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  • In general fish has a limited

    2021-09-17

    In general, fish has a limited ability for digestion and metabolism of carbohydrate though carbohydrate is the cheapest dietary energy and carbon source to animals and the best candidate for replacing dietary protein (Enes et al., 2009, Kamalam et al., 2017, Wilson, 1994). Furthermore, the analysis of carbohydrate metabolic mechanism at molecular level is relatively lacked. Golden pompano (Trachinotus ovatus) is a typical carnivorous fish that can accept formulated feed under commercial culture conditions (Lin et al., 2012, Othman, 2008, Zhou et al., 2015). Optimal dietary carbohydrate for golden pompano is significantly lower than herbivorous or omnivorous categories (Zhou et al., 2015). As a representative of the carnivorous fish, the information of carbohydrate metabolism in golden pompano will provide reference for other fish. Thus, to investigate the physiological function of GK gene in golden pompano through analysis the regulation of GK in carbohydrate metabolism is in request. Our previous study indicated that the level of dietary carbohydrate utilization for juvenile golden pompano was about 11.2–16.8% (Zhou et al., 2015). Therefore, we choose the lowest, middle and highest dietary carbohydrate levels to analyze the hepatic enzyme activity and gene expression of GK in present study. The aims of the present study were to achieve the complete coding sequence (CDS) of TO-GK from golden pompano liver, to analysis the tissue distribution of TO-GK in golden pompano and to investigate the regulation of TO-GK on gene expression and enzyme activity by addition of different dietary carbohydrate levels in diets.
    Materials and methods
    Results
    Discussion The GK is essential for the utilization of dietary carbohydrate which is the first enzyme to phosphorylate glucose in excess in different key tissues (Panserat et al., 2014, Panserat et al., 2000). In the present study, the cDNA sequence of a novel To-GK was first cloned and characterized in golden pompano by PCR and RACE techniques. The full-length cDNAs of To-GK is 2151bp, encoding a 478bp Aliskiren peptide. The calculated molecular mass and pI of the To-GK were 53kDa and 4.91, respectively. The molecular mass of To-GK in golden pompano is similar to other species (Andreone et al., 1989, Cheng et al., 2012). The deduced amino acid sequence of golden pompano shared high identities with other fish species. Like other known GK (Cheng et al., 2012, Panserat et al., 2000), golden pompano To-GK had a conserved hexokinase signature sequence Leu158-Phe183. All residues are conserved in all species. Besides, two potential N-linked glycosylation sites (Asn178 and Asn216), a cell attachment sequence (Arg204-Asp206), and a glycosaminoglycan attachment site (Ser457-Gly460) are also much conserved in all vertebrates including golden pompano. The To-GK amino acid sequence from golden pompano contains several functional sites, including the ATP-binding domain, the glucose binding sites and the regulatory protein binding sites. As for the glucose binding sites, the oxygen atoms of glucose form hydrogen bond interactions with the side chains of glucokinase residues, which is in common with hitherto mammalian (Mahalingam et al., 1999, Veiga-da-Cunha et al., 1996), grass carp (Ctenopharyngodon idella) (Cheng et al., 2012), gilthead seabream (Sparus aurata), rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) (Panserat et al., 2000). The presence of ATP and glucose binding sites in To-GK is in favor of the existence of functional GK in these species. As in mammals, GK were also expressed in liver and brain in rainbow trout (Panserat et al., 2000). GK plays crucial roles in converting excess glucose to lipid and glycogen in liver (Panserat et al., 2001), while other study in fish confirmed that GK in the brain can also be a glucose sensor in fish (Polakof et al., 2008b, Polakof et al., 2007). In the present study, To-GK mRNA was expressed in the liver the highest followed by brain, while there seemed to be a lower expression level in the spleen. These data demonstrate that GK gene expression is highly specific to liver. Similar results were found in other fish species, such as zebrafish (Gonzalez-Alvarez et al., 2009), grass carp (Cheng et al., 2012).