Whole-genome microarray analysis identi?es up-regulation of Nr4a nuclear receptors in muscle and liver from diet-restricted rats
Radu C.Oita a ,1,Dawn J.Mazzatti a ,1,*,Fei Ling Lim a ,Jonathan R.Powell a ,Brian J.Merry b
a Unilever R&D Colworth,Colworth Science Park,Sharnbrook,Bedfordshire MK441LQ,United Kingdom b
School of Biological Sciences,University of Liverpool,Liverpool L697ZB,United Kingdom
1.Introduction
For over 70years,dietary restriction (DR)has been recognised to slow/delay the ageing process and to extend maximum lifespan achieved [recently reviewed in (Merry,2005)].In fact,DR remains the only intervention repeatedly demonstrated to increase lifespan and delay age-associated diseases in mammalian species (Masoro,1988,2000).Since this discovery,a number of studies have sought to identify the mechanisms by which DR retards ageing in laboratory models.These proposed mechanisms include decreased oxidative stress (Sohal and Weindruch,1996),decreased damage to subcellular components (Kristal and Yu,1992),decreased body temperature and hypometabolic state (Walford and Spindler,1997),neuroendocrine changes (Nelson et al.,1995)and altera-tions in gene expression and protein degradation (Van Remmen et al.,1996).
DR-induced alterations in gene expression undoubtedly play a signi?cant role in its anti-ageing actions.To date,many studies
have reported the effects of DR on gene expression pro?ling in a number of tissue-types and laboratory animals.All of these studies were performed using Affymetrix TM partial-genome (i.e.6000transcripts)or cDNA arrays.However,since the sequencing was completed on the rat genome,whole-genome arrays have become commercially available providing a powerful tool to measure genome-wide expression.Recently,Fu et al.(2006)used Affyme-trix TM GeneChips with approximately 12,000genes to describe global gene expression changes in heart,liver,and hypothalamus of young and old ad libitum and DR-fed mice.Whilst this study uncovered a plethora of genes affected by ageing across different tissues in mouse,it remains possible that more genes and ESTs whose function have not previously been described in the context of ageing or DR –or whose function is completely unknown –may be discovered using gene arrays that cover the entire rodent genome.
In the current study,we used Agilent TM whole-genome rat arrays with more than 40,000probes to investigate the effects of DR on gene expression in rat skeletal muscle of old (28months)rats fed the ad libitum or DR-diet (45%restricted from controls)from 1.5months of age.In addition to known alterations in stress response,protein metabolism and energy metabolism,we identi?ed a novel class of genes,Nr4a nuclear receptors,that
Mechanisms of Ageing and Development 130(2009)240–247
A R T I C L E I N F O Article history:
Received 27June 2008
Received in revised form 13November 2008Accepted 12December 2008
Available online 27December 2008Keywords:
Dietary restriction Longevity Ageing
Nuclear orphan receptor NR4A Nur77
A B S T R A C T
One of the most conserved methods to signi?cantly increase lifespan in animals is through dietary restriction (DR).The mechanisms by which DR increases survival are controversial but are thought to include improvements in mitochondrial function concomitant with reductions in reactive oxygen species production and alterations in the insulin signalling pathway,resulting in global metabolic adaptation.In order to identify novel genes that may be important for lifespan extension of Brown Norway rats,we compared gene expression pro?les from skeletal muscle of 28-month-old animals fed ad libitum or DR diets using whole-genome arrays.Following DR,426transcripts were signi?cantly down-regulated whilst only 52were up-regulated.Included in the up-regulated transcripts were three functionally related previously unidenti?ed DR-regulated genes:Nr4a1,Nr4a2,and Nr4a3.Up-regulation of all three Nr4a receptors was also observed in liver –but not brain –of DR-fed animals.Furthermore,RT-PCR revealed up-regulation of several NR4A transcriptional targets (Ucp-3,Ampk-g 3,Pgc-1a and Pgc-1b )in skeletal muscle of DR animals.Due to the proposed roles of the NR4A nuclear receptors in sensing and responding to changes in the nutritional environment and in regulating glucose and lipid metabolism and insulin sensitivity,we hypothesise that these proteins may contribute to DR-induced metabolic adaptation.
?2009Elsevier Ireland Ltd.All rights reserved.
*Corresponding author.Tel.:+44234222372;fax:+441234222161.E-mail address:Dawn.Mazzatti@https://www.doczj.com/doc/102045679.html, (D.J.Mazzatti).1
These authors contributed equally to the work.
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Mechanisms of Ageing and Development
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were differentially regulated not only in skeletal muscle,but also in liver but not brain of animals fed the DR diet compared to ad libitum-fed age-matched animals.Due to the proposed roles of the NR4A subfamily of nuclear receptors in sensing and responding to environmental changes and in regulating glucose and lipid metabolism and insulin sensitivity,we hypothesise that these proteins may play a novel role in DR-induced metabolic adaptation.
2.Materials and methods
2.1.Animals
Male Brown Norway rats(Substrain BN/SsNOlaHSD)were obtained from Harlan UK at21–28days of age and maintained on a CRM diet supplied by Dietex International,Witham,UK as previously described(Merry et al.,2008)Rats were maintained under barrier conditions in which sentinel animals were routinely screened for microbiological states to ensure a consistent health status throughout the duration of the study.Rats were maintained on a12:12-h light/dark cycle (08:00–20:00)at22?18C and caged in groups of four and fed ad libitum until1.5 months of age.After this time they were transferred to single housing and assigned randomly to either control or DR feeding regimes.DR-fed animals were fed the CRM diet at55%the daily food intake of age-matched control rats in order to maintain their body weight at approximately55%that of control,ad libitum-fed animals.The diet of animals maintained on a DR regime was supplied daily as pre-weighed rations between10:30and11:00h.Survival trajectories were determined(data not shown)to ensure that the DR feeding regime induced a signi?cant effect on survival.The survival data,analysed by the log-rank(Peto)test,showed that the DR feeding regime induced a signi?cant extension in survival(p<0.0001).All animal husbandry procedures were carried out in accordance with the provisions of the United Kingdom Animals (Scienti?c Procedures)Act1986.
Six animals were randomly selected from both diet groups to provide tissues for the microarray analysis.The initial group sizes from which the animals were selected were control(102)and DR(75).Hind-limb gastrocnemius muscles were sampled from each animal.Tissues were taken immediately at death and snap-frozen in liquid N2.
2.2.Microarrays
Microarray experimentation was performed as previously described(Mazzatti et al.,2008)with the exception of600ng input RNA for labelling and the use of Agilent rat oligonucleotide arrays(Wokingham,Berkshire,UK).The slides were scanned with the Agilent G2565BA Microarray Scanner System.For data extraction and quality control,the Agilent G2567AA Feature Extraction Software(v.9.1)was used.
2.3.Bioinformatics analysis
Extracted data were analysed using GeneSpring GX7.3.1(Agilent Technologies, CA,USA).Agilent standard scenario normalisations for FE1-colour arrays were applied to all data sets.A subset of genes(from an initial41,105probes)for data interrogation was generated that excluded controls,spots of poor quality(absent calls),and gene probes that were present or marginal in less than6out of12of experimental samples.This yielded20,023probes for analysis.From these20,023 probes,relative expression in animals fed the ad libitum diet was compared to animals on the dietary restriction(DR)diet.Genes differentially regulated by greater than 2.0-fold were selected.One-way,parametric,ANOVA tests were performed followed by Benjamini and Hochberg multiple test correction with a false discovery rate of0.05,in order to decrease the number of false positives (Pawitan et al.,2005).Microsoft Excel templates were prepared containing genes that were over-and under-expressed following DR.
2.4.Ingenuity TM Pathway Analysis(IPA)
Ingenuity TM Pathway Analysis(IPA)3.0(Ingenuity TM Systems,CA,USA)was utilised to assemble functional networks altered by DR.Ingenuity entry tool systematically encodes?ndings presented in peer-reviewed scienti?c publications into ontologies,or groups of genes/proteins related by common function.Molecular networks of direct physical,transcriptional,and enzymatic interactions were computed from this knowledge base using the genes differentially regulated by DR. The resulting networks contain molecular relationships with a high degree of connectivity and each gene in the network is supported by published literature.
2.5.Real-time quantitative reverse-transcription PCR(Q-PCR)
RNA was prepared from cells as described and cDNA synthesis was performed using1m g total RNA and oligo dT primers(Invitrogen,Paisley,UK).Taqman (Applied Biosystems,UK)probes used for RT-PCR validation of microarrays are listed in Table2.Probes not listed in Table2but used for RT-PCR analysis include nuclear receptor subfamily4,group A,member1:Rn00666995_m1;nuclear receptor subfamily4,group A,member3:Rn00581189_m1;nuclear receptor subfamily4,group A,member2:Rn00570936_m1;peroxisome proliferator activator receptor delta:Rn00565707_m1;peroxisome proliferator activated receptor gamma:Rn00440945_m1;peroxisome proliferator activated receptor alpha:Rn00566193_m1;uncoupling protein3:Rn00565874_m1;fatty acid binding protein4:Rn00670361_m1;protein kinase,AMP-activated,gamma3 non-catalytic subunit:Rn01400861_g1;peroxisome proliferative activated recep-tor,gamma,coactivator1beta:Rn00598552_m1;peroxisome proliferative activated receptor,gamma,coactivator1alpha:Rn00580241_m1;glyceralde-hyde-3-phosphate dehydrogenase:Rn99999916_s1;actin,beta:Rn00667869_m1. The Bio-rad I-Cycler(Bio-rad,Hercules,CA)with FAM-490system detection was used for real-time RT-PCR.PCR thermocycler conditions were508C for2min,908C for2min followed by45cycles of958C for15s and608C for60s.All samples were run in triplicate with both primer sets and the control genes Gapdh and b-actin to control for differences in amount of starting material.Fold changes were calculated using the2àDD Ct method.Statistical analyses were performed using Student’s t-test.
A p-value of less than0.05was considered signi?cant.
3.Results
3.1.DR-responsive genes and ontologies
The primary aim of this study was to identify novel genes that are differentially regulated by DR in rats using whole-genome arrays.In order to examine the effect of long-term DR on gene expression pro?les,RNA samples from skeletal muscle of six ad libitum-fed and six DR-fed animals were subjected to whole-genome microarray analysis.Stringent data analysis was per-formed in order to ensure that the genes identi?ed through microarray analysis were truly differentially expressed.After exclusion of absent calls and probes that were expressed (?ags=present or marginal)in less than half of experimental samples,20,023out of an initial41,105probes(50%)were considered for analysis.Only probes with a differential expression of at least2.0-fold and which passed ANOVA testing at p<0.05 were considered to be truly differentially expressed by DR.Gene expression pro?ling of muscle from ad libitum-and DR-fed animals demonstrated that a total of478probes were differentially expressed.These are listed in Supplementary Table1.Of these probes,52were up-regulated and426were down-regulated by DR.The478signi?cantly regulated probes were subjected to gene cluster analysis using a Pearson correlation.A heatmap was generated where each row represents one of478probes and each column represents one skeletal muscle sample from one animal. Expression of the478probes in skeletal muscle of each animal is shown in Fig.1.The intensity of colour in a cell represents the normalised expression of the probe,whilst green and red colouring depicts low and high expression,respectively.Expression of each probe in samples from the six animals fed the DR diet and six animals fed ad libitum are shown from left to right.The observed inter-animal variation in gene expression is likely due to differences in the relative proportion of fast and slow muscle ?bre-types in individual animals.
Biological processes affected by DR were investigated initially using Gene Ontology(GO)analysis of the genes signi?cantly regulated in DR-compared to ad libitum-fed animals.Table1 shows the20most signi?cantly regulated GO biological processes in dietary restricted muscle with associated p-value.Although functions known to be affected by DR(including glucose and lipid metabolism)are not among the top20most-regulated ontologies, several metabolic ontologies were found to be signi?cantly regulated(p<0.05):glycogen metabolism,aspartate metabolism, protein metabolism,nucleoside metabolism,fat cell differentia-tion,and mitochondrial biogenesis.Additionally,we observed DR-dependent signi?cant regulation of cell-cycle,growth and apoptosis-related ontologies.In an expanded list of genes with less stringent selection criteria(differentially regulated at least 1.5-fold and p<0.05)consisting of greater than3000probes,we
R.C.Oita et al./Mechanisms of Ageing and Development130(2009)240–247241
observed signi?cant differences in multiple ontologies related to glucose,lipid,and glycogen metabolism,synthesis,and usage (data not shown).These data con?rm results observed in previously investigated diet-and/or calorie-restriction models.3.2.Validation of arrays using real-time RT-PCR
In order to validate results obtained using the microarrays we arbitrarily selected 14genes and assessed mRNA expression by real-time RT-PCR.Table 2compares DR-regulated gene expression in skeletal muscle by gene array and RT-PCR.GenBank gene identi?er,Taqman probe reference (Applied Biosystems,UK),common gene name,and fold-change in gene expression in muscle of DR-compared to ad libitum -fed animals,as assessed by gene array and RT-PCR,are shown.All 14genes selected for validation demonstrated similar expression patterns for the microarray and real-time RT-PCR measurements,yet with different magnitudes,thus validating the array results.
3.3.Expression of Nr4a1,Nr4a2and Nr4a3in skeletal muscle,liver and brain of ad libitum and DR animals
Included in the transcripts found to be differentially regulated in skeletal muscle by DR were three functionally related previously unidenti?ed DR-regulated genes:Nr4a1,Nr4a2,and Nr4a3.These three genes were up-regulated by 2.2-fold,3.0-fold,and 3.7-fold,respectively,in skeletal muscle from DR animals compared to control.The NR4A subfamily of nuclear receptors was ?rst identi?ed as early responsive genes for growth factors (Milbrandt,1988;Law et al.,1992)and have since been implicated in regulating the response to insulin (Fu et al.,2007).These proteins include NR4A1(Nur77/TR3/NGFI-B/NAK-1),NR4A2(Nur1/NOT)and NR4A3(NOR1/MINOR).In order to determine if these receptors were differentially expressed in a tissue-speci?c manner following DR,we further assessed the gene expression of these receptors in skeletal muscle,liver and whole brain of DR-and ad libitum -fed animals.Fig.2shows mRNA expression of Nr4a1,Nr4a2,and Nr4a3by real-time RT-PCR.Tissue-speci?c differential expression of Nr4a receptors was observed.In muscle and liver,up-regulation of all three Nr4a receptors (Nr4a1,Nr4a2and NR4a3)was observed in dietary restricted animals compared to control.In contrast,in brain,dietary restriction did not signi?cantly
alter
Fig.1.Hierarchical clustering of DR-sensitive gene probes.The 478gene probes signi?cantly differentially regulated in skeletal muscle samples from DR compared to ad libitum -fed animals were subjected to gene cluster analysis using a Pearson correlation.A heatmap was generated where each row represents one of 478probe sets and each column represents one skeletal muscle sample from an individual animal.The intensity of colour in a cell represents the normalised expression of the probe,where green and red colouring depicts low and high expression,respectively,compared to the average expression of each probe across all samples.n =6animals in each group.
Table 1
Top 20signi?cantly DR-regulated ontologies and associated p -values.Ontology
p -value GO:48589:developmental growth 3.11E à06GO:6952:defence response
9.57E à06GO:9607:response to biotic stimulus 2.56E à05GO:6955:immune response
2.95E à05GO:6878:copper ion homeostasis
3.05E à05GO:7517:muscle development 8.58E à05GO:6936:muscle contraction 0.000104GO:19882:antigen presentation
0.000131GO:42535:positive regulation of tumour necrosis factor-alpha biosynthesis
0.000142GO:42534:regulation of tumour necrosis factor-alpha biosynthesis
0.000142GO:42533:tumour necrosis factor-alpha biosynthesis 0.000142GO:48668:collateral sprouting
0.000264GO:48669:collateral sprouting in the absence of injury 0.000264GO:48738:cardiac muscle development
0.000264GO:48739:cardiac muscle ?bre development
0.000264GO:45736:negative regulation of cyclin-dependent protein kinase activity
0.000333GO:30333:antigen processing 0.000397GO:9611:response to wounding
0.00044GO:16198:axon choice point recognition
0.000784GO:16199:axon midline choice point recognition
0.000784
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expression of any Nr4a receptor.However,this appeared to result from high intra-animal variation most likely due to the fact that expression was measured in whole-brain and not in speci?c sub-regions.These data highlight the complex regulation of these receptors and suggest that each NR4A receptor may exert diverse functions in different tissues.This is unsurprising as it has recently been hypothesised that NR4A target genes and function may be both regulated in a cell type-speci?c manner and be developmen-tally programmed (Fu et al.,2007;Pei et al.,2006).3.4.Expression of NR4A-interacting genes
Similar to other steroid receptors,each NR4A receptor contains a DNA-binding domain,a transactivation domain,and a putative ligand-binding domain.Initially classi?ed as orphan receptors due to lack of known ligand,it now seems probable that they are true orphan receptors and do not require ligand binding for their physiological function.All three NR4A proteins bind to a consensus NBRE (NGF-IB response element)sequence (AAAGGTCA)as monomers or to a palindromic NurRE sequence (TGATATTT-X6AAATGCCA)found naturally in the pro-opiomelanocortin (POMC)promoter,as homo-or heterodimers (Philips et al.,1997a ).Additionally,NR4A1and A2heterodimerise with retinoid X-receptor (RXR)to mediate response to retinoids (Wallen-Mackenzie et al.,2003).RXR plays an important regulatory role
in metabolic signalling pathways (glucose,fatty acid and cholesterol metabolism)and has been implicated in metabolic disorders such as type 2diabetes,hyperlipidaemia and athero-sclerosis (reviewed in Ahuja et al.,2003).Due to the increased expression of all three Nr4a receptors in skeletal muscle and known interaction of the NR4A proteins with RXR,we hypothe-sised that Rxr expression might be up-regulated by DR,thereby allowing the possibility of increased transcriptional activation of target genes.However,microarray analysis identi?ed no signi?-cant effects of DR on any RXR isoform,including Rxr-a ,-b ,and -g .We next investigated whether DR affected expression of an important class of RXR-interacting proteins that have previously been shown to be altered as a consequence of DR,the peroxisome proliferator-activated receptors (PPARs)(Zhu et al.,2004;reviewed in Masternak and Bartke,2007)by real-time RT-PCR analysis.Fig.3shows relative mRNA expression of Ppar-a ,-b /d ,and -g in muscle,liver and brain from animals on the DR diet compared to control.DR resulted in down-regulation of Ppar-a in muscle concomitant with up-regulation in liver compared to control.In contrast,Ppar-b /d was down-regulated in both liver and
Table 2
Comparison of DR-regulated gene expression by gene array and RT-PCR.GenBank gene identi?er,Taqman probe reference (Applied Biosystems,UK),common gene name,and fold-change in gene expression in DR-compared to ad libitum -fed animals,as assessed by gene array and RT-PCR,are shown.p <0.05for all genes shown.GenBank identi?er Taqman probe reference Common name
Fold-change array Fold-change RT-PCR NM_024362Rn00577590_m1Aryl hydrocarbon receptor nuclear translocator-like à6.58à6.13NM_017325Rn00569082_m1Runt related transcription factor 1
à5.35à9.20NM_145775Rn00595671_m1Nuclear receptor subfamily 1,group D,member 1à2.88à3.05NM_198769Rn02133647_s1Toll-like receptor 2
à2.86à1.20NM_012861Rn00563462_m1O-6-methylguanine-DNA methyltransferase à2.76à2.42NM_017237Rn00568258_m1ubiquitin carboxy-terminal hydrolase L1à3.70à2.30NM_001024791Rn01456494_g1Epsin 3à3.22à8.83NM_001013247Rn02769377_s1Sarcolipin
à6.54à3.03NM_017131Rn00567508_m1Calsequestrin 2
à3.13à1.50NM_031612Rn00581093_m1Apelin,AGTRL1ligand
à2.54à3.15NM_080767Rn00589926_m1Proteosome (prosome,macropain)subunit,beta type 8à2.25à3.00NM_012708Rn00562296_m1Proteosome (prosome,macropain)subunit,beta type 9à2.15à3.25NM_012922Rn00563902_m1Caspase 3,apoptosis related cysteine protease à2.42à3.80NM_001007713
Rn01400508_g1
Transmembrane BAX inhibitor motif containing 1
à3.90
à
1.33
Fig.3.mRNA expression of Ppar-a ,-b /d ,and -g in muscle,liver and brain from animals on the DR diet compared to control.RNA was prepared and in vitro transcription was performed using 1m g total RNA and random hexamer primers (Invitrogen,Paisley,UK).The Bio-rad I-Cycler (Bio-rad,Hercules,CA)with FAM-490system detection was used for real-time RT-PCR.Taqman (Applied Biosystems,Applera,UK)probes were used as detailed in Section 2.Animals were grouped into DR and ad libitum conditions and relative mRNA expression in each group was determined.All samples were run in triplicate with test probes and the control rat genes B-actin and Gapdh to control for differences in amount of starting material.Fold-change in expression was calculated by normalising the test gene crossing threshold (Ct)with the averaged ampli?ed control and then comparing to gene expression in ad libitum-fed animals.Fold-change in mRNA expression of PPAR a ,g ,b /d in muscle (black),liver (light grey)and whole brain (white)in DR animals compared to ad libitum -fed animals is shown.n =6animals per group;asterisk (*)denotes p <
0.05.
Fig.2.Expression of Nr4a1,Nr4a2and Nr4a3in skeletal muscle,liver and brain of ad libitum and DR animals.RNA was prepared and in vitro transcription was performed using 1m g total RNA and random hexamer primers (Invitrogen,Paisley,UK).The Bio-rad I-Cycler (Bio-rad,Hercules,CA)with FAM-490system detection was used for real-time RT-PCR.Taqman (Applied Biosystems,Applera,UK)probes were used as detailed in Section 2.Animals were grouped into DR and ad libitum conditions and relative mRNA expression in each group was determined.All samples were run in triplicate with test probes and the control rat genes B-actin and Gapdh to control for differences in amount of starting material.Fold-change in expression was calculated by normalising the test gene crossing threshold (Ct)with the averaged ampli?ed control and then comparing to gene expression in ad libitum-fed animals.Fold-change in mRNA expression of Nr4a1,Nr4a2,and Nr4a3in skeletal muscle (black),liver (light grey)and whole brain (white)in DR animals compared to ad libitum -fed animals is shown.n =6animals in each group;asterisk (*)denotes p <0.05.
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brain,but not in muscle.No signi?cant change in Ppar-g expression was observed in any of the three tissues investigated.
Whilst we observed decreased expression of only Ppar-a in muscle,Masternak et al.(2005a,b)have previously demonstrated DR-induced down-regulation of Ppar-a ,b /d ,and g expression in mouse muscle.In the same studies,they identi?ed no effect on the expression of Rxr isoforms following DR,which we have recapitulated.Additionally,in liver and heart,Ppar expression was found to be both up-and down-regulated,depending on the isoform and tissue investigated (Masternak et al.,2005a ,2004)suggesting that regulation of these nuclear hormone receptors by DR is complex and occurs in a tissue-speci?c and isoform-speci?c manner.
In order to better understand the signi?cance of altered Nr4a expression in muscle of DR rats,we further investigated known transcriptional targets of NR4A receptors.To date,very few known transcriptional targets of any of the NR4A family in myocytes exist.Attenuation of NR4A1expression in muscle results in decreased expression of genes including uncoupling protein-3(Ucp-3),glucose transporter 4(Glut4),CD36,caveolin 3(Cav3)and AMP-activated protein kinase g 3subunit (Ampk g 3)(Maxwell et al.,2005).Two recent reports have described NR4A3transcriptional targets in muscle.These include fatty acid binding protein-4(Fabp-4),Ucp-2and Ucp-3,PPAR gamma-coactivators alpha and beta (PGC-1a and b ),lipin1a ,and pyruvate dehydrogenase phospha-tase 1(PDHP1)(Pearen et al.,2006,2008).Since microarray analysis did not demonstrate altered expression of any of these genes,we next investigated muscle-speci?c expression of these targets by RT-PCR.Fig.4shows relative mRNA expression of two NR4A1targets (Ucp-3,and Ampk-g 3)and three NR4A3targets (Pgc-1a and Pgc -1b and Fabp-4)in muscle from animals fed ad libitum or
the DR diet.We observed signi?cant up-regulation of Ucp-3,Ampk-g 3Pgc-1a and Pgc -1b with no change in expression of Fabp-4following DR.These data are consistent with increased transcrip-tional activity of NR4A1and NR4A3.
We next used Ingenuity Pathway Analysis TM (IPA)to investigate whether additional NR4A-interacting proteins were regulated by DR.IPA identi?ed one network in which all three NR4A receptors are depicted together with interacting proteins (Fig.5).Common names of these genes,network score (probability of network being assembled by chance alone)and main cellular functions of the network are shown in Table 3.Coloured genes were identi?ed by microarray analysis as differentially up-regulated or down-regulated by DR (red and green colouring,respectively).
IPA analysis of the genes affected by DR identi?ed one homodimeric protein,platelet-derived growth factor-B (PDGFBB)that directly stimulates expression of all three NR4A receptors.PDGFBB is a potent mitogen for vascular smooth muscle cells.PDGFBB was found to inhibit the induction of nitric oxide synthase activity in vascular smooth muscle cells (Schini et al.,1992).Interestingly,the NR4A orphan nuclear receptor NOR1is induced by platelet-derived growth factor and mediates vascular smooth muscle cell proliferation (Nomiyama et al.,2006).Furthermore,a role for NR4A proteins in protecting against vascular disease has recently been hypothesised (Pols et al.,2007).However,neither investigations of PDGFBB in dietary restriction nor in ageing have been reported.Microarray analysis revealed a trend towards decreased expression of Pdgfbb in the DR condition (à1.2-fold compared to ad libitum ),but this was not statistically signi?cant (p =0.20).These data suggest that up-regulation of NR4A receptors following long-term DR is independent of Pdgfbb expression,but it remains possible that expression or activity of the protein may be affected by DR.Additionally IPA identi?ed the direct effects of tumour necrosis factor (TNF)on expression of all three NR4As,and both direct and indirect effects of leucine and RXRG on NR4A1and NR4A3or NR4A1and NR4A2,respectively.Again,we observed no change in the expression of these molecules but their protein expression and/or activity could be altered as a consequence of DR.Thus these factors could potentially be regulators of NR4A receptors in the DR condition.Taken together,pathway analysis identi?ed multiple potential mechanisms of regulation of NR4A expression which may contribute to the effects of DR.4.Discussion
Here,we demonstrate for the ?rst time increased mRNA expression of all three NR4A receptors (Nr4a1,Nr4a2,and Nr4a3)in rat skeletal muscle and liver following DR.No signi?cant changes were observed in brain,demonstrating tissue-speci?city in expression patterns following DR.We further observed up-regulation of several NR4A transcriptional targets -Ucp-3,Ampk-g 3,Pgc-1a and Pgc-1b -indicating increased NR4A activity in muscle following DR.To date,very few studies have investigated the role of NR4A receptors in disease.Initially,the subfamily of receptors (before they were classed as NR4A receptors)was investigated only in brain and immune system;all three NR4A receptors are widely expressed in the central nervous
system
Fig.4.mRNA expression of NR4A targets Ucp-3,Ampk-g 3,Pgc-1a and Pgc -1b and Fabp-4in muscle from animals on the DR diet compared to control.RNA was prepared and in vitro transcription was performed using 1m g total RNA and oligo dT primers (Invitrogen,Paisley,UK).The Bio-rad I-Cycler (Bio-rad,Hercules,CA)with FAM-490system detection was used for real-time RT-PCR.Taqman (Applied Biosystems,Applera,UK)probes were used as detailed in Section 2.Animals were grouped into DR and ad libitum conditions and relative mRNA expression in each group was determined.All samples were run in triplicate with test probes and the control rat genes B-actin and Gapdh to control for differences in amount of starting material.Fold-change in expression was calculated by normalising the test gene crossing threshold (Ct)with the averaged ampli?ed control and then comparing to gene expression in ad libitum-fed animals.Fold-change in mRNA expression of Ucp-3,Ampk-g 3,Pgc-1a and Pgc -1b and Fabp-4in muscle isolated from DR animals compared to ad libitum -fed animals is shown.n =6animals per group;asterisk (*)denotes p <0.05.
Table 3
Genetic network differentially affected by DR.Genes up-regulated and down-regulated following DR compared to ad libitum -fed animals are depicted with upward pointing and downward pointing arrows,respectively.Other genes listed were not signi?cantly regulated.A score of >3was associated with p <0.001.The network is depicted visually in Fig.5.Genes
Network score Associated functions
ACHE,Akt,APLN(#),ARNTL(#),BHLHB2,Calcineurin A,CCND3,CHI3L1,CSF1R,CYP17A1,DBP,FSTL1,HDAC9,HSD3B2(includes EG:3284),inosine,ITGAL,leucine,MGMT(#),MVP,NR1D1(#),NR4A1("),NR4A2("),NR4A3("),PDGF BB,Pkc(s),RUNX1(#),RUNX3,RXRG,S100A9,SLC6A3,TBX21,TGFB1,TLR2(#),TLR6,TNF
25
Immune and lymphatic system development and function,tissue morphology,cell cycle
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(Zetterstrom et al.,1996).These initial studies demonstrated that NR4A receptors are important in cell survival and apoptosis.Table 4summaries the Nr4a transgenic and knockout mouse phenotypes.In addition to promotion of apoptosis in lymphocytes (Zhou et al.,1996;Cheng et al.,1997)and cancer cells (Wu et al.,2002),NR4A receptors have been shown to be required for development and survival of dopaminergic neurons (Zetterstrom et al.,1997;Wagner et al.,1999)and have been linked to Parkinson’s disease (Le et al.,2003;Eells,2003).Furthermore,a recent report has linked NR4A receptors to DNA repair (Pols and de Vries,2008),demonstrating multiple and diverse functions of this class of nuclear receptors.
It is now well established that the NR4A subgroup of receptors respond to diverse stimuli,including fatty acids,stress,prosta-glandins,growth factors,cytokines,peptide hormones,phorbol esters and neurotransmitters (reviewed in Maxwell and Muscat,2006).A major feature of NR4A receptors is their ability to sense and rapidly respond to changes in the cellular environment,including the neuroendocrine environment.The NR4A receptors have been shown to play a key role in regulating the expression of various genes in the hypothalamus–pituitary–adrenal (HPA)axis,including POMC,the pre-cursor to adrenocorticotropic hormone (ACTH)the principle regulator of adrenal glucocorticoid synthesis.In addition,transcriptional agonism between glucocorticoid receptors (GR)and the NR4A proteins has been described (Philips et al.,1997a,b ).Corticotropin releasing hormone (CRH)induces NR4A1(Philips et al.,1997a )and NR4A1activates expression of CRH (Murphy et al.,2001).Alterations to neuroendocrine status is one of the proposed mechanisms by which DR increases lifespan and retards ageing.Nelson et al.demonstrated that food-restricted rats exhibit daily periods of hyperadrenocorticism (Nelson et al.,1995)which may be a major contributor to DR-induced delay of ageing processes and lifespan extension.We hypothesise that NR4A receptors may potentiate this response due to their interactions with GR,but this remains to be tested.
We have previously demonstrated that bene?cial effects of DR on mitochondria are reversed by exogenous administration of insulin,suggesting that down-regulation of
insulin-mediated
https://www.doczj.com/doc/102045679.html,work of genes differentially expressed in skeletal muscle of DR animals compared to ad libitum -fed animals.NR4A-interacting genes were analysed by the Ingenuity Pathway Analysis TM tool.The network shown was signi?cantly associated with immune and lymphatic system development and function,tissue morphology,and cell cycle (p <0.001).Coloured genes were identi?ed by microarray analysis as differentially up-regulated (red colouring)or down-regulated (green colouring)in the DR condition compared to control.These genes are listed in Table 3.Fold-change values were rounded to the nearest half-integer.Other nodal genes in the network are directly or indirectly associated with the differentially expressed genes.The meaning of the nodal shapes and connecting lines is indicated in the ?gure legend.
Table 4
Nr4a transgenic and knockout mouse phenotypes.Orphan receptor Transgenic phenotype Knockout phenotype
References
Nr4a1Massive thymocyte apoptosis Minimal phenotype
Lee et al.(1995),Cheng et al.(1997)Nr4a2NT Developmental lethal (die right after birth);lacking dopamine-producing neurons
Zetterstrom et al.(1997)
Nr4a3
NT
Embryonic lethal;defect in the proliferation of semi-circular canals of the mouse inner ear
Ponnio et al.(2002),DeYoung et al.(2003)
NT:Not tested.
R.C.Oita et al./Mechanisms of Ageing and Development 130(2009)240–247245
signalling is at least partially responsible for the lifespan extending effects of DR(Lambert et al.,2004).These data demonstrate the importance of DR-dependent regulation of the insulin signalling pathway in mediating the observed phenotype.Of interest,NR4A1 is regulated by D-glucose(Susini et al.,1998),suggesting a role for the protein in sensing-or responding to changes in glucose(or insulin)levels and/or action.Additionally,a recent report has demonstrated that NR4A receptors can modulate insulin sensi-tivity(Fu et al.,2007).In this report,both NR4A1and NR4A3were induced by both insulin and the insulin-sensitising thiazolidine-diones.Furthermore,expression of both receptors was reduced in models of insulin resistance due to aberrant insulin signalling.These?ndings provide evidence for a link between NR4A receptors and the complex metabolic‘shift’observed in animals following DR.
Very recent reports indicate that NR4receptors mediate a broad spectrum of metabolic responses.Attenuation of NR4A1expression in muscle results in decreased expression of genes involved in energy expenditure and lipid/glucose homeostasis(Chao et al., 2007),namely through regulation of uncoupling protein-3(Ucp-3), glucose transporter4(Glut4),CD36,caveolin3(Cav3)and AMP-activated protein kinase g3subunit(Ampk g3)(Maxwell et al., 2005).Here we observed signi?cant up-regulation of Ucp-3,Ampk-g3Pgc-1a and Pgc-1b in muscle following DR.These data are consistent with increased activity of both NR4A1and NR4A3. Interestingly,increased expression of all three Nr4a receptors is also associated with skeletal muscle recovery following endurance exercise(Mahoney et al.,2005),further providing evidence for their involvement in muscle fuel utilisation and physiology.In addition to their function in muscle metabolism,NR4A receptors are also thought to be involved in adipose and liver metabolic regulation.In 3T3-LI adipocytes,Nr4a1,2,and3are rapidly and transiently induced by the PPAR g-agonist rosiglitazone-and dexamethasone/ IBMX/insulin-induction of adipocyte differentiation(Fu et al.,2005). Thus it appears as though NR4A receptors mediate diverse metabolic effects in multiple tissues.Since adipose,liver and muscle are major sites of lipid and glucose metabolism and are important targets for metabolic therapies,it is possible that modulation of NR4A receptors may represent a novel strategy for treatment of metabolic disorders. This concept has already been explored(Hsu et al.,2004)and has generated a great deal of interest in the last year in particular due to a number of publications and a recent review on the potential role of NR4A receptors in vascular function(Pols et al.,2007).However, further investigation is necessary to determine whether any of the NR4A receptors represent suitable targets for pharmaceutical intervention.
Finally,it remains important to elucidate the regulation of NR4As and how they might‘sense’the nutritional environment and exert their metabolic effects.A recent report by Pei et al.(2006) demonstrated that NR4A receptors act as downstream mediators of cAMP action in vitro and in response to glucagon or fasting,in vivo.These data support our observation up-regulation of NR4A receptors in muscle and liver following DR and suggest that these receptors respond to nutritional cues such as insulin levels via an as yet unknown mechanism to regulate whole-body metabolism. This hypothesis is consistent with the previously described role of mammalian target of rapamycin(mTOR)-dependent nutrient signalling/sensing in mediating the effects of calorie restriction (Linford et al.,2007).To our knowledge,no direct links between mTOR and NR4A receptors have been made.However,crosstalk between AMPK and mTOR has been established(reviewed Hardie, 2008).Because NR4A receptors were found to be regulated by cAMP,it is possible that the receptors sense changes in AMP:ATP ratio similar to both AMPK and uncoupling proteins,and that NR4A receptors may be involved in mediating this response.These observations would link NR4As with AMPK and mTOR signalling.In support of this hypothesis we observed increased expression of both Ucp-3and Ampk-g3in the DR condition.However,further studies need to be undertaken to understand the complex relationship between the NR4A receptors and metabolic and regulatory pathways including AMPK-,mTOR-and insulin-signalling.Taken together these data suggest that multiple nutrient-sensing and signalling mechanisms may contribute to the complex metabolic adaptations that occur following DR and suggest a novel role for the NR4A receptors in this process. Acknowledgements
We thank Ann Scarborough and Andrew White for technical assistance with the microarrays.R.C.O.is supported by the NucSys Marie Curie Research Training Network funded by the European Union(contract number:MRTN-CT-019496).B.J.M.was supported by the BBSRC under the ERA Initiative(ERA Project grant16417). Appendix A.Supplementary data
Supplementary data associated with this article can be found,in the online version,at doi:10.1016/j.mad.2008.12.004.
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