Domestic Animal Endocrinology 28 (2005) 105–110

Short communication

Differential expression of peroxisomeproliferator-activated receptors alpha and gamma

gene in various chicken tissues

H. Menga,b, H. Li b,∗, J.G. Zhaob, Z.L. Gub

a School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 201101, PR Chinab College of Animal Science and Technology, Northeast Agricultural University, No. 59 Mucai Street,

Xiangfang District, Harbin, Heilongjiang, 150030, PR China

Received 29 February 2004; accepted 14 May 2004

Abstract

The peroxisome proliferator-activated receptors (PPARs) are the members of superfamily of nu-clear hormone receptors. A great number of studies in rodent and human have shown that PPARswere involved in the lipids metabolism. The goal of the current study was to investigate the expres-sion pattern of PPAR genes in various tissues of chicken. The tissue samples (heart, liver, spleen,lung, kidney, stomach, intestine, brain, breast muscle and adipose) were collected from six ArberAcres broilers (8 weeks old, male and female birds are half and half). Semi-quantitative RT-PCRand Northern blot were used to characterize the expression of PPAR-� and PPAR-� genes in theabove tissues. By semi-quantitative RT-PCR, the results showed the expression level of PPAR-�gene was higher in brain, lung, kidney, heart and intestine, medium in stomach, liver and adiposethan in spleen, and it did not express in breast muscle. The expression level of PPAR-� gene washigher in adipose, medium in brain and kidney than in spleen, heart, lung, stomach and intestine,but it did not express in liver and breast muscle. Northern blot results showed that PPAR-� geneexpressed in heart, liver, kidney and stomach, and the intensity of hybridization signal was thestronger in liver and kidney than in other tissues, however, PPAR-� gene only expressed in adiposeand kidney tissues. The results of this study showed the profile of PPAR gene expression in thechicken was similar to that in rodent, human and pig. However the expression profile of chickenalso have its own specific trait, i.e. compared with mammals, PPAR-� gene can not be detectedin skeletal muscle and PPAR-� gene can be stronger expressed in kidney tissues. This work willprovide some basic data for the PPAR genes expression and lipids metabolism of birds.© 2004 Elsevier Inc. All rights reserved.

Keywords: PPARs; Differential expression; Semi-quantitative RT-PCR; Northern blot; Chicken tissues

∗ Corresponding author. Tel.:+86 451 55191416; fax:+86 451 55103336.E-mail address: lihui@neau.edu.cn (H. Li).

0739-7240/$ – see front matter © 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.domaniend.2004.05.003

106 H. Meng et al. / Domestic Animal Endocrinology 28 (2005) 105–110

1. Introduction

Peroxisome proliferator-activated receptors (PPARs) are the members of superfamily ofnuclear hormone receptors[1] and can be activated by peroxisome proliferators, such ashypolipdemic drugs of fabrates class and some fatty acids[2,3]. The PPARs regulate avariety of target genes which involved in intra- and extracellular lipid metabolism suchas fatty acid absorbed by membrane, fatty acid binding to cell, the transportation andcombination of lipid protein, particularly those involved in peroxisome�-oxidation[4,5].These receptors are critical determinants of adipocyte differentiation[6] and are also di-rect targets of antidiabetic drugs of the thiazolidinedione class[7]. So far, three PPARsubtypes have been characterized: PPAR-�, PPAR-� (also called PPAR� and NUC1),and PPAR-�, all encoded by separate genes and with distinct tissue distribution invertebrates[5,8,13], therefore information from PPARs expression patterns is thefirst step to understand the biological function of different PPAR isotypes and iso-forms.

The metabolism of animal lipid is very complex, and there are many differences in lipidmetabolism between birds and mammals. In chicken, lipogenesis occurs essentially in theliver, and the adipose tissue only being as storage tissue[17,18], but the regulation oflipid metabolism is not yet clearly understood. PPARs have been shown as a potential keyregulator of the lipid metabolism and adipose cell differentiation. The primary aim of thecurrent study was to explore the PPAR genes tissue patterns of expression, and the resultsof this study can be the basis of lipid metabolism studies in poultry and the potential factorsin breeding of low abdominal fat chicken.

2. Materials and methods

2.1. Total RNA isolation

Six broilers (Arber Acres, 8 weeks, three males and three females) were slaughtered andthe heart, liver, abdominal fat, breast muscle, spleen, lung, kidney, muscle stomach, intestineand brain were collected and stored at−80◦C. Total RNA of each tissue were extracted byTRIZOL Reagent kit (GIBICOL, BRL), the RNA concentrations were adjusted to 1�g/�lby measuring the OD value, and stored at−80◦C.

2.2. Oligonucleotide primers sequences

These primers were designed by the software of primer premier 5.0 and the positionsare referred to chicken PPAR-� (accession number: AF163809), PPAR-� (accession num-ber: AF470456), GAPDH (accession number: K01458). These primers are: PPAR-� F:5′-TGGACGAATGCCAAGGTC-3′, R: 5′-GATTTCCTGCAGTAAAGGGTG-3′; PPAR-�F: 5′-AGCCCAGTGGATCTGTCTGC-3′, R: 5′-TGTTCCTGCAGTGGTGATGC-3′;GAPDH F: 5′-TGACGTGCAGCAGGAACAC-3′, R:5′-CAGTTGGTGGTGCACGATG-3′. These primers were used for the semi-quantitative RT-PCR as well as production of theprobes that used for Northern blot.

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2.3. Semi-quantitative RT-PCR

Using BcaBEST RNA PCR Kit Ver.1.1 (Takara, Japan), reverse transcriptions (RT) wereperformed with 1�g of total RNA extracted from chicken heart, liver, abdominal fat, breastmuscle, spleen, lung, kidney, muscle stomach, intestine, brain. The cDNA was amplifiedby polymeras chain reaction (PCR) in a 25�l reaction volume containing 2.5�l of 10 ×PCR buffer, 1�l of each 2.5 mM dNTP mixture, 1 unit of Ex Taq DNA polymerase (Takara,Japan), 1�l of cDNA, 1 �l of each 10 pM primers. The condition of PCR amplificationwere: one cycle at 97◦C for 5 min, 30 (26) cycles at 95◦C for 30 s, 55◦C for 30 s and 72◦Cfor 1 min, and a final extension cycle at 72◦C for 8 min. Prior to do the semi-quantitativeRT-PCR experiments, products of RT-PCR amplified with liver and fat cDNA as templatewere cloned to T-vector and sequenced to find if the products are consistent with targetgenes and then used for semi-quantitative RT-PCR as well as Northern blot. Furthermore, forsemi-quantitative comparisons, PCR reactions for PPAR-� and PPAR-� gene were restrictedto the linear range of amplification by limiting the cycle number to 30. The exception tothis procedure was amplification of the GAPDH control fragment, which, owing to itshigher level of expression, was subjected to only 26 cycles. PCR primers were designedto flank known or putative introns, preventing amplification of any contaminating genomicDNA. PCR-amplified fragments were run beside molecular weight markers on 2% agarosegels stained with ethidium bromide. Gels were photographed using the electrophoresis gelimaging system (UVP). The semi-quantitative measure of gene expression was using theratios of PPAR/GAPDH absorption density of bands on a gel.

2.4. Northern blotting

The RT-PCR products of PPAR-� (872 bp) and PPAR-� (766 bp) were separated by 1.2%agarose gel, recycled by the gel recycle kit (Huashun, China) and used as probes for Northernblot. The probes were labelled with�-P32dCTP by Random Primer DNA Labeling Kit Ver.2 (Takara, Japan). The total RNA of each tissue samples of the six birds was equal mixed fora pool and then used for Northern blot. Ten micrograms of RNA from each tissue sampleswere resolved through electrophoresis in 1.2% agarose gels containing 3% formaldehydeand ethidium bromide at a final concentration of 0.5 mg/ml. The nylon membranes that hadtransferred nucleotides were prehybridized at 2 h in 5× SSC, 0.2% SDS, 5× Denhardt,50 mM PBS (pH 7.0), 500�g/ml salmon sperm DNA, 50%formamide. The probes weredenaturalized in 100◦C water for 5 min and hybridized at 42◦C overnight in 5× SSC, 0.2%SDS, 5× Denhardt, 20 mM PBS (pH 7.0), 100�g/ml salmon sperm DNA, 50% formamide.The hybridized nylon membranes were washed twice (2× SSC, 0.5%S DS) for 15 min at37◦C and once (0.1× SSC, 0.1% SDS) for 30 min at 65◦C. Then the membranes werevisualized by autoradiography.

3. Results

The semi-quantitative RT-PCR were carried out with the total RNA from chicken 10tissues to characterize the PPAR gene expression. Our results showed PPAR-� gene express

108 H. Meng et al. / Domestic Animal Endocrinology 28 (2005) 105–110

Fig. 1. Expression character of PPAR genes in various tissues of broilers by RT-PCR. (A) Electrophoresis ofexpression of PPAR-� genes in various tissues of broilers. (B) Histogram of expression of PPAR-� genes invarious tissues of broilers. (C) Electrophoresis of expression of PPAR-� genes in various tissues of broilers. (D)Histogram of expression of PPAR-� genes in various tissues of broilers. Mean expression of PPAR-�/GAPDH(B)and PPAR-�/GAPDH(D) mRNA in various tissues of broilers (n = 6). (1) Heart; (2) liver; (3) spleen; (4) lung;(5) kidney; (6) stomach; (7) small intestine; (8) brain; (9) breast muscle; (10) abdominal fat.

in almost whole tissues other than breast muscle, and the expression intensity was abundantin brain, lung, kidney, heart than intestine, stomach, liver, fat, spleen (Fig. 1A and B).PPAR-� gene expressed in almost whole tissues other than liver and muscle, The expressionintensity was abundant in fat, brain, kidney and weakly in spleen, heart, lung, stomach andintestine (Fig. 1C and D).

The tissues patterns of expression of the chicken PPAR-� and PPAR-� mRNA wereanalyzed by Northern blot. The results showed PPAR-� gene expression can be detectedin heart, liver, kidney and stomach, and the expression intensities in liver and kidney were

Fig. 2. Expression of PPAR genes in various tissues of broilers by Northern blot analysis. (A) Northern bolt analysisof expression of PPAR-� gene in tissues of broiler. (B) Northern blot analysis of expression of PPAR-� genes intissues of broiler. (1) Heart; (2) liver; (3) spleen; (4) lung; (5) kidney; (6) stomach; (7) small intestine; (8) brain;(9) breast muscle; (10) abdominal fat.

H. Meng et al. / Domestic Animal Endocrinology 28 (2005) 105–110 109

higher than in other tissues (Fig. 2A). We also found the PPAR-� gene expression could bedetected in adipose and kidney (Fig. 2B).

4. Discussion

To summarize, the tissue pattern of expression of the PPAR-� mRNA in poultry wasnamely expressed widely in various tissues and very similar to that in rodents (adult ratand mouse) and human as well as livestock (pig and chicken). PPAR-� expressed highly inbrown adipose tissue, liver, kidney, heart, stomach, duodenum mucous membrane, retina,adrenal gland, skeletal muscle and pancreas in rodents[8]; In human, PPAR-� was expressedhighly in heart, kidney, skeletal muscle and large intestine and lowly in liver than in rodent[9]. The Northern blot results in pig showed that PPAR-� was expressed highly in liver andkidney, medium in heart, skeletal muscle and intestine[15]. Inspiringly, Diot and Duairereported that the PPAR-� gene was expressed very low amounts in muscle, abdominaladipose in chicken[10]. Further more, there were no signals of PPAR-� gene expressionin muscle by RT-PCR and Northern Blot analysis in chicken in our research results, whichwas different with its expression in rodents, human and pig. Maybe this is the functiondifference of PPAR-� gene between the avian and mammals.

The chicken PPAR-� gene expression only can be detected in part of the tissues and atconsiderably higher level in adipose tissue than other tissues, this result was in accordancewith previous studies in other species (rodents, human and bovine) that highly expressedin adipose tissue while a differential expression was found in other tissues investigated. Inrodents and human, PPAR-� gene was mainly expressed in adipose tissue[11], intestinemucous membrane, lymphatic tissue such as spleen, and also lowly expressed in retina andskeletal muscle[8,12]; in human, hPPAR-� 1 and hPPAR-� 2 were highly expressed inadipose tissue, lowly expressed in skeletal muscle. The hPPAR-� 1 can also be detectedin the liver and heart[13,14]. The highest expression was detected in adipose tissue withabout equal amounts of the bovine PPAR-� 1 and PPAR-� 2 transcripts while a differentialexpression was found in other tissues investigated. PPAR-� 1 was expressed at relativelyhigh levels in bovine spleen and lung and to a lower extent in ovary, mammary gland, andsmall intestine. The amount of PPAR-� 2 was apparently lower than that of PPAR-� 1 inspleen, lung, and ovary[16]. Interestingly, our results showed that PPAR-� gene could bedetected in chicken kidney but not in other species. Further study was needed to verifyif chicken PPAR-� gene has its special expression character or play an important role inkidney.

In addition, the results of expression of PPARs mRNA were difference in some tissueby RT-PCR and Northern Blot, for example, PPAR-� highly expressed in brain, lung onlyby RT-PCR but not by Northern blot. It may be showed the difference of the sensitivitybetween the two methods in some degree.

Conclusions acquired by the studies as follows: (1) The PPAR-� expression in chickenwas very similar to that of rodents and human, i.e. it expressed widely in various tissues, butdid not express in muscle. This result was different from the other reports in other species.(2) The chicken PPAR-� gene was expressed in some specific tissues, and highly expressedin adipose. (3) The chicken PPAR-� gene was highly expressed in the kidney.

110 H. Meng et al. / Domestic Animal Endocrinology 28 (2005) 105–110

Acknowledgements

This research was supported by National Natural Science Foundation of China (GrantNo. 30270950) and the Foundation of the Outstanding Youth of Heilongjiang Province. Wethank Dr. Yule Pan (The Semex Alliance, Canada) for his useful comments.

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