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J. Anim Sci. 2008. 86:3367-3376. doi:10.2527/jas.2008-0850
© 2008 American Society of Animal Science
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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Identification of genes downregulated during differentiation of porcine mesenteric adipocytes1

S. Suzuki*,2, S. Sembon*, M. Iwamoto{dagger}, D. Fuchimoto* and A. Onishi*

* Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba 305-0901, Japan; and {dagger} Prime Tech Ltd., Tsuchiura 300-0841, Japan


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Adipose tissue development is a process that comprises not only hypertrophy, but also hyperplasia, of adipocytes. Although the proliferation of undifferentiated preadipocytes plays an important part in hyperplasia, this process is less well understood than the post-proliferation differentiation process. Despite the potential importance of porcine visceral adipose tissue to both meat production and biomedical research, there has been little study of this tissue and, in particular, its development and differentiation. To detect the genes involved in the maintenance of porcine visceral preadipocytes in an undifferentiated state or in the inhibition of adipocyte differentiation, we performed suppression subtractive hybridization using mesenteric preadipocytes in which fragments of the genes that are downregulated at 2 d of differentiation were enriched. We selected 672 clones and subjected them to differential screening and semiquantitative reverse transcription (RT)-PCR. As a result, we identified 34 downregulated genes. Among these, the detailed expression patterns of 6 genes were examined using real-time RT-PCR in both preadipocytes during in vitro differentiation and cell fractions directly isolated from pig mesenteric adipose tissue. The expressions of connective tissue growth factor, AXL receptor tyrosine kinase, stromal membrane-associated protein 1-like, and retinoic acid-induced 14 were significantly downregulated during adipocyte differentiation in vitro (P < 0.05), and the expressions of Rho/Rac guanine nucleotide exchange factor 2 and secreted frizzled-related protein 4 also tended to be decreased, although not significantly. Furthermore, all 6 genes showed significantly greater expression in stromal vascular cells, which contain preadipocytes, than in mature adipocytes (P < 0.05), raising the possibility that these genes are involved in adipocyte differentiation in vivo as well as in vitro.

Key Words: differentiation • gene expression • mesenteric adipocyte • mesenteric adipose tissue • porcine • suppression subtractive hybridization


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The development of adipose tissue involves hypertrophy and hyperplasia of adipocytes (Hausman et al., 2001Go). Hyperplasia results from the commitment of precursor cells to the adipocyte lineage, and the proliferation and differentiation of the committed preadipocytes. In the pig, analyses of gene expression (Ding et al., 1999Go; McNeel et al., 2000Go), and a comprehensive search for genes relevant to the differentiation process has been made (Wang et al., 2006Go). However, the commitment and proliferation processes that accompany the maintenance of preadipocytes in an undifferentiated state have been less well studied.

Visceral fat tissue is currently attracting interest, because visceral obesity is a major component of metabolic syndrome (Licata et al., 2006Go; Matsuzawa, 2006Go). In the pig, differences have been reported in lipid metabolism among fats originating in different depots (Rule et al., 1989Go; Budd et al., 1994Go), but visceral adipogenesis has been scarcely studied. An understanding of porcine visceral adipogenesis may lead to the production of higher quality meat at less cost through regulation of regional adiposity. Moreover, because of its susceptibility to arteriosclerosis, the pig is a potential model for human metabolic syndrome (Moghadasian et al., 2001Go), and an understanding of porcine adipogenesis may also contribute to the development of a genetically modified porcine model that can mimic the disease conditions of human metabolic syndrome.

In this study, to identify genes that may be relevant to the maintenance of porcine visceral preadipocytes in an undifferentiated state, we detected the genes down-regulated at an early phase of mesenteric adipocyte differentiation using suppression subtractive hybridization. Then, expression patterns of selected genes during in vitro adipocyte differentiation were examined by quantitative real-time PCR. The expression patterns of cells directly isolated from mesenteric adipose tissue were also examined.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Protocols for the use of animals in this study were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences.

Cell Isolation

Mesenteric adipose tissue was obtained from 2-yr-old female pigs (Landrace or crossbred) which had an average BW of 225 ± 8 kg (range, 210 to 260 kg). Mature fat cells and stromal vascular (SV) cells were isolated essentially as described (Ramsay et al., 1992Go). Briefly, adipose tissue was washed with PBS(–) supplemented with 100 units/mL penicillin, 100 mg/mL of streptomycin, and 250 ng/mL of amphotericin B (Nacalai Tesque, Kyoto, Japan), and minced finely. The minced tissue was then incubated with gentle shaking in a digestion buffer comprising Dulbecco’s modified Eagle medium (DMEM), 100 mM HEPES, and 1.5% (wt/vol) BSA (fatty acid free, fraction V; A6003, Sigma, St. Louis, MO) supplemented with 0.2% (wt/vol) type II collagenase (C6885, Sigma) for 1 h at 37°C. The digested tissue was filtered through a 150-µm stainless steel mesh and centrifuged at 150 x g for 5 min to separate the floating adipocytes from the pellet of SV cells. The floating adipocytes were collected and subjected to ceiling culture (Sugihara et al., 1986Go) or total RNA extraction. The SV cells were incubated with an erythrocytelysis buffer (0.154 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) at room temperature for 10 min followed by centrifugation at 150 x g for 5 min. The pelleted cells were washed with DMEM and subjected to total RNA extraction.

Preparation of Preadipocytes by Ceiling Culture

Primary preadipocytes were established by the ceiling culture method (Yagi et al., 2004Go; Miyazaki et al., 2005Go). Floating adipocytes collected as described above were washed twice with digestion buffer and seeded in 12.5 cm2 culture flasks (Falcon 353107; Becton Dickinson, Franklin Lakes, NJ) completely filled with DMEM containing 20% (vol/vol) fetal bovine serum (FBS; Biowest, Nuaillé, France) at a density of about 105 cells/flask. The flasks were incubated upside down in a humidified atmosphere of 5% CO2 in air at 37°C for 6 or 7 d. After we confirmed the presence of fibroblastic cells attached to the inner ceiling surface, the flask was turned upside down. The cells were subsequently cultured in DMEM containing 10% FBS until subjected to the differentiation procedure.

Differentiation of Preadipocytes

The preadipocytes were seeded into 10-cm dishes or 12-well plates pre-coated with gelatin (G1890, Sigma) and grown until they reached confluence. At confluence (d 0), adipogenic differentiation was induced by culturing the cells for 8 d in DMEM containing 10% newborn calf serum (NBCS; Gibco, Invitrogen, Carlsbad, CA), 5 µg/mL insulin (I5500, Sigma), 0.25 µM dexamethasone (D4902, Sigma), 33 µM biotin (Nacalai Tesque), 17 µM pantothenate (Nacalai Tesque), 5 mM octanoate (Wako Pure Chemicals, Osaka, Japan), 100 units/mL of penicillin, 100 mg/mL of streptomycin and 250 ng/mL of amphotericin B (Nacalai Tesque; Nakajima et al., 2003Go). The cells were cultured further in DMEM containing 10% NBCS for another 2 d. To confirm the accumulation of lipid droplets, cells were fixed in 10% neutral buffered formalin (Nacalai Tesque) and stained with Oil red O (O0625, Sigma). The total RNA was extracted on d 0 and 2 for suppression subtractive hybridization (SSH) and on d 0, 2, 6, and 10 for quantitative reverse transcription (RT)-PCR.

Total RNA and PolyA(+) RNA Extraction

Total RNA was extracted from the isolated or cultured cells using Isogen (Nippon Gene, Tokyo, Japan). The resulting total RNA was treated with RNase-free DNase I (Takara Bio, Otsu, Japan) at 37°C for 30 min. For SSH, polyA(+) RNA was isolated from the total RNA by using an Oligotex dT30 (super) mRNA purification kit (Takara Bio) following the manufacturer’s instructions.

Suppression Subtractive Hybridization

Suppression subtractive hybridization was performed using a Clontech PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. Briefly, 1.5 µg of poly(A)+ RNA extracted from preadipocytes at d 0 (tester) and d 2 (driver) of differentiation was reverse-transcribed to synthesize double-stranded cDNA. After digestion with RsaI, the tester cDNA population was divided in half and ligated with different adaptors. An aliquot of the tester cDNA population was ligated with both adaptors to serve as an unsubtracted control. The first hybridization was performed by addition of the driver cDNA to each tube, followed by heat denaturation, and, subsequently, the annealing process. The 2 separate samples were then combined simultaneously with the addition of the freshly denatured driver cDNA and subjected to a second hybridization. The hybrids with different adaptors on each end represented differentially expressed cDNA fragments. Two rounds of PCR amplification were then performed to enrich the differentially expressed cDNA fragments. The subtracted cDNA fragments were cloned into a pTA2 vector (Toyobo, Osaka, Japan) and transformed into competent JM109 E. coli cells (Toyobo). Reverse subtraction was also performed by reversing the tester and driver, although the resulting cDNA fragments were not subcloned.

Differential Screening

The differential screening procedure was performed according to the PCR-Select Differential Screening Kit User Manual (Clontech) to eliminate false-positive clones. In brief, cDNA inserts were amplified by PCR using vector primers (T7 and T3 primer) and Ampli-TaqGold (Applied Biosystems, Foster City, CA). The PCR products were denatured by adding an equal volume of 0.6 M NaOH and manually spotted onto Hybond N+ membranes (GE Healthcare, Piscataway, NJ). The membranes were soaked in 0.5 M Tris-Cl (pH 7.5) for 5 min and washed with distilled water. The DNA on the membranes was fixed by a UV crosslinker. The membranes were hybridized with 32P-labeled probes individually derived from forward- and reverse-subtracted cDNA as well as unsubtracted tester and driver cDNA. The membranes were then washed with 2x sodium chloride/sodium citrate buffer/0.1% SDS at room temperature and 0.1x sodium citrate buffer/0.1% SDS at 60°C, and exposed on an Imaging Plate (Fujifilm, Tokyo, Japan), followed by analysis using a FLA-3000G image analyzer (Fujifilm) to select the differentially expressed clones.

Sequencing

Plasmids from the selected clones were isolated using an automatic DNA isolation system PI50 (Kurabo, Osaka, Japan), and cDNA inserts were sequenced on an ABI Genetic Analyzer 370 (Applied Biosystems) using a BigDye terminator cycle sequencing kit version 1.1 (Applied Biosystems). The obtained sequence data were compared and analyzed for homology using the basic local alignment search tool (BLAST: http://www.ncbi.nlm.nih.gov/blast/).

Semiquantitative RT-PCR

Semiquantitative RT-PCR analysis was carried out to confirm the differential expression of selected transcripts. Aliquots of 1 µg of total RNA were reverse-transcribed into cDNA using PowerScript Reverse Transcriptase (Clontech) with random hexamer primers. The subsequent PCR amplification was performed using KOD plus version 2 (Toyobo) under the following conditions: initial denaturation at 94°C for 2 min, then cycles of denaturation for 10 s at 98°C and annealing/extension at 68°C for 45 s. The primers used were designed based on the sequences of the cloned cDNA fragments. The optimal number of cycles was determined in the linear amplification region. The 18S ribosomal RNA (18S) served as an internal control for normalization of sample variation. The primers for 18S were 18S-F (5'-AGCTTATGACCCGCACTTAC-3') and 18S-R (5'-ACCAAAGTCTTTGGGTTCCG-3'). The PCR products were resolved on 2% agarose gel and visualized with ethidium bromide.

Real-time RT-PCR

Real-time quantitative RT-PCR of transcripts of interest was performed to examine the detailed expression pattern using the Lightcycler instrument (Roche, Basel, Switzerland). The PCR amplification was performed in a 20-µL reaction mixture consisting of 1 µL of cDNA, 0.4 µM of each primer, and 10 µL of SYBR Green Realtime PCR Master Mix (Toyobo). Cycling conditions were 95°C for 3 min, followed by 50 cycles of 95°C for 0 s, 60°C for 5 s, and 72°C for 30 s. The primers used are indicated in Table 1Go. To identify nonspecific products, a melt-curve analysis was performed at the end of each run. All quantifications were carried out based on the time point of the log-linear increase of amplified DNA during the PCR using the fit point option of LightCycler software version 3.5. A standard curve was generated by the amplification of serially diluted cDNA using the fit point option of the LightCycler software for target genes and the hypoxanthine phosphoribosyl-transferase 1 (HPRT1) gene that was used as an internal control individually. The threshold fluorescence level was determined within the geometric region of the semilog view of the amplification plot. The relative quantification of target gene expression was calculated from the standard curve and normalized against the expression of HPRT1.


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  		Table 1. Primer sequences used in real-time quantitative reverse transcription-PCR
 
Statistical Analysis

Statistical analysis was performed using Student’s t-test. Differences were considered significant when P values were <0.05.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
We used preadipocytes obtained by a ceiling culture method for SSH during the adipocyte differentiation process. The preadipocytes, which exhibited a fibroblast-like shape under growth conditions, began to accumulate small lipid droplets after exposure to insulin, dexamethazone, octanoate, biotin, and pantothenate. The lipid droplets increased in number and size as differentiation proceeded. Most of the preadipocytes had differentiated into mature adipocytes at 10 d of differentiation (Figure 1Go).


Figure 1
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  		Figure 1. Differentiation of preadipocytes. Cells were subjected to adipogenic differentiation and stained with Oil red O at (A) d 0 (undifferentiated), (B) d 2, (C) d 6, and (D) d 10. Scale bar represents 50 µm.

 
To identify the genes that were downregulated during the early phase of adipocyte differentiation, we performed SSH so that mRNA from preadipocytes at d 2 of differentiation was subtracted from that at d 0 (pre-stimulation). After SSH, 672 clones were randomly selected and subjected to differential screening by dot-blot hybridization. We selected 240 clones that appeared to be expressed more abundantly at d 0 than at d 2. The selected clones were then sequenced and searched for in the GenBank database by BLAST analysis, which revealed that these clones have cDNA fragments that belong to 94 known genes and 3 unknown genes. Next, these 97 genes were further screened by semiquantitative RT-PCR using cDNA at d 0 and d 2 of differentiation. We selected the clones that were consistently found to be expressed more abundantly at d 0 than at d 2 in 3 independent populations of preadipocytes established from 3 different pigs, and thereby identified 34 genes corresponding to 170 clones (Table 2Go).


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  		Table 2. Genes downregulated at d 2 of porcine mesenteric adipocyte differentiation
 
To further confirm the differential expression and examine the change of expression level during the time-course of adipogenic differentiation in vitro, we performed quantitative real-time RT-PCR analysis of 6 of the identified genes that are considered to be involved in signal transduction [connective tissue growth factor (CTGF), AXL receptor tyrosine kinase (AXL), Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2), stromal membrane-associated protein 1-like (SMAP1L), secreted frizzled-related protein 4 (SFRP4), and retinoic acid-induced 14 (RAI14); Figure 2Go]. Expression of CTGF, AXL, SMAP1L, and RAI14 were significantly reduced to 10% to 40% during adipogenic differentiation. Likewise, the expression of SFRP4 and ARHGEF2 tended to be decreased, although not significantly. The expression of all genes examined was greatly decreased within the first 2 d of differentiation and remained at roughly comparable levels for the subsequent 8 d, confirming the results of SSH.


Figure 2
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  		Figure 2. Relative changes in mRNA expression of 6 genes selected from downregulated genes identified by suppression subtractive hybridization (SSH) during in vitro adipocyte differentiation. The 6 genes are (A) connective tissue growth factor, (B) AXL receptor tyrosine kinase, (C) Rho/Rac guanine nucleotide exchange factor 2, (D) stromal membrane-associated protein 1-like, (E) secreted frizzled-related protein 4, and (F) retinoic acid-induced 14. The mRNA expressions at d 0 (prestimulation), 2, 6, and 10 of differentiation were measured by real-time reverse transcription-PCR performed in duplicate for each cDNA sample with hypoxanthine phosphoribosyltransferase 1 used as an internal control for amounts of RNA used in each reaction. The values are shown as a percentage of d 0 of differentiation (100%). The experiments were repeated 3 times using cell populations isolated from 3 individual pigs, and data are expressed as mean ± SE. The asterisks show statistically significant values (**P < 0.01, *P < 0.05) compared with values of d 0.

 
To examine the expression of these 6 genes in adipose tissue in vivo, we performed quantitative real-time RT-PCR analysis using cDNA from mature adipocytes and SV cells containing preadipocytes isolated directly from pig mesenteric adipose tissue. All 6 genes examined were found to be expressed 2 to 100 times more abundantly in SV cells than in mature adipocytes (Figure 3Go), suggesting that these genes are expressed predominantly in undifferentiated preadipocytes in vivo as well as in vitro.


Figure 3
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  		Figure 3. Relative mRNA expressions of 6 genes selected from downregulated genes identified by suppression subtractive hybridization (SSH) in stromal-vascular cells (SV: black bars) and mature adipocytes (AD: dotted bars). The 6 selected genes are connective tissue growth factor (CTGF), AXL receptor tyrosine kinase (AXL), Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2), stromal membrane-associated protein 1-like (SMA-P1L), secreted frizzled-related protein 4 (SFRP4), and retinoic acid-induced 14 (RAI14). The mRNA expression was measured by real-time reverse transcription-PCR performed in duplicate for each cDNA sample with hypoxanthine phosphoribosyltransferase 1 used as an internal control for amounts of RNA used in each reaction. The values are shown as the percentage of SV cells (100%). The experiments were performed using cells from 3 pigs, and data are expressed as mean ± SE. The asterisks show statistically significant values (*P < 0.05).

 

   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In the study of porcine adipocyte differentiation, SV cells are frequently used as the source of preadipocytes. However, SV cells contain a heterogeneous population of cells, including fibroblasts, endothelial cells, and macrophages, in addition to preadipocytes (Ramsay et al., 1992Go); thus, gene expression analysis can be affected by the behaviors of contaminating cells other than preadipocytes. For stricter analysis of gene expression, we used a preadipocyte population established through ceiling culture of mature adipocytes. This population is considered to be comparatively uniform because it originated from dedifferentiated mature adipocytes (Yagi et al., 2004Go) or cells tightly attached to mature adipocytes (Miyazaki et al., 2005Go).

To identify the genes that are downregulated during the early phase of mesenteric adipocyte differentiation in the pig, we performed SSH to detect genes expressed more abundantly in preadipocytes at d 0 than at d 2 of differentiation. The SSH technique we employed is useful for discovering differentially expressed genes, irrespective of whether their sequences are known or unknown. Nevertheless, it should be noted that this technique would miss differentially expressed genes that lack some specific restriction enzyme sites. Moreover, faintly expressed genes are also likely to be missed given the limitation on the number of colonies identified. Of the 34 genes we identified, 13 are classified as encoding components of the cytoskeleton or extracellular matrix. This is considered to be a reflection of the dramatic morphological changes that accompany adipocyte differentiation. On the other hand, the genes considered to be involved in signal transduction are likely to be inhibitors of adipocyte differentiation or regulators of the maintenance of preadipocytes in an undifferentiated state. Therefore, we selected 6 of the identified genes, mainly from those that are considered to be involved in signal transduction, and examined their expression patterns during in vitro adipocyte differentiation. Indeed, downregulation of these genes accompanied adipocyte differentiation, although we could not exclude the possibility that their altered expression was an in vitro artifact due to the culture conditions, including merely the effects of serum and/or differentiation-inducing reagents. Therefore, we also examined expression of these 6 genes in mature adipocytes and SV cells, which include preadipocytes, isolated directly from the mesenteric adipose tissue. As expected, we found that these 6 genes showed more abundant expression in SV cells than in mature adipocytes; this raised the possibility that at least these 6 genes were relevant to adipocyte differentiation in vivo as well as in vitro.

Secreted frizzled related protein (SFRP) 4 is a member of the SFRP family, which includes SFRP1 to SFRP5. All members of the SFRP family function as soluble antagonists for the Wnt signaling pathway (Kawano and Kypta, 2003Go). The Wnt signaling pathway plays an inhibitory role in adipogenesis, and expression of Wnt10b is suppressed during adipogenesis in murine 3T3-L1 preadipocytes (Ross et al., 2000Go; Bennett et al., 2002Go). In addition, 3T3-L1 preadipocytes spontaneously differentiate into adipocytes when exposed to SFRP1 or SFRP2 (Bennett et al., 2002Go). However, SFRP2 is expressed predominantly in undifferentiated preadipocytes in humans and rodents (Hu et al., 1998Go), and our observations of SFRP4 are consistent with this. These apparently contradictory findings might be explained by the idea that Wnt signaling functions in the cell-fate specification of undifferentiated preadipocytes. Indeed, the Wnt signaling pathway plays positive roles in osteogenesis (Gaur et al., 2005Go) and chondrogenesis (Gaur et al., 2006Go). Presumably, precise modulations of Wnt signaling are required for normal development of mesenchymal tissues, including adipose tissue, and the SFRP play an important part in that modulation.

The Roh/Rac guanine nucleotide exchange factor 2 (ARHGEF2) gene encodes a protein, guanine nucleotide exchange factor (GEF)-H1, which activates the Rho family of small GTPases such as Rho and Rac (Ren et al., 1998Go). Rho GTPase has an anti-adipogenic and promyogenic effect, and Rho GTPase and its negative regulator, p190-B Rho GTPase-activating protein (GAP), regulate the decision on cell fate between adipogenesis and myogenesis (Sordella et al., 2003Go). Furthermore, the dominant negative form of RhoA protein promotes adipogenesis, and the constitutively active form promotes osteogenesis, in human mesenchymal stem cells (McBeath et al., 2004Go). In addition, Rac GT-Pase, another member of the Rho family, negatively regulates the differentiation of 3T3-L1 preadipocytes (Liu et al., 2005Go). Finally, GEFT, another GEF of the Rho family, induces myogenesis in C2C12 myogenic cells and inhibits adipogenesis in 3T3-L1 preadipocytes (Bryan et al., 2005Go). Considering these findings, appropriate modulation of Rho GTPase signaling activity would be a prerequisite to maintaining the undifferentiated state of preadipocytes, and GEF-H1 would be involved in this modulation through the activation of Rho GTPase(s).

Stromal membrane associated protein 1-like (SMA-P1L) may be involved in retrograde transport from the early endosome to the trans-Golgi network (TGN) in an inhibitory fashion by modulating ADP-ribosylation factor 1 small GTPase (Natsume et al., 2006Go). In insulin-responsive cells such as adipocytes and muscle cells, intracellular glucose transporter 4 (GLUT4) is rapidly translocated to the plasma membrane by insulin stimulation and enhances uptake of glucose into the cells. An intracellular transport loop between the TGN and the endosome may participate in this translocation system as the mechanism for intracellular retention of GLUT4 under unstimulated conditions (Shewan et al., 2000Go, 2003Go; Palacios et al., 2001Go; Bryant et al., 2002Go). If the establishment of this transport loop between TGN and the endosome augments the insulin-stimulation–dependent glucose uptake that precedes lipid accumulation, then the downregulation of SMAP1L that we observed here and the concomitant and feasible promotion of retrograde transport might be important for the lipid accumulation process that accompanies adipocyte differentiation.

Connective tissue growth factor (CTGF) is an extra-cellular-matrix–associated secreted protein that modulates diverse cellular functions (Brigstock, 2003Go; Perbal, 2004Go).The multifunctional nature of this protein may be attributable to interaction with other diverse proteins (Perbal, 2004Go). Interestingly, Abreu et al. (2002)Go reported that CTGF is able to antagonize BMP4 signaling; this signaling induces the commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage (Tang et al., 2004Go; Bowers and Lane, 2007Go), and to enhance TGF-β signaling, which inhibits adipocyte differentiation (Choy and Derynck, 2003Go). Therefore, CTGF may contribute to the maintenance of preadipocytes in an undifferentiated state.

The functional role of retinoic acid–induced 14 (RAI14) protein is still unclarified. Since the expression of RAI14 is induced by retinoic acid (Kutty et al., 2001Go), this gene may contribute to the inhibition of adipogenesis by retinoic acid (Stone and Bernlohr, 1990Go; Suryawan and Hu, 1997Go).

The relevance of Axl receptor tyrosine kinase (AXL) to adipocytes was suggested by a report about its major ligand, growth arrest-specific 6 (GAS6). The Gas6 knockout mice accumulated less adipose tissue than the wild type when kept on a high fat diet (Maquoi et al., 2005Go). In addition, GAS6 is preferentially expressed at undifferentiated confluent and post-confluent phases in 3T3-L1 (Shugart et al., 1995Go) and 3T3-F442A preadipocytes (Maquoi et al., 2005Go). These phenomena have raised the possibility that GAS6-AXL signaling serves to maintain the undifferentiated state of preadipocytes and allows them to respond to adipogenic stimuli that promote their proliferation and/or differentiation. Because GAS6 has been previously shown to increase β-catenin stability in contact-inhibited cells (Goruppi et al., 2001Go), GAS6-AXL signaling might inhibit adipogenic differentiation by augmenting the anti-adipogenic effect of Wnt signaling. Moreover, AXL interacts with C1 domain-containing phosphatase and TENsin homolog (C1-TEN) (Hafizi et al., 2002Go), which inhibits the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway (Hafizi et al., 2005Go). Because Akt signaling is essential for adipocyte differentiation (Peng et al., 2003Go), AXL signaling may attenuate adipocyte differentiation by inhibiting Akt signaling through C1-TEN.

Lipid metabolism in porcine adipocytes is regionally heterogenic (Rule et al., 1989Go; Budd et al., 1994Go). Although these variations among fat deposits are considered to be due mainly to extrinsic factors (such as hormonal environment, local nutrient availability, and anatomic constraints), an innate difference might also exist, because both the capacity of human preadipocytes to differentiate and their gene expression profiles depend on the origin of the cells (Tchkonia et al., 2002Go, 2007Go). However, whether the capacity of pig preadipocytes to differentiate varies by fat deposit origin is largely unknown. Comparative analysis of the genes we identified here might be useful to unravel this issue. Recently, visceral adipose tissue has received much attention because visceral obesity is a major component of metabolic syndrome in humans (Licata et al., 2006Go; Matsuzawa, 2006Go). The pig is potentially a good model for metabolic syndrome in humans because it is susceptible to arteriosclerosis (Moghadasian et al., 2001Go), a major consequence of that syndrome. Although the occurrence of metabolic syndrome in pigs has not been reported, except in the rare breed Ossabaw (Spurlock and Gabler, 2008Go), there remains a possibility that we can produce such a disease in pigs by inducing and exacerbating visceral obesity by gene modification via somatic cell nuclear transfer technology, which is readily available in our laboratory (Watanabe et al., 2005Go). This study and subsequent studies on the differentiation of porcine visceral adipocytes may provide clues to identification of the genes that need to be modified for the development of porcine models of visceral obesity and metabolic syndrome in humans.

In conclusion, we identified 34 genes that were down-regulated during adipocyte differentiation. Moreover, we observed that 6 of these genes were expressed more abundantly in undifferentiated preadipocytes than in differentiated adipocytes both in vitro and in vivo, raising the possibility that they are significant in pig mesenteric adipose development. Future elucidation of the functions of these genes will be useful to unravel the entire system of pig adipose tissue development.


   Footnotes
 
1 The authors thank the staff of the Swine Management Section of the National Institute of Livestock and Grassland Science (Tsukuba, Japan) for care of the pigs, and N. Watanabe and K. Iijima (Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan) for technical assistance. This study was supported by the Special Fund from the National Institute of Agro-biological Sciences. Back

2 Corresponding author: shunsuzu{at}affrc.go.jp

Received for publication January 8, 2008. Accepted for publication July 17, 2008.


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


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