Molecular cloning and nutrient regulation analysis of long chain acyl-CoA synthetase 1 gene in grass carp, Ctenopharyngodon idella L

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Abstract

Long chain acyl-CoA synthetase 1 (ACSL1), a key regulatory enzyme of fatty acid metabolism, catalyzes the conversion of long-chain fatty acids to acyl-coenzyme A. The full-length cDNAs of ACSL1a and ACSL1b were cloned from the liver of a grass carp. Both cDNAs contained a 2094 bp open reading frame encoding 697 amino acids. Amino acid sequence alignment showed that ACSL1a shared 73.5% sequence identity with ACSL1b. Each of the two ACSL1s proteins had a transmembrane domain, a P-loop domain, and L-, A-, and G-motifs, which were relatively conserved in comparison to other vertebrates. Relative expression profile of ACSL1 mRNAs in different tissues indicated that ACSL1a is highly expressed in heart, mesenteric adipose, and brain tissues, whereas ACSL1b is highly expressed in heart, white muscle, foregut, and liver tissues. Nutrient regulation research showed that the expression levels of ACSL1a and ACSL1b were significantly down-regulated when 3, 6, and 9% fish oil were added in diet of grass carp as compared to the control group. However, no significant difference in the levels of ACSL1 mRNA was observed between the experimental groups. This study demonstrated the relationship between ACSL1a and ACSL1b genes in grass carp and laid a foundation for further research on ACSL family members in other species.

Introduction

In order for the free fatty acids (FFA) to be utilized by animals, they must be esterified with acyl-CoA synthetase (ACS) by a process called fatty acid activation (Piccini et al., 1998). Only CoA esters of fatty acids can carry out a variety of lipid metabolic processes, such as β-oxidation, desaturation, and production of glycerides, cholesteryl esters, and phospholipids. As a result, ACS plays an essential role in the catabolism and anabolism of fatty acids. ACS is encoded by multigene families; in humans, ACS families are composed of 26 members (Watkins et al., 2007). Based on the carbon chain length of the substrate, ACSs can be divided into four subfamilies—very long chain acyl-CoA synthetase (ACSVL), long chain acyl-CoA synthetase (ACSL), medium-chain acyl-CoA synthetase (ACSM), and short-chain acyl-CoA synthetase (ACSS) (Soupene and Kuypers, 2008, Watkins et al., 2007).

Long chain acyl-CoA synthetase (ACSL, EC 6.2.1.3) is an ATP-dependent enzyme that catalyzes long-chain fatty acids to acyl-coenzyme A (acyl-CoA) (Coleman et al., 2000, Mashek et al., 2004, Soupene and Kuypers, 2006). The cDNA encoding ACSL was first cloned from the liver of rats (Suzuki et al., 1990). In 2004, the names of ACSL genes were revised by the human and mouse gene nomenclature committees—both ACSL1 and ACSL2 were renamed as ACSL1, and other members in the ACSL family were named as ACSL3, ACSL4, ACSL5, and ACSL6 according to the order of their discovery (Mashek et al., 2004, Watkins et al., 2007). In humans, as many as three different splice variants for each of the five ACSL genes have been identified because of mRNA alternative splicing (Soupene and Kuypers, 2006). A recent study showed that a novel uncharacterized ACSL-like gene, which was designated as ACSL2, was found in teleosts, suggesting that ACSL2 was conserved in teleosts, but lost in the tetrapod lineage. This was strongly supported by the detailed analysis of ACSL2 teleost gene locus (Lopes-Marques et al., 2013).

Among ACSL subfamilies, ACSL1 was discovered first. In mammals, ACSL1 is located in the endomembrane of the cells. It is highly expressed in the major energy-metabolizing tissues, such as fat, liver, and skeletal muscles, and is associated with the synthesis of triglycerides and uptake of fatty acids (Coleman et al., 2000, Lewin et al., 2001, Mashek et al., 2006). ACSL1 preferentially uses both saturated and mono unsaturated C16 to C18 fatty acids (FAs) (Lopes-Marques et al., 2013). A recent research has provided evidence for an important role of ACSL1 in heart metabolism by demonstrating that it is required to synthesize the acyl-CoAs that are oxidized by the heart. However, in the absence of ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids lead to mTOR activation and cardiac hypertrophy without lipid accumulation or immediate cardiac dysfunction (Ellis et al., 2011). Another research showed that in myocardium of rats, the ACSL1 gene expression occurs at the lowest level during prenatal period, increases gradually after birth and reaches the highest level in adulthood (de Jong et al., 2007). In comparison with mammals and plants, relatively few studies related to ACSL1 have been conducted so far in fish. ACSL1 genes of five Antarctic and sub-Antarctic notothenioid fish (Chaenocephalus aceratus, Gobionotothen gibberifrons, Notothenia coriiceps, N. angustata, and Eleginops maclovinus) were cloned to determine the substrate specificity of fat oxidation (Grove and Sidell, 2004). Furthermore, with the completion of whole-genome sequencing of the fish species Danio rerio, Pseudosciaena crocea, Cynoglossus semilaevis, Stegastes partitus, Poecilia reticulata, P. formosa and Oreochromis niloticus, their ACSL1 cDNA sequences were predicted. In most of the fish, three transcript variants were predicted for ACSL1 mRNAs. In addition, due to the third round of genome duplication in some fish, such as D. rerio, ACSL1 can be produced by two different genes, ACSL1a and ACSL1b (Lopes-Marques et al., 2013).

Grass carp (Ctenopharyngodon idella L.) is an important aquaculture fish in China. With the development of intensive aquaculture, some diseases associated with abnormal lipid metabolism, such as fatty liver, have become a major problem in grass carp in recent years. Nutritional imbalance in their diet is the major cause of fatty liver; however, the molecular mechanism of the nutrient regulation of their fat metabolism is unclear. In this study, we cloned two full-length ASCL1 cDNAs (ACSL1a and ACSL1b) from the liver of grass carp and analyze their expression level in different tissues. In addition, we determined the effect of different fat levels in the diet of grass carp on the expression of ACSL1 genes to understand their roles in fat metabolism. These results would help in optimizing the diet formula of aquaculture grass carp, and provide theoretical support for the treatment of fatty liver in them.

Section snippets

Experimental fish

A total of 10 grass carp, with body weight of 1300 ± 12 g were obtained from a commercial fish farm in Ganyu district, Lianyungang, Jiangsu province, China. The fish were transported to laboratory and reared in a 240 L tank for two weeks. During the feeding period, the water temperature ranged from 20 to 23 °C, and the fish were fed with commercial diets twice a day. The daily feeding rate was 3% of the body weight of grass carp. The fish were sampled 6 h after the last meal.

RNA extraction and synthesis of the cDNA first strand

Three fish were killed by

Molecular characterization of ACSL1a and ACSL1b genes

The full-length of ACSL1a cDNA was 3129 bp (GenBank accession no. KP262348), and the ORF was 2094 bp encoding 697 amino acids with a deduced molecular weight of 77.9 kDa and a theoretical isoelectric point (pI) of 8.25. The predicted amino acid sequence contains a transmembrane domain (Y21–A45), an ATP-binding motif (T275–K283; also known as P-loop), a fatty acid channel named G-motif (D313-X4-Y-LPLAH-X2-E326), a fatty acyl and/or CoA-binding motif (D553–I558; also known as L-motif), and an

Differences between ACSL1a and ACSL1b genes

In some fish species, such as D. rerio, Oryzias latipes and Tetraodon fluviatilis, there are two kinds of ACSL1 transcripts—ACSL1a and ACSL1b (Lopes-Marques et al., 2013). In D. rerio, it was obvious that they were encoded by different genes as ACSL1a (XP_005168406) was located on chromosome 1 and ACSL1b (NP_001003569) on chromosome 14. In our study, two ACSL1 genes of grass carp were named as ACSL1a and ACSL1b after comparing their amino acid sequence with those of D. rerio (Table 3). Further

Acknowledgments

This research was supported by the National Natural Science Foundation of China (grant no. 31272636); Major Project of the Natural Science Foundation of Jiangsu Educational Commission (grant no. 10KJA240002); Natural Science Foundation of Jiangsu Province (grant no. BK2012664); the Open Foundation of Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang, Jiangsu, China (grant no. 2009HS15); Major Project of Zhejiang Agricultural New Varieties Breeding

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    These authors contributed equally to this work.

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