Specific regulation of low-abundance transcript variants encoding human Acyl-CoA binding protein (ACBP) isoforms.

Despite intensive efforts on annotation of eukaryotic transcriptoms, little is known about the regulation of low-abundance transcripts. To address this question, we analysed the regulation of novel low-abundance transcript variants of human acyl-CoA binding protein (ACBP), an important multifunctional housekeeping protein, which we have identified by screening of human expressed sequence tags in combination with ab initio gene prediction. By using RT-, real-time RT- and rapid amplification of cDNA ends-PCR in five human tissues, we find these transcripts, which are generated by a consequent use of alternative promoters and alternate first or first two exons, to be authentic ones. They show a tissue-specific distribution and intrinsic responsiveness to glucose and insulin. Promoter analyses of the corresponding transcripts revealed a differential regulation mediated by sterol regulatory element-binding protein-2, hepatocyte nuclear factor-4α and nuclear factor κB (NF-κB), central transcription factors of fat and glucose metabolism and inflammation. Subcellular localization studies of deduced isoforms in liver HepG2 cells showed that they are distributed in different compartments. By demonstrating that ACBP is a target of NF-κB, our findings link fatty acid metabolism with inflammation. Furthermore, our findings show that low-abundance transcripts are regulated in a similar mode than their high-abundance counterparts.

Identification of a novel human Acyl-CoA binding protein isoform with a unique C-terminal domain.

Seven isoforms of the multifunctional human Acyl-coenzyme A binding protein (ACBP) have been characterized so far. Through ab initio analysis of expressed sequence tag (ESTs), we identified a novel high-abundant ACBP splice variant ACBP1e encoding an ACBP isoform with a unique C-terminus of 81 amino acid residues. Bioinformatic analysis shows that this domain is evolutionary conserved and shares no significant homology with other known proteins, and its function is not known. Quantitative RT-polymerase chain reaction (PCR) revealed that ACBP1e is predominantly expressed in adipose tissue and hippocampus. Protein expression studies showed perinuclear clustering of ACBP1e. These clusters were not seen in ACBP1e mutants with an altered putative subtilisin/kexin isozyme-1 cleavage site within the C-terminus, indicating that this domain is required for proper localization of ACBP1e. Conclusively, we identified a novel ACBP isoforms with an unique C-terminal domain encoded by a high-abundant splice variant.

ACBP knockdown leads to down-regulation of genes encoding rate-limiting enzymes in cholesterol and fatty acid metabolism.

The human Acyl-CoA binding protein (ACBP) is a structural and functional highly conserved protein. As an intracellular pool former and carrier of acyl-CoAs, ACBP influences overall lipid metabolism. Its nuclear abundance and physical interaction with hepatocyte nuclear factor 4alpha suggested a gene regulatory function of ACBP. To identify ACBP target genes we performed genome-wide transcript profiling under siRNA-mediated ACBP knockdown in human liver HepG2 cells. Based on a single sided permutation T-test (p<0.05) we identified 256 down-regulated and 198 up-regulated transcripts with a minimal fold change of 1.32 (log 0.5). Gene annotation enrichment analysis revealed ACBP-mediated down-regulation of 18 genes encoding key enzymes in glycerolipid (i.e. mitochondrial glycerol-3-phosphate acyltransferase), cholesterol (i.e. HMG-CoA synthase and HMG-CoA reductase) and fatty acid (i.e. fatty acid synthase) metabolism. Integration of these genes in common pathways suggested decreased lipid biosynthesis. Accordingly, saturated (16:0) and monosaturated (16:1, 18:1) fatty acids were significantly reduced to 75% in ACBP-depleted cells. Taken together, we obtained evidence that ACBP functions in lipid metabolism at the level of gene expression. This effect seems to be translated into certain metabolites. The identified 454 ACBP regulated genes present a first reference for further studies to define the ACBP regulon in mammalian cells.

Analysis of the transcriptional regulation of the FABP2 promoter haplotypes by PPARgamma/RXRalpha and Oct-1.

Variants of the human intestinal fatty acid binding protein 2 gene (FABP2) are associated with traits of the metabolic syndrome. Relevant FABP2 promoter polymorphisms c.-80_-79insT, c.-136_-132delAGTAG, c.-168_-166delAAGinsT, c.-260G>A, c.-471G>A, and c.-778G>T result in two haplotypes A and B. Activation of haplotypes by rosiglitazone stimulated PPARgamma/RXRalpha leads to 2-fold higher activity of haplotype B than A. As shown by chimeric FABP2 promoter constructs, the higher responsiveness of FABP2 haplotype B is mainly but not solely determined by polymorphism c.-471G>A. As shown by EMSA and promoter-reporter assays, Oct-1 interacts with the -471 region of FABP2 promoters, induces the activities of both FABP2 promoter haplotypes and abolishes the different activities of haplotypes induced by rosiglitazone activated PPARgamma/RXRalpha. In conclusion, our findings suggest a functional role of PPARgamma/RXRalpha and Oct-1 in the regulation of the FABP2 gene.

PYK10, a beta-glucosidase located in the endoplasmatic reticulum, is crucial for the beneficial interaction between Arabidopsis thaliana and the endophytic fungus Piriformospora indica.

Piriformospora indica, an endophyte of the Sebacinaceae family, promotes growth and seed production of many plant species, including Arabidopsis. Growth of a T-DNA insertion line in PYK10 is not promoted and the plants do not produce more seeds in the presence of P. indica, although their roots are more colonized by the fungus than wild-type roots. Overexpression of PYK10 mRNA did not affect root colonization and the response to the fungus. PYK10 codes for a root- and hypocotyl-specific beta-glucosidase/myrosinase, which is implicated to be involved in plant defences against herbivores and pathogens. Expression of PYK10 is activated by the basic helix-loop-helix domain containing transcription factor NAI1, and two Arabidopsis lines with mutations in the NAI1 gene show the same response to P. indica as the PYK10 insertion line. PYK10 transcript and PYK10 protein levels are severely reduced in a NAI1 mutant, indicating that PYK10 and not the transcription factor NAI1 is responsible for the response to the fungus. In wild-type roots, the message level for a leucine-rich repeat protein LRR1, but not for plant defensin 1.2 (PDF1.2), is upregulated in the presence of P. indica. In contrast, in lines with reduced PYK10 levels the PDF1.2, but not LRR1, message level is upregulated in the presence of the fungus. We propose that PYK10 restricts root colonization by P. indica, which results in the repression of defence responses and the upregulation of responses leading to a mutualistic interaction between the two symbiotic partners.

Type 2 diabetes-associated fatty acid binding protein 2 promoter haplotypes are differentially regulated by GATA factors.

The human intestinal fatty acid binding protein 2 (FABP2) mediates fat absorption by binding and intracellular trafficking of long-chain free fatty acids. Studies with knockout mice and association analysis of polymorphisms revealed that FABP2 is a susceptibility gene for type 2 diabetes (noninsulin dependent diabetes mellitus [NIDDM]) and related traits. Relevant FABP2 promoter polymorphisms c.-80_-79insT (rs5861422), c.-136_-132delAGTAG (rs5861423), c.-168_-166delAAGinsT (rs1973598), c.-260G>A (rs6857641), c.-471G>A (rs2282688), and c.-778G>T (rs10034579) result in two haplotypes A and B, whereby B possesses two- to three-fold lower transcriptional activity than A. We show in luciferase reporter gene assays by a series of chimeric FABP2 promoter constructs in intestinal Caco-2 cells that polymorphism c.-80_-79insT essentially determines different activities of the FABP2 promoter. In accordance, in electrophoretic mobility shift assays (EMSAs), transcriptional factors GATA-5 and -6 bind with higher binding affinities to the FABP2 promoter region containing the -80A allele compared to B. As functional consequence, haplotype A is twice as much more activated by GATA factors than haplotype B in liver Huh7 cells. Additionally, a construct bearing the -80B allele in the background of haplotype A reversed the activity from A to B. Thus, the GATA mediated differential activation of FABP2 haplotypes depends on polymorphism c.-80_-79insT. This provides the molecular basis for the variant specific transcriptional regulation of the diabetes type 2-associated FABP2 gene.

Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene.

The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers.

Transcriptional regulation of the fatty acid binding protein 2 (FABP2) gene by the hepatic nuclear factor 1 alpha (HNF-1alpha).

The human fatty acid binding protein (FABP2) is involved in intestinal absorption and intracellular trafficking of long-chain fatty acids. Here we investigate transcriptional regulation of FABP2 by the endodermal hepatic nuclear factor 1 alpha (HNF-1alpha). In electromobility shift and supershift assays we show the presence of two adjacent HNF-1alpha binding sites within the FABP2 promoter regions -185 to -165 and -169 to -149. HNF-1alpha activates an FABP2 promoter luciferase construct by 3.5 and 20-fold in Caco-2 and Hela cells, respectively. Mutational analysis of HNF-1alpha elements resulted in about 50% reduction of basal and HNF-1alpha induced activity of FABP2 promoter constructs, predominantly caused by deletion of the -185 to -165 site. Thus, our data suggest a major role of HNF-1alpha in control of FABP2 expression in intestine via a functional HNF-1alpha recognition element within FABP2 promoter region -185 to -165.

Transcriptional regulation of HMG-CoA synthase and HMG-CoA reductase genes by human ACBP.

The acyl-CoA binding protein (ACBP) is an ubiquitary expressed multi-functional protein which regulates basic cellular functions such as fatty acid and steroid metabolism. Since ACBP is described to interact with the transcription factor hepatocyte nuclear factor 4 alpha (HNF-4alpha), we investigated the role of human ACBP on transcriptional regulation of the putative HNF-4alpha target gene HMG-CoA synthase 1 (HMGCS1). As shown by promoter-reporter assays ACBP represses the HNF-4alpha-induced activity of a 617bp HMGCS1 promoter fragment by approximately 80% in HepG2 cells as well as in non-endodermal HeLa cells devoid of HNF-4alpha. Interestingly, reporter assays without co-transfection of HNF-4alpha revealed that ACBP reduces the activity of the HMGCS1 promoter by about 60 to 80% in both cell lines. Activities of 417bp and 317bp HMGCS1 promoter fragments were 2.5 to 4 fold decreased by ACBP. Concordantly, the levels of HMGCS1-mRNA and -protein were diminished to 60% and 70% in ACBP-expressing HeLa cells, respectively. Additionally, ACBP reduces the promoter activity and the mRNA levels of the cholesterogenic HMG-CoA reductase (HMGCR). In conclusion, we provide evidence that ACBP is a transcriptional regulator of the HMGCS1 and HMGCR genes encoding rate-limiting enzymes of cholesterol synthesis pathway.

MTTP variants and body mass index, waist circumference and serum cholesterol level: Association analyses in 7582 participants of the KORA study cohort.

The microsomal triglyceride transfer protein (MTTP) is a key regulator in the assembly and secretion of chylomicrons and very low density lipoprotein (VLDL) in the intestine and in liver. Associations between MTTP variants and traits of the metabolic syndrome are carried out in relatively small cohorts and are not consistent. We analysed MTTP polymorphisms in 7582 participants of the KORA study cohort. Seven htSNPs covering a 52kb region of the MTTP locus and two cSNPs (I128T, H297Q) were selected. A MTTP haplotype containing the minor allele of H297Q showed a significant decrease of -0.636 (95% CI: -1.226, -0.046; p=0.035) BMI units in females but not in males. In comparison to homozygous H-carriers for the major allele of the MTTP H297Q polymorphism, homozygous Q297Q carriers showed a significant decrease in BMI of -0.425B MI units (95% CI: -0.74, -0.12; p=0.007), in waist circumference of -0.990 cm (95% CI: 1.74, -0.24; p=0.01) and in total cholesterol of -0.039 mmol/l (95% CI: -0.07, 0; p=0.03). Heterozygous Q-carriers displayed a reduction in BMI of -0.183 BMI unit (95% CI: -0.33, -0.04; p=0.012), in waist circumference of -0.45 cm (95% CI: 0.8, -0.1; p=0.01) and in total cholesterol of -0.103 mmol/l (95% CI: -0.18, -0.03; p=0.01). Gender stratified statistics revealed a significant reduction of -0.657 BMI units (95% CI: -1.14, -0.18; p=0.007), -1.437 cm waist circumference (95% CI: -2.55, -0.32; p=0.01) and -0.052 mmol/l total cholesterol (95% CI: -0.1, -0.01; p=0.03) for females homozygous for the Q297Q polymorphism. Females carrying the Q-allele showed a decrease of -0.259 BMI unit (95% CI: -0.48, -0.04; p=0.023), -0.662 cm waist circumference (95% CI: -1.18, -0.14; p=0.01) and -0.111 mmol/l total cholesterol (95% CI: -0.21, -0.01; p=0.03). Our association analysis in a large population based study cohort provides evidence that the minor allele of the MTTP H297Q polymorphism is associated with lower BMI, waist circumference and total cholesterol in females but not in males.

Association of prostaglandin E synthase 2 (PTGES2) Arg298His polymorphism with type 2 diabetes in two German study populations.

CONTEXT: On the basis of its chromosomal localization and its role in the synthesis of the antilipolytic compound prostaglandin E(2), the prostaglandin E synthase 2 (PTGES2) is a candidate gene for type 2 diabetes. OBJECTIVE: The aim of the present study was to investigate whether genetic variants in the PTGES2 gene are associated with type 2 diabetes. RESULTS: Sequencing of the PTGES2 gene revealed one nonsynonymous coding single-nucleotide polymorphism (SNP) (Arg298His, rs13283456) and a previously unknown promoter SNP g.-417G>T. Both SNPs and additional haplotype tagging SNPs (rs884115, rs10987883, rs4837240) were genotyped in a nested case-control study of 192 incident type 2 diabetes subjects and 384 controls (European Prospective Investigation into Cancer and Nutrition-Potsdam). Carriers of the minor allele of Arg298His had a lower risk to develop the disease [odds ratio (OR) 0.63, 95% confidence interval (CI) 0.41-0.97, P = 0.04], compared with homozygous individuals with the common allele. The PTGES2 Arg298His polymorphism was reinvestigated in a population-based cross-sectional study (Cooperative Health Research in the Augsburg Region) consisting of 239 individuals with impaired glucose tolerance, 226 with type 2 diabetes, and 863 normoglycemic controls. In this study population, the Arg298His polymorphism was significantly associated with impaired glucose tolerance (OR 0.68, 95% CI 0.50-0.93, P = 0.007) and type 2 diabetes (OR 0.61, 95% CI 0.43-0.86, P = 0.004). A pooled analysis of data from both study populations revealed reduced risk of type 2 diabetes (OR 0.62, 95% CI 0.47-0.81, P = 0.0005) in PTGES2 298His allele carriers. CONCLUSION: We obtained evidence from two Caucasian study populations that the His298-allele of PTGES2 Arg298His confers to reduced risk of type 2 diabetes.

Association analyses of GIP and GIPR polymorphisms with traits of the metabolic syndrome.

Glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin release via interaction with its pancreatic receptor (GIP receptor (GIPR)). GIP also acts as vasoactive protein. To investigate whether variations in GIP and GIPR genes are associated with risk factors of the metabolic syndrome we sequenced gene regions and identified two coding SNPs (GIP Ser103Gly, GIPR Glu354Gln) and one splice site SNP (GIP rs2291726) in 47 subjects. Interestingly, in silico analyses revealed that splice site SNP rs2291726 results in a truncated protein and classified GIPR variant Glu354Gln as a functional amino acid change. Association analyses were performed in a case-cohort study of incident cardiovascular disease (CVD) nested in the EPIC-Potsdam cohort. No significant associations between incident CVD and GIP Ser103Gly and rs2291726 were found. For GIPR Glu354Gln, we obtained a nominal association of heterozygous minor allele carrier with CVD in a codominant model adjusted for BMI, sex, and age (OR: 0.67, CI: 0.50-0.91, p = 0.01) or additional covariates of CVD (OR: 0.72, CI: 0.52-0.97, p = 0.03). In conclusion, we identified a common splice site mutation (rs2291726) of the GIP gene which results in a truncated protein and provide preliminary evidence for an association of the heterozygous GIPR Glu354Gln genotype with CVD.

Association of acyl-CoA-binding protein (ACBP) single nucleotide polymorphisms and type 2 diabetes in two German study populations.

The human acyl-CoA-binding protein (ACBP) is a potential candidate gene of type 2 diabetes (T2D), since it plays a central role in determining the intracellular concentration of activated fatty acids which contribute to insulin resistance. The aim of our study was to evaluate whether single nucleotide polymorphisms (SNPs) of the ACBP gene are associated with risk of T2D. Genotyping of eight SNPs (rs2084202, rs3731607, rs8192501, rs8192504, rs2244135, rs2276596, rs8192506, rs2289948) was performed in 192 incident T2D subjects and 384 matched controls of the European Prospective Investigation into Cancer and Nutrition-Potsdam cohort. A putative promoter SNP (rs2084202) of splice variant ACBP 1c showed decreased risk of T2D (odds ratio (OR) 0.63, 95% CI 0.41-0.96). The haplotype, that contained the mutant base of rs2084202 showed similar evidence for the association with disease risk as single SNP rs2084202. In a second population-based study, Cooperative Health Research in the Augsburg Region of 226 individuals with T2D and 863 control subjects a borderline significant association between rs2084202 and T2D (OR 0.72, 95% CI 0.51-1.01) was observed. In summary, we obtained evidence from two Caucasian study populations that the minor allele of ACBP rs2084202 might be associated with reduced risk of T2D.

Candidate gene association study of type 2 diabetes in a nested case-control study of the EPIC-Potsdam cohort - role of fat assimilation.

To search for common variants etiological for type 2 diabetes, we screened 15 genes involved in fat assimilation for sequence variants. Approximately 55 kb in promoter and coding regions, and intron/splice sites were sequenced by cycle sequencing. In the set of 15 genes, 71 single nucleotide polymorphisms (SNPs) were detected. 33 SNPs were presumed to be functionally significant and were genotyped in 192 incident type 2 diabetes subjects and 384 matched controls from the European Prospective Investigation into Cancer and Nutrition-Potsdam cohort. A total of 27 SNPs out of 15 genes showed no statistical association with type 2 diabetes in our study. Six SNPs demonstrated nominal association with type 2 diabetes, with the most significant marker (FABP6 Thr79Met) having an adjusted odds ratio of 0.45 (95% CI 0.22-0.92) in homozygous Met allele carriers. Evidence for an association with disease status was also found for a novel Arg109Cys (g.2129C > T) variant of colipase, 5'UTR (rs2084202) and Met71Val (rs8192506) variants of diazepam-binding inhibitor, Arg298His (rs13283456) of PTGES2, and a novel promoter variant (g.-1324G > A) of SLC27A5. The results presented here provide preliminary evidence for the association of common variants in genes involved in fat assimilation with the genetic susceptibility of type 2 diabetes. However, they definitely need further verification.

The L513S polymorphism in medium-chain acyl-CoA synthetase 2 (MACS2) is associated with risk factors of the metabolic syndrome in a Caucasian study population.

Enzymes of the medium-chain acyl-CoA synthetase (MACS) family catalyze the ligation of medium chain fatty acids with CoA to produce medium-chain-acyl-CoA. At least four members of the MACS gene family are clustered on human chromosome 16p12. Association studies in the Japanese Suita cohort of MACS polymorphisms and various phenotypes revealed the contribution of the Leu513Ser polymorphism in MACS2 to multiple risk factors of the metabolic syndrome. Here, we investigated the association between this polymorphism and different risk factors in the Caucasian Metabolic Intervention Cohort Kiel. Seven hundred and sixteen male subjects aged 45-65 years were recruited for a standard oral glucose tolerance test and the postprandial assessment of metabolic parameters after an oral metabolic tolerance test (oMTT; 1017 kcal, 51.6% fat, 29.6% carbohydrates, 11.9% protein). The MACS2 Leu513Ser polymorphism was determined by TaqMan-Assay in 705 subjects. Postprandial triglyceride levels following oMTT [area under the curve (AUC)] were significantly higher in subjects carrying the Ser allele compared to subjects homozygous for the Leu allele (1690 +/- 100 mg x h/dL versus 1514 +/- 39 mg x h/dL, p = 0.04). Significant differences between genotype groups were also found for fasting (108 +/- 1.9 mg/dL versus 104 +/- 0.66 mg/dL, p = 0.04) and postprandial (AUC 535 +/- 11 versus 512 +/- 4.0, p = 0.02) glucose levels as well as for high-density-lipoprotein, body mass index, waist circumference, systolic and diastolic blood pressure. Carriers of the Ser allele also show an increased risk of impaired glucose metabolism (OR: 1.48, 95% confidence interval: 0.98-2.27, p = 0.07), adiposity (1.8, 1.16-2.81, p = 0.01) and hypertension (1.5, 0.99-2.17, p = 0.06). In conclusion, our results suggest an involvement of the MACS2 Leu513Ser polymorphism in the development of the metabolic syndrome in Caucasian population. Additionally, the higher triglyceride and glucose levels after an oMTT support a possible functional impact of the polymorphism in vivo.

Identification of new acyl-CoA binding protein transcripts in human and mouse.

The ubiquitously expressed acyl-CoA binding protein (ACBP) is involved in lipid metabolism and is regulated by hormones and feeding status via transcription factors such as sterol regulatory element-binding protein 1 and peroxisome proliferator-activated receptor-gamma (PPARgamma). In humans, two transcripts encoding proteins of 86 and 104 amino acids are known, whereas in mouse only one protein of 86 amino acids is described. We identified new transcripts in human and mouse tissues, that had been generated by alternative first exon usage. Quantitative RT-PCR analyses showed a high expression of the new human transcript, ACBP-1c, in adipose tissue. By promoter reporter gene assays, specific regulation of this transcript by PPARgamma2 was revealed, implicating the usage of an alternative promoter that contains a PPARgamma responsive element. Subcellular localizations of the known human proteins and the new variant showed an occurrence in cytoplasma and nucleus. Reported studies concerning ACBP gene regulation should be re-evaluated with respect to a new ACBP gene model. Given the fact that the new variant is highly expressed in adipose tissue and a PPARgamma target, it might be relevant for diseases like diabetes and obesity.

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4alpha.

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4alpha (HNF-4alpha), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4alpha binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4alpha by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4alpha, that are both candidate genes for diabetes type 2, may be a powerful approach.

Analysis of PGC-1alpha variants Gly482Ser and Thr612Met concerning their PPARgamma2-coactivation function.

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a cofactor involved in adaptive thermogenesis, fatty acid oxidation, and gluconeogenesis. Dysfunctions of this protein are likely to contribute to the development of obesity and the metabolic syndrome. This is in part but not definitely confirmed by results of population studies. The aim of this study was to investigate if common genetic variants rs8192678 (Gly482Ser) and rs3736265 (Thr612Met) in the PGC-1alpha gene lead to a functional consequence in cofactor activity using peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) as interacting transcription factor. Reporter gene assays in HepG2 cells with wildtype and mutant proteins of both PGC1alpha and PPARgamma2 (Pro12Ala, rs1801282) using the acyl-CoA-binding protein (ACBP) promoter showed no difference in coactivator activity. This is the first study implicating that the Gly482Ser and Thr612Met polymorphisms in PGC-1alpha and Pro12Ala polymorphism in PPARgamma2 do not affect the functional integrity of these proteins.

Expression analysis of genes involved in fat assimilation in human monocytes.

The metabolic syndrome X is characterized by a group of risk factors such as obesity, atherogenic dyslipidemia, hypertension, and insulin resistance. To study the functional alterations resulting from genetic variations, ex vivo studies are one option to be carried out. Since it is not an easy procedure to obtain cells from the related tissues ex vivo, the aim of the present study was to investigate whether monocytes can serve as model cells. The purpose was to check if monocytes are insulin target cells or not and to elucidate the expression of genes involved in fat assimilation. Human monocytes were drawn from venous blood of healthy donors, aged 25 - 30, using density gradient separation and antibody-based magnetic cell sorting of CD14-positive cells. An expression analysis of genes was performed using RT-PCR and Western Blot. Transcripts of the three splice-variants of the Acyl-CoA binding protein (ACBP), the Medium-chain Acyl-CoA Synthetase 1 (MACS1), the Insulin Receptor (INSR) and the Peroxisome Proliferator-activated Receptor gamma (PPARgamma) are consistently expressed in monocytes of all donors. Differences in gene expression between donors are found for two other members of the MACS-family, the fatty acid transport protein 3 (FATP3) and the FATP4. On protein level, we tested for ACBP expression. The ACBP protein is consistently expressed in monocytes of all donors. Human monocytes are insulin target cells and capable of fatty acid metabolism to some extent. Ex vivo-derived monocytes could be used in additional studies for analyzing differences in genotype-dependent expression levels of genes involved in fat assimilation such as ACBP, MACS1 or PPARgamma.