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Dexamethasone sensitizes the neonatal intestine to fructose induction of intestinal fructose transporter (Slc2A5) function.

ABSTRACT: The recent dramatic increase in fructose consumption is tightly correlated with an equally dramatic surge in the incidence of type 2 diabetes and obesity in children, but little is known about dietary fructose metabolism and absorption in neonates. The expression of the rat intestinal fructose transporter GLUT5 [Slc2A5, a member of the glucose transporter family (GLUT)] can be specifically induced by its substrate fructose, but only after weaning begins at 14 d of age. In suckling rats younger than 14 d old, dietary fructose cannot enhance GLUT5 expression. The aim of this study was to identify the mechanisms allowing fructose to stimulate GLUT5 during weaning. After intestines were perfused with fructose or glucose (control), using microarray hybridization we showed that of 5K genes analyzed in 10-d-old pups, only 13 were fructose responsive. Previous work found approximately 50 fructose-responsive genes in 20-d-old pups. To identify fructose-responsive genes whose expression also changed with age, intestines of 10- and 20-d-old littermate pups perfused with fructose were compared by microarray. Intestines of 10- and 20-d-old pups perfused with glucose were used to segregate age- but not fructose-responsive genes. About 28 genes were up- and 22 down-regulated in 20- relative to 10-d-old pups, under conditions of fructose perfusion, and many were found, by cluster analysis, to be regulated by corticosterone. When dexamethasone was injected into suckling pups before fructose perfusion, the expression of GLUT5 but not that of the sodium glucose cotransporter (SGLT) 1 and of GLUT2, as well as the uptake of fructose but not of glucose increased dramatically. Thus, dexamethasone, which allows dietary fructose to precociously stimulate intestinal fructose absorption, can mimic the effect of age and modify developmental timing mechanisms regulating GLUT5.

SUBMITTER: Douard V

PROVIDER: S-EPMC2194616 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Dexamethasone sensitizes the neonatal intestine to fructose induction of intestinal fructose transporter (Slc2A5) function.

Douard Veronique V Cui Xue-Lin XL Soteropoulos Patricia P Ferraris Ronaldo P RP

Endocrinology 20071018 1

The recent dramatic increase in fructose consumption is tightly correlated with an equally dramatic surge in the incidence of type 2 diabetes and obesity in children, but little is known about dietary fructose metabolism and absorption in neonates. The expression of the rat intestinal fructose transporter GLUT5 [Slc2A5, a member of the glucose transporter family (GLUT)] can be specifically induced by its substrate fructose, but only after weaning begins at 14 d of age. In suckling rats younger t ...[more]

PMID: 17947353

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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.

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:BackgroundHigh intakes of fructose are associated with metabolic diseases, including hypertriglyceridemia and intestinal tumor growth. Although small intestinal epithelia consist of many different cell types, express lipogenic genes, and convert dietary fructose to fatty acids, there is no information on the identity of the cell type(s) mediating this conversion and on the effects of fructose on lipogenic gene expression.ObjectivesWe hypothesized that fructose regulates the intestinal expression of genes involved in lipid and apolipoprotein synthesis, that regulation depends on the fructose transporter solute carrier family 2 member a5 [Slc2a5 (glucose transporter 5)] and on ketohexokinase (Khk), and that regulation occurs only in enterocytes.MethodsWe compared lipogenic gene expression among different organs from wild-type adult male C57BL mice consuming a standard vivarium nonpurified diet. We then gavaged twice daily for 2.5 d fructose or glucose solutions (15%, 0.3 mL per mouse) into wild-type, Slc2a5-knockout (KO), and Khk-KO mice with free access to the nonpurified diet and determined expression of representative lipogenic genes. Finally, from mice fed the nonpurified diet, we made organoids highly enriched in enterocyte, goblet, Paneth, or stem cells and then incubated them overnight in 10 mM fructose or glucose.ResultsMost lipogenic genes were significantly expressed in the intestine relative to the kidney, liver, lung, and skeletal muscle. In vivo expression of Srebf1, Acaca, Fasn, Scd1, Dgat1, Gk, Apoa4, and Apob mRNA and of Scd1 protein increased (P < 0.05) by 3- to 20-fold in wild-type, but not in Slc2a5-KO and Khk-KO, mice gavaged with fructose. In vitro, Slc2a5- and Khk-dependent, fructose-induced increases, which ranged from 1.5- to 4-fold (P < 0.05), in mRNA concentrations of all these genes were observed only in organoids enriched in enterocytes.ConclusionsFructose specifically stimulates expression of mouse small intestinal genes for lipid and apolipoprotein synthesis. Secretory and stem cells seem incapable of transport- and metabolism-dependent lipogenesis, occurring only in absorptive enterocytes.

Dataset Information

Dexamethasone sensitizes the neonatal intestine to fructose induction of intestinal fructose transporter (Slc2A5) function.

Publications

Dexamethasone sensitizes the neonatal intestine to fructose induction of intestinal fructose transporter (Slc2A5) function.

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