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Identification of genes downregulated during differentiation of porcine mesenteric adipocytes¹

S. Suzuki^*,2, S. Sembon^*, M. Iwamoto, D. Fuchimoto^* and A. Onishi^*

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

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

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