Comment[ArrayExpressAccession] E-GEOD-11289 Investigation Title Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells Comment[Submitted Name] Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells Publication Title Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells reveals an individual gene expression profile response. Publication Author List Bouwens M, Afman LA, Müller M Publication DOI PubMed ID 18518955 Experimental Design Experimental Design Term Source REF Public Release Date 2008-06-06 Experiment Description Background: Peripheral blood mononuclear cells (PBMCs) are relatively easily obtainable cells in humans. Gene expression profiles of PBMCs have been shown to reflect the pathological and physiological state of a person. Recently, we showed that the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) has a functional role in human PBMCs during fasting. However, the extent of the role of PPARα in human PBMCs remains unclear. In this study, we therefore performed gene expression profiling of PBMCs incubated with the specific PPARα ligand WY14,643. Results: Incubation of PBMCs with WY14,643 for 12 hours resulted in a differential expression of 1,373 of the 13,080 genes expressed in the PBMCs. Gene expression profiles showed a clear individual response to PPARα activation between six healthy human blood donors, which was not the result of the nutritional status of the donors. Pathway analysis showed that genes in fatty acid metabolism, primarily in β-oxidation were up-regulated upon activation of PPARα with WY14,643, and genes in several amino acid metabolism pathways were down-regulated. Conclusions: This study shows that PPARα in human PBMCs regulates fatty acid and amino acid metabolism. In addition, PBMC gene expression profiles show individual responses to WY14,643 activation. We show that PBMCs are a suitable model to study changes in PPARα activation in healthy humans. Keywords: metabolic state analysis PBMCs from six healthy Caucasian male blood donors, aged between 30 and 48 yr, were isolated directly after arrival of the buffy coat (max. 8 hours after donation) by Ficol-paque Plus density gradient centrifugation (Amersham Biosciences, Roosendaal, the Netherlands). PBMCs were incubated in RPMI1640 medium with 2 mmol/L L-glutamine, 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in the presence of 5% CO2 at 37°C. at 1.0 x 106 cells per ml with either WY14,643 (50 μM) or vehicle (DMSO, 0.1%) for 12 hours. Total RNA from PBMCs was labeled using a one-cycle cDNA labeling kit (Affymetrix Inc, Santa Clara, CA) and hybridized to Affymetrix Human whole genome U133 plus 2.0 arrays (Affymetrix). Sample labeling, hybridization to chips and image scanning was performed according to the manufacturer’s GeneChip Expression Analysis Technical Manual (Affymetrix). Date of Experiment Term Source Name EFO Term Source Version Term Source File http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl Person Last Name Hooiveld Bouwens Afman Müller Person First Name Guido Mark Lydia Michael Person Mid Initials A Person Email guido.hooiveld@wur.nl Person Affiliation Wageningen University Person Phone Person Fax Person Address Div. Human Nutrition, Wageningen University, Bomenweg 2, Wageningen, Netherlands Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE11289-1 P-GSE11289-6 P-GSE11289-5 P-GSE11289-2 P-GSE11289-3 P-GSE11289-4 P-GSE11289-7 Protocol Description ID_REF =
VALUE = RMA signal (log2 expression value) Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G, according to the standard protocols of Affymetrix Arrays were hybridized on an Affymetrix 450 fluidics stations according to the protocol Midi_euk2v3_450, as described by Affymetrix PBMCs were incubated in RPMI1640 medium with 2 mmol/L L-glutamine, 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in the presence of 5% CO2 at 37°C. at 1e6 cells per ml with either WY14,643 (50 μM) or vehicle (DMSO, 0.1%) for 12 hours. PBMCs were lysed in Qiagen RLT buffer and homogenized using seringes with 0.4 mm needles, whereafter purified total RNA was isolated using Qiagen RNEasy columns Total RNA from PBMCs (5µg) was labeled using the Affymetrix One-cycle Target Labeling Assay Expression estimates were calculated using RMA Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation image_aquisition hybridization specified_biomaterial_action nucleic_acid_extraction labeling feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Experimental Factor Term Accession Number Comment[SecondaryAccession] GSE11289 Comment[GEOLastUpdateDate] 2008-06-10 Comment[GEOReleaseDate] 2008-06-05 Comment[ArrayExpressSubmissionDate] 2008-04-28 SDRF File E-GEOD-11289.sdrf.txt