Molecular cloning and characterization of GLUT2 from pompano (Trachinotus ovatus), and its gene expression in response to exogenous enzymes supplementation in high carbohydrate diet

Abstract

Glucose transport across membranes mediated by glucose transporter protein (GLUTs) is a momentous process in tissue glucose metabolism and blood glucose regulation. In this experiment, the glucose transporter 2 of pompano, Trachinotus ovatus (ToGLUT2) was cloned and identified. A 56-day breeding experiment was performed to study the effects of exogenous enzymes supplementation in high carbohydrate diets on the mRNA expression of ToGLUT2. The pompano (38.50 ± 0.50 g) used in this experiment were fed five diets (46% protein, 5.8% lipid), one of which was the control group (non-enzyme) and the remaining four groups were supplemented with four kinds of exogenous enzymes (amylase, glucoamylase, pullulanase and complex enzymes). The experimental results showed that the cDNA full length of 2280 bp, a 3′-UTR of 610 bp, a 5′-UTR of 32 bp, an open reading frame of 1638 bp, and a polypeptide of 546 amino acids are characteristic of ToGLUT2 (GenBank accession No: MK649830). Analysis of the phylogenetic tree revealed that the highest similarity was found between ToGLUT2 and Dicentrarchus labrax GLUT2. The results of qRT-PCR showed that ToGLUT2 gene was ubiquitously observed among tissues tested and was expressed at the highest level in the liver, closely followed by the intestine and brain. However, the poorest expression of ToGLUT2 was detected in the muscle, kidney and heart; the findings indicated that the glucokinase (GK) and 6-phosphofructo kinase − 1 (PFK-1) activities were significantly highest in the high carbohydrate group (H) (P < 0.05), followed by the amylase group (A) (P < 0.05), and finally the complex enzymes group (C) (P < 0.05); to respects of digestive enzymes, the intestinal amylase (AMS) in the amylase group (A) was not significantly difference compared to the high carbohydrate group (H) (P > 0.05), and was significantly lower in all other groups (P < 0.05). The trypsin (TRP) in the complex enzymes group (C) was significantly highest compared to the high carbohydrate group (H), followed by the amylase group (A), and finally in the glucoamylase group (G). The lipase (LPS) was significantly highest in the amylase group (A) compared to the high carbohydrate group (H) (P < 0.05), while it was significantly lower in the glucoamylase (G) and pullulanse groups (P) (P < 0.05), with no significant difference in the complex enzyme group (C) (P > 0.05). Hence, the activity of digestive enzymes can be improved by the addition of carbohydrase in feed; in terms of serum biochemical parameters, there was no significant difference in glucose concentration for all treatment groups (P > 0.05). Insulin concentration was not significant difference in the pullulanase group (P) compared to the high carbohydrate group (H) (P > 0.05), while it was significantly lower in all other groups (P < 0.05). Liver glycogen in the amylase group (A), glucoamylase group (G) and pullulanase group (P) was significantly higher compared to the high carbohydrate group (H) (P < 0.05), with no significant difference in the complex enzyme group (C) (P > 0.05). Muscle glycogen was not significantly different in the glucoamylase group (G) compared to the high carbohydrate group (H) (P > 0.05), while it was significantly lower in all other groups (P < 0.05); GLUT2 mRNA expression from liver and muscle was significantly up-regulated in the glucoamylase group (G) compared to the high carbohydrate group (H) (P < 0.05), while it was significantly lowest in the pullulanase group (P) and the complex enzyme group (C) (P < 0.05). In conclusion, GLUT2 from pompano showed the typical structure of glucose transporter protein family with high sequence homology with other species, which could improve the utilization of enhanced carbohydrates; the addition of carbohydrase to the high carbohydrate diet had different degrees of impacts on the growth performance, digestive enzymes, glucose metabolism and GLUT2 gene expression of pompano trevally, especially the glucoamylase group, which could significantly improve the utilization of carbohydrate.