Dataset Information

Molecular analysis of the fructose transporter gene (GLUT5) in isolated fructose malabsorption.

ABSTRACT: Fructose, a naturally occurring monosaccharide, is increasingly used as an added sweetener in processed foods in the form of high fructose corn syrup. Increased fructose intake combined with the identification of children with clinical evidence of isolated fructose malabsorption (IFM) has stimulated interest in possible disorders of fructose absorption. The intestinal absorption of fructose is carried out by the facilitative hexose transporter, which has been designated as GLUT5. Functional properties and tissue distribution of GLUT5 suggest that IFM might be due to mutations in the GLUT5 gene. To test this hypothesis, we screened the GLUT5 gene for mutations in a group of eight patients with IFM and in one subject with global malabsorption, as compared with 15 healthy parents of subjects and up to 6 unrelated controls. No mutations were found in the protein coding region of this gene in any of the subjects. A single G to A substitution in the 5' untranslated region of exon 1 was identified in the subject with global malabsorption. This subject and her healthy mother were heterozygous for the variant sequence, suggesting that it was unlikely to be clinically significant. In addition, sequence analysis of each of the 12 GLUT5 exons was performed in the index case and confirmed the negative single-strand conformation polymorphism findings. These studies demonstrate that IFM does not result from the expression of mutant GLUT5 protein.

SUBMITTER: Wasserman D

PROVIDER: S-EPMC507692 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Molecular analysis of the fructose transporter gene (GLUT5) in isolated fructose malabsorption.

Wasserman D D Hoekstra J H JH Tolia V V Taylor C J CJ Kirschner B S BS Takeda J J Bell G I GI Taub R R Rand E B EB

The Journal of clinical investigation 19961101 10

Fructose, a naturally occurring monosaccharide, is increasingly used as an added sweetener in processed foods in the form of high fructose corn syrup. Increased fructose intake combined with the identification of children with clinical evidence of isolated fructose malabsorption (IFM) has stimulated interest in possible disorders of fructose absorption. The intestinal absorption of fructose is carried out by the facilitative hexose transporter, which has been designated as GLUT5. Functional prop ...[more]

PMID: 8941659

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Project description:Recent studies suggest that the jejunal/kidney-type facilitative glucose transporter (GLUT5) functions as a high-affinity D-fructose transporter. However, its precise role in the small intestine is not clear. In an attempt to identify the fructose transporter in the small intestine, we measured fructose uptake in Xenopus oocytes expressing jejunal mRNA from five species (rat, mouse, rabbit, hamster and guinea-pig). Only jejunal mRNA from the rabbit significantly increased fructose uptake. We also cloned a rabbit GLUT5 cDNA from a jejunal library The predicted amino acid sequence of the 487-residue rabbit GLUT5 showed 72.3 and 67.1% identity with human and rat GLUT5 respectively. Northern-blot analysis revealed GLUT5 transcripts in rabbit duodenum, jejunum and, to a lesser extent, kidney. After separation of rabbit jejunal mRNA on a sucrose density gradient, the fractions that conferred D-fructose transport activity in oocytes also hybridized with rabbit GLUT5 cDNA. Hybrid depletion of jejunal mRNA with a GLUT5 antisense oligonucleotide markedly inhibited the mRNA-induced fructose uptake in oocytes. Immunoblot analysis indicated that GLUT5 (49 kDa) is located in the brush-border membrane of rabbit intestinal epithelial cells. Xenopus oocytes injected with rabbit GLUT5 cRNA exhibited fructose uptake activity with a Km of 11 mM for D-fructose. D-Fructose transport by GLUT5 was significantly inhibited by D-glucose and D-galactose. D-Fructose uptake in brush-border membrane vesicles shows a Km similar to that of GLUT5, but was not inhibited by D-glucose or D-galactose. Finally, cytochalasin B photolabelled a 49 kDa protein in rabbit brush-border-membrane preparations that was immunoprecipitated by antibodies to GLUT5. Our results suggest that GLUT5 functions as a fructose transporter in rabbit small intestine. However, biochemical properties of fructose transport in Xenopus oocytes injected with GLUT5 cRNA differed from those in rabbit jejunal vesicles.

Project description:BackgroundFructose is an abundant source of carbon and energy for cells to use for metabolism, but only certain cell types use fructose to proliferate. Tumor cells that acquire the ability to metabolize fructose have a fitness advantage over their neighboring cells, but the proteins that mediate fructose metabolism in this context are unknown. Here, we investigated the determinants of fructose-mediated cell proliferation.MethodsLive cell imaging and crystal violet assays were used to characterize the ability of several cell lines (RKO, H508, HepG2, Huh7, HEK293T (293T), A172, U118-MG, U87, MCF-7, MDA-MB-468, PC3, DLD1 HCT116, and 22RV1) to proliferate in fructose (i.e., the fructolytic ability). Fructose metabolism gene expression was determined by RT-qPCR and western blot for each cell line. A positive selection approach was used to "train" non-fructolytic PC3 cells to utilize fructose for proliferation. RNA-seq was performed on parental and trained PC3 cells to find key transcripts associated with fructolytic ability. A CRISPR-cas9 plasmid containing KHK-specific sgRNA was transfected in 293T cells to generate KHK-/- cells. Lentiviral transduction was used to overexpress empty vector, KHK, or GLUT5 in cells. Metabolic profiling was done with seahorse metabolic flux analysis as well as LC/MS metabolomics. Cell Titer Glo was used to determine cell sensitivity to 2-deoxyglucose in media containing either fructose or glucose.ResultsWe found that neither the tissue of origin nor expression level of any single gene related to fructose catabolism determine the fructolytic ability. However, cells cultured chronically in fructose can develop fructolytic ability. SLC2A5, encoding the fructose transporter, GLUT5, was specifically upregulated in these cells. Overexpression of GLUT5 in non-fructolytic cells enabled growth in fructose-containing media across cells of different origins. GLUT5 permitted fructose to flux through glycolysis using hexokinase (HK) and not ketohexokinase (KHK).ConclusionsWe show that GLUT5 is a robust and generalizable driver of fructose-dependent cell proliferation. This indicates that fructose uptake is the limiting factor for fructose-mediated cell proliferation. We further demonstrate that cellular proliferation with fructose is independent of KHK.

Dataset Information

Molecular analysis of the fructose transporter gene (GLUT5) in isolated fructose malabsorption.

Publications

Molecular analysis of the fructose transporter gene (GLUT5) in isolated fructose malabsorption.

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