J. Anim Sci. 2007. 85:2445-2456. doi:10.2527/jas.2007-0142
© 2007 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Patterns of gene expression in pig adipose tissue: Transforming growth factors, interferons, interleukins, and apolipoproteins1
G. J. Hausman*,2,
C. R. Barb* and
R. G. Dean
* USDA-ARS, Russell Agricultural Research Center, Athens, GA; and
University of Georgia, Athens, GA
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Abstract
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Although cDNA microarray studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, cDNA microarray studies of adipose tissue from growing pigs have not been reported. Total RNA was collected at slaughter from outer s.c. adipose tissue (OSQ), middle s.c. adipose tissue (MSQ), ovary, uterus, hypothalamus, and pituitary tissue samples from gilts at 90, 150, and 210 d (n = 5/age). Dye-labeled cDNA probes were hybridized to custom microarrays (70 mer oligonucleotides) representing approximately 600 pig genes involved in growth and reproduction. Expression intensity ratios revealed little change in expression of 27 cytokines and 4 apolipoproteins with age in OSQ and MSQ from pigs at 90, 150, and 210 d of age. Distinct patterns of relative gene expression were evident within apolipoproteins, IL, interferons, and transforming growth factor ß family members in adipose tissue from growing pigs (90-, 150-, and 210-d-old pigs). Patterns of gene expression within apolipoproteins, IL, interferons, and transforming growth factor ß family members distinguished OSQ and MSQ depots in growing pigs. We also demonstrated, for the first time, the expression of several major cytokine and apolipoprotein genes in pig adipose tissue, including small inducible cytokine A5 (RANTES), IL-1B, IL-1A, IL-12A, IL-1 receptor antagonist, and apolipoproteins A1 and E with microarray and reverse transcription-PCR assays or reverse transcription-PCR assays alone. These studies demonstrate that expression of major cytokine and apolipoprotein genes in pig adipose tissue are not influenced by age in growing pigs but may be influenced by location or depot.
Key Words: adipose tissue • fat cell • gene microarray • cytokine
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INTRODUCTION
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Adipose tissue secretes a variety of proteins that include leptin, IL-6, IL-8 (reviews, Gimeno and Klaman, 2005
; Hauner, 2005; Jacobi et al., 2006), IL-15 (Ajuwon et al., 2004), and the IL-1 receptor agonist (Juge-Aubry et al., 2003, 2004; Sopasakis et al., 2005). Additional factors secreted by adipose tissue include apolipoproteins (APO), transforming growth factor (TGF)-ß, and adiponectin (Trayhurn and Wood, 2004; Sopasakis et al., 2005; Taleb et al., 2006). Secretion of these factors by adipose tissue impacts a number of physiological and metabolic processes (reviews, Hauner, 2005; Trayhurn et al., 2006). Gene expression of adipose tissue-secreted factors has been studied with cDNA microarrays (Gomez-Ambrosi et al., 2004; Taleb et al., 2006), but this approach has not been employed in studies of adipose tissue in growing meat animals. We report herein the first integrated view of the cytokine gene expression pattern for adipose tissue from growing pigs in gene microarray and reverse transcription (RT)-PCR studies.
We previously identified APO and many cytokines at the gene and protein level in 90-d fetal stromal-vascular cultures and subcutaneous adipose tissue from 105 d of gestation fetuses and 5-d-old pigs (Hausman et al., 2006). In the current study we examined the expression of these APO and cytokines (Hausman et al., 2006) at the gene level in adipose tissue from growing pigs. Furthermore, we compared outer s.c. adipose tissue (OSQ) and middle s.c. adipose tissue (MSQ) depots in terms of their relative gene expression patterns for APO, IL, interferons, and TGFß family members, given that cellular and metabolic development distinguish these depots (Anderson et al., 1972) and only several genes have been compared in studies of MSQ and OSQ (Omi et al., 2005; Ramsay and Richards, 2005). The studies presented represent initial efforts to identify potential adipose tissue-secreted factors involved in regulating puberty and growth in pigs.
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MATERIALS AND METHODS
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Animals
All procedures were approved by the Richard B. Russell Agriculture Research Center Committee on Animal Care and Use.
Five PIC composite (Pig Improvement Company, Hendersonville, TN), lean phenotype gilts were utilized at 90, 150, and 210 d, for a total of 15 pigs. Mean BW (kg) were 78 ± 11 at 90 d, 180 ± 11 at 150 d, and 280 ± 11 at 210 d (least squares means ± SEM, n = 5), which were significantly different from each other (P < 0.0001; 1-way ANOVA). In this herd, the expected time of puberty was 210 d, and changes in expression of pituitary and hypothalamic genes associated with reproduction distinguished 210- and 150- from 90-d-old pigs. Therefore, 90-, 150-, and 210-d-old pigs represented distinct stages of pig pubertal development (unpublished observations). Before slaughter, gilts were penned in an environmentally controlled building and exposed to a constant temperature of 21°C and an artificial 12:12-h light:dark photoperiod and fed a corn-soybean meal ration (14% CP, as fed) on an ad libitum basis, according to the National Research Council guidelines.
Gene Expression Analysis
Tissue Collection and RNA Preparation.
Five pigs at 90, 150, and 210 d of age were euthanized (a total of 15 pigs) with an overdose of 10% sodium thiopental, and OSQ, MSQ, ovary, uterus, hypothalamus, and pituitary tissues samples were collected at slaughter. Tissue samples were flash-frozen in liquid nitrogen and stored at –80°C until RNA isolation. Total RNA was isolated from frozen tissues using Qiazol reagent and RNA Maxi Kits (Qiagen, Valencia, CA), according to the manufacturer’s procedure. Total RNA quality was determined using spectrophotometry at the UV absorbency of 260 nm by microfluidic technology on an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA). Quantity of the RNA was determined by spectrophotometry on a µQuant Microplate Spectrophotometer (BioTek Instruments, Winooski, VT). To correct for dye-specific bias during hybridization, a dye swap design was used, as follows: each total RNA sample was split and transcribed into cDNA, and the replicate cDNA were labeled with Cy3 or Cy5 in direct reactions using a Cyscribe First-Strand cDNA labeling kit (oligo-dT primers, Cy3- dCTP, Cy5-dCTP; Amersham Bioscience, Piscataway, NJ). Labeled targets were purified with a QIAquick PCR purification kit (Qiagen). The dye swap replicates were then hybridized to different array slides.
Pig Adipose Tissue Microarrays.
Custom cDNA microarrays (Telechem International Inc., ArrayIt.com, Sunnyvale, CA) were prepared by spotting 70-mer oligonucleotides that were designed from 560 pig gene sequences, as described (Hausman et al., 2006). Oligonucleotides were produced from sequenced expressed sequence tags that had at least 90% homology to known genes in The Institute of Genome Research pig gene index: http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=pig (last accessed Aug. 23, 2007). Slides (microarrays) were prehybridized in a 25% formamide solution containing 0.10 mg/mL of sonicated salmon sperm DNA at 42°C for 45 min. Hybridization to the labeled targets was performed under coverslips in individual chambers, using a 25% formamide solution, at 42°C overnight. Microarrays were scanned on a ScanArray 5000 (GSI Lumonics Inc., Billerica, MA), and signal output was reported with QuantArray image analysis software (GSI Lumonics Inc.). Background fluorescent intensity determination was based on a local background method. Areas were chosen surrounding each spot and used to compute a number that represented the background for that spot. Normalization among arrays, which allows comparison of data across multiple microarray slides, was performed with a version of a global mean normalization procedure, as described (Hausman et al., 2006). Microarray intensities were adjusted for local background intensities, which averaged 1,531 ± 100 and 1,754 ± 100 for OSQ and MSQ microarrays, respectively (ages combined; means ± SEM of 30 means).
Relative Expression of Normalized Microarray Gene Intensities.
Normalized microarray intensities for each set of triplicate spots per gene, using the values from the Cy3- and Cy5-labeled dye swap replicates, were averaged, resulting in 1 normalized gene intensity per pig and 5 average intensities (i.e., 5 pigs) per gene per tissue at each age. A gene within each of the 4 groups (i.e., APO, IL, interferons, and TGFß gene group) was arbitrarily designated as a reference gene for that gene group and given a value 100. Percentages of the average intensity of the reference gene were calculated for the other genes in that group, resulting in 1 percentage per pig and 5 percentages per gene at each age. Subsequently, we compared relative gene expressions (percentages) with each other within a gene group at each age for MSQ and OSQ depots. Analysis of percentages from 90-, 150-, and 210-d-of-age microarray data for age effects showed no significant changes with age for MSQ and only several significant but small magnitude changes (
30% change) for OSQ. We combined gene percentages from 90-, 150-, and 210-d-old pig adipose tissue microarrays and compared relative gene percentages with each other within a gene group for OSQ and MSQ. For each gene group, percentages were also computed with a second reference gene to determine if the choice of a reference gene influenced gene expression patterns. Collectively, percentages of a total of 120 genes in 20 gene groups were computed and analyzed from 90-, 150-, and 210-d-of-age OSQ, MSQ, ovary, uterus, hypothalamus, and pituitary microarray data.
Determination of Gene Expression Ratios Among the 90 to 150, 150 to 210, and 90 to 210 d of Age Groups.
Microarrays were scanned, and the signal output was reported as described above. Microarray intensities from each of 5 pigs/age group were utilized with 2 data points/pig (Cy3, Cy5 dye swap replicates) totaling 10 microarray intensities/age. Each gene was spotted in triplicate, so for each gene for each age group there were 30 microarray intensities. Fluorescent intensities were corrected for local background, and if present, negative values were excluded. The data were normalized by a global mean normalization as follows: the total of the signal intensities of each channel on each microarray was calculated; the average of these totals was calculated and each total was divided by the average total to yield a set of normalization factors. For each channel on each microarray, every signal measurement was multiplied by the normalization factor for that particular channel and microarray. The normalized values (30 total intensity values) were averaged for each gene and age. These averages were used to compute expression ratios between the ages for each gene. The accession numbers and identity of the genes examined in these studies are shown in Table 1.
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Table 1. Identities of cytokines and apolipoproteins (APO) examined in outer and middle subcutaneous adipose tissue microarrays from 90-, 150-, and 210-d-old pig and neonatal subcutaneous adipose tissue microarrays
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Quantitative Real-Time Reverse Transcription-PCR.
Real-time, reverse transcription PCR was performed by the University of Georgia Functional Genomics Resource Facility and carried out using a 7900HT Sequence Detection System (Applied Biosystems). Primers and probes were custom-designed, based on GenBank sequences of target genes (Applied Biosystems; Table 2). Porcine 18S rRNA was amplified as an endogenous control. Total RNA (500 ng) was transcribed into cDNA in a volume of 20 µL with a High Capacity cDNA Archive Kit (Applied Biosystems; cat. No. 4322171), which uses random primers, following the manufacturer’s protocol (25°C for 10 min, 37°C for 2 h). The cDNA was diluted 1:20 (vol/vol) for use in the amplification reaction. The amplification reaction consisted of 1 µL of diluted cDNA, 6.25 µL of 2x TaqMan (Applied Biosystems) Universal PCR Master Mix, 0.625 µL of 20x custom TaqMan Gene Expression Assay [forward primer, reverse primer, and 6FAM dye (MGB labeled probe)], and water up to a total volume of 12.5 µL. Thermal cycling parameters were as follows: an initial denaturing step (95°C for 15 min), followed by 40 cycles of denaturing-annealing/elongating (95°C for 15 s, then 60°C for 1 min) in a 384-well optical plate. Applied Biosystems SDS 2.1 software was used to report the values.
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Table 2. Primer pairs for each selected gene, reporter 1 sequence, and GenBank accession number from which the primer pairs were selected.
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Analysis of Quantitative, Real-Time, RT-PCR Data.
Threshold cycle (Ct) values were determined using 7900HT Version 2.2.1 Sequence Detection Systems software (Applied Biosystems), in relative quantification study mode, with thresholds set manually. The Ct values (within-sample triplicate wells averaged) were then exported and subjected to 2 analyses, as described in the following 2 paragraphs.
2–
Ct Method.
The Ct values for replicates of each age group were averaged; Ct was calculated as the difference in Ct between the target gene and the 18S endogenous control. Each Ct was calculated as the difference in Ct between the sample of interest and a calibrator sample; in this case, the sample of interest was the older age and the calibrator sample was the younger age. The differential expression ratio of the older to younger age groups for each gene was calculated as 2–Ct. Ratios are reported as 2–Ct if upregulated or –1 divided by 2–Ct if downregulated to correct for the nonlinear appearance, with positive numbers indicating upregulation and negative numbers indicating downregulation (Livak and Schmittgen, 2001).
REST Method.
For calculating probe efficiencies within the relative expression software tool (REST), pooled cDNA of all available pig samples were run in quantitative, real-time, PCR reactions using 1/20, 1/40, 1/60, and 1/80 dilutions (vol/vol) of Taqman probes. The Ct values for the experimental samples, along with Ct values for the probe efficiencies, were imported into the REST software tool. Replicates of each age group were not averaged. The REST software tool determined differential gene expression ratios of the older to younger age groups for each gene, which were corrected for varying probe efficiencies, as calculated by the software. The REST estimated P-values using the software’s statistical model, a pairwise fixed reallocation randomization test. The software reports ratios as 2–Ct if upregulated or 1 divided by 2–Ct if downregulated. The equation used within the software was
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where E = probe real-time PCR efficiency; target = target gene; and ref = endogenous control gene, in this case 18S (Pfaffl et al., 2002). Mean Ct from the REST analysis are shown to demonstrate gene expression.
Real-Time RT-PCR Reactions Performed on Middle and Outer Subcutaneous Adipose Tissue Microarray mRNA.
Complementary DNA templates were designed to study a total of 38 genes present on the gene microarrays and were chosen based on expression in microarrays of neonatal adipose tissue and fetal stromal-vascular cell cultures (Hausman et al., 2006). These genes include receptors, transcription factors (TF), and growth factors (GF), in addition to cytokines and APO. Reactions for RT-PCR were performed on 90 and 210 d of age MSQ mRNA for 3 GF, 5 TF, 4 receptors, 8 cytokines, and 4 apolipoproteins, totaling 62 (31 for each age), with 13 RT-PCR reactions performed on 150-d-of-age MSQ mRNA. In addition, RT-PCR reactions were performed on 90 and 210 d of age OSQ mRNA for 2 GF, 2 TF, 1 apolipoprotein, and 3 cytokines, totaling 26 (13 for each age), with 9 RT-PCR reactions performed on 150-d-of-age OSQ mRNA. Complementary DNA templates were also designed to study 4 genes [IL-8, IL-1RN, RANTES, and adiponectin)] not on our gene microarrays but identified in a study of media conditioned by pig preadipocyte cultures (Hausman et al., 2006). A grand total of 42 genes were analyzed, with 122 RT-PCR reactions performed (3 pigs/age/gene) on MSQ and OSQ mRNA in this study.
Statistics.
The relative expression levels (percentages) of genes within 3 groups or families of genes were analyzed with a 2-way ANOVA for the effects of age, gene, and age x gene interaction using PROC GLM (SAS Inst. Inc., Cary, NC) or with a 1-way ANOVA for comparing relative gene expressions (percentages) with each other within a gene group at each age using PROC GLM. Relative gene expression data from all 3 age groups were combined and analyzed with a 1-way ANOVA for comparing relative gene expressions to each other within a gene group using PROC GLM. Differences between means were determined by the least squares contrasts using PROC GLM. Pearson’s product moment correlation and simple linear regression (P. Wessa, 2006, Free Statistics Software, Office for Research Development and Education, version 1.1.18, http://www.wessa.net/; last accessed 1 August 2007) were used to examine the relationships between real-time RT-PCR and microarray gene expression data after log10 transformation. Differences in gene expression determined by real-time RT-PCR were analyzed using a pairwise, fixed allocation, randomization test, utilizing the relative expression software tool (REST version 2). All means were normalized to the endogenous control 18S rRNA expression. Statistical significance was set at P < 0.05.
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RESULTS
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Gene Expression Studies of Neonatal Adipose Tissue and Adipose Tissue from 90-, 150-, and 210-d-Old Pigs
Microarray Studies.
Our microarrays were designed for studies of several tissues including ovary, uterus, pituitary, hypothalamus and adipose. As a result, our microarrays contain genes, which can be considered unrelated controls relative to adipose tissue because they are primarily expressed by another tissue or group of tissues. For instance, several "hypothalamus" genes and the "pituitary" genes preprolactin and thyroid stimulating hormone beta and others were expressed at very low levels in neonatal (fetal + young pig) adipose tissue arrays (Hausman et al., 2006). The relative expression of preprolactin, thyroid stimulating hormone beta, and porcine GH was 13 ± 3 fold greater in pituitary microarrays (unpublished observations) than in microarrays of MSQ of growing pigs (mean ± SEM of the 90-, 150-, and 210-d fold increases). Conversely, the relative expression of adipocyte fatty acid binding protein and lipoprotein lipase was 17 ± 2 fold higher in MSQ microarrays than in pituitary microarrays (mean ± SEM of the 90-, 150-, and 210-d fold increases).
Adipose Tissue Cytokine and Apolipoprotein Gene Expression Between 90 to 150, 150 to 210, and 90 to 210 d Age Groups.
Expression ratios clearly indicate little change in cytokine and APO gene expression with age in MSQ and OSQ because an expression ratio of 1 indicates no change (Table 3). One of the greatest expression ratios detected was 2.1 for leptin between 90 and 210 d in the MSQ depot. We chose to report the expression ratios as averages of the gene groups because ratios of individual genes in each age group were similar with very little variation around 1 as indicated by the small standard deviations (Table 3). Despite the longer time period, gene expression between 90 and 210 d was similar to gene expression between 90 and 150 and 150 and 210 d (Table 3). Analysis of these microarray data with either a LOWESS normalization based or an ANOVA normalization based method showed that expression of these genes was not significantly influenced by age (G. J. Hausman, C. R. Barb, R. G. Dean, and R. Rekaya, 2007, University of Georgia, Athens, unpublished observations).
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Table 3. Average expression ratios of 4 gene groups for 3 age comparisons of middle (MSQ) and outer (OSQ) subcutaneous adipose tissue microarray data1
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Patterns of Cytokine and APO Gene Expression in Subcutaneous Adipose Tissue from 90-, 150-, and 210-d-Old Pigs.
We calculated expression of each gene relative to a reference gene for each group [i.e., APO, IL, interferons, and TGFß (TGF) group] after relative gene expressions or percentages from 90-, 150-, and 210-d pig adipose tissue microarrays were combined for OSQ and MSQ (Table 4). Comparing the relative expression of genes with each other within each group and comparing MSQ and OSQ expression percentages helped to establish patterns of expression for the APO and cytokine gene groups (Table 4). Relative expression or percentages of many TGF group members, several APO and IL, and fewer interferons were different from percentages of other genes in that gene group within MSQ or OSQ depots. Comparison of OSQ and MSQ depots (Table 4) showed that many TGF group members, APO, and interferons were influenced by depot (Tables 4 and 5).
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Table 4. The relative expression patterns of IL, interferons, transforming growth factor (TGF) family members, and apolipoproteins (APO) for middle (MSQ) and outer (OSQ) adipose tissue based on combined microarray data from 90-, 150-, and 210-d-old pigs1
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Table 5. Relative expression of IL-5, IL-6, IL-18, IL-12, IL-IB, and IL-10 in microarrays of middle (MSQ) and outer (OSQ) subcutaneous adipose tissue from growing pigs (90-, 150-, and 210-d-of-age data combined)1
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The IL expression pattern for MSQ from growing pigs consisted of IL-15 and IL-6 percentages significantly different from each other, IL-15 different from all other IL and IL-6 different from nearly all other IL percentages, regardless of the reference gene (Table 5
; Figure 1). This IL pattern was similar for the OSQ depot, but IL-15 was not significantly separated from IL-6 (Table 5; Figure 1).
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Figure 1. Relative expression of IL-1A, IL-1B, IL-4, IL-5, IL-6, IL-7, IL-10, and IL-15 in middle (MSQ) and outer (OSQ) adipose tissue microarrays from growing pigs (90-, 150-, and 210-d data combined). Expression level percentages (levels of IL-2 were arbitrarily set at 100) included IL-1A/IL-2, IL-1B/IL-2, IL-4/IL-2, IL-5/IL-2, IL-6 IL-2, IL-7/IL-2, IL-10/IL-2, and IL-15/IL-2. Least squares means ± SEM of 15 percentages of relative normalized fluorescent intensities from each of 15 MSQ and 15 OSQ adipose tissue arrays because the data from 90-, 150-, and 210-d-old pigs were combined. a–eMeans (bars) within a depot (MSQ, OSQ) that do not have a common superscript differ (P < 0.01).
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Patterns of TGF gene percentages and the TGF percentages were depot dependent in growing pigs because the TGFß3 percentage was increased and growth/differentiation factor 8 (GDF8, myostatin), BMP-15, and BMP-4 percentages decreased for OSQ relative to MSQ (Table 4). The TGFß3 percentage was significantly distinguished from all other TGF percentages for only the OSQ depot (Table 4). As we found with IL gene percentage patterns, using another gene as a reference (i.e., TGFß3) did not influence the patterns of TGF gene percentages. Patterns of interferon and APO gene expression and gene percentages were also depot dependent in growing pigs (Table 4).
Age-Independent Cytokine and Apolipoprotein Gene Expression in Middle Subcutaneous Adipose (MSQ) and Outer Subcutaneous Adipose (OSQ) from Growing Pigs.
Mean Ct generated during REST analysis were used to demonstrate cytokine and APO gene expression that was not influenced by age (Tables 6 and 7). The Ct values are inversely related to gene expression. Expression of APO- R, IL- 15, IL-6, IL-12A, and IL-1RN was detected in OSQ and MSQ depots (Tables 6 and 7) and expression of APO- E, APO-A1, IL-8, and interferon 
was detected in the MSQ depot (Table 6). Note that RT-PCR data (Table 7) substantiates or supports the pattern of IL expression for the MSQ depot (Table 5; Figure 1).
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Table 6. Cytokine and apolipoprotein gene expression in middle (MSQ) and outer (OSQ) subcutaneous adipose in growing pigs that is not influenced by age [90 to 210 d of age; real-time reverse transcription-PCR crossing points (CP = Ct), and expression ratios (ratios)]
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Table 7. Interleukin real-time RT-PCR crossing points (Ct) and normalized microarray intensities for middle subcutaneous adipose tissue from 90- and 210-d of age pigs1,2
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Age-Dependent Cytokine and Adiponectin Gene Expression in MSQ And OSQ in Growing Pigs.
Expression ratios and associated Ct revealed age-dependent expression of RANTES and leptin in OSQ and ISQ depots and IL-1A, IL-1B, and adiponectin in the MSQ depot between 90 and 210 d (Table 8). Leptin, adiponectin, and IL-1A expression increased with age, whereas expression of RANTES and IL-1B decreased with age (Table 8).
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Table 8. Cytokine and adiponectin gene expression influenced by age in middle (MSQ) and outer (OSQ) adipose tissue in growing pigs: real-time reverse transcription-PCR analysis
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Pearson Correlation Coefficients and Simple Linear Regression Analysis.
Regression analysis demonstrated a significant linear regression (Figure 2; r2 = 0.79, P < 0.0001) between 75 real-time RT-PCR assay Ct and matching microarray intensities for 28 different genes from subcutaneous adipose tissue samples from growing pigs. Thirty-nine percent of these samples came from 90- and 210-d-old pigs each and 22% from 150-d-old pigs. A strong relationship between RT-PCR Ct and matching microarray intensities was also demonstrated by a significant (P < 0.0001) Pearson correlation coefficient of 0.88. The data used in these analyses were primarily MSQ depot data and represented 28 genes including apolipoproteins, cytokines, TF, GF, and receptors.
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Figure 2. A simple linear regression (P < 0.0001) of 75 real-time, reverse transcription (RT)-PCR assay crossing thresholds (Ct) vs. matching microarray intensities generated from middle and outer subcutaneous adipose tissue samples from 90-, 150-, and 210-d-old pigs. Data were log10 transformed before analysis. A total of 28 genes, including cytokines, transcription factors, growth factors, and receptors, were represented. These RT-PCR assay Ct and matching microarray intensities were significantly correlated (i.e., the Pearson correlation coefficient, r = 0.88, P < 0.0001).
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A strong relationship between RT-PCR Ct and matching microarray intensities was also observed in gene groups. For instance, Pearson correlation coefficients were 0.97 (P < 0.005) for 210-d IL (n = 5 matching data sets for 5 genes; Table 7), 0.89 (P < 0.001) for 210- and 90-d IL data (n = 10 matching data sets for 5 genes; Table 7) and 0.93 (P < 0.001) for 90- and 210-d APO (n = 8 matching data sets for 3 genes). Furthermore, Pearson correlation coefficients were also significant (P < 0.0001) for receptor gene (0.92, n = 17 matching data sets for 6 genes) and transcription factor gene data (0.82, n = 15 matching data sets for 5 genes). Regression analysis demonstrated significant linear regressions for APO, IL, receptor, and transcription factor gene data (r2 = 0.78 to 0.84, P < 0.001).
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DISCUSSION
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In the current study, we report, for the first time, gene expression of 7 cytokines and APO-E and APO-A1 in s.c. adipose tissue from growing pigs utilizing a combination of gene microarrays and RT-PCR assays or RT-PCR assays alone. Furthermore, a number of cytokines and APO-E and APO-A1 identified earlier at the protein level in media conditioned by neonatal pig adipocyte cultures (Hausman et al., 2006) were detected, herein, at the gene level in s.c. adipose tissue of growing pigs. Therefore, our collective results (present study; Hausman et al., 2006) indicate that a number of cytokines and 2 apolipoproteins are expressed and possibly secreted by pig adipose tissue throughout growth. This group of cytokines includes RANTES, IL- 8, IL-6, IL-1B, IL-1A, IL-12A, and IL-1RN. Pig studies of these and other cytokines are few and include studies of IL-6 gene expression by adipose tissue from Yucatan mini-pigs (Sebert et al., 2005), primary cultures of pig adipocytes (Ajuwon et al., 2004; Ajuwon and Spurlock, 2005), and studies of IL-8 and IL-6 gene expression by adipose tissue from young pigs (Brix-Christensen et al., 2005a,b). Studies of APO-E and APO-A1 gene expression by pig adipose tissue are few and include a study of APO-A1 in various tissues (no expression in adipose tissue; Birchbauer et al., 1993) and our gene microarray study of APO-A1 and APO-E in neonatal adipose tissue and stromal-vascular cells (Hausman et al., 2006). Therefore, the current study provides the first evidence of RANTES, IL-IRN, IL-1B, IL-1A, IL-12A, IFNG, and APO-E and APO-A1 gene expression by pig adipose tissue. Furthermore, this is the first report of IL-1A, RANTES, and APO-A1 gene expression by adipose tissue regardless of species (Sopasakis et al., 2005; reviews: Hauner, 2005; Gimeno and Klaman, 2005; Trayhurn et al., 2006).
A combination of gene microarray and RT-PCR analysis or RT-PCR alone demonstrated that for most of the 27 cytokines studied gene expression did not appreciably change with age in OSQ and MSQ depots from growing pigs. However, RT-PCR analysis showed that, despite a limited number of replicates, RANTES and IL-1B expression significantly decreased whereas adiponectin, leptin, and IL-1A expression increased in adipose tissue from 210-d pigs relative to 90-d adipose tissue. There are no comparable integrated or comprehensive studies on adipose tissue cytokine gene expression in any species. Nevertheless, adipose tissue IL-6 gene expression was also not changed with age in Yucatan mini-pigs (Sebert et al., 2005). Furthermore, an age-associated increase in adipose tissue leptin gene expression in pigs has been reported (Qian et al., 1999; Zhou et al., 2004). An increase in adipose tissue adiponectin expression is consistent with reported increases in serum adiponectin levels with age in pigs (Jacobi et al., 2004). An age-associated decrease in IL-1B gene expression (present study) was also detected in a comparison of fetal and neonatal adipose tissue by microarray analysis (Hausman et al., 2006). The decrease in IL-1B gene expression may be permissive to adipocyte development in the growing pig because IL-1 generally inhibits lipogenic and adipogenic gene expression (Memon et al., 1998) and IL-1B blocks human preadipocyte differentiation (Simons et al., 2005).
Comparison of the relative expression of genes within IL, TGF, interferon, and APO gene groups was used to establish patterns of gene expression for MSQ and OSQ from growing pigs. The pattern of IL gene expression served to distinguish MSQ from OSQ in growing pigs given that distinction of IL-6 and IL-15 expression from expression of other IL was more pronounced in MSQ than OSQ. It is important to note that IL-15 and IL-6 genes are expressed by isolated pig adipocytes (Ajuwon et al., 2004; Ajuwon and Spurlock, 2005) and IL-6 is expressed by human adipocytes (Wang and Trayhurn, 2006) and preadipocytes (Wang et al., 2005) in culture. Furthermore, several lines of evidence indicate that IL-6 expression is correlated with adipocyte size in humans in vivo and in vitro (Sopasakis et al., 2004). Therefore, the MSQ pattern of IL expression may reflect an adipocyte development associated increase in IL-6 expression relative to IL-15 expression. The current study substantiates and extends the reports of expression of IL-15 and IL-6 genes by pig adipocytes (Ajuwon et al., 2004; Ajuwon and Spurlock, 2005) by demonstrating IL-15 and IL-6 expression in MSQ and OSQ depots from 90- and 210-d-old pigs.
The IL-I system includes IL-1A, IL-1B, IL-1RN, and IL-1 receptors and is best known as a major mediator or regulator of inflammation (review, Gerard et al., 2004). It is noteworthy that adipose tissue is a major source of circulating IL-1RN (Juge-Aubry et al., 2003). We detected the secretion of IL-1RN by cultured neonatal pig adipocytes (Hausman et al., 2006) and IL-1RN gene expression by OSQ and MSQ from growing pigs (present study). Recent studies show that the IL-1 system can markedly influence lipid metabolism and adipose tissue deposition (Matsuki et al., 2003; Somm et al., 2006; Garcia et al., 2006). Therefore, the adipose tissue IL-1 system in growing pigs may also be linked to adipogenesis in response to inflammation or other stimuli. In this regard it is important to note that adipose tissue IL-1B gene expression was decreased during early (Hausman et al., 2006) and later growth (present study) in pigs. However, future studies are necessary to examine the regulation of secretion of IL-1 system component proteins per se.
Comparison of the relative expression of genes within the TGF gene group established TGF gene expression patterns that distinguished MSQ from OSQ in growing pigs. There is no apparent report of adipose tissue or adipocyte expression of BMP-15, GDF8, or BMP-4. Nevertheless, the distinction between OSQ and MSQ in growing pigs based on TGFß3 expression may reflect greater fibroblast growth in OSQ (Ellis and Schor, 1998). Regardless, the TGF gene expression patterns demonstrated in the current study are the first reported for adipose tissue.
We have validated with RT-PCR analysis that interferon- was expressed by MSQ, which is relevant to studies which showed that interferon- was a regulator of adipocyte IL-15 expression (Ajuwon and Spurlock, 2004; Ajuwon et al., 2004). Therefore, interferon- may be considered a depot-dependent autocrine or paracrine regulatory factor in porcine adipose tissue. Further discussion is limited because there are no relevant or published reports of porcine adipose tissue interferon gene expression.
A combination of gene microarray and RT-PCR analysis demonstrated that apolipoprotein gene expression did not appreciably change with age in OSQ and MSQ depots from growing pigs. However, the expression pattern of APO distinguished OSQ and MSQ depots in growing pigs. It should be pointed out that the significant correlations and regressions between apolipoprotein microarray and RT-PCR data substantiate the apolipoprotein gene expression patterns. The distinction between OSQ and MSQ based on APO-A1 expression may reflect enhanced adipocyte development or metabolism in MSQ because neonatal studies showed a relationship between APO-A1 protein secretion and adipogenesis in vitro (Hausman et al., 2006). Furthermore, adipose tissue expression of both the APO-A1 gene and protein neonatally (Hausman et al., 2006) coupled with APO-A1 gene expression during later growth indicate a possible developmental role for APO-A1 (review, Sacks, 2006). Given that APO-E expression studies have been limited to mouse and human adipocytes and adipose tissue (Zechner et al., 1991; Shih et al., 2000; Yue et al., 2004), our studies (present study; Hausman et al., 2006) are the first studies of meat animal adipose tissue APO-E gene expression. Recent studies show that adipose tissue APO-E may play a role in adipocyte development and metabolism (Chiba et al., 2003; Yue et al., 2004; Huang et al., 2006). Therefore, APO-E may have a role in porcine adipocyte development because the APO-E gene is expressed neonatally (Hausman et al., 2006) and later in growth (present study) and secreted APO-E protein is detected neonatally (Hausman et al., 2006). However, further studies are necessary to determine whether and how APO-A1 and APO-E influence porcine adipose tissue. Like APO-E and APO-A1, adipose tissue APO-R gene expression was detected neonatally (Hausman et al., 2006) and later in growth (present study). The APO-R gene was recently detected as an abundantly expressed gene in a porcine adipose tissue cDNA library in an expressed sequence tag study (Chen et al., 2006).
Considerable evidence indicates that nonadipocytes are responsible for a large portion of the cytokines secreted from adipose tissue. Except for leptin and adiponectin, over 90% of adipokine release from adipose tissue could be attributable to nonadipocytes (Fain et al., 2004). Therefore, in adipose tissue from growing pigs, nonadipocyte cell types may represent a major source of secreted factors (Hausman et al., 2006; review, Hauner, 2005).
The significant correlations and linear regressions detected between microarray and matching RT-PCR data serve to validate the microarray data in general. Furthermore, significant correlations and linear regressions were also detected between microarray and matching RT-PCR data in smaller gene groups. However, age-related changes in IL-1A and IL-1B expression detected with RT-PCR assays were not confirmed by microarray data. This is expected because RT-PCR assays usually do not validate or confirm all of the microarray detected changes in studies like these.
The current evidence indicates that of all the cytokines examined in this study IL-6, IL-8, IL-1B, IL-10, IL-1RN, tumor necrosis factor (TNF) 
, leptin and adiponectin are considered secreted endocrine factors primarily based on studies of obese humans (reviews: Gimeno and Klaman, 2005; Hauner, 2005; Trayhurn et al., 2006). The secretion of cytokines by adipose tissue from nonobese humans [i.e., IL-6, IL-8, and IL-1RN (Sopasakis et al., 2005)] and pigs (35 to 40 kg) [i.e., IL-8, IL-6, IL-10 and TNF (Brix-Christensen et al., 2005a,b)] has also been demonstrated. As pointed out previously (Hausman et al., 2006), IL-1B, IL-6, IL-8, or TNF can decrease food intake in rodents when administered centrally. Therefore, our studies (present study; Hausman et al., 2006) demonstrate that pig adipose tissue may secrete many endocrine factors, which could impact growth and performance. Further research is necessary to examine adipose tissue secretion of cytokines at the protein level. Regardless, our studies greatly expand the list of pig adipose tissue secreted factors.
Compared with other species, there is little information on cytokines expressed by adipose tissue from growing pigs. Porcine adipose tissue expresses numerous cytokines, many of which can influence fat cells and fat cell development at the local level. These studies reveal several fat tissue cytokines potentially involved in pubertal development and the inflammatory response in pigs. However, it will be important to examine adipose tissue cytokines at the protein level during growth and pubertal development in the pig.
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Footnotes
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1 Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. The authors would like to gratefully acknowledge Laura-Lee Rutherford for her excellent technical assistance and support, which made these studies possible. 
2 Corresponding author: Gary.Hausman{at}ars.usda.gov
Received for publication March 6, 2007.
Accepted for publication June 28, 2007.
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Abstract
INTRODUCTION
