cAMP-dependent protein kinase A in grass carp Ctenopharyngodon idella: Molecular characterization, gene structure, tissue distribution and mRNA expression in endoplasmic reticulum stress-induced adipocyte lipolysis

Highlights

• Nine isoforms of the PKA family were cloned and characterized in grass carp.

• The gene structure and functional domains of the nine PKA isoforms were conserved.

• The abundance of each PKA (mRNA) showed tissue-dependent expression patterns.

• Grass carp PRKACAb and PRKACBa may be involved in ER stress-mediated lipolysis.

Abstract

Protein kinase A (PKA), one of the most widely studied protein kinases, has many functions in cells, including regulating the metabolism of sugar and lipid. Here we identified nine isoforms of the PKA family in grass carp Ctenopharyngodon idella and obtained their complete coding sequences (CDS), including PRKACAa, PRKACAb, PRKACBa, PRKACBb, PRKAR1A, PRKAR1B, PRKAR2Aa, PRKAR2Ab and PRKAR2B, and PRKACA, PRKACB and PRKAR2A, which may experience fish-specific genome duplication. Sequence analysis showed that the predicted protein structures of PKA gene family members in grass carp were different. Grass carp PRKACAa, PRKACAb, PRKACBa, and PRKACBb contained serine/threonine protein kinases, while PRKAR1A, PRKAR1B, PRKAR2Aa, PRKAR2Ab and PRKAR2B contained two cyclic nucleotide-monophosphate binding domains. PRKACAa, PRKACBa, PRKACBb, PRKAR1A, PRKAR1B and PRKAR2Aa contained 10 coding exons, while PRKACAb and PRKAR2Ab consisted of 12 coding exons and 5 coding exons, respectively. The messenger RNA (mRNA) of the nine PKA isoforms was detected in a wide range of tissues, but their abundance showed tissue-dependent expression patterns. In tunicamycin-induced adipocyte lipolysis, only the mRNA levels of PRKACAb and PRKACBa showed a significant increase in adipocyte (p < .05), indicating that nine PKA isoforms may serve somewhat different roles in endoplasmic reticulum (ER) stress-mediated lipolysis in fish. These results suggested that nine grass carp PKA isoforms may play different roles in tissues, and their expression levels were differently modulated by ER stress in adipocyte.

Introduction

In fish species, lipids and proteins are the main organic constituents that possess a wide diversity of functions in structure and biological process through interactions. (Tocher, 2003; Sul and Smith, 2008). However, excessive accumulation of lipids in adipose tissue of cultured fish can lead to fatty liver, reduced stress resistance, decreased quality of fish, and even cause a large number of fish deaths and reduce the benefits of aquaculture. Adipose tissue is a special connective tissue. As a primary energy bank and endocrine organ, it can synthesize some bioactive compounds that regulate the homeostasis of metabolism (Rosen and Spiegelman, 2014). Therefore, adipose tissue is considered to be the key regulatory site for improving the healthy growth and meat quality of fish. Adipocytes are the main cells of adipose tissue, which store excessive lipids in the form of triglyceride (TG). As metabolic demand (exercise or cold stress) increases, adipocytes mobilize these stores and release fatty acids as energy substrates for other tissues (Sun et al., 2016). In this process, the hydrolysis of triglycerides in adipocytes is regulated by a series of lipases, mainly including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol hydrolase (MGL) (Cerk et al., 2017).

Endoplasmic reticulum (ER), the central organelle for protein synthesis, folding and transportation, contains many regulatory proteins and enzymes involved in lipid metabolism. In adipocytes, the formation of lipid droplets and the maintenance of lipid homeostasis are closely related to endoplasmic reticulum (Zha and Zhou, 2012; Torre-Villalvazo et al., 2018). The homeostasis of ER is very important for cell function and proper protein production. However, the accumulation of unfolded or misfolded proteins in the ER leads to the imbalance of the homeostasis of ER, which induce the ER stress (Schröder and Kaufman, 2005; Flamment et al., 2012; Han and Kaufman, 2016). To neutralize the protein burden and restore homoeostasis, the ER triggers a group of intracellular signaling pathways called unfolded protein response (UPR) by activating three transmembrane receptors in the ER membrane: PRKR-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) (Schuck et al., 2009; Wang and Kaufman, 2016; Han and Kaufman, 2016). Ozcan et al. (2004) found that ER stress is closely related to obesity, and certain specific medicine can inhibit the ER stress caused by obesity, which can alleviate the UPR response, thus having a relieving effect on type 2 diabetes in mice (Ozcan 2006). However, little is known about the relationship between ER sress and lipolysis in fish adipocytes.

The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity (Londos et al., 1999; Honnor et al., 1985). Studies have shown that the PKA activator forskolin can improve the survival of INS-1 cells and rat pancreatic β-cells under thapsigargin-induced ER stress, which implicates cAMP/PKA signaling is involved in the regulation of unfolded protein response to ER stress (Yusta et al., 2006; Cunha et al., 2009). And in the downstream lipolysis cascade, the key to activating lipolysis by PKA is phosphorylated both lipid droplet-associated protein perilipin A (Brasaemle, 2007; Sztalryd et al., 2003; He et al., 2006; Greenberg et al., 1991; Zhang et al., 2003) and HSL, as well as promoting translocation of phosphorylated HSL from cytoplasm to lipid droplet surface (Anthonsen et al., 1998; Brasaemle et al., 2000; Sztalryd et al., 2003). In addition, ATGL, an important lipase (Zimmermann et al., 2004), can also be indirectly activated in the process of lipolysis regulated by PKA (Schweiger et al., 2006; Rydén et al., 2007). Hence, PKA may mediate the lipolysis induced by ER-stress.

Grass carp (C. idella) is a herbivorous freshwater fish which has become an important fish culture in China because of its high market value (Wang et al., 2015). And because grass carp storages excessive fat in the liver and adipose tissue, it can be used as a model to further study the mechanism of lipid metabolism in fish, especially the adipocyte lipolysis and its molecular regulation mechanism. Additionally, the draft genome of grass carp released previously is a useful tool for identifying genomic structure of genes involved lipid metabolism. Numerous pharmacological agents interfere with the normal functioning of the ER, resulting in ER dysfunction and ER stress. Tunicamycin, an antibiotic isolated from Streptomyces sp. that inhibits N-linked glycosylation of proteins and lipids within the ER (Takatsuki and TAMURA, 1971; Yamamoto et al., 2010), has been widely used to induce ER stress in vivo and in vitro (Bogdanovic et al., 2015). In order to determine whether the PKA gene of adipocyte is involved in the process of ERs- mediated lipolysis in grass carp, we used tunicamycin to treat adipocytes and detected the expression level of mRNA. This study will help to reveal the mechanism of ER maintaining lipid homeostasis from a new perspective, and provides basis and research ideas for the regulation of lipid accumulation in fish.