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First published online December 3, 2004
Journal of Experimental Biology 207, 4697-4706 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01346

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Sequence and expression of a constitutive, facilitated glucose transporter (GLUT1) in Atlantic cod Gadus morhua

Jennifer R. Hall, Tyson J. MacCormack, Catherine A. Barry and William R. Driedzic^*

Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, A1C 5S7, Canada

View larger version (21K): [in a new window]   Fig. 1. Phylogenetic analysis of Atlantic cod GLUT1. A phylogenetic tree of protein sequences from cod GLUT1 and GLUTs 1-4 from other vertebrates. SwissProt accession numbers are as follows: GLUT1 common carp (AAF75683, GLUT1 rainbow trout (AAF75681, GLUT1 Atlantic cod (AAS17880, (AAB02037, GLUT1 chicken GLUT1 human (AAA52571, GLUT1 rabbit (P13355), GLUT1 cow (P27674), GLUT1 rat (P11167), GLUT1 mouse (AAA37752, GLUT2 chicken (Q90592), GLUT2 rainbow trout (AAK09377, GLUT2 human (AAA59514, GLUT2 mouse (P14246), GLUT2 rat (P12336), GLUT3 cow (AAK70222, GLUT3 dog (P47842), GLUT3 human (AAB61083, GLUT3 mouse (AAH34122, GLUT3 rat (Q07647), GLUT3 chicken (AAA48662, GLUT4 cow (Q27994), GLUT4 human (AAA59189, GLUT4 mouse (P14142), GLUT4 rat (P19357), GLUT4 coho salmon (AAM22227, GLUT4 brown trout (AAG12191, GLUT grass carp (AAP03065, GLUT pacific hagfish (AAL27090. The 28 GLUT protein sequences were aligned using the CLUSTAL W alignment mode in AlignX. The alignment was imported into MEGA version 2.1 and the phylogenetic tree constructed using the neighbor-joining method with Poisson correction. Bootstrap analysis was performed with 1000 replicates. Scale bar shows Tamura Nei distances.

View larger version (78K): [in a new window]   Fig. 2. Amino acid alignment of cod GLUT1 with GLUT1s from human (hGLUT1; AAA52571, common carp (ccGLUT1; AAF75683 and rainbow trout (rtGLUT1; AAF75681. Amino acids are represented by the single letter code. Dots are inserted where gaps are present in the amino acid sequences. Numbers correspond to the human GLUT1 sequences. Asterisks indicate residues that are identical among all four sequences shown.

View larger version (47K): [in a new window]   Fig. 3. Sequence analysis of Atlantic cod GLUT1. Amino acids are represented by the single letter code and numbered on the right. Boxes numbered with Roman numerals represent the putative 12 transmembrane helices. Residues that are highly conserved in all glucose transporters are highlighted in gray, while those that are specific to class I glucose transporters are highlighted in black. It should be noted that the PETKGR motif varies in Atlantic cod GLUT1 in that the K is substituted by an R.

View larger version (50K): [in a new window]   Fig. 4. Tissue distribution of Atlantic cod GLUT1. (A) Northern blot analysis of total RNA from various cod tissues hybridized with a 664 bp antisense RNA probe specific for Atlantic cod GLUT1. Top panel shows the 4560 bp GLUT1 mRNA transcript; bottom panel the 18S rRNA bands, as detected by ethidium bromide staining. MW, RNA marker (kb). (B) RT-PCR analysis. Top panel shows a 642 bpfragment amplified using primers located within the Atlantic cod GLUT1 ORF; bottom panel a 617 bp fragment amplified using cod ß-actin-specific primers. MW, DNA marker (bp).

View larger version (26K): [in a new window]   Fig. 5. Developmental expression patterns for Atlantic cod GLUT1. Two sets of Atlantic cod (A,B) were analyzed for GLUT1 expression at various stages of development as indicated, from eggs to small fish, by RT-PCR. GLUT1 is expressed at all developmental stages examined. Letters above age of post-hatch fish refer to food offered: T-ISO, T-ISO enriched rotifers; AER, algamac 2000 enriched rotifers; ART, enriched artemia; DF, dry food (see Materials and methods for details). MW, DNA marker (bp).

View larger version (63K): [in a new window]   Fig. 6. The effects of hypoxia on GLUT1 expression in heart (A), brain (B), gill (C) and kidney (D). Atlantic cod were exposed to 6 h of normoxia (N=6) or hypoxia (40% DO2; N=7). GLUT1 expression in tissues that highly express GLUT1 was analyzed by northern blot and normalized to 28S rRNA levels.

View larger version (21K): [in a new window]   Fig. 7. The effects of temperature on GLUT1 expression in heart. Atlantic cod were either held at 8°C (N=10) or allowed to follow ambient seawater temperatures (N=10). Fish were sampled in early June when ambient seawater temperature had risen to 4°C after an average temperature of –0.5°C over the winter months. GLUT1 mRNA levels were analyzed by northern blot and normalized to 28S rRNA levels.

View larger version (22K): [in a new window]   Fig. 8. The effects of food deprivation on GLUT1 expression in heart. Atlantic cod (N=10) were food-deprived for 2 months and GLUT1 mRNA levels compared (by northern blot analysis) with that of Atlantic cod (N=10) fed a commercial diet over the same time period. GLUT1 levels were normalized to 28S rRNA.

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