Molecular characterization and expression analysis of glucokinase from herbivorous fish Megalobrama amblycephala subjected to a glucose load after the adaption to dietary carbohydrate levels

摘要

A full-length cDNA coding glucokinase (GK) was cloned from Megalobrama amblycephala by RT-PCR and RACE approaches. The cDNA obtained covered 2090 bp with an open reading frame of 1431 bp encoding 476 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (84-99%) among most fish and higher vertebrates, retaining one hexokinase signature sequence, one ATP-binding domain, two N-linked glycosylation sites and several glucose-binding sites. The highest GK expression was observed in the liver followed by intestine, whereas relatively low values were detected in the muscle, kidney and brain. Then, plasma glucose levels and liver GK activities and expressions were determined in fish subjected to a glucose load after being fed two dietary carbohydrate levels (30% and 42%) for 11 weeks. Plasma glucose levels decreased significantly after high-carbohydrate adaption. Glucose load induced a remarkable increment of glycemia in both groups with the maximum level being attained at 1 h. Thereafter, it decreased significantly to the basal value at 6 h and kept constant afterward. Hepatic GK expressions in both groups increased significantly with the maximum value being attained at 2 h after the glucose load, whereas no statistical difference was observed in enzymatic activities during the first 4 h. Then, both the activities and expressions decreased significantly with further increasing time. In addition, an inducible liver GK activity and expression by dietary carbohydrate levels were both observed. The results indicated that the GK gene of Megalobrama amblycephala showed a typical structure of the hexokinase family, and shared a high similarity with that of the other vertebrates. Hepatic GK expressions and activities of this species were highly inducible by glucose administration, as favored a short postprandial period of hyperglycemia. Furthermore, high dietary carbohydrate improved its glucose tolerance through the enhanced GK expressions and activities.