CIDEA and CIDEC are regulated by CREB and are not induced during fasting in grass carp Ctenopharyngodon idella adipocytes

Highlights

• CIDEA, CIDEB and CIDEC were identified in grass carp.

• The gene structure of CIDEA, CIDEB and CIDEC was conserved in teleosts.

• CIDEA and CIDEC rather than CIDEB were regulated by CREB in grass carp adipocytes.

• CIDEA and CIDEC were not induced during fasting in grass carp adipocytes.

Abstract

Cell death-inducing DNA fragmentation factor 45-like effector family proteins, including CIDEA, CIDEB and CIDEC, play an important role in energy metabolism. In the present study, CIDEA, CIDEB and CIDEC cDNAs were firstly isolated and characterized from grass carp Ctenopharyngodon idella, encoding peptides of 205, 208 and 238 amino acids, respectively. Analysis of the exon–intron structures clarified that grass carp CIDEA, CIDEB and CIDEC consisted of 5 coding exons, 5 coding exons and 6 coding exons, respectively, which is similar with human and mouse. Both CIDE family genes mRNAs were expressed in a wide range of tissues, but the abundance of each CIDE family gene mRNA showed the tissue-dependent expression patterns. Time-course analysis of CIDE family expressions indicated that their expression were enhanced significantly from day 0 to day 8 after differentiation. Forskolin caused an increase in CIDEA and CIDEC expression, and the effects were attenuated by treatment with CREB inhibitor, revealing that CIDEA and CIDEC are regulated by CREB. Further study found that CIDEA and CIDEC mRNA levels did not show significant changes during fasting. These results provide the groundwork to elucidate the gene structure and physiological function of CIDE family in fish.

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 resulting from excessive fat accumulation have been reported in many fish species (Tacon, 1996); however, the mechanisms involved in fat metabolism in fish has not been revealed clearly. Lipid homeostasis is crucial and excess fatty acids (FAs) can be harmful to cells. Cells convert excess FAs into neutral lipids for storage in organelles called lipid droplets (LDs) to prevent such lipotoxicity. The traditional view think that LDs serve simply as lipid storage organelles, but LDs are now generally believed to be highly dynamic organelles (Wang, 2015). Because of its central role in energy homeostasis, studies involved how LDs maintain lipid homeostasis in adipocytes have been given more and more research concerns in recent years.

LD surfaces are decorated by specific proteins, and many of these contribute to lipid droplet formation and function in lipid metabolism (Wilfling et al., 2014). Cell death-inducing DFF45-like effector (CIDE) family proteins including CIDEA, CIDEB and CIDEC are known as LD and endoplasmic reticulum-associated proteins. Systematic analyses in mouse have demonstrated that CIDE proteins play important roles in regulating various aspects of lipid homeostasis, such as lipid storage, lipolysis and lipid secretion (Puri et al., 2008; Li et al., 2007a, Li et al., 2007b; Nishino et al., 2008; Toh et al., 2008; Ye et al., 2009). CIDEA-deficient mice have increased lipolysis and decreased levels of serum triacylglycerols (TAGs) and free fatty acids (Zhou et al., 2003a, Zhou et al., 2003b). In human adipocytes, CIDEA knockdown also markedly elevates lipolysis (Puri et al., 2008). Studies on CIDEB-deficient mice have suggested that CIDEB may play an alternative role in controlling very low density lipoprotein triacylglycerols (VLDL-TAG) secretion in the liver (Ye et al., 2009). CIDEC is expressed at high levels in white adipose tissue (Nishino et al., 2008). CIDEC-deficient mice have drastically decreased fat storage, increased lipolysis and smaller fat pads throughout the body (Nishino et al., 2008). CIDEB promotes lipid storage under normal diet conditions, whereas CIDEA and CIDEC are responsible for liver steatosis under fasting (Xu et al., 2016). Overall, research has demonstrated that CIDE proteins are vital regulators of lipid metabolism in adipose tissue or liver. However, information is very scarce about the function of CIDE family 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 in lipid metabolism. In this study, all three CIDE family genes were cloned and their tissue-specific expressions and mRNA levels in preadipocytes during differentiation were determined in grass carp. Furthermore, the effects of forskolin and fasting on CIDE family genes mRNA expression were evaluated in vitro in this fish species. The present study will extend our understanding on the physiological function of CIDE family genes in adipocyte of fish.