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J. Anim. Sci. 2006. 84:1059-1066
© 2006 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

The expression of genes related to adipocyte differentiation in pigs1

H. C. Wang, Y. H. Ko, H. J. Mersmann2, C. L. Chen and S. T. Ding3

Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The purpose of this study was to detect differential expression of genes related to adipocyte differentiation in pigs by suppression subtractive hybridization. Adipocytes and stromal vascular cells (a fraction containing preadipocytes) from pig adipose tissue were isolated for mRNA extraction. The cDNA from preadipocytes was subtracted from the cDNA from adipocytes. The subtracted gene fragments were cloned into pGEM-T Easy TA cloning vector. We selected 384 clones for gene sequence determination and for further analysis. These genes were subjected to a differential screening procedure to confirm the differential expression of genes between the 2 cell types. We found that at least 36 genes were highly expressed in the adipocytes compared with preadipocytes. Among these, 6 genes including 2 novel genes with the greatest differences were selected and confirmed by Northern analysis. We found that angiotensin I-converting enzyme (ACE), ataxia-telangiectasia mutated protein (ATM), calpain 1, and stearoyl coenzyme A desaturase 1 (SCD1) were highly expressed in adipocytes compared with preadipocytes (P < 0.05). The relative mRNA abundance of ACE, ATM, calpain 1, SCD1, and 2 novel genes discovered in the current study was increased at the later stages of adipocyte differentiation (P < 0.05). The results confirmed that the genes involved in lipid metabolism and adipocyte differentiation were highly expressed in porcine adipocytes. However, further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

Key Words: adipocyte • adipose tissue • calpain 1 • gene • porcine • suppression subtraction hybridization


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The major function of adipose tissue is to accumulate excess energy as fat in animals. Recent research indicates that adipose tissue is also an endocrine and paracrine tissue that secretes leptin, adiponectin, visfatin, and other factors into the blood to regulate energy homeostasis (Mohamed-Ali et al., 1998Go; Havel, 2002Go, Fukuhara et al., 2005Go). Porcine adiponectin and leptin mRNA is abundant in adipose tissue and differentiating adipocytes (Spurlock et al., 1998Go; McNeel et al., 2000Go; Wang et al., 2004Go). These findings indicate that the adipose tissue in pigs also acts as an endocrine tissue and expresses genes involved in regulating metabolism and other physiological functions.

The molecular events that lead preadipocytes to differentiate to adipocytes are complicated. Several transcription factors [e.g., peroxisome proliferator-activated receptor {gamma}(PPAR{gamma}) and CCAAT enhancer binding proteins (C/EBP) {alpha}, ß, and {delta}] have been shown to play major roles in regulating this process (reviewed in Cowherd et al., 1999Go; Hausman et al., 2001Go). In an elegant microarray experiment using 3T3-L1 as a model, Burton et al. (2002Go, 2004)Go showed the expression level of more than 285 genes increased during the first 24 h of adipocyte differentiation. The result demonstrated the complexity of molecular events happening during adipocyte differentiation. Therefore, more efforts will be needed to clarify which genes are central to the differentiation process.

In the current study, experiments were designed to detect differential expression of genes between porcine preadipocytes and adipocytes using a suppression subtractive hybridization technique (SSH). Selected genes related to adipocyte differentiation were confirmed by Northern analysis. Their relative mRNA abundance in other tissues was also determined.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Animals and Tissue
The animal protocol was approved by the Animal Care and Use Committee of the National Taiwan University. Three Lee-Sung pigs (2 mo old; 6.9 ± 1.9 kg) were killed by electrocution combined with exsanguination. Heart, intestine, kidney, LM, liver, lung, ovary, spleen, and subcutaneous adipose tissue were dissected and frozen quickly in liquid nitrogen and then stored at –70°C until RNA extraction. Additional adipose tissue was removed under sterile conditions from the dorsal subcutaneous depot for adipocyte and stromal vascular cell (S/V cell; containing preadipocytes) isolation.

Cell Isolation
Within 5 min from death, 0.66-mm slices of adipose tissue were prepared under sterile conditions. The slices were digested with collagenase (C6885, Sigma, St. Louis, MO) in sterile Krebs Ringer bicarbonate buffer supplemented with 5.6 mM glucose, 100 U of penicillin/mL, and 100 mg of streptomycin/mL (Suryawan and Hu, 1997Go; Ding et al., 1999Go; Hsu and Ding, 2003Go) at 37°C for 90 min. The digested tissue was passed through a 200-mesh filter to remove tissue residue, and the cells were separated by centrifugation at 800 x g for 10 min. The floating cells were defined as adipocytes and washed 3 times with a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with NaHCO3, 100 U of penicillin/mL, and 100 mg of streptomycin/mL (catalogue # 03-033-1B; Gibco-BRL, In-vitrogen Corporation, Carlsbad, CA). The S/V cell fraction, containing the preadipocytes, was isolated by collecting the pellet, which was then washed 3 times with DMEM supplemented with NaHCO3, 100 U of penicillin/mL, and 100 mg of streptomycin/mL. The adipocytes and preadipocytes were used to extract total RNA for further analysis of mRNA concentration, or were subjected to SSH analysis.

Cell Culture
The S/V cells were resuspended in DMEM/F12 containing 10% fetal bovine serum and plated at a concentration of 6 x 104 cells/cm2. Three pigs were used to collect S/V cells for 3 individual culture experiments. To allow cell attachment, the S/V cells were then cultured at 37°C in air containing 5% CO2 for 24 h. After 24 h of incubation, the medium was removed and replaced by serum-free differentiation medium (DMEM/ F12 containing 1 µM bovine insulin, 100 nM hydrocortisone, 10 µg of transferrin/mL, 1 nM thyronine, 1 µM rosiglitazone, 33 µM biotin, and 17 µM pantothenic acid) to induce adipocyte differentiation for 3 d (Hauner et al., 1989Go). After 3 d of culture, the medium was changed to differentiation medium without rosiglitazone for another 5 d. The mRNA from cultured cells was extracted at 0, 2, 4, 6, and 8 d of incubation for transcript analysis.

Suppression Subtractive Hybridization
The SSH procedure utilized the PCR Select kit from Clontech (BD Biosciences, Mountain View, CA). Briefly, mRNA from each of the 2 cell types (adipocytes and S/ V cells) was reverse-transcribed to synthesize double-stranded cDNA. After RsaI restriction enzyme digestion, the tester DNA (cDNA from adipocytes) was divided into 2 groups and ligated with adaptor 1 (tester 1) or adaptor 2 (tester 2), respectively. The driver DNA (cDNA from S/V cells) was not ligated with any adaptor. Tester 1 or tester 2 DNA was denatured at 95°C for 10 min and hybridized with denatured driver DNA in separate tubes. After hybridization, any single-stranded DNA with adaptor 1 or adaptor 2 represented genes expressed in adipocytes but not in preadipocytes, whereas the single-stranded DNA without adaptors represented genes expressed in preadipocytes but not in adipocytes. The resulting 2 populations were pooled for a second hybridization with fresh denatured drivers. The resulting molecules with both adaptor 1 and 2 represented gene sequences preferentially expressed in adipocytes. These molecules were amplified after a 14-cycle PCR using a pair of nested primer sequences from adaptor 1 and 2.

The differentially expressed gene fragments were then cloned into pGEM-T Easy TA cloning vector (Promega, Madison, WI). The resulting clones were selected for sequence analysis by a genetic analyzer (ABI 3730; Applied Biosystems, Foster City, CA). We selected 384 clones for forward subtraction (genes expressed in preadipocytes were subtracted from those in adipocytes) for expression-level analysis. These genes were subjected to further differential screening and Northern analysis to confirm the differential expression of genes between adipocytes and preadipocytes. The genes for Northern analysis were selected on the basis of high expression and known significance. Individual gene fragments preferentially expressed in preadipocytes (reverse subtraction) were not sequenced.

Differential Screening
The differential screening procedure followed that described by the Clontech PCR-Select Differential Screening Kit User’s manual (BD Biosciences). In brief, the transformed Escherichia coli were transferred to a nylon membrane and grown on an LB agar plate. The plate with the membrane containing the bacterial colonies was incubated for 12 h at 37°C. The membrane then was treated with 10% SDS before being treated with denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 5 min. The membrane was then neutralized by neutralizing buffer (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4) for 5 min. The DNA on the membrane was then fixed on the membrane by UV-crosslinking and baking at 80°C for 30 min. The membranes were used for hybridization to detect differentially expressed genes. The DNA from forward subtraction and reverse subtraction (genes expressed in adipocytes were subtracted from those in preadipocytes) were used as probes for the screening.

Transcript Analysis
Total RNA was extracted from the cells by the guanidinium-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987Go), modified by McNeel and Mersmann (1999)Go and Hsu et al. (2004)Go. The mRNA concentrations of angiotensin I-converting enzyme (ACE), ataxia-telangiectasia mutated protein (ATM), calpain 1, stearoyl coenzyme A desaturase 1 (SCD1), and 3 novel genes, and the concentration of 18S ribosomal RNA (18S) were quantified by the Northern blot analysis procedure previously described by Ding et al. (1999Go, 2003Go, 2004)Go. The source of the probes for 18S rRNA is stated in Ding et al. (2002)Go. The probes for the other porcine genes were generated from gene fragments discovered in the current study (Table 1Go).


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  		Table 1. Genes expressed at greater levels in adipocytes than in stromal vascular cells
 
Twenty micrograms of total RNA from each sample was electrophoresed under denaturing conditions, blotted to a nylon membrane, and hybridized with a radiolabeled probe synthesized by PCR. Each RNA sample was represented on 2 different membranes. To allow direct comparison within an experiment, all membranes for a given experiment were hybridized at the same time using the same-labeled probe. The relative mRNA concentration of each gene was determined by phosphor-image analysis (BAS-1500; Fujifilm, Kanagawa, Japan) and a quantification software program. To correct for extraction, sampling, gel loading, and membrane transfer variation, the density value for each sample was normalized to the density value for 18S rRNA in the same sample.

Statistical Analysis
The first experiment was a complete randomized design with 2 treatments, and Student’s t-test was performed. The tissue distribution of various transcripts was analyzed by ANOVA (SAS Inst. Inc., Cary, NC). Significance was considered to be P ≤ 0.05. The mean and SE for each transcript are presented.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Differentially Expressed Genes
The differentially expressed genes resulting from the SSH subtraction were cloned and preserved as subtractive libraries. Three hundred eighty-four colonies each with a subtracted fragment of various genes were subjected to differential screening and gene sequencing. Because there were lots of repeated gene fragments in the fragments analyzed, especially after 192 colonies, 384 colonies were sequenced. At least 122 cDNA fragments were proven to be differentially expressed between S/V cells and adipocytes (Table 1Go). These cDNA fragments belong to 21 known genes and 15 novel genes. According to their functions analyzed with Gene Ontology (http://www.geneontology.org), these genes were cataloged into 6 categories. The majority were involved in cellular physiological processes (9 of the 21 known genes) and metabolism (6 of the 21 known genes), indicating the possible function of these genes to support adipose tissue development and cellular metabolism. Among these genes, 4 known and 2 novel genes, with the greatest differential expression or with an important function, were selected for Northern analysis to confirm their differential expression. We found that ACE, ATM, calpain 1, SCD1, and 2 adipocyte-expressed unknown genes (AEUG1 and AEUG3) were all highly expressed in the adipocytes compared with preadipocytes (Figure 1Go).


Figure 1
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  		Figure 1. The expression of genes related to adipocyte differentiation in Lee-Sung pigs. Total RNA was extracted from porcine adipocytes and stromal vascular cells. The relative mRNA abundance for angiotensin I-converting enzyme (ACE), ataxia-telangiectasia mutated protein gene (ATM), calpain 1 light subunit (calpain 1), stearoyl coenzyme A desaturase 1 (SCD1), and 2 adipocyte-expressed unknown genes (AEUG) were determined by Northern analysis. AD = adipocytes; S/V = stromal vascular cells (preadipocyte-enriched). The results represent data from 3 pigs (n = 3).

 
Tissue Distribution of Porcine Genes
The ACE transcript was highly expressed in the adipose tissue and kidney of Lee-Sung pigs (Table 2Go). It was also detected in the heart and lung but was not detectable in the LM, liver, ovary, or spleen. The ATM transcript was universally expressed in all the tissues that were measured in this study (Table 2Go), suggesting that all tissues require the gene for DNA repair. The greatest expression levels were observed in the porcine adipose tissue and spleen. The calpain 1 transcript was highly expressed in the adipose tissue and kidney and to a lesser extent in the lung, LM, liver, and spleen of Lee-Sung pigs (Table 2Go). It was not detectable in the intestine. The SCD1 transcript was highly expressed in porcine adipose tissue and, to a much lesser extent, in the liver (Table 2Go). It was not detectable in the heart, spleen, ovary, kidney, lung, and LM. The novel gene AEUG1 was specifically expressed in adipose tissues and was not detectable in any other tissue. The AEUG3 was highly expressed in adipose tissue and was detected at lower levels in the intestine and kidney.


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  		Table 2. The mRNA tissue distribution of genes highly expressed in adipocytes1
 
Transcript Abundance in the Differentiating Adipocytes
At d 0, transcripts for ACE, SCD1, AEUG1, and AEUG3 were not detectable (Figure 2Go). There was a gradual increase in the steady-state relative abundance of these transcripts between d 0 and d 8. There was detectable level of transcripts for calpain 1 and the relative abundance of the transcript was increased at the later stages of adipocyte differentiation. In contrast, the relative transcript abundance of ATM was high at d 0 but very low at 2 and 4 d of incubation and increased to a greater level during the later stages of adipocyte differentiation (Figure 2Go). The great relative abundance of the transcripts for these genes during the later stages of adipocyte differentiation suggests a role of these genes in the adipocytes.


Figure 2
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  		Figure 2. The relative mRNA abundance of genes related to adipocyte differentiation in pigs. Pig S/V cells were cultured in Dulbecco’s modified Eagle’s medium/ F12 (DMEM/F12) with 10% fetal bovine serum for 24 h, at which time they had reached confluence. After the 24 h incubation, the medium was removed and replaced by serum-free differentiation medium (DMEM/F12 containing 1 µM bovine insulin, 100 nM hydrocortisone, 10 µg of transferrin/mL, 1 nMthyronine, 1 µM rosiglitazone 33 µM biotin, and 17 µM pantothenic acid) for 3 d to induce adipocyte differentiation. After 3 d of culture, the medium was changed to differentiation medium without rosiglitazone for another 5 d. The mRNA from cultured cells was extracted at 0, 2, 4, 6, and 8 d of incubation for transcript analysis. The relative mRNA concentrations for angiotensin I-converting enzyme (ACE), ataxia-telangiectasia mutated protein gene (ATM), calpain 1 light subunit (calpain 1), stearoyl coenzyme A desaturase 1 (SCD1), and 2 adipocyte-expressed unknown genes (AEUG1 and 3) were determined by Northern analysis. A: Relative mRNA abundance in differentiating adipocytes. Each individual gene was normalized to the value of 18S rRNA in the same sample, and the normalized value of d 8 was set at 100%. B: Typical images of Northern blots. n = 3 replicates/treatment. a–dMeans without a common superscript are significantly different (P < 0.05).

 

   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Gene Expression in Adipocytes
Although utilizing the SSH technique to explore the differential gene expression between preadipocytes and adipocytes was demonstrated to be useful in the current experiment, it was not as powerful as microarray technique (Guo and Liao, 2000Go; Burton et al., 2002Go, 2004Go). Fewer differentially expressed genes were discovered in the current experiment than were observed in the microarray experiments (Guo and Liao, 2000Go; Burton et al., 2002Go, 2004Go). Both groups used 3T3-L1 mouse clonal cell lines as models and identified more than 200 genes expressed more than 5-fold difference during early or late adipocyte differentiation. During the first day of adipocyte differentiation, the genes that involved in initiating adipocyte differentiation, e.g., C/EBPß and {delta}, are highly expressed (Burton et al., 2002Go). In the well-differentiated adipocytes, an adipocyte-specific transcription factor, PPAR{gamma}, is highly expressed (Guo and Liao, 2000Go). However, several adipocyte specific abundant genes, i.e., SCD1 and ACE [identified by the current study and others (Ailhaud et al., 2002Go; Wang et al., 2004Go)], were not identified in the microarray study (Guo and Liao, 2000Go; Burton et al., 2002Go). Therefore, for a genome-wide gene profiling study, microarray may be the most powerful choice, but the SSH technique is useful for discovering some differentially expressed genes that were not detected by the microarray techniques. It is important to know that the SSH technique will miss some of the genes expressed due to the lack of some specific restriction enzyme cutting sites in those genes. But such defects can be avoided by using other restriction enzymes before the hybridization subtraction procedure. Such an approach will increase the extent of analyses, however. Because we used only 1 restriction enzyme in the current study, some of the adipocyte-specific genes such as leptin and PPAR{gamma} were not observed. In the following paragraphs, several of the genes found to be differentially expressed between porcine preadipocytes and adipocytes were discussed.

Angiotensin I-converting enzyme (enzyme class 3.4.15.1) belongs to a zinc metalloproteinase family. It contains 2 catalytic domains, N- and C-terminal exo-and endo-carboxypeptidase activities. The ACE is a type I integral membrane glycoprotein enzyme that hydrolyzes angiotensin I to produce angiotensin II, which is an octapeptide potent in the control of blood pressure and fluid and electrolyte homeostasis (Corvol et al., 2004Go). Thus, ACE plays an important role in cardiovascular homeostasis, and its inhibitors are effective and widely used drugs for the treatment of hypertension and heart failure (Acharya et al., 2003Go). In the current study, ACE is expressed in many tissues with the greatest expression in kidney, lungs, and adipose tissue similar to what was observed in mice and pigs (Ehler and Riordan, 1989Go; Matsui and Takahashi, 2002Go; Corvol et al., 2004Go). We also found that it is specifically expressed in adipocytes but not in S/V cells, indicating that adipocytes may be able to regulate blood pressure through release of angiotensin II by increasing the expression of ACE. Recent studies indicate that adipocytes also express ACE and that it is involved in inducing adipocyte differentiation and hypertrophy through the production of angiotensin II and activation of rennin-angiotensin system (Engeli et al., 2000Go; Ailhaud et al., 2002Go; Sibley, 2003Go). Whether there is any other specific function of ACE in adipocytes awaits further studies to demonstrate.

The major function of ATM protein is in signaling DNA damage that leads to DNA repair to maintain the integrity of a genome (Shiloh, 2001Go). Recent findings indicate that deficiency of ATM triggers zinc-finger transcription factors, Sp1 and WT1-mediated reduction of the expression of IGF-I receptor, and phosphorylation of insulin receptor subtrate-1 to decrease the function of IGF-I and insulin (Shahrabani-Gargir et al., 2004Go). Therefore, the ATM gene may be also involved in adipocyte function through mediating IGF and insulin function. In the current study, we found that ATM is highly expressed in well-differentiated adipocytes compared with the newly isolated preadipocytes, indicating its possible involvement in adipocyte differentiation.

Calpains, a family of nonlysosomal cysteine proteases, cleave a wide variety of proteins to modify the functions of these proteins. Calpains are able to influence the binding of C/EBPß to increase the expression of C/EBP{alpha} and increase adipocyte differentiation (Patel and Lane, 1999Go). In 3T3-L1 adipocytes, calpains may also act by increasing the translocation of glucose transporter 4 (Glut4), thereby increasing the use of glucose (Paul et al., 2003Go). However, a recent study showed that Glut4 expression in 3T3-L1 adipocytes is repressed by protease inhibition but not by inhibition of calpains (Cooke and Patel, 2005Go). Therefore, the role of calpains on adipocyte functions requires further study. We demonstrated that calpain 1 is highly expressed in differentiated adipocytes and the transcript abundance is associated with the degree of adipocyte differentiation. A high level of calpain 1 was observed in the differentiating 3T3-L1 adipocytes (Patel and Lane, 1999Go), suggesting a role of this gene in the adipocytes.

The SCD1 is a rate-limiting enzyme catalyzing the formation of an n-9 double bond of palmitoleate and oleate from palmitate and stearate, respectively. Its products may also upregulate the expression of enzymes in lipogenic pathway and downregulate enzymes in lipid oxidation (Ntambi et al., 2002Go); therefore the enzyme is important in regulating metabolism in adipocytes. In the current study, tissue distribution of porcine SCD1 in Lee-Sung pigs was similar to that reported in mice (Ntambi et al., 1988Go), genetically selected pigs (Smith et al., 1999Go), and crossbred pigs (Wang et al., 2004Go). In the current study, there was a relatively high SCD1 mRNA abundance in porcine adipose tissue and a significant increase in SCD1 mRNA abundance when preadipocytes differentiated into adipocytes. It was shown that the SCD1 mRNA concentration associated well with the degree of porcine adipocyte differentiation (Wang et al., 2004Go). Because the SCD1 is one of the major lipogenic genes, the increased expression of SCD1 in differentiating porcine adipocytes indicates the requirement for greater lipogenic capacity during differentiation.

The function of the novel genes (AEUG1 and 3) is not known, but they were all highly expressed in the adipose tissue, suggesting the possible involvement of these genes in porcine adipocyte function. Moreover, the expression of AEUG1 was specific to the adipose tissue and was not expressed in preadipocytes, suggesting that this gene may have an important function related to porcine adipocytes.

General Discussion
In the literature, many studies on gene expression during adipocyte differentiation used mouse clonal cell lines as study models (Guo and Liao, 2000Go; Burton et al., 2002Go, 2004Go). These models require heavy doses of hormones and other factors to initiate adipocyte differentiation; therefore the gene expression patterns may just be results of the hormonal influence and differ from adipocyte differentiation in vivo. We attempted to use freshly isolated S/V cell (enriched with preadipocytes) and adipocyte fractions to study the differentially expressed genes between these 2 cell groups. We have found at least 2 known genes that were highly expressed in the adipocytes, i.e., SCD1 and ACE, confirming previous reports (Ailhaud et al., 2002Go; Wang et al., 2004Go). We have also found 2 novel genes that were highly expressed in porcine adipose tissue and highly associated with adipocyte differentiation in the current study. These data demonstrate that the approach in the current study is appropriate for our purposes. Some may argue that cultured S/V cells at d 0 might be a better representation for preadipocytes; however, that has yet to be demonstrated.

In conclusion, we have demonstrated by an SSH method that there are many genes that are specifically expressed in the porcine adipocytes compared with preadipocytes. We have also demonstrated the tissue distribution of 6 of the differentially expressed genes. Most of these genes were expressed in greater quantity in adipose tissue as compared with other tissues, indicating important roles of these genes in porcine adipose tissue. Further demonstration of the functions of these genes discovered in the current study will add great value to the understanding of porcine adipocyte biology.


   Footnotes
 
1 This work was supported by National Science Council in Taiwan (grant number 93-2313-B-002-023). We thank the Experimental Farm at National Taiwan University for providing the experimental animals. Back

2 Visiting scientist at National Taiwan University. Back

3 Corresponding author: sding{at}ntu.edu.tw

Received for publication September 30, 2005. Accepted for publication December 26, 2005.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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