Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human and moise primary hepatocytes from 12 donors were treated with PPARI? agonist Wy14643

ABSTRACT: Studies in mice have shown that PPARÎ± is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARÎ± in human liver. Here we set out to compare the function of PPARÎ± in mouse and human hepatocytes via analysis of target gene regulation. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARÎ± agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARÎ± expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARÎ± in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARÎ± targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARÎ± in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARÎ± targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARÎ± activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARÎ± as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARÎ± regulates a mostly divergent set of genes in mouse and human hepatocytes. GSE17250: Comparative analysis of gene regulation by the transcription factor PPARÎ±_mouse; GSE17251: Comparative analysis of gene regulation by the transcription factor PPARÎ±_human Experiment Overall Design: Refer to individual Series

ORGANISM(S): Mus musculus

SUBMITTER: Guido Hooiveld

PROVIDER: E-GEOD-17254 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human.

Rakhshandehroo Maryam M Hooiveld Guido G Müller Michael M Kersten Sander S

PloS one 20090827 8

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARalpha agonist Wy14643 and gene expres ...[more]

PMID: 19710929

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Project description:Studies in mice have shown that PPARÎ± is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARÎ± in human liver. Here we set out to compare the function of PPARÎ± in mouse and human hepatocytes via analysis of target gene regulation. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARÎ± agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARÎ± expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARÎ± in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARÎ± targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARÎ± in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARÎ± targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARÎ± activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARÎ± as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARÎ± regulates a mostly divergent set of genes in mouse and human hepatocytes. Experiment Overall Design: Primary hepatocytes from 6 mice from 6 different strains were treated with the PPARÎ± agonist Wy14643 for 6 and 24 hours, and gene expression profiling was performed using Affymetrix GeneChips. Mouse primary hepatocytes were isolated by two-step collagenase perfusion from 6 different strains of mouse; NMRI, SV129, FVB, DBA, BALB/C and C57BL/6J. Cells were plated on collagen-coated six-well plates. Viability was determined by Trypan Blue exclusion, and was at least 75%. Hepatocytes were suspended in William's E medium (Lonza Bioscience, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum, 20 m-units/mL insulin, 10 nM dexamethasone, 100 U/mL penicillin, 100 Î¼g/mL of streptomycin, 0.25 Î¼g/mL fungizone and 50 Î¼g/mL gentamycin. After four hours the medium was discarded and replaced with fresh medium. The next day, cells were incubated in fresh medium in the presence or absence of Wy14643 (10 microM) dissolved in DMSO for 6 and 24 hours, followed by RNA isolation. A 5-fold lower concentration of Wy14643 was used in mouse primary hepatocytes to take into account the higher affinity of Wy14643 for mouse PPARÎ± compared to human PPARÎ±.

Project description: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. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARalpha agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARalpha expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARalpha in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARalpha targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARalpha in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARalpha targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARalpha activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARalpha as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARalpha regulates a mostly divergent set of genes in mouse and human hepatocytes. Experiment Overall Design: Primary hepatocytes from 6 human subjects were treated with the PPARalpha agonist Wy14643 for 6 and 24 hours, and gene expression profiling was performed using Affymetrix GeneChips. Human hepatocytes and Hepatocyte Culture Medium Bulletkit were purchased from Lonza Bioscience (Verviers, Belgium). Primary hepatocytes were isolated from surgical liver biopsies obtained from six individual donors who underwent surgery after informed consent was obtained for surgery with subsequent use of samples in experiments. Hepatocytes were isolated with two-step collagenase perfusion method and the viability of the cells was over 80%. Cells were plated on collagen-coated six-well plates and filled with maintenance medium. Upon arrival of the cells, the medium was discarded and was replaced by Hepatocyte Culture Medium (HCM) with additives. The additives included Gentamicin sulphate/Amphotercin-B, Bovine serum albumin (Fatty acid free), Transferrin, Ascorbic acid, Insulin, Epidermal growth factor, Hydrocortisone hemisuccinate. The next day, cells were incubated in fresh medium in the presence or absence of Wy14643 (50 microM) dissolved in DMSO for 6 and 24 hours, followed by RNA isolation.

Project description:Background: Studies in mice have shown that PPARÎ± is an important regulator of lipid metabolism in liver and a key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARÎ± in human liver. Here we set out to study the function of PPARÎ± in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARÎ± agonist Wy14643. Results: Quantitative PCR indicated that PPARÎ± is well expressed in human liver and human liver slices and that the classical PPARÎ± targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARÎ± activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARÎ± activation (q value<0.05). Many genes induced by PPARÎ± activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARÎ± activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon Î³-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARÎ± is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARÎ± activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARÎ± were identified that were commonly induced by PPARÎ± activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L. Conclusion: Our manuscript demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARÎ± in human liver. Our data underscore the major role of PPARÎ± in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARÎ± in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease. Precision-cut liver slices, prepared from liver biopsies obtained from obese subjects undergoing bariatric surgery, were incubated with the peroxisome proliferator-activated receptor alpha (PPARÎ±) agonist Wy14643 or vehicle for 24hrs, after which gene expression was profiled by array.

Project description:PPARÎ± is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARÎ± in hepatic lipid metabolism, many PPARÎ±-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARÎ±-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARÎ± target genes, livers from several animal studies in which PPARÎ± was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARÎ±-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARÎ±-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein Î² polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Since Pnpla2, Lipe, and Mgll contribute to hepatic triglyceride hydrolysis, gene regulation was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARÎ± agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARÎ±. Our study illustrates the power of transcriptional profiling to uncover novel PPARÎ±-regulated genes and pathways in liver. Experiment Overall Design: Primary rat and mouse hepatocytes were incubated in the presence (10 uM) or absence of the synthetic PPARÎ± ligand Wy14643 for 24 hours. Primary human hepatocytes were incubated in the presence (50 uM) or absence of Wy14643 for 12 hours. Total RNA was pooled within groups and hybridized onto Rat Genome 230 2.0 Arrays, Mouse Genome 430 2.0 Arrays or Human Genome U133 Plus 2.0 Arrays.

Project description:Background: The selective absorption of nutrients and other food constituents in the small intestine is mediated by a group of transport proteins and metabolic enzymes, often collectively called âintestinal barrier proteinsâ. An important receptor that mediates the effects of dietary lipids on gene expression is the peroxisome proliferator-activated receptor alpha (PPARÎ±), which is abundantly expressed in enterocytes. In this study we examined the effects of acute nutritional activation of PPARÎ± on expression of genes encoding intestinal barrier proteins. To this end we used triacylglycerols composed of identical fatty acids in combination with gene expression profiling in wild-type and PPARÎ±-null mice. Treatment with the synthetic PPARÎ± agonist WY14643 served as reference. Results: We identified 74 barrier genes that were PPARÎ±-dependently regulated 6 hours after activation with WY14643. For eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and oleic acid (OA) these numbers were 46, 41, and 19, respectively. The overlap between EPA-, DHA-, and WY14643-regulated genes was considerable, whereas OA treatment showed limited overlap. Functional implications inferred form our data suggested that nutrient-activated PPARÎ± regulated transporters and phase I/II metabolic enzymes were involved in a) fatty acid oxidation, b) cholesterol, glucose, and amino acid transport and metabolism, c) intestinal motility, and d) oxidative stress defense. Conclusion: We identified intestinal barrier genes that were PPARÎ±-dependently regulated after acute activation by fatty acids.This knowledge provides a better understanding of the impact dietary fat has on the barrier function of the gut, identifies PPARÎ± as an important factor controlling this key function, and underscores the importance of PPARÎ± for nutrient-mediated gene regulation in intestine. Experiment Overall Design: Pure bred wild-type (129S1/SvImJ) and PPARÎ±-null (129S4/SvJae) mice were treated for 6 hours with the synthetic triacylglycerols triolein [oleic acid (OA, C18:1)], trieicosapentaenoin [eicosapentaenoic acid (EPA, C20:5)] or tridocosahexaenoin [ocosahexaenoic acid (DHA, C22:6)], or the potent PPARÎ± agonist WY14,643. Two weeks before the start of the experiment, mice were put on a modified AIN76A diet, in which corn oil was replaced by olive oil. At the day of the experiment mice were fasted for four hours. At 9 AM mice were dosed by oral gavage with 400 Âµl of a 0.1% WY14643 suspension in 0.5% carboxymethyl cellulose, or 400 Âµl of the synthetic triacylglycerols. Six hours after the gavage the mice were anaesthetized, small intestines were removed, flushed with ice-cold PBS and remaining fat and pancreatic tissue was carefully removed. Total RNA was then isolated. RNA of 4-5 biological replicates was hybridized to Affymetrix 430-2.0 plus arrays. Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

Project description:PPARÎ± is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARÎ± in hepatic lipid metabolism, many PPARÎ±-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARÎ±-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARÎ± target genes, livers from several animal studies in which PPARÎ± was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARÎ±-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARÎ±-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein Î² polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Since Pnpla2, Lipe, and Mgll contribute to hepatic triglyceride hydrolysis, gene regulation was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARÎ± agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARÎ±. Our study illustrates the power of transcriptional profiling to uncover novel PPARÎ±-regulated genes and pathways in liver. Experiment Overall Design: 3-5 months old male pure bred wild-type (129S1/SvImJ) and PPARÎ±-null (129S4/SvJae) mice were used. Experiment Overall Design: Wild-type and PPARÎ±-null mice fasted for 4 hours received a single dose of WY14643 (400 Î¼l of 10 mg/ml WY14643) or vehicle and were killed 6 hours later (n=3-5 per group). Liver total RNA from biological replicates was hybridized onto Affymetrix mouse genome 430 2.0 GeneChip arrays. Experiment Overall Design: Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

Project description:PPARÎ± is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARÎ± in hepatic lipid metabolism, many PPARÎ±-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARÎ±-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARÎ± target genes, livers from several animal studies in which PPARÎ± was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARÎ±-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARÎ±-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein Î² polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Since Pnpla2, Lipe, and Mgll contribute to hepatic triglyceride hydrolysis, gene regulation was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARÎ± agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARÎ±. Our study illustrates the power of transcriptional profiling to uncover novel PPARÎ±-regulated genes and pathways in liver. Experiment Overall Design: 3-5 months old male pure bred wild-type (129S1/SvImJ) and PPARÎ±-null (129S4/SvJae) mice were used. Experiment Overall Design: Wild-type and PPARÎ±-null mice were treated with the synthetic PPARÎ± ligand Wy14643 (0.1% w/w) mixed in the food, or normal food (control) for 5 days (n=4 per group). Liver total RNA from biological replicates was hybridized onto Affymetrix mouse genome 430 2.0 GeneChip arrays. Experiment Overall Design: Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

Project description:PPARÎ± is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARÎ± in hepatic lipid metabolism, many PPARÎ±-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARÎ±-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARÎ± target genes, livers from several animal studies in which PPARÎ± was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARÎ±-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARÎ±-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein Î² polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Since Pnpla2, Lipe, and Mgll contribute to hepatic triglyceride hydrolysis, gene regulation was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARÎ± agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARÎ±. Our study illustrates the power of transcriptional profiling to uncover novel PPARÎ±-regulated genes and pathways in liver. Experiment Overall Design: 3-5 months old male pure bred wild-type (129S1/SvImJ) and PPARÎ±-null (129S4/SvJae) mice were used. Experiment Overall Design: Wild-type and PPARÎ±-null mice were treated with the synthetic PPARÎ± ligand WY14643 (0.1% w/w) mixed in the food, or normal food (control) for 5 days (n=5 per group). Liver total RNA of pooled samples (within groups) was hybridized onto Affymetrix MOE430A GeneChip arrays. Experiment Overall Design: Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

Dataset Information

Transcription profiling of human and moise primary hepatocytes from 12 donors were treated with PPARI? agonist Wy14643

Publications

Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human.

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