenoic acid [DHA; 22:6(n-3)]. ARA, EPA, and DHA play central roles in infant growth, neural development, and immune function. The maternal ARA, EPA, and DHA status in gestation influences maternal-to-infant transfer and breast milk provides fatty acids for infants after birth. We determined if single nucleotide polymorphisms in FADS1 and FADS2 influence plasma phospholipid and erythrocyte ethanolamine phosphoglyceride (EPG) (n-6) and (n-3) fatty acids of women in pregnancy or their breast milk during lactation. We genotyped rs174553, rs99780, rs174575, and rs174583 in the FADS1 FADS2 gene cluster and analyzed plasma and erythrocyte fatty acids and dietary intake for 69 pregnant women and breast milk for a subset of 54 women exclusively breast-feeding at 1 mo postpartum. Minor allele homozygotes of rs174553(GG), rs99780(TT), and rs174583(TT) had lower ARA but higher LA in plasma phospholipids and erythrocyte EPG and decreased (n-6) and (n-3) fatty acid product:precursor ratios at 16 and 36 wk of gestation. Breast milk fatty acids were influenced by genotype, with significantly lower 14:0, ARA, and EPA but higher 20:2(n-6) in the minor allele homozygotes of rs174553(GG), rs99780(TT), and rs174583(TT) and lower ARA, EPA, 22:5(n-3), and DHA in the minor allele homozygotes G/G of rs174575. We showed that genetic variants of FADS1 and FADS2 influence blood lipid and breast milk essential fatty acids in pregnancy and lactation. Show less

The rapidly evolving field of metabolomics aims at a comprehensive measurement of ideally all endogenous metabolites in a cell or body fluid. It thereby provides a functional readout of the physiological state of the human body. Genetic variants that associate with changes in the homeostasis of key lipids, carbohydrates, or amino acids are not only expected to display much larger effect sizes due to their direct involvement in metabolite conversion modification, but should also provide access to the biochemical context of such variations, in particular when enzyme coding genes are concerned. To test this hypothesis, we conducted what is, to the best of our knowledge, the first GWA study with metabolomics based on the quantitative measurement of 363 metabolites in serum of 284 male participants of the KORA study. We found associations of frequent single nucleotide polymorphisms (SNPs) with considerable differences in the metabolic homeostasis of the human body, explaining up to 12% of the observed variance. Using ratios of certain metabolite concentrations as a proxy for enzymatic activity, up to 28% of the variance can be explained (p-values 10(-16) to 10(-21)). We identified four genetic variants in genes coding for enzymes (FADS1, LIPC, SCAD, MCAD) where the corresponding metabolic phenotype (metabotype) clearly matches the biochemical pathways in which these enzymes are active. Our results suggest that common genetic polymorphisms induce major differentiations in the metabolic make-up of the human population. This may lead to a novel approach to personalized health care based on a combination of genotyping and metabolic characterization. These genetically determined metabotypes may subscribe the risk for a certain medical phenotype, the response to a given drug treatment, or the reaction to a nutritional intervention or environmental challenge. Show less

Atherogenic dyslipidemia associated with elevated plasma triacylglycerol concentrations and reduced HDL is improved by both caloric restriction and reduced carbohydrate consumption. We aimed to identify the molecular pathways responsive to both caloric restriction and dietary composition within adipose tissue by monitoring transcriptional expression. Subcutaneous adipose tissue biopsy specimens were obtained from 131 moderately overweight men [body mass index (in kg/m2): 29.2 +/- 2.0] after 1 wk of a basal diet [54%, 16%, and 30% carbohydrate, protein, and fat, respectively; 7% saturated fat), after 3 wk with 1 of 4 randomized diets [basal diet; moderate-carbohydrate (39%) diet; low-carbohydrate (26%) and low-saturated-fat (9%) diet; low-carbohydrate (26%) and high-saturated-fat (15%) diet], after 5 wk of acute weight loss with a randomized diet (-1103.0 +/- 216.5 kcal/d, which resulted in a loss of 10.0 +/- 3.3 lb, or 4.5 +/- 1.5 kg), and after 4 wk of being stabilized at a reduced weight. Transcriptional response was identified by using expression array analysis and was confirmed by using real-time polymerase chain reaction analysis. Of the 1473 transcripts significantly decreased in expression in response to acute weight loss, 30 were responsive to isocaloric alterations in dietary composition, including stearoyl-coenzyme A desaturase (SCD), fatty acid desaturases 1 and 2 (FADS1 and FADS2), and diacylglycerol transferase 2 (DGAT2). Response was confirmed by real-time polymerase chain reaction analysis for these genes (P < 0.003). SCD expression in response to isocaloric dietary change was most strongly correlated with carbohydrate intake (P = 0.019) and, with the low-carbohydrate diet, SCD expression was inversely correlated with saturated fat intake (P = 0.05). Triacylglycerol responses to changes in dietary composition were independently correlated with SCD (P = 0.003) and DGAT2 (P = 0.05) responses. SCD expression in adipose tissue is independently regulated by weight loss and by carbohydrate and saturated fat intakes. Moreover, SCD and DGAT2 expression may be involved in dietary regulation of systemic triacylglycerol metabolism. Show less

The genetic background of hepatocellular carcinoma (HCC) has yet to be completely understood. Here, we describe the application of suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis for the isolation and identification of differential expression of genes in HCC. Twenty-six known genes were validated as up-regulated and 19 known genes as down-regulated in HCC. The known genes identified were found to have diverse functions. In addition to the overexpression of AFP, these genes (increased in the presence of HCC) are involved in many processes, such as transcription and protein biosynthesis (HNRPDL, PABPC1, POLR2K, SRP9, SNRPA, and six ribosomal protein genes including RPL8, RPL14, RPL41, RPS5, RPS17, RPS24), the metabolism of lipids and proteins (FADS1, ApoA-II, ApoM, FTL), cell proliferation (Syndecan-2, and Annexin A2), and signal transduction (LRRC28 and FMR1). Additionally, a glutathione-binding protein involved in the detoxification of methylglyoxal known as GLO1 and an enzyme which increases the formation of prostaglandin E(2) known as PLA2G10 were up-regulated in HCC. Among the underexpressed genes discovered in HCC, most were responsible for liver-synthesized proteins (fibrinogen, complement species, amyloid, albumin, haptoglobin, hemopexin and orosomucoid). The enzyme implicated in the biotransformation of CYP family members (LOC644587) was decreased. The genes coding enzymes ADH1C, ALDH6A1, ALDOB, Arginase and CES1 were also found. Additionally, we isolated a zinc transporter (Zip14) and a function-unknown gene named ZBTB11 (Zinc finger and BTB domain containing 11) which were underexpressed, and seven expression sequence tags deregulated in HCC without significant homology reported in the public database. Essentially, by using SSH combined with a cDNA microarray we have identified a number of genes associated with HCC, most of which have not been previously reported. Further characterization of these differentially expressed genes will provide information useful in understanding the genes responsible for the development of HCC. Show less

To identify novel methylation-silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome-wide search for genes upregulated by treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC). After 5-aza-dC treatment of a gastric cancer cell line (AGS) 579 genes were upregulated 16-fold or more, using an oligonucleotide microarray with 39,000 genes. From these genes, we selected 44 known genes on autosomes whose silencing in gastric cancer has not been reported. Thirty-two of these had CpG islands (CGI) in their putative promoter regions, and all of the CGI were methylated in AGS, giving an estimated number of 421+/-75 (95% confidence interval) methylation-silenced genes. Additionally, we analyzed the methylation status of 16 potential tumor-related genes with promoter CGI that were upregulated four-fold or more, and 14 of these were methylated in AGS. Methylation status of the 32 randomly selected and 16 potential tumor-related genes was analyzed in 10 primary gastric cancers, and 42 genes (ABHD9, ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, ULBP2, WIF1, ZNF177 and ZNF559) were methylated in at least one primary gastric cancer. A metastasis suppressor gene, MTSS1, was located in a genomic region with frequent loss of heterozygosity (8q22), and was expressed abundantly in the normal gastric mucosa, suggesting its role in gastric carcinogenesis. (Cancer Sci 2006; 97: 64 -71). Show less

Fatty acids, in particular omega-3 fatty acids, have been found to affect behavior and cognition both directly and indirectly. Evidence to suggest a link with attention-deficit/hyperactivity disorder (ADHD) derives from three key areas: 1) animal dietary restriction studies observed increased locomotive hyperactivity and reduced cognitive ability in offspring; 2) animal dietary studies indicate alterations in the dopamine pathway; and 3) human studies report reduced plasma omega-3 fatty acids in ADHD subjects. We investigated three genes that encode essential enzymes (desaturases) for the metabolism of fatty acids by scanning for genetic association between 45 single nucleotide polymorphisms (SNPs) and ADHD. Our findings suggest a significant association of ADHD with SNP rs498793 (case-control p = .004, odds ratio [OR] 1.6, 95% confidence interval [CI] 1.15-2.23; transmission disequilibrium test [TDT] p = .014, OR 1.69) in the fatty acid desaturase 2 (FADS2) gene. As alcohol is known to decrease the activities of these desaturase enzymes, we also tested for interactions between ADHD subjects' genotypes and maternal use of alcohol during pregnancy. Two SNPs in the fatty acid desaturase 1 (FADS1) gene were nominally associated with ADHD only in the prenatal alcohol-exposed group of children; formal test for interaction was not significant. These preliminary findings are suggestive of an association between FADS2 and ADHD. Show less

Fatty acid composition in membranes plays an important role in cellular processes and has shown to be associated with the aetiology of several complex diseases in humans. We report strong associations between variants in the human delta-5 and delta-6 desaturase genes FADS1 FADS2 and fatty acid composition in serum phospholipids. Eighteen polymorphisms located in this gene cluster were genotyped in 727 adults from Erfurt, a German centre of the European Community Respiratory Health Survey. The cluster is located at chromosome 11q12-11q13.1, a region repeatedly found to be linked with atopy and other complex diseases. Polymorphisms and statistically reconstructed haplotypes of FADS1 and the upstream region of FADS2 showed strongest associations with the level of the direct precursor of inflammatory eicosanoids, the n-6 fatty acid arachidonic acid (C20:4n-6), also strong associations with levels of the n-6 fatty acids C18:2n-6, C18:3n-6, C20:2n-6, C20:3n-6, C22:4n-6 and of the n-3 fatty acids C18:3n-3, C20:5n-3 and C22:5n-3 (P-values < 1.0 x 10(-13)). Carriers of the rare alleles of several SNPs and their respective haplotypes had a lower prevalence of allergic rhinitis and atopic eczema. No association was found for total and specific IgE levels. Show less

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis. Show less

The insertion of double bonds into specific positions of fatty acids is achieved by the action of distinct desaturase enzymes. Here we report the cloning and characterization of three members of the fatty acid desaturase (FADS) gene family in humans. Initially identified as cDNA fragments by direct cDNA selection within a defined 1.4-Mb region in 11q12-q13.1, full-length fatty acid desaturase-1 (FADS1) and fatty acid desaturase-2 (FADS2) transcripts were obtained by EST sequence assembly. A third member, fatty acid desaturase-3 (FADS3), was identified in silico revealing 62 and 70% nucleotide sequence identity with FADS1 and FADS2, respectively. The three genes are clustered within 92 kb of genomic DNA located 2 kb telomeric to FEN1 and 50 kb centromeric to VMD2 and are likely to have arisen evolutionarily from gene duplication as they share a remarkably similar exon/intron organization. Protein database searches identified FADS1, FADS2, and FADS3 as fusion products composed of an N-terminal cytochrome b5-like domain and a C-terminal multiple membrane-spanning desaturase portion. Show less

The cranial ultrasound features of two neonates with neuronal migration disorders are described. One infant had lissencephaly and the other polymicrogyria in conjunction with the Pena-Shokeir syndrome type 1. A third infant is described who was born extremely prematurely and with Down's syndrome, who had similar ultrasound features. By two weeks of age, however, the scan of this third infant had become normal, which illustrates the need for caution in diagnosing migrational disorders in very preterm babies and those with Down's syndrome. The disorders of neuronal migration are discussed. Show less

This report describes the sonographic diagnosis of the Pena-Shokeir syndrome type 1 during the second trimester of a pregnancy which was electively terminated. The mother had previously delivered a macerated, hydropic infant with multiple congenital anomalies. The diagnosis was based on the recurrence of hydramnios and nonimmune hydrops in a fetus with normal chromosomes, normal amniotic fluid alpha-fetoprotein, normal fetal echocardiography, and lack of evidence of a lysosomal storage disease. These observations suggest that serial sonography during the second trimester in pregnancies at risk may allow for the prenatal diagnosis of the Pena-Shokeir syndrome type 1. Without further experience, it would not be prudent to suggest to couples at risk that the prenatal diagnosis of a recurrence can be assured with a high degree of accuracy. Show less