Chicken FTO gene: Tissue-specific expression, brain distribution, breed difference and effect of fasting

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Abstract

Fat mass and obesity-associated (FTO) gene is widely expressed in central and peripheral tissues of mammals, and exhibits a range of functions, especially in energy balance. However, basic knowledge of FTO in the chicken is lacking. Therefore, we studied the tissue distribution, age and breed dependent changes, brain localization, as well as the impact of fasting on FTO mRNA expression in the chicken. FTO mRNA was expressed in all the tissues studied, and generally, with high expression in hypothalamus, liver, visceral fat and cerebellum. However it exhibited breed-specific patterns: in broilers, the highest expression was seen in the liver, while in layers, hypothalamus and cerebellum showed relatively higher FTO mRNA expression. One-week-old broilers expressed markedly higher FTO mRNA in liver compared with the layers of the same age (P < 0.01), while the breed difference was reversed in visceral fat and cerebellum (P < 0.05). Compared with newly hatched chicks (one week of age), adult layers expressed higher FTO mRNA in liver and visceral fat, while adult broilers showed higher expression in hypothalamus and cerebellum. In situ hybridization demonstrated distribution of FTO mRNA in paraventricularis magnocellularis (PVN), nucleus ventromedialis hypothalami (VMN), nucleus lateralis hypothalami (LHy), nucleus dorsomedialis hypothalami (DMN) of the hypothalamus and nucleus habenularis medialis (HM) and stratum cellulare externum (SCE) of the thalamus. Breed-specific expression of FTO mRNA was shown in PVN, but not in VMN, with higher abundance in broilers compared to layers. The decrease in FTO mRNA levels after 24 h of fasting was seen only in VMN of layer chickens. These results may provide some intriguing hints for further investigation of FTO function in the chicken.

Introduction

Fat mass and obesity-associated (FTO) gene is the first gene contributing to common forms of human obesity (Loos and Bouchard, 2008). It was originally identified in the fused toes mutant mouse (van der Hoeven et al., 1994). However, FTO did not receive much attention until 2007 when a cluster of single nucleotide polymorphisms (SNPs) located in the first intron of FTO was found to be closely correlated with obesity-related traits in human (Frayling et al., 2007, Scuteri et al., 2007). Since then, this correlation has been confirmed by several independent large-scale genome association studies in various ethnical populations including European (Al-Attar et al., 2008, Gonzalez-Sanchez et al., 2009, Zimmermann et al., 2009), Asian (Cha et al., 2008, Chang et al., 2008, Hotta et al., 2008), American (Scuteri et al., 2007, Do et al., 2008), and Australian (Cornes et al., 2009) cohorts, although there are a few exceptions (Li et al., 2008, Hennig et al., 2009).

To date, considerable progress has been made in understanding the function of FTO, especially its impact on energy balance. Recent findings indicate that the SNPs of the FTO gene are associated with resting metabolic rate (Do et al., 2008, Hubacek et al., 2010). In addition, FTO-deficient mice are lean and protected from obesity, which can be attributed to their elevated metabolic rate and enhanced energy expenditure (Fischer et al., 2009). Moreover, FTO is proposed to participate in the regulation of food intake. Histological studies revealed that the localization of FTO mRNA and protein in the hypothalamic nucleus is of critical importance for feed intake regulation in mice (Gerken et al., 2007, Fredriksson et al., 2008, Olszewski et al., 2009, Olszewski et al., 2011b). Furthermore, hypothalamic FTO expression is associated with neuropeptides related to appetite control, showing a negative correlation with galanin and galanin-like peptide, yet a positive correlation with neuropeptide Y (NPY) and oxytocin (Fredriksson et al., 2008, Olszewski et al., 2011a). Immunohistochemistry co-localization studies clearly confirmed abundant FTO overlapping with anorexigenic neuropeptide pro-opiomelanocortin (POMC) and oxytocin in mice hypothalamus (Fischer et al., 2009, Olszewski et al., 2011a).

The increased susceptibility to obesity in carriers of the risk allele of FTO SNPs seems to be a result of increased food intake (Wardle et al., 2008, Wardle et al., 2009, Church et al., 2010), or impaired energy expenditure (Berentzen et al., 2008, Haupt et al., 2008). Recently, it is reported that selective manipulation of FTO levels in the hypothalamus affects food intake in rats (Tung et al., 2010). FTO mutant mice demonstrate immediate postnatal growth retardation which is associated with reduced serum levels of IGF-1. Moreover, brain-specific deletion of FTO results in similar phenotypes as the whole body deletion, indicating the role of brain FTO in postnatal growth regulation (Gao et al., 2010).

Furthermore, FTO expression is influenced by nutritional status. Hypothalamic FTO mRNA was significantly up-regulated after food deprivation in mice (Fredriksson et al., 2008, Olszewski et al., 2009). In weaning sheep, hypothalamic FTO gene expression was increased by prenatal nutrient restriction (Sebert et al., 2010). However, some studies revealed opposite findings that hypothalamic FTO mRNA and protein expression were decreased by fasting or energy restriction in mice (Stratigopoulos et al., 2008, Wang et al., 2011). A very recent study suggested that FTO may play a role in sensing the availability of essential amino acids (Cheung et al., 2012).

Layer and broiler chickens have been intensively selected over generations for egg and meat production respectively, and differ greatly in feed intake, growth efficiency, body composition and behavior. For example, the feed consumption was two times higher in broilers than in layers, and the proportion of time spent feeding was also higher in broilers compared with layers (Hocking et al., 1997). Therefore, these two chicken breeds are excellent models to investigate the function of FTO in birds. In the present study, we determined FTO gene tissue-specific expression in broiler and layer chickens of different ages. We investigated FTO mRNA distribution pattern in hypothalamic and thalamic of layer chicken by in situ hybridization, and compared PVN and VMN FTO mRNA expression by Real-time PCR under different feed intake status and breeds.

Section snippets

Animals and experimental design

One-day-old male Ross broilers and male Leghorn layers (Gallus gallus) were purchased from Lohmann (Lohmann, Animal Production; 27472 Cuxhaven, Germany) and reared at standard conditions of light and temperature. Feed and water were provided ad libitum up to day 6. On day 6, half of the chicks from each breed were deprived of feed whereas the other chicks continued to be fed ad libitum until day 7. Both fed and fasted chicks had free access to water. The fed chicks were also used for

Tissue-specific expression in young and adult layer and broiler chickens

According to the Real-time PCR results, FTO gene was expressed in all tested tissues, and generally, with high expression in hypothalamus, liver, visceral fat and cerebellum (Fig. 1). However, the patterns of tissue-specific expression were breed-dependent: in broilers, the highest expression was seen in the liver, while in layers, hypothalamus and cerebellum showed relatively higher FTO mRNA expression. As a result, one-week-old broilers expressed markedly higher FTO mRNA levels in liver than

Discussion

Examination of tissue-specific gene expression of FTO together with its developmental and breed differences is the first step to assess its physiological functions in chickens. We found that the FTO gene was expressed in peripheral and central tissues of two breeds of chickens at two different ages. Taken together, our results showed that FTO is highly expressed in chicken hypothalamus, liver, visceral fat and cerebellum, which is consistent with mammalian studies that have demonstrated high

Acknowledgment

The first author is extremely grateful to Ute Beermann for the excellent technical assistance and Els Willems for her review and suggestions. This work was supported by the NSFC-Guangdong Joint Fund (project no. U0931004), the Sino-German Cooperation in Agriculture, project no. 28/04-05CHN7 (2010–2011), the Special Fund for Agro-scientific Research in the Public Interest (201003011) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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