Rosendale AJ et al. (2014), Cloning, characterization, and expression of gl...

XB-ART-48408

Biochim Biophys Acta 2014 Jun 01;18406:1701-11. doi: 10.1016/j.bbagen.2013.12.011.

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Cloning, characterization, and expression of glucose transporter 2 in the freeze-tolerant wood frog, Rana sylvatica.

Rosendale AJ , Philip BN , Lee RE , Costanzo JP .

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The essential role of glucose transporter 2 (GLUT2) in glucose homeostasis has been extensively studied in mammals; however, little is known about this important protein in lower vertebrates. The freeze-tolerant wood frog (Rana sylvatica), which copiously mobilizes glucose in response to freezing, represents an excellent system for the study of glucose transport in amphibians. GLUT2 was sequenced from northern and southern phenotypes of R. sylvatica, as well as the freeze-intolerant Rana pipiens. These proteins were expressed and functionally characterized in Xenopus oocytes. Abundance of GLUT2 in tissues was analyzed using immunoblotting techniques. GLUT2s cloned from these anurans encoded proteins with high sequence homologies to known vertebrate GLUT2s and had similar transport properties, although, notably, transport of the glucose analog 3-O-methyl-d-glucose (3-OMG) was strongly inhibited by 150mM urea. Proteins from all study subjects had similar affinity constants (~12mM) and other kinetic properties; however, GLUT2 abundance in liver was 3.5-fold greater in northern R. sylvatica than in the southern conspecific and R. pipiens. Our results indicate that amphibian GLUT2s are structurally and functionally similar to their homologs in other vertebrates, attesting to the conserved nature of this transport protein. The greater abundance of this protein in the northern phenotype of R. sylvatica suggests that these transporters contribute importantly to freezing survival. This study provides the first functional characterization of any GLUT isoform from an anuran amphibian and novel insights into the role of these proteins in glucose homeostasis and cryoprotectant mobilization in freeze-tolerant vertebrates.

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Species referenced: Xenopus Xenopus tropicalis Xenopus laevis
Genes referenced: omg slc2a1 slc2a2 slc2a3 slc2a4

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	Fig. 1. Sequences of newly-identified, ranid glucose transporter 2 (GLUT2) in relation to GLUTs known from other taxa. A: Deduced amino acid sequences of newly-identified GLUT2s from Ohioan Rana sylvatica (OH), Alaskan R. sylvatica (AK), and R. pipiens (Rp), aligned with GLUT2 from Xenopus laevis (Xl; accession no. NP_001084982) and Homo sapiens (Hs; accession no. NP_000331) using ClustalW in BioEdit v7.0.9.0. Gaps in the amino acid sequences are indicated with a dash (–). Amino acids that are identical (*), highly conserved (:), or moderately conserved (.) among all five sequences are identified. Box highlights a putative N-glycosylation site; horizontal bars and numbers indicate the putative 12 transmembrane regions. Amino acids that are specific to members of class 1 glucose transporters are highlighted with black; residues conserved among all GLUTs are highlighted with gray; and the HVA motif characteristic of mammalian GLUT2 is underlined [9]. B: Three-dimensional ribbon model of ranid GLUT2s, X. laevis GLUT2, and H. sapiens GLUT2 demonstrating structural alignment of multiple proteins (STAMP) tool within the Visual Molecular Dynamics (VMD) program. Structurally-conserved regions (blue) were found primarily within the membrane-spanning regions; whereas, less structural conservation (red) was found in the connecting loops.
	Fig. 2. Phylogenetic tree demonstrating the relationships among the newly-identified, functional ranid GLUT2s, Ohioan Rana sylvatica (Rs OH), Alaskan R. sylvatica (Rs AK), and R. pipiens (Rp); and functionally-characterized and/or predicted GLUTs from other vertebrates including Homo sapiens (Hs), Mus musculus (Mm), Rattus norvegicus (Rn), Gallus gallus (Gg), Xenopus laevis (Xl), Gadus morhua (Gm), Oncorhynchus mykiss (Om), Ctenopharyngodon idella (Ci), and Salmo trutta (St). Accession numbers: H. sapiens GLUT1 (AAA52571), M. musculus GLUT1 (AAA37752), R. norvegicus GLUT1 (P11167), G. gallus GLUT1 (AAB02037), X. laevis GLUT1 (NP_001088068), O. mykiss GLUT1 (AAF75681), G. morhua GLUT1 (AAS17880), H. sapiens GLUT2 (AAA59514), M. musculus GLUT2 (P14246), R. norvegicus GLUT2 (P12336), G. gallus GLUT2 (Q90592), X. laevis GLUT2 (NP_001084982), O. mykiss GLUT2 (AAK09377), G. morhua GLUT2 (AAV63984), R. sylvatica OH GLUT2 (KF270880), R. sylvatica AK (KF270881), R. pipiens (KF270882), H. sapiens GLUT3 (AAB61083), M. musculus GLUT3 (AAH34122), R. norvegicus GLUT3 (Q07647), G. gallus GLUT3 (AAA48662), X. laevis GLUT3 (NP_001079713), C. idella GLUT3 (AAP03065), G. morhua GLUT3 (AAT67456), H. sapiens GLUT4 (AAA59189), M. musculus GLUT4 (P14142), R. norvegicus GLUT4 (P19357), X. laevis GLUT4 (NP_001085607), S. trutta GLUT4 (AAG12191), and G. morhua GLUT4 (AAZ15731). The tree was constructed using the neighbor-joining method with Poisson correction. The number of substitutions per amino acid site is represented in the scale bar. Bootstrap proportions (10,000 replicates) are indicated above the nodes.
	Fig. 3. Kinetics of glucose transport in Xenopus oocytes expressing RsOH-GLUT2 (▲, dashed line), RsAK-GLUT2 (●, dotted line), or Rp-GLUT2 (■, solid line). Prior to the assay, oocytes were injected with 50 nl of GLUT2 cRNA (300 ng μl− 1). A: 3-O-methyl-d-glucose (3-OMG) uptake measured under zero-trans conditions at various 3-OMG concentrations. Uptake values reflect measured 3-OMG concentrations minus average values obtained for respective sham-injected oocytes. B: Lineweaver–Burk plot from the linearization of data acquired in zero-trans kinetic studies. Values are means ± SEM. (n = 8–10 oocytes). C: 3-OMG export was measured under zero-trans conditions at various times. Export values reflect measured 3-OMG concentrations minus average values obtained for respective sham-injected oocytes. Values are means ± SEM (n = 8–10 oocytes).
	Fig. 4. Inhibition of 3-O-methyl-d-glucose (3-OMG) uptake in Xenopus oocytes injected with RsOH-GLUT2 cRNA. A: Inhibition of 3-OMG uptake in RsOH-GLUT2-expressing oocytes pre-incubated with phloretin or cytochalasin B. B: Substrate-specificity of Rs-GLUT2 assessed by competition assay with d- and l-glucose, galactose, mannose, and fructose. Uptake of 3-OMG is expressed as a percentage of uptake into oocytes incubated without inhibitors or hexoses. Uptake values reflect measured 3-OMG concentrations minus average values obtained for respective sham-injected oocytes. Values are means ± SEM (n = 8–10 oocytes). Letters that differ from one another indicate significant differences between group means (P < 0.05).
	Fig. 5. Effect of urea on 3-O-methyl-d-glucose (3-OMG) transport in Xenopus oocytes injected with RsOH-GLUT2, RsAK-GLUT2, or Rp-GLUT2 cRNA. A: Inhibition of 3-OMG uptake by urea. Oocytes were preincubated with 0 mM (black bars), 50 mM (gray bars), or 150 mM (dark gray bars) urea prior to 3-OMG uptake experiments. Uptake of 3-OMG is expressed as a percentage of uptake into oocytes incubated without urea. B: Inhibition of 3-OMG export by urea in oocytes expressing RsOH-GLUT2. Oocytes were pre-incubated with 3-OMG, then transferred to MBS containing 0 mM (black bars), 50 mM (gray bars), or 150 mM (dark gray bars) urea for 0, 5, or 10 min. 3-OMG values reflect measured 3-OMG concentrations minus average values obtained for respective sham-injected oocytes. Values are means ± SEM (n = 8–10 oocytes). Mean values identified by different letters were statistically distinguishable (P < 0.05).
	Fig. 6. Tissue distribution of GLUT2 protein from Ohioan Rana sylvatica examined by immunoblotting. A: Immunoblot of GLUT2 protein in Ohioan R. sylvatica liver using an antibody (Ab) designed against the ranid GLUT2; a single band was detected at 54 kDA. Ab specificity was confirmed by probing Xenopus oocytes injected with RsOH-GLUT2 (RsOH), RsAK-GLUT2 (RsAK), Rp-GLUT2 (Rp) cRNA, or nuclease-free water (sham). B: Select immunoblots of tissues (20 μg protein) probed with the anti-GLUT2 antibody, showing immunoreactive bands at 54 kDa (bands not intended to show relative abundance). Relative abundance of GLUT2 protein, standardized to total protein, among tissues was determined by densitometry. Intensity values are mean ± SEM (n = 6).
	Fig. 7. Relative abundance of GLUT2 protein in the liver of Ohioan Rana sylvatica (Rs OH), Alaskan R. sylvatica (Rs AK), and R. pipiens (Rp) determined by immunoblotting. Tissues (20 μg protein) were probed with an antibody designed against the ranid GLUT2 and relative abundance of GLUT2 protein, standardized to total protein, was determined by densitometry. Intensity values are mean ± SEM (n = 6). Different letters indicate significant differences among groups (P < 0.001).