Domestic Animal Endocrinology 28 (2005) 105–110
Short communication
Differential expression of peroxisome proliferator-activated receptors alpha and gamma gene in various chicken tissues H. Meng a,b , H. Li b,∗ , J.G. Zhao b , Z.L. Gu b a b
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 201101, PR China 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 nuclear hormone receptors. A great number of studies in rodent and human have shown that PPARs were involved in the lipids metabolism. The goal of the current study was to investigate the expression 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 Arber Acres broilers (8 weeks old, male and female birds are half and half). Semi-quantitative RT-PCR and Northern blot were used to characterize the expression of PPAR-␣ and PPAR-␥ genes in the above 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 adipose than in spleen, and it did not express in breast muscle. The expression level of PPAR-␥ gene was higher 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-␣ gene expressed in heart, liver, kidney and stomach, and the intensity of hybridization signal was the stronger in liver and kidney than in other tissues, however, PPAR-␥ gene only expressed in adipose and kidney tissues. The results of this study showed the profile of PPAR gene expression in the chicken was similar to that in rodent, human and pig. However the expression profile of chicken also have its own specific trait, i.e. compared with mammals, PPAR-␣ gene can not be detected in skeletal muscle and PPAR-␥ gene can be stronger expressed in kidney tissues. This work will provide 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
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1. Introduction Peroxisome proliferator-activated receptors (PPARs) are the members of superfamily of nuclear hormone receptors [1] and can be activated by peroxisome proliferators, such as hypolipdemic drugs of fabrates class and some fatty acids [2,3]. The PPARs regulate a variety of target genes which involved in intra- and extracellular lipid metabolism such as fatty acid absorbed by membrane, fatty acid binding to cell, the transportation and combination of lipid protein, particularly those involved in peroxisome -oxidation [4,5]. These receptors are critical determinants of adipocyte differentiation [6] and are also direct targets of antidiabetic drugs of the thiazolidinedione class [7]. So far, three PPAR subtypes have been characterized: PPAR-␣, PPAR- (also called PPAR␦ and NUC1), and PPAR-␥, all encoded by separate genes and with distinct tissue distribution in vertebrates [5,8,13], therefore information from PPARs expression patterns is the first step to understand the biological function of different PPAR isotypes and isoforms. The metabolism of animal lipid is very complex, and there are many differences in lipid metabolism between birds and mammals. In chicken, lipogenesis occurs essentially in the liver, and the adipose tissue only being as storage tissue [17,18], but the regulation of lipid metabolism is not yet clearly understood. PPARs have been shown as a potential key regulator of the lipid metabolism and adipose cell differentiation. The primary aim of the current study was to explore the PPAR genes tissue patterns of expression, and the results of this study can be the basis of lipid metabolism studies in poultry and the potential factors in 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 and the heart, liver, abdominal fat, breast muscle, spleen, lung, kidney, muscle stomach, intestine and brain were collected and stored at −80 ◦ C. Total RNA of each tissue were extracted by TRIZOL Reagent kit (GIBICOL, BRL), the RNA concentrations were adjusted to 1 g/l by 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 positions are referred to chicken PPAR-␣ (accession number: AF163809), PPAR-␥ (accession number: 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 -CAGTTGGTGGTGCACGATG3 . These primers were used for the semi-quantitative RT-PCR as well as production of the probes 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) were performed with 1 g of total RNA extracted from chicken heart, liver, abdominal fat, breast muscle, spleen, lung, kidney, muscle stomach, intestine, brain. The cDNA was amplified by 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 amplification were: one cycle at 97 ◦ C for 5 min, 30 (26) cycles at 95 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 1 min, and a final extension cycle at 72 ◦ C for 8 min. Prior to do the semi-quantitative RT-PCR experiments, products of RT-PCR amplified with liver and fat cDNA as template were cloned to T-vector and sequenced to find if the products are consistent with target genes and then used for semi-quantitative RT-PCR as well as Northern blot. Furthermore, for semi-quantitative comparisons, PCR reactions for PPAR-␣ and PPAR-␥ gene were restricted to the linear range of amplification by limiting the cycle number to 30. The exception to this procedure was amplification of the GAPDH control fragment, which, owing to its higher level of expression, was subjected to only 26 cycles. PCR primers were designed to flank known or putative introns, preventing amplification of any contaminating genomic DNA. PCR-amplified fragments were run beside molecular weight markers on 2% agarose gels stained with ethidium bromide. Gels were photographed using the electrophoresis gel imaging system (UVP). The semi-quantitative measure of gene expression was using the ratios 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 Northern blot. The probes were labelled with ␣-P32 dCTP by Random Primer DNA Labeling Kit Ver. 2 (Takara, Japan). The total RNA of each tissue samples of the six birds was equal mixed for a pool and then used for Northern blot. Ten micrograms of RNA from each tissue samples were resolved through electrophoresis in 1.2% agarose gels containing 3% formaldehyde and ethidium bromide at a final concentration of 0.5 mg/ml. The nylon membranes that had transferred 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 were denaturalized 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 at 37 ◦ C and once (0.1 × SSC, 0.1% SDS) for 30 min at 65 ◦ C. Then the membranes were visualized by autoradiography.
3. Results The semi-quantitative RT-PCR were carried out with the total RNA from chicken 10 tissues to characterize the PPAR gene expression. Our results showed PPAR-␣ gene express
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Fig. 1. Expression character of PPAR genes in various tissues of broilers by RT-PCR. (A) Electrophoresis of expression of PPAR-␣ genes in various tissues of broilers. (B) Histogram of expression of PPAR-␣ genes in various 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 abundant in 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 expression intensity was abundant in fat, brain, kidney and weakly in spleen, heart, lung, stomach and intestine (Fig. 1C and D). The tissues patterns of expression of the chicken PPAR-␣ and PPAR-␥ mRNA were analyzed by Northern blot. The results showed PPAR-␣ gene expression can be detected in 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 analysis of expression of PPAR-␣ gene in tissues of broiler. (B) Northern blot analysis of expression of PPAR-␥ genes in tissues 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.
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higher than in other tissues (Fig. 2A). We also found the PPAR-␥ gene expression could be detected in adipose and kidney (Fig. 2B).
4. Discussion To summarize, the tissue pattern of expression of the PPAR-␣ mRNA in poultry was namely expressed widely in various tissues and very similar to that in rodents (adult rat and mouse) and human as well as livestock (pig and chicken). PPAR-␣ expressed highly in brown adipose tissue, liver, kidney, heart, stomach, duodenum mucous membrane, retina, adrenal gland, skeletal muscle and pancreas in rodents [8]; In human, PPAR-␣ was expressed highly 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 and kidney, medium in heart, skeletal muscle and intestine [15]. Inspiringly, Diot and Duaire reported that the PPAR-␣ gene was expressed very low amounts in muscle, abdominal adipose in chicken [10]. Further more, there were no signals of PPAR-␣ gene expression in muscle by RT-PCR and Northern Blot analysis in chicken in our research results, which was different with its expression in rodents, human and pig. Maybe this is the function difference of PPAR-␣ gene between the avian and mammals. The chicken PPAR-␥ gene expression only can be detected in part of the tissues and at considerably higher level in adipose tissue than other tissues, this result was in accordance with previous studies in other species (rodents, human and bovine) that highly expressed in adipose tissue while a differential expression was found in other tissues investigated. In rodents and human, PPAR-␥ gene was mainly expressed in adipose tissue [11], intestine mucous membrane, lymphatic tissue such as spleen, and also lowly expressed in retina and skeletal muscle [8,12]; in human, hPPAR-␥ 1 and hPPAR-␥ 2 were highly expressed in adipose tissue, lowly expressed in skeletal muscle. The hPPAR-␥ 1 can also be detected in the liver and heart [13,14]. The highest expression was detected in adipose tissue with about equal amounts of the bovine PPAR-␥ 1 and PPAR-␥ 2 transcripts while a differential expression was found in other tissues investigated. PPAR-␥ 1 was expressed at relatively high levels in bovine spleen and lung and to a lower extent in ovary, mammary gland, and small intestine. The amount of PPAR-␥ 2 was apparently lower than that of PPAR-␥ 1 in spleen, lung, and ovary [16]. Interestingly, our results showed that PPAR-␥ gene could be detected in chicken kidney but not in other species. Further study was needed to verify if chicken PPAR-␥ gene has its special expression character or play an important role in kidney. In addition, the results of expression of PPARs mRNA were difference in some tissue by RT-PCR and Northern Blot, for example, PPAR-␣ highly expressed in brain, lung only by RT-PCR but not by Northern blot. It may be showed the difference of the sensitivity between the two methods in some degree. Conclusions acquired by the studies as follows: (1) The PPAR-␣ expression in chicken was very similar to that of rodents and human, i.e. it expressed widely in various tissues, but did 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 expressed in adipose. (3) The chicken PPAR-␥ gene was highly expressed in the kidney.
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Acknowledgements This research was supported by National Natural Science Foundation of China (Grant No. 30270950) and the Foundation of the Outstanding Youth of Heilongjiang Province. We thank Dr. Yule Pan (The Semex Alliance, Canada) for his useful comments.
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