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

https://doi.org/10.1016/j.ygcen.2014.12.020Get rights and content

Highlights

  • Two PPARγ isoforms were identified in fish, generated from alternative promoter of PPARγ gene.

  • The differential expression profiles of two PPARγ isoforms were determined in different tissues.

  • The differential expression profiles of two PPARγ isoforms were determined during different developmental stages.

  • Intraperitoneal injection of insulin in vivo stimulated the PPARγ1 expression, but not PPARγ2.

  • Incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1 and PPARγ2.

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426 bp and 2537 bp, respectively, with an open reading frame (ORF) of 1584 bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein’s domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55–76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish.

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).

References (54)

  • E. Grindflek et al.

    Characterization of porcine peroxisome proliferator-activated receptors γ1 and γ2: detection of breed and age differences in gene expression

    Biochem. Biophys. Res. Commun.

    (1998)
  • M. Hendlich et al.

    Ligsite: automatic and efficient detection of potential small molecule-binding sites in proteins

    J. Mol. Graph. Model.

    (1997)
  • M. Hojo et al.

    Expression patterns of the chicken peroxisome proliferator-activated receptors (PPARs) during the development of the digestive organs

    Gene Expr. Patterns

    (2006)
  • A. Ibabe et al.

    Expression of peroxisome proliferator-activated receptors in the liver of gray mullet (Mugil cephalus)

    Acta Histochem.

    (2004)
  • T. Ikonen et al.

    Interaction between the amino-and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators

    J. Biol. Chem.

    (1997)
  • H. Kondo et al.

    Ligand-dependent transcriptional activities of four torafugu pufferfish Takifugu rubripes peroxisome proliferator-activated receptors

    Gen. Comp. Endocrinol.

    (2007)
  • M.J. Leaver et al.

    A peroxisomal proliferator-activated receptor gene from the marine flatfish, the plaice (Pleuronectes platessa)

    Mar. Environ. Res.

    (1998)
  • S. Liebel et al.

    Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure

    Toxicol. In Vitro

    (2011)
  • R. Mukherjee et al.

    Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPARγ2 versus PPARγ1 and activation with retinoid X receptor agonists and antagonists

    J. Biol. Chem.

    (1997)
  • C.M. Press et al.

    The morphology of the immune system in teleost fishes

    Fish Shellfish Immunol.

    (1999)
  • D. Raingeard et al.

    Cloning and expression pattern of peroxisome proliferator-activated receptors, estrogen receptor α and retinoid X receptor α in the thicklip grey mullet (Chelon labrosus)

    Comp. Biochem. Physiol.

    (2009)
  • E.D. Rosen et al.

    PPARγ: a nuclear regulator of metabolism, differentiation, and cell growth

    J. Biol. Chem.

    (2001)
  • H. Sundvold et al.

    Characterization of bovine peroxisome proliferator-activated receptors γ1 and γ2: genetic mapping and differentiation expression of the two isoforms

    Biochem. Biophys. Res. Commun.

    (1997)
  • H. Sundvold et al.

    Identification of a novel allele of peroxisome proliferator-activated receptor gamma (PPARG) and its association with resistance to (Aeromonas salmonicida) in Atlantic salmon (Salmo salar)

    Fish Shellfish Immunol.

    (2010)
  • M. Todorcevic et al.

    Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes: effects of n-3 and n-9 fatty acids

    BBA Mol. Cell Res.

    (2008)
  • P. Tontonoz et al.

    Stimulation of adipogenesis in fibroblasts by PPAR [gamma] 2, a lipid-activated transcription factor

    Cell

    (1994)
  • M.L. Tsai et al.

    Cloning of peroxisome proliferators activated receptors in the cobia (Rachycentron canadum) and their expression at different life-cycle stages under cage aquaculture

    Gene

    (2008)
  • Cited by (26)

    • Agonistic and potentiating effects of perfluoroalkyl substances (PFAS) on the Atlantic cod (Gadus morhua) peroxisome proliferator-activated receptors (Ppars)

      2022, Environment International
      Citation Excerpt :

      As anticipated, the gmPpar proteins share the common structural features found in the nuclear receptor superfamily, including an N-terminal transactivation domain, a DNA-binding domain, a hinge region, and a C-terminal ligand binding domain (LDB) (Fig. 1). Other features are also conserved in the gmPpar aa sequences, including the AF-2 motif important for coregulator interaction in the C-terminal part of the LBD (Andersen et al., 2000, Batista-Pinto et al., 2005), the linker region that allows flexibility between the DBD and LBD upon ligand- and DNA binding (Andersen et al., 2000), as well as helices 1, 3, and 12 (H1, H3, and H12) that form and stabilize the ligand-binding pocket (Fig. 1) (Zheng et al., 2015). Human PPARs have been extensively studied for therapeutic purposes and the amino acid residues important for binding certain ligands have been identified.

    • Molecular characterization, expression and functional analysis of large yellow croaker (Larimichthys crocea) peroxisome proliferator-activated receptor gamma

      2022, Fish and Shellfish Immunology
      Citation Excerpt :

      Binding of natural or synthesized ligands, for example, docosahexaenoic acid (DHA) and thiazolidinediones, to PPARs results in the dissociation of corepressors and the recruitment of coactivators; this multiprotein complexes further recognize and bind to specific PPAR response elements in promoter regions to regulate gene transcriptions [4,5]. To date, PPARs have been identified in species ranging from basal vertebrates (e.g., teleost fish) to mammals with key structures remaining relatively conserved across evolution [6,7]. In mammals, three isotypes (i.e., PPARα/β/γ) that encoded by separate genes have been identified and PCR analysis shown PPARs are predominantly expressed in metabolically active tissues and thus, their roles in fatty acid metabolism, including biosynthesis, storage, mobilization, activation and oxidation of fatty acids and their derivatives, have been well established [8–10].

    • The morphological changes and molecular biomarker responses in the liver of fluoride-exposed Bufo gargarizans larvae

      2018, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      Thus, this finding suggests that in the setting of fluoride exposure, mRNA expression of ACC and FAE is decreased, thereby triggering impairment of the ability of fatty acid synthesis and elongation. PPARα, a member of the nuclear hormone receptor superfamily, is a key modulator of peroxisomal and mitochondrial fatty acid β-oxidation (Cho et al., 2012; Zheng et al., 2015). Several studies have reported that high PPARα mRNA expression resulted in high β-oxidation capacity in tissues such as heart and muscle (Batista-Pinto et al., 2005; Leaver et al., 2005; Tsai et al., 2008; Zheng et al., 2013).

    View all citing articles on Scopus
    1

    These authors equally contributed to the work.

    View full text