Forkhead box O1 in grass carp Ctenopharyngodon idella: Molecular characterization, gene structure, tissue distribution and mRNA expression in insulin-inhibited adipocyte lipolysis

doi:10.1016/j.cbpa.2016.11.011

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology

Volume 204, February 2017, Pages 76-84

https://doi.org/10.1016/j.cbpa.2016.11.011 Get rights and content

Abstract

Factor forkhead box O1 (FoxO1) is a transcription factor and plays an important role in insulin-mediated lipid metabolism. In the present study, two distinct FoxO1 cDNAs, designated

FoxO1a and FoxO1b, were firstly isolated and characterized from grass carp Ctenopharyngodon idella, encoding peptides of 654 and 631 amino acids, respectively. Phylogenetic and synteny analyses suggested that FoxO1a and FoxO1b were derived from paralogous genes that could be originated from teleost-specific genome duplication (TSGD) event. Analysis of the exon–intron structures clarified that grass carp FoxO1a and FoxO1b comprise 3 coding exons and contain a extra intron compared with human and mouse FoxO1. Both FoxO1a and FoxO1b mRNAs were expressed in a wide range of tissues, but the abundance of each FoxO1 mRNA showed the tissue- dependent expression patterns. Time-course analysis of FoxO1 expressions indicated that the level of FoxO1a mRNA reached almost maximal level at day 2, while that of FoxO1b mRNA reached almost maximal level at day 4 during grass carp primary preadipocyte differentiation. In insulin-inhibited adipocyte lipolysis, only FoxO1a showed a significant decrease in adipocyte, indicating that two FoxO1 isoforms may serve somewhat different roles in the regulation of lipolysis by insulin. These results suggested that grass carp FoxO1a and FoxO1b may play different roles in tissues, and their expression levels were differently modulated by insulin in adipocyte.

Introduction

Lipids are the predominant source of energy for fish and fatty acids are very important substrates used to maintain metabolic homoeostasis (Weil et al., 2013). In aquaculture, metabolic disorders resulted from excessive fat accumulation has been reported in many fish species (Tacon, 1996); however, the mechanisms involved in fat metabolism in fish have not been revealed clearly. Adipose tissue is characterized as a form of connective tissue that plays a major role in nutrient homeostasis and functions as an endocrine organ at the center of energy homeostasis (Rosen and Spiegelman, 2014). The excessive fat deposited in the adipose tissue is closely related to metabolic disease (Eckel et al., 2005). Because of its central role in energy homeostasis, interest in “solving” the mechanisms involved in adipocyte lipid metabolism has been given more and more research concerns in fish in recent years.

Many transcription factors are involved in the lipid metabolism in adipose tissue. FoxO1, a member of the evolutionarily conserved FoxO subfamily of forkhead transcription factors, functions in adipose cells to couple insulin signalling to adipogenesis, which involves switching preadipocytes from proliferation to terminal differentiation (Birkenkamp and Coffer, 2003, Nakae et al., 2003). FoxO1 is expressed in tissues involved in energy metabolism such as liver, muscle, and adipose tissue (Farmer, 2003). FoxO1 proteins promote triacylglycerol (TAG) catabolism in adipose tissue by stimulating the expression of adipose TAG lipase (ATGL) (Chakrabarti and Kandror, 2009), which mediates the first step in lipolysis, and thereafter other lipases, including hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MGL), promote the removal of additional fatty acids from the glycerol backbone of TAG (Coleman and Mashek, 2011, Zechner et al., 2009). FoxO1 protein interacts with response elements in the ATGL gene promoter and stimulates ATGL gene expression in adipose tissue (Chakrabarti and Kandror, 2009). However, information is very scarce about the function of FoxO1 gene in fish.

The development of adiposity is regulated by many factors. Insulin is an important anabolic hormone that can promote cell functions and signals in mammals, including glycogen synthesis, gene transcription and protein synthesis (Cheatham and Kahn, 1995). Insulin stimulates adipocyte lipogenesis and inhibits adipocyte lipolysis (Choi et al., 2010). In fish, insulin has almost no effect on lipolysis in sea bream (Sparus aurata) adipocytes (Albalat et al., 2005b), while insulin decreases the lipolysis in large yellow croaker (Pseudosciaena crocea) adipocytes (Wang et al., 2012). Information regarding the insulin-mediated control of lipolysis in fish is still limited. Recent study indicated that FoxO1 controls insulin-dependent ATGL expression and lipolysis in 3T3-L1 adipocytes (Chakrabarti and Kandror, 2009). Despite of insulin's important role in lipolysis, the ability of insulin to regulate FoxO1 remains poorly characterized in fish.

Grass carp (Ctenopharyngodon idella), a herbivorous freshwater fish, is an important farmed fish in China for its delicious meat and high market value (Wang et al., 2015). It is considered as a good model for the study of lipid metabolism because grass carp store excess fat in liver and visceral adipose tissue. Additionally, the draft genome of grass carp (Wang et al., 2015) released recently is a useful tool for identifying genomic structure of genes involved lipid metabolism. In this study, two different FoxO1 genes were cloned and their tissue-specific expressions and mRNA levels in preadipocytes during differentiation were determined in grass carp. Furthermore, the effects of insulin on adipocyte lipolysis and FoxO1 mRNA expression were evaluated in vitro in this fish species. The present study will extend our understanding on the physiological function of FoxO1 gene in adipocyte of fish.

Section snippets

Fish culture and sampling

Experimental grass carp were obtained from the local fish farm. Animals were acclimated to laboratory conditions for at least 4 weeks prior to experiments. Fish were assigned randomly to one of six treatment groups in 150-L circular tanks (approximately16–20 fish per tank) with a flow-through water supply at 28 °C under a 12L:12D photoperiod. They were fed a commercial pellet diet (crude protein: 35%; crude lipid: 7%) three times a day and provided with continuous aeration to maintain the

Identification of FoxO1 in grass carp

We obtained two FoxO1 genes from grass carp by in silico screening and named FoxO1a (Genbank accession No. KP325483.1) and FoxO1b (Genbank accession No. KP325484.1). Grass carp FoxO1a and FoxO1b encoded a predicted protein of 654 and 631 amino acids, respectively. FoxO1a and FoxO1b contained two major structural domains, a fork head domain at the N-terminal domain and a transactivation domain at the C-terminal domain. The fork head domain is highly conserved between species (Fig. 1). Based on

Discussion

FoXO1, a member of the Forkhead family proteins of the O subclass, is particularly known to be an important downstream target of the insulin signalling pathway and has a role in longevity, cell cycle control and apoptosis, fertility, stress response and multiple metabolic pathways (Gross et al., 2009, Lettieri Barbato et al., 2014). In adipocytes, the transcription factor FoxO1 plays a leading role in integrating dietary conditions and insulin signalling to maintain metabolic balance (Lettieri

Acknowledgments

This work was financially supported by the National Basic Research Program of China (973 program 2014CB138603).

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