Glucose transporter 2 in common carp (Cyprinus carpio L.): molecular cloning, tissue expression, and the responsiveness to glucose, insulin, and glucagon.

Glucose transporter 2 (glut2) has been studied in mammals, aves, and several fish, while the comparative studies of glut2 in common carp are still lacking. In this study, glut2 was firstly isolated and characterized from the liver of common carp. The full-length cDNA of glut2 was 2351 bp with an open reading frame (ORF) of 1512 bp, encoding 503 amino acids. Alignment of glut2 amino acid sequences from different species revealed that common carp glut2 showed higher sequence identity with teleosts, and lower homology with mammals and amphibians. Tissue distribution demonstrated that glut2 mRNA level was mainly expressed in liver, foregut, and midgut. To investigate the actions of glut2 on glucose metabolism, the level of glut2 mRNA was detected after intraperitoneal injection of glucose, human insulin and glucagon (100 ng/g), respectively. Following glucose administration, glut2 gene expression was significantly upregulated at 3 h in the foregut. However, no change was found in hepatic glut2 mRNA level, indicating that glut2 may have a role in intestinal glucose uptake rather than in the liver. Following insulin treatment, the expression of glut2 was markedly downregulated at 3 h and 6 h in the liver, and at 3 h in the foregut, respectively. Furthermore, glut2 mRNA expression was unaffected by glucagon injection in the liver and foregut. These results suggested that the expression of glut2 regulated by pancreatic hormones was different. Taken together, our studies firstly revealed the structure of the glut2 gene and its potential functions in glucose metabolism of common carp.

Purification and characterization of mammalian glucose transporters expressed in Pichia pastoris.

The major bottleneck to the application of high-resolution techniques such as crystallographic X-ray diffraction and spectroscopic analyses to resolve the structure of mammalian membrane proteins has been the ectopic expression and purification of sufficient quantities of non-denatured proteins. This has been especially problematic for members of the major facilitator superfamily, which includes the family of mammalian glucose transporters. A simple and rapid method is described for the purification of milligram quantities of recombinant GLUT1 and GLUT4, two of the most intensively studied GLUT isoforms, after ectopic expression in Pichia pastoris. The proteins obtained were >95% pure and exhibited functional transport and ligand-binding activities.

High human GLUT1, GLUT2, and GLUT3 expression in Schizosaccharomyces pombe.

In this study, three subfamily members of the human 12-transmembrane-domain cell-surface receptors GLUT1, GLUT2, and GLUT3 were heterologously expressed in the fission yeast Schizosaccharomyces pombe utilizing GST-GLUT fusion proteins. These fusion proteins were driven by the full-length nmt1 promoter (Pnmt1) derived from S. pombe. The transcription levels of the GST-GLUT fusion proteins were very high upon induction by removing thiamine from the media. One-step purification of the recombinant fusion proteins was achieved by GST-affinity chromatography. Approximately 300 microg of highly purified fusion protein were obtained from 3 g of wet cell paste (1 liter of cell culture), indicating that human membrane proteins can be efficiently expressed and purified in the fission yeast. With its available extensive genetic information and ease of genetic manipulation, the fission yeast is potentially a highly efficient host to express eukaryotic membrane proteins.