Molecular cloning and tissue distribution of glucokinase and glucose-6-phosphatase catalytic subunit paralogs in largemouth bass Micropterus salmoides: Regulation by dietary starch levels and a glucose load

Abstract

The dysregulation of glucose-G6P (glucose-6-phosphate) interconversion is thought to be one of the main reasons for the low glucose disposal of carnivorous fish, but is not yet well understood in largemouth bass Micropterus salmoides (LMB). In this study, the full length cDNA sequences of genes encoding glucokinase (Gck, catalyzing glucose phosphorylation) and glucose-6-phosphatase catalytic subunit (G6pc, catalyzing glucose dephosphorylation) were cloned by the RACE method from the liver of LMB. Subsequently, the distribution of g6pc and gck as well as their transcriptional regulation by dietary starch levels and a glucose load were investigated. Only one gck gene was identified, while the tandem duplication of g6pca.1 gene was named as g6pca.2 in LMB. The full cDNA sequences of g6pca.1, g6pca.2 and gck in LMB were 1585, 1813 and 2115 bp in length, encoding 478, 352 and 359 amino acids, respectively. Gck was predicted to contain two hexokinase domains, an ATP-binding domain and multiple functional sites, while G6pca.1 and G6pca.2 contained nine transmembrane helices, a PAP2 (type-2 phosphatidic acid phosphatase) domain and multiple functional amino acid sites. Both g6pca.1 and g6pca.2 were predominantly distributed in the liver and to some extent in the intraperitoneal fat, intestine and pyloric caeca, while gck was mainly transcribed in the liver and to some extent in the heart, intestine and brain. Both feeding a high starch diet and a glucose load stimulated the mRNA expression of gck in the liver of LMB. An increase of dietary starch from 9% to 14% down-regulated the transcription of g6pca.1 in the liver of LMB. However, both the mRNA levels of hepatic g6pca.1 and g6pca.2 were sharply up-regulated in LMB during 1-3 h after a glucose load. Overall, the results of this study suggested that the functions of G6pc (G6pca.1 and G6pca.2) and Gck in LMB were highly conserved in evolution. Though hepatic glucose-G6P interconversion was well regulated at the transcript level in LMB fed high starch diets, a futile cycle between glucose and G6P was induced in the liver after a glucose load.

Keywords: Gene expression; Glucokinase; Glucose homeostasis; Glucose-6-phosphatase; Largemouth bass.