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GENETICS |
Gene: A Key Regulator of Adipocyte Differentiation in Chickens1College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, P. R. China
3 Corresponding author: lihui{at}neau.edu.cnorlihui645{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is regarded as a "master regulator" of adipocyte differentiation in mammals. The current study was designed to investigate the function and regulatory mechanism of PPAR in chicken adipogenesis by RNA interference. Preadipocytes were isolated from the abdominal fat tissue of 12-d-old chickens and cultured. Small-interference PPAR RNA (siPPAR) was synthesized by in vitro transcription and transfected into chicken preadipocytes by using liposomes. The suppressive effect of siPPAR was detected by real-time reverse-transcription PCR and reverse-transcription PCR. The results showed that transient transfection with siPPAR significantly inhibited differentiation and enhanced proliferation of chicken preadipocytes (P < 0.05). The adipogenesis-associated adipocyte fatty acid-binding protein gene was down-regulated when PPAR was silenced. The current work indicates that PPAR is a key regulator of chicken preadipocyte differentiation.
Key Words: chicken preadipocyte • proliferation • differentiation • peroxisome proliferator-activated receptor- • RNA interference
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adipose tissue mass can be increased by the multiplication of new fat cells through adipogenesis, by increased deposition of triglycerides in the cytoplasm, or both (Soukas et al., 2001). In mammals, adipocyte differentiation has been studied extensively in vitro by using a number of preadipocyte cell lines such as mouse 3T3-L1 cells (Green and Meuth, 1974). These studies have led to the identification of key regulatory genes, including peroxisome proliferator-activated receptor- (PPAR), which are known to permit, or to be necessary for, the transition of preadipocytes into adipocytes in vitro (MacDougald and Lane, 1995).
The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear hormone receptor superfamily of transcription factors. They bind small molecular weight ligands and regulate the expression of various genes involved in intra- and extracellular lipid metabolism pathways, such as absorption of fatty acids through membranes and their intracellular binding, and the formation and transport of associated proteins, particularly those involved in peroxisome β-oxidation (Dreyer et al., 1993; Wahli et al., 1995; Desvergne and Wahli, 1999). Three different subtypes of PPAR have been identified: PPAR
, PPARβ and PPAR. Peroxisome proliferator-activated receptor- is mainly expressed in white and brown adipose tissues and is a key transcription factor in adipocyte differentiation in mammals (Tontonoz et al., 1994; Lehmann et al., 1995). The critical role of PPAR in adipogenesis has been demonstrated in PPAR-knockout animals (Barak et al., 1999; Kubota et al., 1999; Rosen et al., 1999); in mammals, it was induced early during adipocyte differentiation (Brun et al., 1996a). The early expression of PPAR is logical, given its subsequent involvement in terminal differentiation by activating adipocyte-specific genes such as adipocyte fatty acid-binding protein (A-FABP). During the early stage of adipocyte differentiation, PPAR was expressed at low but detectable levels in preadipocytes, and its expression increased rapidly after hormonal induction of differentiation (Brun et al., 1996a). Tonotoz et al. (1995) reported that PPAR was highly expressed in adipocytes and that its ectopic expression could trigger the entire program of adipogenesis in fibroblast and muscle cells. These findings led to the hypothesis that PPAR regulates the development of the adipocyte lineage.
The regulation of lipid metabolism, especially the function of PPAR, in chicken lipogenesis is not yet clearly understood. Elucidating the pattern of function and gene networks of such transcription factors will provide clues that will ultimately clarify the mechanism of regulation of chicken adipogenesis and development.
The chicken PPAR gene has been investigated by our group in recent years. Meng et al. (2005a) found this gene to be highly expressed in chicken adipose tissues; the distribution frequency of 3 genotypes derived from the single nucleotide polymorphism (C297T) of the PPAR gene differed significantly among Arber Acres broilers, Hyline layers, and native Chinese breeds (Shiqiza, Beijing You, Bai’er; P < 0.01; Meng et al., 2005b). These results suggest that the PPAR gene may be a major influence on chicken fat deposition.
Knockdown by RNA interference has been shown to have great promise for basic research into loss of gene function (Tetsuji et al., 2005). It inactivates the target gene and has been successfully deployed in cultured cells similar to gene knockout at the cellular level (Julian, 2004; Kuwabara et al., 2004; Tetsuji et al., 2005). The primary aim of the present work is to elucidate the function and mechanism of regulation of PPAR in the proliferation and differentiation of chicken preadipocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chicken preadipocytes were cultured according to the methods of Cryer and Ramsay (Cryer et al., 1987; Ramsay and Rosebrough, 2003) with several modifications. Abdominal adipose tissue was excised from 12-d-old birds and digested. Stromal-vascular cells (including preadipocytes) were seeded at a density of 1 x 105 cells/cm2 in medium and maintained at 37°C in a humidified, 5% CO2 atmosphere until confluence (d 3 to 4). Oleate (Sigma, St. Louis, MO) was added to the culture system after 48 h to induce preadipocyte differentiation (Ramsay and Rosebrough, 2003).
Three parallel groups of cells were cultured separately for the 3 main corresponding experiments. One experiment was designed to detect the mRNA expression level of the chicken PPAR gene by real-time reverse-transcription PCR (RT-PCR) after suppression; the other 2 were used to confirm the down-regulation of the PPAR gene by RT-PCR and to detect the silencing effects of the PPAR gene, including proliferation, differentiation of chicken preadipocytes, and expression level of the chicken A-FABP gene. In each individual experiment, there were 3 groups: the small-interfering (si)RNA-treated group, the negative control group, and the control group. Within each group, there were 3 biological repeats. Direct comparisons were provided between every 2 groups in each experiment.
siRNA Synthesis
Three 21-nucleotide double-stranded RNA targeting chicken PPAR (GenBank accession no. AF470456) were designed online according to the principles suggested by Holen et al. (2002). They were synthesized by using an siRNA in vitro Transcription T7 Kit (TaKaRa, Dalian, China). The sequence of the negative siRNA has no homology to any known chicken gene (Table 1
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Chicken preadipocytes were plated on 6-well or 12-well plates in Dulbecco’s modified Eagle’s medium-F12 without antibiotics. Transfections were carried on at more than 80% confluence by using lipofectamine 2000 (Invitrogen, Carlsbad, CA).
Chicken preadipocytes cultured in 6-well plates were transfected with siPPAR (4.0 µg per well) to detect gene expression, and cells of the same group in 12-well plates were tranfected with siPPAR (1.6 µg per well) to monitor preadipocyte proliferation and differentiation.
Detection of Gene Expression
The levels of PPAR and A-FABP gene expression in preadipocytes transfected with siPPAR were tested after 48 h by quantitative real-time PCR and RT-PCR. Primers were designed to amplify a fragment containing sequences from 2 adjacent exons to avoid amplification of contaminating genomic DNA. Chicken β-actin, considered to be a housekeeping gene, was used as an internal reference. The sequences of the primers used to analyze gene expression and the corresponding sizes of the products are shown in Table 2.
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Real-time RT-PCR was used to detect PPAR expression by using SYBR Premix Ex Taq (TaKaRa) with PPAR1 primers. Reaction mixtures were incubated in an ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA) programmed to conduct 1 cycle at 95°C for 10 s and 40 cycles at 95°C for 5 s and at 60°C for 31 s. Dissociation curves were analyzed by Dissociation Curve 1.0 software (Applied Biosystems) for each PCR reaction to detect and eliminate possible primer-dimer artifacts. Results (fold changes) were expressed as 2–
Ct in which
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where Ct PPAR and Ct β-actin are, respectively, the cycle thresholds for chicken PPAR and β-actin genes in the different treated groups; e is the experimental group; and c is the control group.
Reverse-transcription PCR was performed by using cDNA as a template with PPAR2 and A-FABP primers. Expression of the chicken β-actin gene was used as a control. For the PPAR gene, the 25-µL PCR reaction volumes included 5 pmol of each primer, 0.2 mM each deoxy nucleotide 5'-triphosphate, and 1 U of Taq polymerase in 1x Taq buffer with the above reverse-transcription mixture (2 to 0.2 µL). The PCR reaction conditions were as follows: 94°C for 3 min, followed by 29 cycles of 94°C for 30 s, 63°C for 1 min, and 72°C for 1 min. For the A-FABP gene, the same PCR volume was used, and the PCR reaction conditions were 94°C for 10 min, followed by 24 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with an extension at 72°C for 10 min. The PCR volume and reaction conditions of the β-actin gene were the same as those for the A-FABP gene. The PCR products were loaded on a 1% agarose gel and stained with ethidium bromide. Images of ethidium bromide-stained gels were obtained by using the UVP system LabworksTM3.0 (UVP, Upland, CA), and densitometric analyses were performed by using a laboratory imaging and analysis system (UVP).
Chicken Preadipocyte Proliferation
Chicken preadipocytes were seeded in culture plates (more than 1 x 104 cells per well) and cultured under normal conditions. Cell confluence of approximately 90% was reached. The medium was then discarded and the preadipocytes were trypsinized and resuspended in Dulbecco’s modified Eagle’s medium plus Trypan blue. Cells were counted with a hematocytometer under an inverted microscope.
Chicken Preadipocyte Differentiation
Oil Red O staining was used to evaluate the extent of preadipocyte differentiation. In brief, preadipocytes were washed in PBS and stained with double-filtered Oil Red O solution to show lipid accumulation. Oil Red O was extracted from the cells by using 100% isopropanol and measured at 500 nm by using an Ultraviolet Spectrophotometer 1000 (Pharmacia, Piscataway, NJ), and its concentration was determined against a cell-free well with the same treatment.
Statistical Analysis
Data were subjected to 1-way ANOVA by using JMP 5.0 (SAS Institute, Cary, NC). Results were given as mean ± SE. Differences were considered significant at P < 0.05 unless otherwise specified.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene in Chicken Preadipocytes at Different Levels of Differentiation
Chicken preadipocytes (Figure 1) were induced by oleate, and the expression patterns of the PPAR gene at 2 levels of differentiation were clearly distinguished. Reverse-transcription PCR analysis showed a significant difference in PPAR expression between high and low levels of preadipocyte differentiation (Figure 2). Expression levels of the chicken PPAR gene were 0.75 ± 0.1006 and 0.4495 ± 0.0069 in highly differentiated and poorly differentiated preadipocytes, respectively. It was obvious that more of the PPAR gene was expressed in the highly differentiated preaipocytes than in the less differentiated ones (P < 0.05).
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Expression in Cultured Chicken Preadipocytes
Selection of siPPAR.
Reverse-transcription PCR was developed to select the most powerful siPPAR (Figure 3). Three candidate siPPAR were ready to be selected. The silencing efficiency was 35.5, 32.8, and 49.8%, corresponding to siPPAR783, siPPAR1028, and siPPAR1337, respectively. The siPPAR1337 had the highest silencing efficiency (Figure 3) and was used in the following experiments.
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Expression in Cultured Chicken Preadipocytes.
Real-time RT-PCR analysis showed that the PPAR gene was markedly suppressed in the siPPAR-treated group. The expression level of the PPAR gene in the siPPAR1337-treated group was 1.18 ± 0.1066, which was significantly lower than the 3.94 ± 0.7021 of the negative control group and the 3.51 ± 0.0820 of the cell control group (P < 0.05). To confirm the silencing effects of siPPAR, another 2 groups of chicken preadipocytes were cultured. Reverse-transcription PCR analysis showed that PPAR gene expression was suppressed consistently in each siPPAR-treated group (Table 3).
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on Chicken Preadipocyte Proliferation.
The proliferation of chicken preadipocytes was evaluated 48 h after transfection with siPPAR. The cell numbers in the siPPAR-treated groups were higher than in the negative control groups (P < 0.05) and lower than in the cell control groups (P < 0.05; Table 4).
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on Chicken Preadipocyte Differentiation.
During the differentiation process, chicken preadipocytes initiated the storage of energy in the form of triacylglycerol-rich lipid droplets. The degree of preadipocyte differentiation was evaluated by measuring the accumulation of intracellular lipids after staining with Oil Red O (Figure 4). Oil Red O was extracted from preadipocytes in the different cell groups 48 h after transfection with siPPAR. There was a significant difference in Oil Red O content between the siPPAR group and the negative control groups (or P < 0.05; Table 5).
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in Cultured Chicken Preadipocytes.
The level of A-FABP gene expression was monitored 48 h after transfection with siPPAR. The results showed that chicken A-FABP mRNA levels decreased with the suppression of PPAR (P < 0.05; Table 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is a master regulator of adipogenesis and plays an important role in the regulation of insulin sensitivity and glucose homeostasis in mammals (Tontonoz et al., 1994; Wu et al., 1998; Kubota et al., 1999; Berger and Moller, 2002). Peroxisome proliferator-activated receptor- is the most adipose-specific of the PPAR and is induced before the transcription of most adipocyte genes is activated (Tonotoz et al., 1995). The expression of PPAR is sufficient to induce growth arrest as well as to initiate adipogenesis in exponentially growing fibroblast cell lines, demonstrating its critical role in the regulation of adipocyte differentiation in mammals (Hu et al., 1995; Tonotoz et al., 1995; Altiok et al., 1997). Moreover, retrovirally mediated ectopic expression of PPAR in the presence of PPAR activators decreases myoblast-specific gene expression and directs cells into the adipocyte lineage in mammals (Hu et al., 1995). In mice, activated PPAR induces exit from the cell cycle and triggers the expression of adipocyte-specific genes, resulting in increased delivery of energy to the cells (Fajas et al., 1998). Gene ablation experiments in mice showed that PPAR is required for the development of adipose tissue (Barak et al., 1999).
To elucidate the biological role of PPAR in adipose tissue, the effect of RNA interference on the PPAR gene was investigated in mouse 3T3-L1 cells. Expression of PPAR mRNA was effectively suppressed and the differentiation of 3T3-L1 preadipocytes to adipocytes was inhibited concurrently (Tetsuji et al., 2005). Xu et al. (2006) examined the potential of siRNA against human PPAR to suppress adipocyte differentiation (adipogenesis) in human preadipocytes, and found that transient transfection with siPPAR resulted in significant inhibition.
With the development of a chicken preadipocyte culture system, factors responsible for regulating adipocyte differentiation in chickens have gradually been characterized. Regulatory factors have been identified, and species-specific mRNA expression of the factors involved has recently been analyzed. The expression profiles of genes involved in regulating chicken adipocyte differentiation, such as PPAR, CCAAT/enhancer binding protein, β, 
, and sterol response element-binding protein-1, were studied. Rapid increases in PPAR expression were observed 9 and 12 h after culturing in differentiation medium. These results suggest that PPAR may be a key regulator of the early stages of chicken preadipocyte differentiation (Matsubara et al., 2005).
There is an obvious augmentation of PPAR gene expression from the early stage of differentiation to mature adipocytes in mammals. Peroxisome proliferator-activated receptor- was easily detectable during the second day of 3T3-L1 adipocyte differentiation, and maximal levels of expression were attained in mature adipocytes (Brun et al., 1996b). In the current study, the chicken PPAR gene was significantly more highly expressed in highly differentiated preadipocytes than in less differentiated ones (P < 0.05). This showed that, as in mammals, expression of chicken PPAR increases during differentiation of preadipocytes to adipocytes, indicating that PPAR is an important regulator of chicken adipocyte differentiation.
In the current study, lipofectamine 2000 was used to transfect siPPAR. During this process, the chicken preadipocyte membrane may be damaged by the transfection reagent, resulting in some cell death (Hollon and Youshimura, 1989). In view of this, comparison of results between the siPPAR-treated and negative control groups to evaluate the efficiency of siPPAR will be more precise.
When PPAR mRNA expression was down-regulated, proliferation was significantly greater in the siPPAR-treated groups than in the negative control ones (P < 0.05), indicating that the suppression of PPAR led to enhancing chicken preadipocyte proliferation. Simultaneously, chicken preadipocyte differentiation was influenced by the silencing of PPAR. There was noticeably less Oil Red O in the siPPAR groups than in the negative control groups (P < 0.05), implying that there was less triglyceride in chicken preadipocytes with low PPAR expression levels. The current results indicated that chicken preadipocyte proliferation was increased and differentiation was decreased by suppression of the PPAR gene.
In mammals, the involvement of PPAR in the terminal differentiation of preadipocytes was demonstrated by the activation of adipocyte-specific genes such as A-FABP. In the current study, chicken A-FABP gene expression was also examined by RT-PCR in the 3 parallel cell groups. The results showed that A-FABP expression was significantly decreased following the suppression of PPAR (P < 0.05). Adipocyte fatty acid-binding protein, a gene specifically expressed in adipose tissue, may be regulated by PPAR, subsequently participating in chicken adipogenesis.
The results from the current study indicated that suppression of the chicken PPAR gene led to the inhibition of chicken preadipocyte differentiation and the enhancement of chicken preadipocyte proliferation, and directly regulated the adipocyte-specific gene A-FABP. We concluded that PPAR is probably a key regulator of chicken preadipocyte proliferation and differentiation, and may be a major gene affecting fat deposition, as in mammals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 These authors contributed equally to this work.
Received for publication August 7, 2007. Accepted for publication October 22, 2007.
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