Peroxisome proliferator-activated receptors

Peroxisomes are interior cell organelles involved in fatty acid metabolism and use toxic peroxide as an oxidising agent to protect the cell. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor super-family of transcription factors that consist of receptors for vitamin D and retinoids, thyroid, and steroid hormones (Villalba et al., Reference VILLALBA, JIMENEZ-LARA and ARANDA1996). PPARs are involved in cellular differentiation, insulin sensitisation, cancer, atherosclerosis and several metabolic diseases (Chawla et al., Reference CHAWLA, REPA, EVANS and MANGELSDORF2001).

The PPARs are messengers that make possible the translation of pharmacological, nutritional and metabolic stimuli into changes in the expression of genes involved mainly in lipid metabolism pathways (Schoonjans et al., Reference SCHOONJANS, STAELS and AUWERX1996b).

Three distinct PPAR genes have been recognised as α, δ (also known as PPARβ), and γ. PPARα is expressed mainly in tissues with an active peroxisomal proliferation such as liver, kidney, and heart (Auboeuf et al., Reference AUBOEUF, RIEUSSET and FAJAS1997; Mukherjee et al., Reference MUKHERJEE, JOW, CROSTON and PATERNITI1997). First observations showed that PPARα gene was poorly expressed in brown adipose tissue and muscle (Meng et al., Reference MENG, LI, ZHAO and GU2005). PPARα expression in pre-adipocytes is low but expression is enhanced during adipocyte differentiation (Chawala et al., Reference CHAWALA, SSHWARZ, DIMACULANGAN and LAZAR1994). All the three distinct PPAR genes have been characterised in chickens (Hojo et al., Reference HOJO, TAKADA, KIMURA, FUKUDA and YASUGI2006), however there are only reports on the expression of α and γ PPARs in turkeys (Ding et al., Reference DING, LI, NESTOR, VELLEMAN and MERSMANN2003), ducks (Wu et al., Reference WU, LIU, XIAO and ZHANG2010) and geese (Han et al., Reference HAN, WEI, HE, LIU, WAN, LIU, LI, XU, DU and XU2015).

In chickens PPARδ is mostly expressed in digestive organs whereas PPARα and PPARγ have a limited expression in these organs (Hojo et al., Reference HOJO, TAKADA, KIMURA, FUKUDA and YASUGI2006). Several studies in human subjects have suggested that PPARδ, which is the main isotype expressed in skeletal muscle (Chevillotte et al., Reference CHEVILLOTTE, RIEUSSET, ROQUES, DESAGE and VIDAL2001), controls the lipid metabolism in this tissue. Activation of PPARδ in brown adipose cells increases fatty acid burning and exerts a protective action against dietary- or genetically-induced obesity (Luquet et al., Reference LUQUET, LOPEZ-SORIANO, HOLST, GAUDEL, JEHL-PIETRI, FREDENRICH and GRIMALDI2004).

cPPARγ gene characteristics

The most important metabolic effect of PPARγ is its role in adipogenesis (Luquet et al., Reference LUQUET, LOPEZ-SORIANO, HOLST, GAUDEL, JEHL-PIETRI, FREDENRICH and GRIMALDI2004). PPARγ is a central gene regulator in adipose tissue and stimulate the expression of several genes involved in adipogenesis such as insulin-dependent glucose transporter 4 (GLUT4) (Wu et al., Reference WU, XIE, MORRISON, BUCHER and FARMER1998), adipocyte fatty acid binding protein (aP2) (Tontonoz et al., Reference TONTONOZ, HU, GRAVES, BUDAVARI and SPIEGALMAN1994a), lipoprotein lipase (LPL) (Schoonjans et al., Reference SCHOONJANS, PEINADO-ONSURBE and LEFEBVRE1996a) and the fatty acid translocase (CD-36/FAT) (Sato et al., 2002) and in this manner activates adipocyte differentiation (Tontonoz et al., Reference TONTONOZ, HU, GRAVES, BUDAVARI and SPIEGALMAN1994a; Reference TONTONOZ, HU and SPIEGALMAN1994b). PPARγ exists in two isotypes: PPARγ1 and -γ2 which are expressed from the same gene by different promoter usage and mRNA joining (Fajas et al., Reference FAJAS, AUBOEUF and RASPE1997). Human and mouse are include two isoforms γ1 and γ2 (Ding et al., Reference DING, GAO, WANG and LI2011).

The chicken PPARγ (cPPARγ) is encoded by the Gallus gallus PPARγ gene (Ensembl ID: ENSGALG00000004974) and include one isoform which is close to mammalian PPARγ2 (Ding et al., Reference DING, GAO, WANG and LI2011). The chicken PPARγ structural organisation is not conserved and, as compared to humans and mouse, the cPPARγ gene is shorter and spans over 50 kb of the genomic sequence on chromosome 12 and includes six exons (Takada and Kobayashi, Reference TAKADA and KOBAYASHI2013). Human and mouse PPARγ gene is located on chromosome 3 and 6, consists of eight and seven exons and extends across more than 160 and 120kb of genomic DNA respectively (Takada and Kobayashi., 2013). cPPARγ gene has same-type organisation similar to squirrel, hedgehog, Chinese softshell turtle, and Anole lizard PPARγ genes (Takada and Kobayashi, Reference TAKADA and KOBAYASHI2013).

Sato et al. (Reference SATO, FUKAO, SEKI and AKIBA2004) and Takada and Kobayashi (Reference TAKADA and KOBAYASHI2013) could not find cPPARγ2 in the chicken adipose tissues and suggested that chickens may lack a PPARγ2 protein isoform. However, Duan et al. (Reference DUAN, SUN, ZHANG, ZHANG, ZHANG, ZHANG, WANG, WANG, LENG, LI and WANG2015) reported the expression of cPPARγ1 and cPPARγ2 isoforms, which vary only in their N-terminal regions. The cPPARγ1 matches the formerly reported cPPARγ protein (NP 001001460.1). The cPPARγ2 has six more N-terminal amino acids, compared with the cPPARγ1. The protein homology survey confirmed that the cPPARγ1 protein is similar to the mouse and human PPARγ1 proteins. But, the cPPARγ2 protein isoform was different from mouse or human PPARγ2, and showed a high variation in its N-terminus.

Previous researches have shown that the human PPARγ2 isoform is a more potent ligand independent activator than the human PPARγ1 (Werman et al., Reference WERMAN, HOLLENBERG, SOLANES, BJORBAEK, VIDAL-PUIG and FLIER1997) and has the main role in the adipogenesis (Zhang et al., Reference ZHANG, FU, CUI, XIONG, XU, ZHONG, XIAO, FLOYD, LIANG, LI, SONG and CHEN2004). Whether chicken PPARγ1 and PPARγ2 have different acts in adipogenesis and adipose development need more research (Duan et al., Reference DUAN, SUN, ZHANG, ZHANG, ZHANG, ZHANG, WANG, WANG, LENG, LI and WANG2015).

cPPARγ and fat metabolism

Lipogenesis in birds mostly takes place in the liver (which has 20 times more lipogenesis capacity compared to adipose tissue), and adipose only acts as the main fat storage tissue (Sato et al., Reference SATO, AKIBA, CHIDA and TAKAHASHI1999). cPPARγ expression in the liver was correlated to relative abdominal fat content despite the comparable expression in adipose tissue of fat and lean chicks. (Larkina et al., Reference LARKINA, SAZANOVA, FOMICHEV, BARKOVA, MALEWSKI, JASZCZAK and SAZANOV2011), a situation similar to PPARγ hyper-expression in humans obesity (Hindle et al., Reference HINDLE, KOURY, McCAFFREY, FU and BRODY2009).

Hyperplasia and hypertrophic growth of adipocytes are essential factors for the development of chicken adipose tissue in the abdominal fat pad mass (Hood, Reference HOOD1982). cPPARγ plays an important role in these growth and development of adipose tissue (Wang et al., Reference WANG, MU, LI, DING, WANG, WANG, WANG and WANG2008). cPPARγ activation in the hyperplasia stage of adipose tissue development manages fatness in broiler chickens. Increase of LPL mRNA expression in the hypertrophic stage of chicken adipose tissue is associated with fat accumulation (Sato et al., Reference SATO, ABE, KONO, YAMAZAKI, NAKASHIMA, KAMADA and AKIBA2009). In the adipogenesis process, PPARγ has an important role in releasing fatty acids from triglycerides, make possible intracellular fatty acid transport, and fatty acid esterification by stimulation specific genes such as fatty acid binding proteins and lipoprotein lipase (Rosen and Spiegelman, Reference ROSEN and SPIEGELMAN2001).

Effects of ligand activation of PPARγ on lipid metabolism in the chicken

The majority of evidence on the effects of ligand activation of PPARγ on lipid metabolism has been reported in mammals, and comparable reports in poultry are limited. Oral administration of troglitazone, a synthetic PPARγ ligand, to five week-old broilers increased PPARγ mRNA expression in the adipose tissue and increased abdominal fat pad weight (Sato et al., Reference SATO, FUKAO, SEKI and AKIBA2004). Sato et al. (Reference SATO, FUKAO, SEKI and AKIBA2004) demonstrated that PPARγ mRNA expression in abdominal fat was higher in broilers fed 8% safflower oil (18:2-rich) or linseed oil (18:3-rich) for seven days compared to broilers fed olive oil (18:1-rich). They suggested that the PPARγ gene expression in broilers fed linoleic acid was higher than those fed oleic acid.

In another study, Sato et al. (Reference SATO, MATSUSHITA, MATSUBARA, KAMADA and AKIBA2008) reported that the PPARγ gene in the abdominal fat pad of seven day old broilers injected with a single intraperitoneal dose of troglitazone at one day old were up-regulated compared to those in control birds. They suggested that a single intra-peritoneal injection of troglitazone to one day old chicks decreased fat deposition in mature birds. These authors suggested that the effects of using PPARγ ligand on the hyperplasia step of chick adipose tissue deposition was initiated from a few days before hatching until around four weeks of age.

Lee et al. (Reference LEE, KIM, AHN, HWANG, KIM, LEE, AN and KANG2010) reported that injection with oestradiol up-regulated the PPARγ gene in the liver of hens and increased the synthesis of fatty acids and triacylglycerols, and the accumulation of hepatic lipids. In the study of Royan et al. (Reference ROYAN, MENG, OTHMAN, SAZILI and NAVIDSHAD2011), the down-regulation of the PPARγ gene in the abdominal fat of broilers fed dietary CLA, fish oil or soybean oil was accompanied by less abdominal fat deposition than those fed dietary palm oil.

Khalaji et al. (Reference KHALAJI, ZAGHARI, GANJKHANLOO and GHAZIANI2013) reported that high-energy diets containing maize oil (rich in linoleic acid), decreased the expression of the PPARγ gene in the hen's liver with an increase in abdominal fat pad and enhanced plasma triglyceride and cholesterol concentrations. Lu et al. (Reference LU, HARPER, ZHAO, CORL, LEROITH and DALLOUL2014) found that the expression of PPARγ was higher in the control group compared to birds fed a high-oxidant diet, supplemented with vitamin E at 10 IU/kg, 3% oxidised oil and 3% PUFA source. The levels of 20:4, 20:5, 22:5, 22:6, and all the n-3 fatty acids were higher in the abdominal fat of the treated broilers than the control group on day 21. Ramiah et al. (Reference RAMIAH, MENG, WEI, KEONG and EBRAHIMI2014) reported that dietary CLA could down-regulate PPARγ and reduce body fat deposition in the chicken.

PPARγ and the immune system

Despite various studies to determine the role of cPPARγ in poultry lipid metabolism, there have been few studies on the relationship between cPPARγ and the chicken immune system. It has been confirmed that both chronic and acute cold stresses could influence PPARγ gene expression in immune organs such as the spleen, thymus, and Bursa of Fabricius tissues (Wang et al., Reference WANG, BRAHMAKSHATRIYA, ZHU, LUPIANI, REDDY, YOON, GUNARATNE, KIM, CHEN, WANG and ZHOU2009). The cPPARγ mRNA expression pattern may determine the cellular functions related to the phenotypic expression of feed efficiency in broilers, so that PPARγ mRNA expression in the duodenum is higher compared to low efficiency animals (Ojano-Dirain et al., Reference OJANO-DIRAIN, TOYOMIZU, WING, COOPER and BOTTJE2007).

Regulation of cPPARγ by ligands (agonists and antagonists)

There are many synthetic and natural exogenous PPARγ agonists. However, rosiglitazone (trade name Avandia, GlaxoSmithKline), troglitazone (Rezulin, banned by the FDA because of causing liver failure), thiazolidinediones (TZDs), pioglitazone (Actos; Takeda Pharmaceutical Ltd) are the most famous PPARγ agonists. Troglitazone can stimulate cPPARγ regulation in broiler chicken adipose tissue and its administration enhanced adipocyte in vivo differentiation (Sato et al., Reference SATO, FUKAO, SEKI and AKIBA2004; Reference SATO, MATSUSHITA, MATSUBARA, KAMADA and AKIBA2008). Oral administration of troglitazone to broiler chickens significantly increased abdominal fat pad weights due to stimulation of PPARγ activity and increases in lipoprotein lipase (LPL) mRNA expression in abdominal adipose tissues (Sato et al., Reference SATO, FUKAO, SEKI and AKIBA2004). Troglitazone injection causes PPARγ activation in the hyperplasia stage of chicken adipose development and decreases fat deposition in chickens after growth (Sato et al., Reference SATO, MATSUSHITA, MATSUBARA, KAMADA and AKIBA2008). Differentiation from pre-adipocyte to mature adipocyte results in cell growth arrest (Ntambi and Young-Cheul, 2000).

Polyacetylenes from N. incisum (Atanasov et al., Reference ATANASOV, BLUNDER, FAKHRUDIN, LIU, NOHA, MALAINER, KRAMER, COCIC, KUNERT, SCHINKOVITZ, HEISS, SCHUSTER, DIRSCH and BAUER2013), honokiol from Magnolia bark (Atanasov et al., Reference ATANASOV, BLUNDER, FAKHRUDIN, LIU, NOHA, MALAINER, KRAMER, COCIC, KUNERT, SCHINKOVITZ, HEISS, SCHUSTER, DIRSCH and BAUER2013), terpenoids such as 20(S)-protopanaxatriol from ginseng (Han et al., Reference HAN, KWON, YANG, JUNG and CHOI2006) and 6-shogaol from ginger (Isa et al., Reference ISA, MIYAKAWA, YANAGISAWA, GOTO, KANG, KAWADA, MORIMITSU, KUBOTA and TSUDA2008), catechin from green tea (Shin et al., Reference SHIN, KIM, LEE, LEE, SONG, PARK, LEE, BAIK, KIM, HONG and NOH2009), activator from Citrus spp. (Kuroyanagi et al., Reference KUROYANAGI, KANG, GOTO, HIRAI, OHYAMA, KUSUDO, YU, YANO, SASAKI, TAKAHASHI and KAWADA2008), and alkaloids such as harmine (Waki et al., Reference WAKI, PARK, MITRO, PEI, DAMOISEAUX, WILPITZ, REUE, SAEZ and TONTONOZ2007) are some of the PPARγ natural exogeneous agonists.

Physiological endogenous agonists include polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) (Allred et al., Reference ALLRED, TALBERT, SOUTHARD, WANG and KILGORE2008), linoleic acid and oxidised fatty acid metabolites (Capobianco et al., Reference CAPOBIANCO, WHITE, HIGA, MARTINEZ and JAWERBAUM2008), cyclopentenone prostaglandin 15 deoxy-Δ12,14 (15d-PGJ2) (Forman et al., Reference FORMAN, TONTONOZ, CHEN, BRUN, SPIEGELMAN and EVANS1995), 8(S)-hydroxyeicosatetraenoic acid (Yu et al., Reference YU, BAYONA, KALLEN, YU, HARDING, RAVERA, McMAHON, BROWN and LAZAR1995), and the ipoxygenase product, 9-hydroxyoctadecadienoic acid (Nagy et al., Reference NAGY, TONTONOZ, ALVAREZ, CHEN and EVANS1998). Conjugated linoleic acid (CLA) is another PPARγ ligand (Belury et al., Reference BELURY, MOYA-CAMAREN, LU, SHI, LEESNITZER and BLANCHARD2002). All of the isomers of CLA activated PPARγ in a manner similar to the linoleic acid (18:2n6) (Belury et al., Reference BELURY, MOYA-CAMAREN, LU, SHI, LEESNITZER and BLANCHARD2002).

PUFAs and CLA isomers have lipogenesis inhibition abilities, unlike the saturated (SFAs) and monounsaturated fatty acids (MUFAs). SFAs have a well-known ability for increasing abdominal fat pad deposition (Sanz et al., Reference SANZ, LOPEZ-BOTE, MENOYO and BAUTISTA2000). PPARγ gene upregulation has been shown in adipose tissue of chickens fed palm oil compared to the birds fed PUFAs or CLA (Royan et al., Reference ROYAN, MENG, OTHMAN, SAZILI and NAVIDSHAD2011). Ramiah et al. (Reference RAMIAH, MENG, WEI, KEONG and EBRAHIMI2014) found that dietary CLA can decrease body fat accumulation in chickens by reducing pre-adipocyte number and abdominal fat cell numbers through cPPARγ and aP2 down-regulation (Ramiah et al., Reference RAMIAH, MENG, WEI, KEONG and EBRAHIMI2014). Very few endogenous PPARγ antagonists have been identified. Prostaglandin F2α (PGF2α) and cyclic phosphatidic acid (cPA) are two natural antagonists and bisphenol A diglycidyl ether (BADGE), dimethyl α-(dimethoxyphosphinyl)-p-chlorobenzyl phosphate, PD068235 (Camp et al., Reference CAMP, CHAUDHRY and LEFF2001), chloro-5-nitrobenzanilide (GW9662) (Leesnitzer et al., Reference LEESNITZER, PARKS, BLEDSOE, COBB, COLLINS, CONSLER, DAVIS, HULL-RYDE, LENHARD, PATEL, PLUNKET, SHENK, STIMMEL, THERAPONTOS, WILLSON and BLANCHARD2002), 2-chloro-5-nitro-N-4-pyridinylbenza (T0070907) are synthetic antagonist of PPARγ.

PPARγ-dimer partner retinoid X receptor (RXR)

The transcription through nuclear receptor homodimers or heterodimers in contact with ligands requires attaching of the nuclear receptors to response elements in the target genes (Glass, Reference GLASS1994). Retinoid X receptor (RXR) is the necessary element for the majority of the heterodimers reported. Actually, RXR is necessary for high-affinity DNA binding, suggesting that one role of RXR is only to act as an auxiliary DNA binding factor (DiRenzo et al., Reference DIRENZO, SODERSTROM, KUROKAWA, OGLIASTRO, RICOTE, INGREY, HORLEIN, ROSENFELD and GLASS1997). There are some heterodimeric partners including (PPARs) for RXR which carry out the task of heterodimeric nuclear receptors (Bardot et al., Reference BARDOT, ALDRIDGE, LATRUFFE and GREEN1993).

While there are some reports that the PPARs can make homodimers and attach to DNA response elements (Okuno et al., Reference OKUNO, ARIMOTO, IKENOBU, NISHIHARA and IMAGAWA2001), it is commonly accepted that the PPARs must heterodimerise with RXR to do most of their roles (Chan et al., 2009). Selvaraj and Klasing (Reference SELVARAJ and KLASING2006) in a study on chickens found that cPPARγ is very sensitive to RXR. In their study, increases in cPPARγ and RXR because of high dietary fat, EPA or lutein were decreased inducible nitric oxide synthase (iNOS) mRNA expression. iNOS are enzymes which produce nitric oxide from L-arginine. Nitric oxide is an important cellular signalling molecule and plays significant roles in host defence (Knowles and Moncada, Reference KNOWLES and MONCADA1994). Upregulation of the PPAR/RXR dimer makes it possible for transcriptional regulation of PPAR/RXR-responsive genes (Kota et al., Reference KOTA, HUANG and ROUFOGALIS2005). Moraes et al. (Reference MORAES, RIBEIRO, SANTIN and KLASING2016) reported that lipopolysaccharide downregulated cPPARγ and RXR in the chicken's spleen.