Mouse Glucose Transporter 9 Splice Variants Are Expressed in Adult Liver and Kidney and Are Up-Regulated in Diabetes

Abstract

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy

“…Thus, Glut9 may be involved in both steps of transepithelial urate transport, mostly in the distal tubule. We also confirmed that Glut9 is expressed at the plasma membrane of all hepatocytes (20) (Fig. S2).…”

“…The long form is targeted to the basolateral membrane and the short form to the apical membrane of polarized epithelial cells (19,20). In the human kidney, Glut9 is present in proximal convoluted tubules, whereas in rodents it is most probably in distal connecting tubules (20). Functional studies confirmed that Glut9 is a urate transporter (18,21) and mutations in its sequence cause hypouricemia in human subjects (22).…”

“…At least two splice variants have been identified differing only in the length of their amino-terminal cytoplasmic domains. The long form is targeted to the basolateral membrane and the short form to the apical membrane of polarized epithelial cells (19,20). In the human kidney, Glut9 is present in proximal convoluted tubules, whereas in rodents it is most probably in distal connecting tubules (20).…”

Uric acid transport and disease

“…GLUT9 has the structure of a type II Glut isoform, with 12 transmembrane domains, a large extracellular loop between the first and second transmembrane domains, and both amino- and carboxyterminal ends on the cytoplasmic side (82). In both humans and mice, GLUT9 exists as two alternatively spliced variants that encode different aminoterminal cytoplasmic tails (83,84). Human GLUT9a has 540 amino acids and is encoded by 12 exons, whereas GLUT9b is 512 amino acids long and encoded by 13 exons, spread over an approximately 250-kb genomic region.…”

“…In polarized epithelial cells, human GLUT9a is expressed in the basolateral membrane, whereas GLUT9b is targeted to the apical pole (84), and in human kidney, GLUT9 is present in the proximal tubule (84). In the mouse, Glut9 is present in the distal convoluted tubule (83), both in the basolateral and apical membranes (87) (Figure 3), but it is not yet known which isoform is present in each pole of the cells, in particular since both mouse isoforms have been reported to be targeted to the basolateral membrane of MDCK cells (83).…”