Rainbow trout glucose transporter (OnmyGLUT1): functional assessment in Xenopus laevis oocytes and expression in fish embryos
1 Institute of Applied Biotechnology, University of Kuopio, PO Box 1627, Kuopio 70211, Finland,
2 Biological Institute, University of Sanct Petersburg, Oranienbaum Chaussee 2, Stary Peterhof, Sanct Petersburg 198504, Russia and
3 Department of Infectious Diseases, St Georges Hospital Medical School, London SW17 0RE, UK
*Author for correspondence (e-mail: krasnov{at}uku.fi)
Accepted May 25, 2001
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Summary |
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Key words: glucose transporter, GLUT1, Xenopus laevis oocyte, development, expression, rainbow trout, Oncorhynchus mykiss, in situ hybridization, kinetics.
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Introduction |
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Fish genes encoding putative glucose transporters have been identified recently. We have cloned cDNAs from the rainbow trout Oncorhynchus mykiss and the common carp Cyprinus carpio that are similar to mammalian and avian GLUT1 (Teerijoki et al., 2000; Teerijoki et al., 2001). Planas et al. (Planas et al., 2000) reported molecular identification of the GLUT of the brown trout Salmo trutta, which is structurally related to mammalian GLUT4. We and S. Panserat (INRA, France) have cloned the rainbow trout GLUT2 (GenBank AF321816). From an analysis of the derived amino acid sequence, the residues involved in glucose transport (Barrett et al., 1999) appear to be well conserved in these proteins. However, direct evidence for the functionality of fish GLUTs has hitherto been lacking.
The importance of facilitative glucose transporters for fish remains undefined. To study its functional properties, we expressed the putative glucose transporter OnmyGLUT1 in Xenopus laevis oocytes. This heterologous expression system has been very useful for characterizing mammalian and protozoan hexose transporters. The relative abundance of OnmyGLUT1 transcripts in rainbow trout embryos suggested that this protein might play an important role in development. In mammals, GLUT1 is referred to as an early isoform because it is expressed at high levels in embryos and foetuses, being gradually substituted for other GLUT types during the course of development (Santalucia et al., 1992; Postic et al., 1994). We have analysed the expression of OnmyGLUT1 at a number of developmental stages (blastula, early and late gastrula, and during somitogenesis and the formation of the vitelline plexus) using whole-mount in situ hybridization. We also measured glucose uptake and determined the ratio of transport to hexokinase activity in embryos and dissociated embryonic cells.
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Materials and methods |
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Synthesis of OnmyGLUT1 mRNA and hexose transport
Xenopus laevis oocytes were prepared and used for hexose uptake analyses as described elsewhere (Penny et al., 1998). In brief, mRNA encoding OnmyGLUT1 was transcribed (MEGAscript SP6 with cap-analog; Ambion, Austin, TX, USA) from template linearised with XbaI (Promega). Oocytes were injected with either mRNA (15ng) or RNase-free water. All studies were carried out 2472h after microinjection. Uptake of labelled sugars was measured under zero-trans conditions. Groups of 810 oocytes were placed into 600µl of Barths medium containing labelled hexose (0.5µCi; 18.5kBq). The reaction was terminated after 20min, and the oocytes were washed three times with ice-cold Barths medium. Oocytes were then placed individually into scintillation counter vials. Uptakes were corrected for the uptake by water-injected controls. To verify the functionality of the transporter, D-[U-14C]glucose, 3-O-methyl-D-[U-14C]glucose (3-OMG) and D-[U-14C]fructose (all from Amersham Pharmacia Biotech, Amersham, UK) were used. To study kinetics, substrate selectivity and sensitivity to inhibitors, the uptake of labelled D-glucose was measured. Concentrations of D-glucose, competitors and inhibitors (all from Sigma) are given in the figures and Table1. To measure transport in Na+-free medium, Na+ was replaced with equimolar choline chloride. Kinetic parameters were estimated by nonlinear regression analysis using a MichaelisMenten model, and Ki was calculated using a one-site competition model (PRISM Ver.2, GraphPad, San Diego, CA, USA).
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In situ hybridization
OnmyGLUT1 in pcDNA3.1-TOPO backbone (Invitrogen) was linearised with KpnI (MBI Fermentas) and used to generate the RNA probe. In vitro transcription with T7 RNA polymerase (MBI Fermentas) was carried out in the presence of DIG-oxigenin-11-UTP (Boehringer Mannheim). Embryos were fixed and processed as described previously (Joly et al., 1993) and the transcript was detected using a Boehringer Mannheim kit according to the manufacturers recommendations. Stained embryos mounted in glycerol were observed and photographed using an Olympus SZX9 stereomicroscope.
Hexose transport
Transport measurements were performed in dissociated embryonic cells using 2-deoxy-D-glucose (2-DOG); this hexose is phosphorylated by hexokinase. Embryos were excised from chorions and washed with phosphate-buffered saline (PBS) to remove yolk. To facilitate dissociation, embryos were incubated in calcium- and magnesium-free PBS for 30min. Embryos were triturated first with a glass Pasteur pipette and then with an automatic pipette equipped with a 1ml plastic tip. Cells were filtered through tissue paper, centrifuged for 30s at 600g and resuspended in PBS. Using the exclusion of erythrosin B as an indicator, over 95% of the cells were found to be alive. Uptake was initiated by adding incubation medium (100µl), which included PBS, 5mmoll-1 2-DOG and 1.0µCi (37.0kBq) of label (2-deoxy-D-[1-3H]glucose; Amersham Pharmacia Biotech). To account for carrier-independent binding of label, cytochalasin B (50µmoll-1) was used in parallel incubations. Cells were incubated with labelled 2-DOG for 20min with periodic shaking. To complete the reaction, the cells were loaded onto the surface of oil (1-bromododecane; Sigma) and centrifuged briefly at 13000g. The medium was removed by aspiration, and the oil surface was washed three times with PBS. After removal of the oil, the cells were lysed in water with 0.2% Triton X-100 and heated to 90°C for 3min. To measure total and non-phosphorylated 2-DOG, lysates were divided into two samples. Phosphorylated sugar was removed using treatment with Ba(OH)2 and Zn(SO)4 (as described by Colville et al., 1993). Equal volumes of water were added to the parallel samples. Protein was determined using a BioRad kit (catalog no. 50001210).
Hexokinase assay
Embryos were homogenized in 5mmoll-1 K2HPO4, 5mmoll-1 KH2PO4 (pH7.8 at 20°C), 0.5mmoll-1 EDTA, 250mmoll-1 sucrose and centrifuged at 900g for 2min to remove cell debris. The homogenate was added to a mixture containing 75mmoll-1 Tris (pH7.4), 7.5mmoll-1 MgCl2, 0.8mmoll-1 EDTA, 1.5mmoll-1 KCl, 0.4mmoll-1 NADP, 2.5mmoll-1 ATP and 0.7i.u.ml-1 glucose-6-phosphate dehydrogenase. The reaction was started by adding glucose to a final concentration of 1mmoll-1. Increases in light absorbance were measured at 340nm.
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Results |
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Hexose transport activity
Cytochalasin B (50µmoll-1) significantly decreased 3-OMG uptake by rainbow trout embryos at all analysed stages except the blastula (data not shown). To assess the contribution of transport by OnmyGLUT1 to hexose uptake, we carried out assays with 2-DOG (Table2). Hexose absorbed by dissociated embryonic cells was separated into phosphorylated and non-phosphorylated moieties, as described in Materials and Methods. Cytochalasin B decreased the content of 2-DOG phosphate (by 75.6%) in cells to a greater extent than that of non-phosphorylated 2-DOG (by 18.1%). This result suggested that transport might be rate-limiting for the delivery of glucose to embryonic fish cells. If the rate of uptake were limited by the rate of phosphorylation, inhibitors of transport (such as cytochalasin B) would have affected the accumulation of 2-DOG to a much greater extent than the accumulation of phosphorylated hexose. Hexokinase activity in rainbow trout embryos (1.127±0.037nmolmin-1µg-1protein, mean ± S.E.M., N=8) was two orders of magnitude greater than that of hexose transport and was therefore not rate-limiting in our experiments.
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Discussion |
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Competitive inhibition analyses (Table1) were designed to address the substrate specificity of OnmyGLUT1. Uptake of D-glucose was not inhibited by L-glucose, which suggested stereospecificity of transport. Transport activity for D-glucose decreased in the presence of 1-DOG, 2-DOG, 3-DOG and 6-DOG, suggesting that the removal of hydroxyl groups from these positions did not eliminate the ability to compete with D-glucose. D-Mannose, the C2 epimer of D-glucose, also decreased D-glucose transport, suggesting an insignificant role of equatorial hydroxyl in C2. Substitution or removal of the C3 hydroxyl group reduced transport activity. For example, OnmyGLUT1 showed higher activity with D-glucose than with 3-OMG. The inhibitory effects of 2-DOG and mannose were greater than those of 3-DOG and 3-OMG. D-Fructose, D-galactose, D-xylose, D-ribose, D-allose, L-sorbitol and mannitol did not compete with D-glucose. Similar results were obtained with human (Gould et al., 1991; Woodrow et al., 2000) and carp (Teerijoki et al., 2000; Teerijoki et al., 2001) GLUT1. Of all the vertebrate facilitative glucose transporters, OnmyGLUT1 is most similar to GLUT1, both from an analysis of its primary sequence and from its functional characteristics. Mammalian GLUT3 and GLUT4 are characterised by a higher affinity for D-glucose. The Km of GLUT2 for D-glucose is greater than that of OnmyGLUT1, and GLUT2 is capable of fructose transport.
We analysed hexose transport activity and expression of OnmyGLUT1 in embryos to investigate its importance for rainbow trout development. Uptake of 3-OMG by embryos was inhibited by cytochalasin B. We carried out assays with 2-DOG to determine the relative contributions of transport and phosphorylation to hexose uptake in embryonic cells. Accumulation of 2-DOG-phosphate was almost completely abolished by cytochalasin B, suggesting that metabolized sugar was absorbed predominantly via the facilitative carrier.
We studied the temporal and spatial patterns of OnmyGLUT1 expression in rainbow trout embryos. In mammals, expression of GLUT1 is markedly increased after fertilization. Both mRNA and GLUT1 protein are accumulated during pre-implantation in accord with an increase in glucose transport activity during development (Hogan et al., 1991; Morita et al., 1992; Dan-Goor et al., 1997). Using RT-PCR, the earliest stage at which OnmyGLUT1 expression was detected was the blastula. In situ hybridization analyses suggested that, at early developmental stages, OnmyGLUT1 was expressed in all cells. Transcripts were found throughout the embryo until the 57 somite stage (Fig.3B), but hybridization was most evident in the germ ring and embryo proper. These structures are known for their high density of cells and active morphogenetic processes. Restricted spatial patterns of OnmyGLUT1 expression were observed beginning from the 1415 somite stage (Fig.3C). In older embryos, expression declined during development along the rostro-caudal axis and was undetectable in the myotomes, which are the derivatives of the somites. Expression of mammalian GLUT1 is also reduced during the differentiation of skeletal muscle. Myogenic differentiation includes the fusion of myoblasts into multinuclear myotubes, with subsequent upregulation of muscle-specific protein expression. Guillet-Deniau et al. (Guillet-Deniau et al., 1994) reported the expression of different GLUT proteins at these stages; GLUT1 was abundant in myoblasts, expression of GLUT3 increased markedly at cell fusion and GLUT4 was found exclusively in myotubes.
Expression of OnmyGLUT1 was stable in embryonic neural tissues of the rainbow trout. The transcripts were abundant at the boundary between the neural plate and lateral ectoderm (Fig.3CF). In vertebrate embryos, this area harbours the cells of the neural crest (Artinger et al., 1999), which is an exceptionally important structure found exclusively in vertebrates (Baker and Bronner-Fraser, 1997; Gorodilov, 2000). The neural crest gives rise to the connective, skeletal and muscle tissues of the head; moreover, it produces skeleton, pigmented cells and nervous roots in the trunk/tail (Gans and Northcutt, 1983; Langille and Hall, 1989; Couly et al., 1993; Kontges and Lumsden, 1996). The detection of OnmyGLUT1 transcripts in putative derivatives of the neural crest (cartilaginous tissue of the pectoral fins, gill arches and upper jaw) confirmed that OnmyGLUT1 expression is likely to distinguish this cell population. Smoak and Branch (Smoak and Branch, 2000) reported a preponderance of GLUT1 protein in the heart and neural tube in mouse embryos during early organogenesis. An increase in GLUT1 expression in response to hypoglycaemia demonstrated the importance of the enzyme for the embryonic heart. We did not detect OnmyGLUT1 transcripts in the heart of rainbow trout embryos. This finding was unexpected since, in adult fish, this gene is expressed predominantly in the cardiac muscle (Teerijoki et al., 2000). Mammalian GLUT1 is expressed in virtually all embryonic and foetal tissues. In contrast, no OnmyGLUT1 transcripts were found in the notochord, kidney, liver and gut of the rainbow trout embryos.
The distribution of OnmyGLUT1 transcripts in rainbow trout embryos was clearly related to active morphogenetic processes. Expression was most stable in the neural crest cells, which are unique in their totipotent characteristics and extensive migration abilities. It is likely that demand for glucose is increased in differentiating cells because of high metabolic activity. Another possibility is that GLUT is substituted for other isoforms upon differentiation. Recent identification of salmonid GLUT4 and GLUT2 will allow this issue to be addressed.
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Acknowledgments |
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References |
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---|
Artinger, K. B., Chitnis, A. B., Mercola, M. and Driever, W. (1999). Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons. Development 126, 39693979.
Baker, C. V. H. and Bronner-Fraser, M. (1997). The origins of the neural crest. II. An evolutionary perspective. Mech. Dev. 69, 1329.[Medline]
Baldwin, S. A. (1993). Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins. Biochim. Biophys. Acta 1154, 1849.
Barrett, M. P., Walmsley, A. R. and Gould, G. W. (1999). Structure and function of facilitative sugar transporters. Curr. Opin. Cell Biol. 11, 496502.[Medline]
Burant, C. F. and Bell, G. I. (1992). Mammalian facilitative glucose transporters: evidence for similar substrate recognition sites in functionally monomeric proteins. Biochemistry 31, 1041410420.[Medline]
Burant, C. F., Sivitz, W. I., Fukumoto, H., Kayano, T., Nagamatsu, S., Seino, S., Pessin, J. E. and Bell, G. I. (1991). Mammalian glucose transporters: structure and molecular regulation. Recent Progr. Horm. Res. 47, 349388.[Medline]
Colville, C. A., Seatter, M. J., Gould, G. W. and Thomas, H. M. (1993). Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors. Biochem. J. 290, 701706.[Medline]
Couly, G. F., Coltey, P. M. and Le Douarin, N. M. (1993). The triple origin of skull in higher vertebrates: a study in quailchick chimeras. Development 117, 409429.
Dan-Goor, M., Sasson, S., Davarashvili, A. and Almagor, M. (1997). Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos. Human Reprod. 12, 25082510.[Abstract]
Gans, C. and Northcutt, R. G. (1983). Neural crest and origin of vertebrates: a new head. Science 220, 268274.
Gorodilov, Y. N. (1989). Comparative analysis of the dynamics of early ontogeny of species of genus Salmo. J. Ichtyol. 29, 1931.
Gorodilov, Y. N. (1996). Description of the early ontogeny of the Atlantic salmon, Salmo salar, with a novel system of interval (state) identification. Env. Biol. Fish. 47, 109127.
Gorodilov, Y. N. (2000). The fate of Spemanns organizer. Zool. Sci. 17, 11971220.
Gould, G. W., Thomas, H. M., Jess, T. J. and Bell, G. I. (1991). Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver and brain isoforms. Biochemistry 30, 51395145.[Medline]
Guillet-Deniau, I., Leturque, A. and Girard, J. (1994). Expression and cellular localization of glucose transporters (GLUT1, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat foetuses. J. Cell Sci. 107, 487496.
Hogan, A., Heyner, S., Charron, M. J., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Thorens, B. and Schultz, G. A. (1991). Glucose transporter gene expression in early mouse embryos. Development 113, 363372.[Abstract]
Joly, J. S., Joly, C., Schulte-Merker, S., Boulekbache, H. and Condamine, H. (1993). The ventral and posterior expression of the even-skipped homeobox gene eve 1 is perturbed in dorsalized and mutant embryos. Development 119, 12611275.
Kayano, T., Burant, C. F., Fukumoto, H., Gould, G. W., Fan, Y. S., Eddy, R. L., Byers, M. G., Shows, T. B., Seino, S. and Bell, G. I. (1990). Human facilitative glucose transporters. Isolation, functional characterization and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J. Biol. Chem. 265, 1327613282.
Köntges, G. and Lumsden, A. (1996). Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development 122, 32293242.
Langille, R. M. and Hall, B. K. (1989). Developmental processes, developmental sequences and early vertebrate phylogeny. Biol. Rev. 64, 7391.[Medline]
Morita, Y., Tsutsumi, O., Hosoya, I., Taketani, Y., Oka, Y. and Kato, T. (1992). Expression and possible function of glucose transporter protein GLUT1 during preimplantation mouse development from oocytes to blastocysts. Biochem. Biophys. Res. Commun. 188, 815.[Medline]
Mueckler, M. (1990). Family of glucose-transporter genes. Implications for glucose homeostasis and diabetes. Diabetes 39, 611.[Abstract]
Penny, J. I., Hall, S. T., Woodrow, C. J., Cowan, G. M., Gero, A. M. and Krishna, S. (1998). Expression of substrate-specific transporters encoded by Plasmodium falciparum in Xenopus laevis oocytes. Mol. Biochem. Parasitol. 93, 8189.[Medline]
Planas, J. V., Capilla, E. and Gutierrez, J. (2000). Molecular identification of a glucose transporter from fish muscle. FEBS Lett. 481, 266270.[Medline]
Postic, C., Leturque, A., Printz, R. L., Maulard, P., Loizeau, M., Granner, D. K. and Girard, J. (1994). Development and regulation of glucose transporter and hexokinase expression in rat. Am. J. Physiol. 266, E548E559.
Santalucia, T., Camps, M., Castello, A., Munoz, P., Nuel, A., Testar, X., Palacin, M. and Zorzano, A. (1992). Developmental regulation of GLUT-1 (erythroid/Hep G2) and GLUT-4 (muscle/fat) glucose transporter expression in rat heart, skeletal muscle and brown adipose tissue. Endocrinology 130, 837846.[Abstract]
Smoak, I. W. and Branch, S. (2000). Glut-1 expression and its response to hypoglycemia in the embryonic mouse heart. Anat. Embryol. 201, 327333.[Medline]
Soengas, J. L. and Moon, T. W. (1995). Uptake and metabolism of glucose, alanine and lactate by red blood cells of the American eel Anguilla rostrata. J. Exp. Biol. 198, 877888.
Stein, W. D. (1990). Channels, Carriers and Pumps: An Introduction to Membrane Transport. San Diego: Academic Press. 137pp.
Teerijoki, H., Krasnov, A., Pitkänen, T. I. and Mölsä, H. (2000). Cloning and characterization of teleost fish rainbow trout (Oncorhynchus mykiss) glucose transporter. Biochim. Biophys. Acta 1493, 290294.
Teerijoki, H., Krasnov, A., Pitkänen, T. I. and Mölsä, H. (2001). Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by facilitative glucose transporter. Comp. Biochem. Physiol. 128B, 483491.
Woodrow, C. J., Burchmore, R. J. and Krishna, S. (2000). Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes. Proc. Natl. Acad. Sci. USA 97, 99319936.