Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2013 Jun 04;86:e68475. doi: 10.1371/journal.pone.0068475.
Show Gene links
Show Anatomy links
FGT-1 is a mammalian GLUT2-like facilitative glucose transporter in Caenorhabditis elegans whose malfunction induces fat accumulation in intestinal cells.
Kitaoka S
,
Morielli AD
,
Zhao FQ
.
???displayArticle.abstract???
Caenorhabditis elegans (C. elegans) is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS), that are conserved in mammalian cells. To explore C. elegans as a model system to study the regulation of the facilitative glucose transporter (GLUT), we have characterized the GLUT gene homologues in C. elegans: fgt-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1 and Y37A1A.3. The exogenous expression of these gene products in Xenopus oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (Km) of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1-mediated 2-deoxy-D-glucose uptake (P < 0.01), indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in C. elegans, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of fgt-1 resulted in increased fat staining in both wild-type and daf-2 (mammalian insulin receptor homologue) mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by fgt-1 knockdown in wild-type or daf-2 mutants. Our results indicated that in C. elegans, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism.
???displayArticle.pubmedLink???
23826391
???displayArticle.pmcLink???PMC3691140 ???displayArticle.link???PLoS One
Figure 2. Amino acid sequence alignments of human GLUT1-4 and C. elegans FGT-1 and R09B5.11.A. Alignments of the deduced amino acid sequences of FGT-1, R09B5.11 and human GLUT1-4 were performed with the Clustal W program with open gap penalty = 10 and gap extension penalty = 0.05. Residues that are highlighted by a black shaded background represent absolutely conserved amino acids, and the gray shaded background indicates four or more conserved residues at those positions. Regions of presumed transmembrane domains (TM) [32] are indicated by numbered dashed lines, and the functionally important residues for glucose uptake activity are indicated by the letter “F” at the top of the sequence alignments. In addition, the highly conserved amino acids are shown on the bottom of the sequence alignment. B. Phylogenetic tree of the aligned sequences in (A) by the Clustal W program. Scale bar indicates relative branch lengths obtained from the Clustal W alignment result.
Figure 3. Glucose transport activity and kinetics of C. elegans GLUT candidates in Xenopus oocytes.A. Analysis of 2-deoxy-D-glucose (2-DG) transport activities of FGT-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1, and Y37A1A.3 in Xenopus oocytes. Water or the cRNA of C. elegans genes or hGLUT1 was injected into oocytes, and the oocytes were incubated in 10 mM 2-DG containing 2-deoxy-D-[1-3H]-glucose ( 3H-2-DG) for 15 min before being counted. Error bars represent SEM (n ≥ 31). Statistical analysis was conducted using Dunnett’s one-way ANOVA before the Tukey-Kramer HSD test, with water-injected samples as the control group (***P < 0.001). B and C. Plasma membrane localization of FGT-1 and R09B5.11. The eGFP fusion proteins of FGT-1 (B) or R09B5.11 (C) were individually expressed in oocytes and were observed under a confocal laser microscope. D. Analysis of 2-DG transport activities of the FGT-1::eGFP and R09B5.11::eGFP fusion proteins in Xenopus oocytes. Water or the cRNA of fgt-1, fgt-1::egfp, R09B5.11 or R09B5.11::egfp was injected into oocytes, and the oocytes were incubated in 10 mM 2-DG containing 3H-2-DG for 15 min before being counted. Error bars represent SEM (n ≥ 27). Statistical analysis was conducted using Dunnett’s one-way ANOVA before the Tukey-Kramer HSD test, with water-injected samples as the control group (***P < 0.001). E. Inhibition of the glucose transport activity of FGT-1 by phloretin. The fgt-1 cRNA-injected oocytes were incubated in 10 mM 2-DG with 3H-2-DG and increasing concentrations of phloretin from 0 µM to 200 µM for 15 min. The 2-DG uptake of fgt-1 cRNA-injected oocytes was corrected by the uptake of water-injected oocytes. Error bars represent SEM (n = 10). F. Kinetic analysis of 2-DG uptake by FGT-1. The fgt-1 cRNA- or water-injected oocytes were exposed to increasing concentrations of 2-DG (0-30 mM) containing 3H-2-DG for 15 min. Points represent 2-DG uptake of fgt-1 cRNA-injected oocytes after correction for the uptake of water-injected oocytes. Error bars represent SEM (n ≥ 13). Michaelis-Menten nonlinear analysis was conducted in GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA).
Figure 4. Hexose sugar substrate specificity of fgt-1.Oocytes injected with either fgt-1 cRNA or water were exposed to 10 mM 2-DG containing 3H-2-DG and 30 mM concentrations of L-glucose, D-glucose, 3-O-methylglucose (3-OMG), D-mannose, D-galactose, or D-fructose for 15 min. 2-DG uptake from water-injected oocytes was subtracted from fgt-1 cRNA-injected oocytes. Error bars represent SEM (n = 40). Statistical analysis was conducted using Dunnett’s one-way ANOVA before the Tukey-Kramer HSD test, with L-glucose as the control group (**P < 0.01, ***P < 0.001).
Figure 5. Cellular and subcellular localizations of FGT-1 in C. elegans.The fgt-1::egfp fusion construct under the control of the fgt-1 promoter was injected into C. elegans, and the expression of the FGT-1::eGFP fusion protein was visualized with a confocal laser microscope. A: whole animal, B: embryos inside of a parental worm, C: higher magnification image of mid-body. For each panel (A to C), GFP fluorescence was shown alone or as a merged picture with differential interference contrast (DIC) images. D–H. Immunostaining of the apical membrane marker IFB-2 for fgt-1::egfp embryos. Subcellular localizations of FGT-1::eGFP (green) and IFB-2 (red) were observed under a confocal laser microscope. D: FGT-1::eGFP, E: IFB-1 immunostaining. F: Merged image of D and E.G: The higher magnification of the white box area in F.H: Merged image of eGFP fluorescence and DIC images. I. Schematic diagram of an intestinal cell with subcellular localizations of eGFP (green) and IFG-2 (red). The potential adherent junction (AJ) is indicated by a dark box.
Figure 6. Cellular localizations of R09B5.11 in C. elegans.The R09B5.11::egfp fusion construct under the control of the R09B5.11 promoter was injected into C. elegans, and the expression of the R09B5.11::eGFP fusion protein was visualized with a confocal laser microscope. GFP images and merged pictures of GFP with DIC images in the L2 stage (A–D) or three-fold embryo (E and F) are shown. Left- and right-lateral strings of seam cells (A and B) and individual seam cells (C and D) are visualized. Arrowheads indicate each seam cell.
Figure 7. Functional analyses of fgt-1 on fat storage, dauer formation and brood size in C. elegans.A. The expression of the fgt-1::egfp fusion protein was detected by Western blot analysis using an anti-GFP antibody in the wild-type worms (wt) injected with (+) or without (-) the fgt-1 double-stranded RNA (dsRNA, RNAi). B. The intensities of the fusion protein in A were quantified by Image Lab Software and were normalized to β-actin levels. Error bars represent SEM (n = 4). C, D, and E. Fat accumulation in the fgt-1 RNAi wt (C) and daf-2(e1370) mutant worms (D), as well as in fgt-1(tm3165) mutant animals (E, no RNAi), was measured by Sudan black B staining. Staining intensity was quantitated using ImageJ software (NIH). Error bars represent SEM (n ≥ 41). Statistical analysis was conducted using Student’s t-test (**P < 0.01, ***P < 0.001). F and G. Comparison of the effects of fgt-1 knockdown on the dauer formation rate (F) and brood size (G). In F, wild-type and daf-2(e1370) worms were incubated at either 20 °C or 25 °C. Error bars represent SEM (n ≥ 9 and 25 for F and G, respectively). Statistical analysis was conducted using Student’s t-test.
Figure 1. Structural schematic representation of GLUT candidate genes in C. elegans, compared with human GLUT4.The amino acid sequence of individual C. elegans genes was obtained from Wormbase (http://www.wormbase.org/). The blue boxes indicate the predicted transmembrane domains by Wormbase, and the dashed boxes in R09B5.11 indicate the missing predicted transmembrane domains. Red filled circles indicate potential N-glycosylation sites that were predicted by NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/). Arrowheads indicate known functionally important residues that were found in human GLUT4: R92, R153, R333/4, and E393 [11]. The predicted conserved long loop 6 is indicated by red dashed circles.
Bentley,
Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes.
2012, Pubmed,
Xenbase
Bentley,
Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes.
2012,
Pubmed
,
Xenbase
Bossinger,
The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413.
2004,
Pubmed
Brenner,
The genetics of Caenorhabditis elegans.
1974,
Pubmed
Cassada,
The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.
1975,
Pubmed
Cheeseman,
GLUT2 is the transporter for fructose across the rat intestinal basolateral membrane.
1993,
Pubmed
Duerr,
The cat-1 gene of Caenorhabditis elegans encodes a vesicular monoamine transporter required for specific monoamine-dependent behaviors.
1999,
Pubmed
Fire,
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
1998,
Pubmed
Fukumoto,
Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein.
1988,
Pubmed
Golden,
The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature.
1984,
Pubmed
Gould,
The glucose transporter family: structure, function and tissue-specific expression.
1993,
Pubmed
Gould,
Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms.
1991,
Pubmed
,
Xenbase
Haber,
Tissue distribution of the human GLUT3 glucose transporter.
1993,
Pubmed
Joost,
The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review).
2001,
Pubmed
Karabinos,
Essential roles for four cytoplasmic intermediate filament proteins in Caenorhabditis elegans development.
2001,
Pubmed
Keller,
Different mammalian facilitative glucose transporters expressed in Xenopus oocytes.
1990,
Pubmed
,
Xenbase
Keller,
Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes. Comparison of kinetic parameters.
1989,
Pubmed
,
Xenbase
Kramer,
The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.
1990,
Pubmed
Lee,
Glucose shortens the life span of C. elegans by downregulating DAF-16/FOXO activity and aquaporin gene expression.
2009,
Pubmed
Leto,
Regulation of glucose transport by insulin: traffic control of GLUT4.
2012,
Pubmed
Maenz,
The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics.
1987,
Pubmed
Mello,
DNA transformation.
1995,
Pubmed
Saltiel,
Insulin signalling and the regulation of glucose and lipid metabolism.
2001,
Pubmed
Schürmann,
Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.
1997,
Pubmed
Shepherd,
Glucose transporters and insulin action--implications for insulin resistance and diabetes mellitus.
1999,
Pubmed
Shepherd,
Distribution of GLUT3 glucose transporter protein in human tissues.
1992,
Pubmed
Sun,
Crystal structure of a bacterial homologue of glucose transporters GLUT1-4.
2012,
Pubmed
Takashima,
Expression profiles and unc-27 mutation rescue of the striated muscle type troponin I isoform-3 in Caenorhabditis elegans.
2012,
Pubmed
Thorens,
Glucose transporters in the 21st Century.
2010,
Pubmed
Togo,
Immunological detection of alkaline-diaminobenzidine-negativeperoxisomes of the nematode Caenorhabditis elegans purification and unique pH optima of peroxisomal catalase.
2000,
Pubmed
Wright,
The Na+/glucose cotransporter (SGLT1).
1992,
Pubmed
Yokota,
Peroxisomes of the nematode Caenorhabditis elegans: distribution and morphological characteristics.
2002,
Pubmed
Zhao,
Functional properties and genomics of glucose transporters.
2007,
Pubmed
