Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: Molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro
Introduction
Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors belonging to the nuclear hormone receptor superfamily. To date, three PPAR isotypes-α, β and γ, encoded by separate genes and showing different tissue distribution patterns have been identified (Desvergne and Wahli, 1999, Escher et al., 2001). Among three PPAR isotypes, PPARγ is highly expressed in adipose tissue and has important roles in lipid metabolism, cell proliferation and inflammation (Tsai et al., 2008). In mammals, two transcripts of PPARγ with different lengths of the N-terminal, γ1 and γ2, have been found in mice (Tontonoz et al., 1994b, Zieleniak et al., 2008). The expression of these two transcripts results from differential promoter use and alternative RNA splicing (Zhu et al., 1995). The PPARγ2 transcript was predominantly expressed in adipose tissue and had a key regulatory role in the induction and maintenance of the adipocyte phenotype (Tontonoz et al., 1994a, Fajas et al., 1997), whereas PPARγ1 was relatively widely expressed (Fajas et al., 1997, Mukherjee et al., 1997).
At present, PPARγ has been identified and cloned in many fish species. For example, a single PPARγ transcript has been identified in Rachycentron canadum (Tsai et al., 2008), Paralichthys olivaceus (Cho et al., 2009), Dicentrarchus labrax (Boukouvala et al., 2004), Salmo trutta (Batista-Pinto et al., 2005), Pleuronectes platessa and Sparus aurata (Leaver et al., 2005), Takifugu rubripes (Kondo et al., 2007), Danio rerio (Ibabe et al., 2005) and Chelon labrosus (Raingeard et al., 2009). However, the exact number of genes and/or the presence of distinct PPARγ have not been determined in fish. In Salmo salar, two PPARγ transcripts that differed in the length due to alternative usage of the multiple polyadenylation were sequenced and Northern blot analysis revealed a third PPARγ transcript that encoded a C-terminally truncated variant (Andersen et al., 2000). A short PPARγ transcript was also found in S. salar, which represented an alternatively spliced form of PPARγ that lacked the first 102 nucleotides of exon 3 (Todorcevic et al., 2008). The further study suggested that PPARγ short was induced during adipocyte differentiation, indicating that this transcript played a role in lipid accumulation in adipocytes, whereas the PPARγ long was induced in the early phase of cultivation and repressed at later stages of differentiation. Studies also suggested that the fish PPARγ gene was not activated by common mammalian PPARγ-specific ligands (Leaver et al., 2005, Kondo et al., 2007), indicating a marked difference in structure and function of PPARγ gene between fish and mammals.
Insulin is a peptide hormone that stimulates cell growth and differentiation, and promotes the storage of substrates in fat, liver and muscle by stimulating lipogenesis, glycogen and protein synthesis, and inhibiting lipolysis, glycogenolysis and protein breakdown (Saltiel and Kahn, 2001). Insulin resistance is a widely pathological disease in humans (Reaven, 1988). Some PPARγ activators such as thiazolidinediones (TZD) are widely reported to improve insulin sensitivity (Benton et al., 2010), suggesting that the regulation of insulin on metabolism could be mediated by PPARγ. However, information on the direct effect of insulin on PPARγ expression is very scarce. Limited studies pointed out that insulin up-regulated both PPARγ1 and PPARγ2 expressions in isolated human adipocytes (Vidal-Puig et al., 1997), and no information was available in fish.
Yellow catfish Pelteobagrus fulvidraco is an omnivorous, freshwater species of fish with increasing interest in Chinese inland aquaculture. As a result of the rapid expansion of intensive aquaculture for yellow catfish, excess lipid deposition in the adipose tissue and liver of the fish species has seriously impacted growth performance and health. Recently, we cloned the partial cDNA sequence of PPARγ and investigated mRNA tissue expression profiles of the single PPARγ gene (Zheng et al., 2013). As a continuation of our studies involved in the structure and functions of the gene, the present study cloned the full-length cDNA sequences of two PPARγ transcripts, and determined their tissue-specific and developmental expression profiles. Meanwhile, the patterns of two PPARγ transcripts mRNA expression under insulin treatment in vivo and in vitro were evaluated in this fish species. The present study would extend our understanding on the physiological function of PPARγ gene in fish.
Section snippets
Materials and methods
Here, two experiments were conducted. The first experiment was involved in the PPARγ cDNA cloning, mRNA expression patterns of various tissues and during different developmental stages. The second experiment was designed to evaluate the regulation of PPARγ by insulin in vivo and in vitro. We assured that the experiments performed on animals and cells followed the ethical guidelines of Huazhong Agricultural University.
PPARγ sequence and molecular characterization
In the present study, by RT-PCR and RACE methods, we successfully obtained full-length cDNA sequences of two PPARγ transcripts for yellow catfish, named as PPARγ1 (GenBank accession No. KF614118) and PPARγ2 (GenBank accession No. KF614119). The sequences were further confirmed by amplifying full-length cDNA sequences using gene-specific primers in 5′UTR. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene (as shown in Fig. 1B). PPARγ1 and PPARγ2 mRNA covered 2426 bp
Discussion
To date, studies have been involved in cloning PPARγ sequence in several fish species, but most of these studies reported only one version of PPARγ subtypes (Maglich et al., 2003, Raingeard et al., 2009). Our recent study cloned the partial cDNA sequence of one single PPARγ from yellow catfish (Zheng et al., 2013). However, Leaver et al. (2005) reported the existence of multiple-alternative splice-variants in PPARγ mRNAs of Atlantic salmon and European plaice. Furthermore, Sundvold et al. (2010)
Acknowledgments
This work was supported by the National Natural Science Foundation of China for excellent young scientists (Grant No. 31422056), Fundamental Research Funds for the Central Universities (Grant Nos. 2014JQ002, 2013PY073), and the Postgraduates Innovation Research Project of Huazhong Agricultural University (Grants No. 2009sc018).
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These authors equally contributed to the work.