Sequence and expression of a constitutive, facilitated glucose transporter (GLUT1) in Atlantic cod Gadus morhua
Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, A1C 5S7, Canada
* Author for correspondence (e-mail: wdriedzic{at}mun.ca)
Accepted 15 October 2004
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Summary |
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Key words: Atlantic cod, glucose transporter, Gadus morhua, GLUT1, heart, hypoxia, larval development
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Introduction |
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The structure and tissue distribution of GLUT1 in fish has been addressed
but requires further resolution. Using antibodies to mammalian GLUT1, Wright
et al. (1998) reported that
the protein was abundant in tilapia heart, present in brain, but undetectable
in skeletal muscle, liver, adipose tissue or islet cells. A putative GLUT1
cDNA was cloned from rainbow trout alevins. The deduced amino acid sequence
was 7779% identical to avian and mammalian GLUT1 proteins and
transcripts were detectable shortly after fertilization of eggs. In
situ hybridization revealed transcript distribution in a variety of cell
types during embryogenesis. Micro injection of this GLUT1 mRNA into
Xenopus laevis oocytes resulted in Na+-independent glucose
transport that was inhibited by cytochalasin B and phloretin. These features
and competitive inhibition studies with a number of other sugars support the
view that the transcript was indeed similar to a mammalian GLUT1. It was also
stated but data were not provided, that in adult rainbow trout, abundant
transcripts were detected in heart with low level of expression in skeletal
muscle, liver, spleen, blood and brain (Teerijoki et al.,
2000
,
2001a
). Capilla et al.
(2002
) further reported low
levels of GLUT1 transcript in red and white muscle of rainbow trout. A glucose
transporter has also been sequenced from the carp cell line, epithelioma
papulosum cyprini, and again, based upon deduced amino acid sequence and
hexose uptake characteristics, is similar to a mammalian GLUT1
(Teerijoki et al., 2001b
).
In this study, we determine the sequence of GLUT1 from Atlantic cod
(Gadus morhua). Expression during development from fertilization to
larval fish, and transcript tissue distribution in adult fish, are assessed.
We also address the impact of hypoxia, fasting and temperature challenge on
gene expression with a particular emphasis on heart. We focus on heart,
because GLUT1 is found in both tilapia and rainbow trout hearts
(Wright et al., 1998;
Teerijoki et al., 2000
),
facilitated glucose transport is essential to the performance and glucose
uptake of Atlantic cod heart under hypoxic conditions via an unknown
glucose transporter (Clow et al.,
2004
), and low temperature favors fatty acids as opposed to
glucose as the preferred fuel of metabolism in the fish heart
(Driedzic et al., 1996
). In
addition, we assess the impact of food deprivation on heart GLUT1 expression
since in the rat heart fasting leads to a substantial decrease in GLUT1 mRNA
level (Kraegen et al.,
1993
).
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Materials and methods |
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Preparation of RNA and DNA from tissues
For RNA preparation, when long-term storage was required, tissues were
stored in RNAlater (QIAGEN Inc., Mississauga, ON, Canada) according
to the manufacturer's protocol. For northern blot analysis, total RNA was
extracted from tissues using Trizol Reagent (Invitrogen, Burlington, ON,
Canada). For expression analysis using RT-PCR, the Trizol generated RNA was
treated with Amplification Grade DNaseI (Invitrogen, Burlington, ON, Canada)
prior to reverse transcription. For cDNA cloning, RNA was immediately
extracted from fresh tissue and concurrently treated with DNaseI using the
RNeasy Mini Kit (QIAGEN Inc., Mississauga, ON, Canada). Poly(A)+
RNA was isolated from total RNA using the Oligotex mRNA Mini Kit (QIAGEN Inc.,
Mississauga, ON, Canada). RNA was quantitated by UV absorption at 260 nm. RNA
purity was determined by calculating the OD260/OD280
ratio.
For genomic DNA preparation, liver was snap frozen in liquid nitrogen and stored at 80°C. Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. DNA was quantitated as described for RNA.
cDNA cloning
Atlantic cod GLUT1 cDNA was cloned using a combination of RT-PCR, RLM-RACE
and genome walking. The sequences of all primers used in cDNA cloning are
presented in Table 1.
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To clone the central portions, total RNA was reverse-transcribed with an
oligo(dT) primer using Superscript II RNase H Reverse
Transcriptase (Invitrogen, Burlington, ON, Canada). PCR amplification was
performed using Taq DNA Polymerase (Invitrogen, Burlington, ON, Canada).
Touchdown PCR was used with 40 cycles of 94°C for 1 min,
65°C0.5°C per cycle for 1.5 min and 72°C for 1 min. To
obtain the first fragment, primers were designed based upon consensus
sequences from conserved areas of aligned vertebrate GLUT1s. Primers 1 and 2
generated a 664-nucleotide product. The PCR product was electrophoresed on a
1.5% agarose gel, excised and purified using the QIAquick Gel Extraction Kit
(QIAGEN Inc., Mississauga, ON, Canada). The product was then subcloned into
pCRII-TOPO (Invitrogen, Burlington, ON, Canada) and sequenced on both strands
at MOBIX, McMaster University, using ABI BigDye terminator chemistry and
ABIPRISM® 3100 Genetic Analyzer. This partial cod GLUT1 cDNA sequence was
aligned with sequence from two other glucose transporters cloned from cod, to
which GLUT1 had 65.3% and 63.2% sequence identity (J.R.H. and W.R.D.,
unpublished data). Primer 3 was designed in an area to specifically amplify
GLUT1 and used in conjunction with the degenerate primer (Primer 4) to obtain
a 702-nucleotide fragment that yielded, in conjunction with the first PCR
product, a total of 1273-nucleotides of ORF sequence data.
The 5' end of cod GLUT1 was cloned with a commercial kit for RLM-RACE, GeneRacer Kit (Invitrogen, Burlington, ON, Canada) using poly(A)+ RNA. PCR amplification was performed at 94°C for 1 min, 68°C for 2 min for 10 cycles followed by 94°C for 1 min, 65°C for 1 min and 68°C for 1.5 min for 30 cycles using Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Burlington, ON, Canada). The gene-specific Primer 5 was used with the GeneRacer 5' primer for the primary PCR and Primer 6 with the GeneRacer 5' nested primer for nested PCR. The 545-nucleotide product contained the 149-nucleotide 5'UTR and the remaining 76 nucleotides of 5' coding sequence.
The 3' end of cod GLUT1 was obtained using a combination of genome walking and 3' RLM-RACE. Attempts to clone the remaining 3' sequences by 3' RACE at this point were unsuccessful. This was not surprising since northern blot analysis indicated that the mRNA for cod GLUT1 was approximately 4.6 kb, leaving about 3.7 kb of unknown 3' sequence. Therefore, genome walking was chosen as it would break up the long 3' UTR into more reasonable fragments based upon the presence of restriction enzyme sites within. GenomeWalker libraries for DraI, EcoRV, MscI, MslI, PvuII, SspI and StuI were constructed using the Universal GenomeWalker Kit (BD Biosciences Clontech, Palo Alto, CA, USA) according to the manufacturer's protocol. The first walk was performed using Primer 7 and the GenomeWalker Adaptor Primer 1 (AP1). The reaction was diluted 1/50 and 1 µl used as the template for nested PCR using Primer 8 and the GenomeWalker Nested Adaptor Primer 2 (AP2). Both the primary and nested PCR amplifications were performed at 94°C for 10 s, 72°C for 3 min for 7 cycles, followed by 94°C for 10 s, 67°C for 30 s, 68°C for 3 min for 32 cycles, using Elongase Enzyme Mix (Invitrogen, Burlington, ON, Canada). Bands were obtained for MslI, DraI, SspI, PvuII and StuI, the largest of which was the 1.6 kb band for StuI. Although this band contained a predicted intron (191 bp), the remaining 218 bp of coding sequence and 1070 bp of the 3' UTR were resolved using GENSCAN.
These data were used to design primers for 3' RACE using the
GeneRacer Kit (Invitrogen, Burlington, ON, Canada) and poly(A)+
RNA. The primary PCR was performed using Primer 9 and the GeneRacer 3'
primer. A 1 µl aliquot of the primary PCR reaction was used as the template
for nested PCR using Primer 10 and the GeneRacer 3' nested primer. PCR
amplification was performed using Touchdown PCR with 40 cycles of 94°C for
30 s, 70°C0.3°C per cycle for 30 s and 72°C for 3.5 min
using DyNAzyme EXT (MJ Research, Waltham, MA, USA). A 1 kb band was generated
that matched the sequence obtained by genome walking. However, as northern
blot analysis indicates that the mRNA transcript is about 4.6 kb, this is a
truncated product. The sequence generated by the StuI walk shows the
presence of an A-rich sequence (8As), which corresponds to the position where
the 3' RACE transcript was terminated. Although this sequence is
preceded by a potential poly(A) site (ATTAAA), the truncated product is most
probably the result of the mispriming of the oligo(dT) to the A-rich sequence
within the mRNA.
To circumvent this truncated RACE transcript and generate further 3' UTR sequence, another genome walk was performed as previously described using Primers 11 and AP1 for the primary PCR and Primers 12 and AP2 for the nested PCR. A band was generated from the DraI library that generated sequence data to position 3431.
A second 3' RACE was then performed using primers downstream of the alternative poly(A) site as previously described. Primer 13 and the GeneRacer 3' primer were used for the primary PCR and Primer 14 and the GeneRacer 3' nested primer for the nested PCR. A band was generated that uses an alternative poly(A) site at position 3996.
Still being about 600 bp from the true poly(A) signal, a third genome walk was performed to circumvent the second alternative poly(A), as previously described. Primer 15 was used with AP1 for the primary PCR and Primer 16 with AP2 for the nested PCR. A band was generated from the SspI library to generate sequence to position 4639. A potential poly(A) signal was present at position 4537 that would generate a mRNA transcript of the size predicted by northern blot.
To determine if this poly(A) site is used, a third 3' RACE was performed using Primer 17 with the GeneRacer 3' primer for the primary PCR and Primer 18 and the GeneRacer 3' nested primer for the nested PCR. A band was obtained which uses this poly(A) signal. Based on the transcript size predicted by northern blot, this is probably the true poly(A) signal. The poly(A) tail is located 15 bp downstream of this site, yielding a total transcript size of 4560 bp.
Sequence analysis
Sequence data was compiled and analyzed using Vector NTI v. 6.0 (Informax
Inc., Bethesda, MD, USA). Alignments were performed using AlignX (Informax
Inc., Bethesda, MD, USA) which uses the CLUSTAL W algorithm
(Thompson et al., 1994). For
phylogenetic and molecular evolutionary analyses, alignments were imported in
MSF format into MEGA version 2.1
(Kumar et al., 2001
).
Phylogenetic trees were constructed using the neighbor-joining method
(Saitou and Nei, 1987
) with
Poisson correction. Bootstrap analysis was performed with 1000 replicates.
Exon/intron boundaries from genome walking sequences were analyzed using
GENSCAN
(http://genes.mit.edu/GENSCAN.html).
Transmembrane helices were predicted using HMMTOP
(http://www.enzim.hu/hmmtop)
(Tusnady and Simon, 1998
,
2001
).
Northern blot analysis
Total RNA (12 µg) was electrophoresed on a formaldehyde-agarose
gel and the ribosomal RNA bands visualized by ethidium bromide staining. The
RNA was then transferred to a positively charged nylon membrane (Roche, Laval,
QC, Canada) using a vacuum blotter (Bio-Rad, Mississauga, ON, Canada) and UV
crosslinked (UV Stratalinker, Stratagene, La Jolla, CA, USA). The membrane was
pre-hybridized at 68°C for 1 h in DIG Easy Hyb (Roche, Laval, QC, Canada).
Antisense RNA probes were generated from cDNA encoding the region from 259 to
923 of the open reading frame that was subcloned into pCRII-TOPO (Invitrogen,
Burlington, ON, Canada). The plasmid was linearized with HindIII and
the RNA labeled in an in vitro transcription reaction with
digoxigenin-11-UTP and T7 RNA polymerase according to the manufacturer's
protocol (DIG Northern Starter Kit, Roche, Laval, QC, Canada). The membrane
was hybridized with denatured probe at 100 ng ml1 in DIG
Easy Hyb at 68°C overnight. The membrane was then processed according to
the manufacturer's protocol (DIG Northern Starter Kit, Roche, Laval, QC,
Canada). The bands were visualized by exposure to X-ray film for 530
min. X-ray films were scanned using the Alpha Imager 1220 Documentation and
Analysis System (Alpha Innotech Corporation, San Leandro, CA, Canada).
Densitometry was performed on the bands using the 1D-Multi Line Densitometry
software program.
RT-PCR Analysis of GLUT1 expression
For RT-PCR, 1 µg of DNaseI-treated total RNA was reverse transcribed
using M-MLV reverse transcriptase (Invitrogen, Burlington, ON, Canada).
For analysis of GLUT1 expression, PCR amplification was performed using Taq DNA Polymerase (Invitrogen, Burlington, ON, Canada) with 40 cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 1 min. A 642 nucleotide PCR product, which encodes the region from 294 to 936 of the open reading frame, was amplified using the forward primer 5'-GTGATCGAGAACTTCATCAACGA-3' and the reverse primer 5'-CATCATCTGCCGGTGCTCGCT-3'. These primers were designed in areas to specifically amplify cod GLUT1 when aligned with sequences from two other cod GLUT transporters (J.R.H. and W.R.D., unpublished data).
For normalization purposes, Atlantic cod actin (GenBank accession number U48856) was amplified using Taq DNA Polymerase (Invitrogen, Burlington, ON, Canada) with 40 (for developmental experiments) or 35 (for tissue distribution experiments) cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 1 min. The 617 nucleotide product was amplified using the forward primer 5'-CCTGAACCCCAAGGCCAACAGA-3' and the reverse primer 5'-TCTGCATGCGGTCAGCGATACC-3'.
In vivo experiments
Developmental expression patterns
GLUT1 expression was monitored throughout the developmental period of
Atlantic cod from egg to larval fish at intervals corresponding to changes in
diet. The post hatch diet consisted of T-ISO enriched rotifers (to day 10),
algamac 2000 enriched rotifers (to day 40), enriched artemia (to day 50) and
then dry food (Shur Gain, Truro, NS, Canada). Eggs were sampled from two
batches at fertilization (fertilization day 0), 7 days post fertilization
(pre-hatch day 7) and upon hatching, approximately 1314 days post
fertilization (day 0). Post-hatch samples were then taken at day 5 (T-ISO
enriched rotifers), day 22 (algamac 2000 enriched rotifers), day 44 (enriched
artemia) and day 55 (dry food). Typical body mass of day 55 larvae was about
100 mg. At each interval, for each batch approximately 100 mg of biomass was
used in the RNA extraction. Samples were pooled, if necessary, to achieve the
required amount of biomass.
Hypoxia challenge
Animals (body mass 396±31 g) were held at 8±0.4°C in a
blackened, temperature-controlled cooler containing 35 l ofwater, in late
spring. Aeration was achieved by gassing with an air pump supplying 100%
O2. Dissolved oxygen was monitored by pumping water from the cooler
to a DO2 electrode (VWR model 4000 DO2 meter) housed in
a Plexiglas chamber, and back to the cooler at 60ml min1.
Dissolved oxygen was reduced to selected values in less than 5 min by gassing
with 100% N2 and maintained within 4% of the desired level. When
necessary, DO2 levels were increased by gassing with 100%
O2.
For hypoxia trails, fish were placed in the chamber under normoxia and held for 1 h before DO2 levels were reduced. After 1 h, oxygen was reduced to 60% and held for 30 min. DO2 was then reduced to 40% and held for 6 h. Observations on temperature and DO2 (mg l1) were collected at the end of each 30 min interval.
For normoxia trials, animals were held under identical conditions to those described above except that DO2 in the chamber was maintained at approximately 100% saturation as described above. Animals were placed in the cooler 1 h before the experiment and observations were made on DO2 and temperature every 60 min for a total of 7 h.
Temperature effects
Atlantic cod were divided into two groups and held in identical aerated,
flow-through seawater 8100 l tanks and followed natural photoperiod. Both
groups were fed a commercial diet (Shur Gain, Truro, NS, Canada). One group
(body mass 298±29 g) was held in 8°C seawater whereas the other
(body mass 195±23 g) was allowed to follow ambient seawater
temperature. Cod were sampled in early June, when ambient seawater temperature
had risen to 4°C from an average temperature of 0.5°C over the
winter months.
Fasting
Atlantic cod (body mass 246±16 g) were divided into two groups and
held in identical aerated, flow-through seawater 2000 l tanks at 8°C and
natural photoperiod. One group was fed a commercial diet (Shur Gain, Truro,
NS, Canada) while the other was deprived of food for 2 months, from January to
March.
Statistical analysis
Expression levels are presented as mean ± S.E. A
two-tailed t-test was used to assess if there was any significant
difference in GLUT1 expression between experimental groups. A P value
less than 0.05 was considered to be significant.
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Results |
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Deduced amino acid sequence of cod GLUT1 protein
The 1470 nucleotide cod GLUT1 encodes a 489 aa protein that has a predicted
molecular mass of 53.5 kDa and an isoelectric point of 8.97.
Phylogenetic analysis (Fig. 1) was performed to determine the relatedness of Atlantic cod GLUT1 to class I GLUTs (14) from other vertebrates, including all those reported in fish. A multiple alignment of 28 GLUT protein sequences was generated using the CLUSTAL W alignment mode in the program AlignX (Informax Inc., Bethesda, MD, USA). The alignment was imported into MEGA version 2.1 and a phylogenetic tree was constructed using the neighbor-joining method with Poisson correction. Bootstrap analysis was performed with 1000 replicates. Atlantic cod GLUT1 clusters with GLUT1s from rainbow trout and carp, as well as GLUT1s from other vertebrates. It does not cluster with other class I GLUTs from other fish including GLUT4s from brown trout and coho salmon, GLUT2 from rainbow trout nor with GLUTs from grass carp and pacific hagfish.
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At the amino acid level, Atlantic cod GLUT1 had the highest degree of sequence identity to two GLUT1s from rainbow trout and common carp, with 85.2% and 79.6% identity, respectively. Atlantic cod GLUT1 shows about 78% amino acid identity with all of the other vertebrate GLUT1s, including human (Fig. 2). When compared with GLUTs 24 from other fish, Atlantic cod GLUT1 shows 61.4%, 60.4%, 61.2%, 55% and 53% identity to GLUT4s from brown trout and coho salmon, pacific hagfish GLUT, grass carp GLUT and rainbow trout GLUT2, respectively.
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Glucose transporters have several structural motifs common to all classes,
as well as class specific motifs (Joost
and Thorens, 2001; Hruz and
Mueckler, 2001
). All glucose transporters are characterized by the
presence of twelve transmembrane helices, seven glycines in the helices (1, 2,
4, 5, 7, 8, 10), two tryptophan residues in helices 6 and 11, two tyrosine
residues in helices 4 and 7, as well as, several charged residues on the
cytoplasmic surface. Highly conserved residues in all GLUTs include the FGY
motif in helix 1, GRK/R motif in intracellular loop 2, glutamate and arginine
residues in intracellular loops 4 and 10, the PESPR motif in intracellular
loop 6, D/ERAGRR motif in intracellular loop 8 and the PETKG motif after helix
12. Motifs specific for class I glucose transporters include an
N-glycosylation site in extracellular loop 1, the QL motif in helix
5, the QLS motif in helix 7, the STSIF motif in extracellular loop 7 and the
PGPIPW motif in helix 10. As indicated in
Fig. 3, the Atlantic cod GLUT1,
identified here, is characterized by the presence of all the conserved amino
acid sequences for all classes of GLUTs and most specifically the sequences
unique to class I GLUTs. The only variation in amino acid sequence is in the
PETKGR motif in which the K is substituted by an R. Analysis of the amino acid
sequence for Atlantic cod GLUT1 using HMMTOP indicates the presence of 12
transmembrane helices. It should be noted that the boundaries predicted for
helix 7 using this algorithm include the STSIF motif, which is a class
I-specific feature of extracellular loop 7. It is most probable that this
motif is in fact part of extracellular loop 7 and not part of helix 7 as
predicted by this program.
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Tissue distribution of Atlantic cod GLUT1
Northern blot analysis of adult Atlantic cod tissues indicates that the
4560 bp Atlantic cod GLUT1 transcript is highly expressed in brain, gill,
heart and kidney. Lower levels were detected in gonad, liver, spleen, stomach,
red muscle and white muscle (Fig.
4A). The tissue expression pattern determined by RT-PCR parallels
that observed by northern analysis (Fig.
4B).
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Developmental expression patterns of Atlantic cod GLUT1
Two batches of Atlantic cod were monitored from eggs to larvae to determine
at which point GLUT1 expression, and hence glucose utilization ability through
this transporter, becomes detectable. Samples were collected at seven time
points throughout development including day 0 fertilized eggs, day 7
fertilized eggs and day 0 hatched eggs. Thereafter, samples were taken while
they were being fed different diets. For each batch, RNA was extracted at each
time point and analyzed for GLUT1 expression by RT-PCR. As shown in
Fig. 5, GLUT1 is expressed at
all developmental stages examined.
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GLUT1 expression under hypoxia challenge
Atlantic cod were held at either normoxic (N=6) or hypoxic (40%
DO2; N=7) conditions for about 6 h. Tissues that showed
the highest levels of GLUT1 expression (heart, brain, gill and kidney) were
analyzed for changes in expression under hypoxic challenge by northern
analysis (Fig. 6). GLUT1
expression was measured by densitometry and normalized to 28S rRNA bands.
GLUT1/28S rRNA ratios were as follows: heart (normoxic 0.97±0.09;
hypoxic 1.01±0.10), brain (normoxic 0.29±0.03; hypoxic
0.25±0.03); gill (normoxic 0.31±0.01; hypoxic 0.36±0.04);
kidney (normoxic 0.35±0.05; hypoxic 0.53±0.14). There was no
significant difference in GLUT1 expression between normoxic and hypoxic fish
in the tissues examined under these conditions.
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The effects of temperature on heart GLUT1 expression
Atlantic cod were held at either 8°C (N=10) or allowed to
follow ambient seawater temperature (N=10). Fish were sampled when
ambient seawater temperature had risen to 4°C after an average temperature
of 0.5°C over the winter months. GLUT1 mRNA levels in heart were
determined by northern analysis and normalized to 28S rRNA levels
(Fig. 7). GLUT1/28S rRNA ratios
were 0.51±0.06 in fish held at high temperature and 0.36±0.05 in
fish allowed to track lower ambient temperatures. There was a tendency for
normalized GLUT1 level to be higher in fish maintained at high temperature
than in fish held at low seasonal temperature (P=0.071).
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The effects of fasting on heart GLUT1 expression
Atlantic cod that were deprived of food for 2 months (N=10) were
compared to fish fed a commercial diet (N=10). GLUT1 expression in
heart was determined by Northern analysis
(Fig. 8). Level of expression
was measured by densitometry and normalized to 28S rRNA bands. GLUT1/28S rRNA
ratios were 0.44±0.03 and 0.58±0.08 in fed and fasted fish,
respectively. There was a tendency for normalized GLUT1 level to be lower in
fed than in fasted fish (P=0.099).
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Discussion |
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The presence of GLUT1 mRNA in at least 10 tissue types of Atlantic cod, at
albeit differing levels, is similar to the ubiquitous distribution in
mammalian and rainbow trout tissues
(Behrooz and Ismail-Beigi,
1999; Teerijoki et al.,
2000
). High expression in heart of Atlantic cod is consistent with
the abundance of this transcript in rainbow trout heart
(Teerijoki et al., 2000
) and
detection of GLUT1 protein in heart of tilapia
(Wright et al., 1998
). Also,
the relatively low level of expression in Atlantic cod skeletal muscle, liver
and spleen matches the pattern reported for rainbow trout
(Teerijoki et al., 2000
).
These findings are in line with the inability to detect GLUT1 protein in
skeletal muscle of tilapia, rainbow trout, American eel or bullhead catfish
using antibodies to mammalian protein
(Wright et al., 1998
;
Legate et al., 2001
). Atlantic
cod and rainbow trout differ with respect to GLUT1 expression in brain, which
is relatively high in Atlantic cod but only weak in rainbow trout. In mammals,
GLUT1 is expressed at highest levels in erythrocytes and brain, where it is
found predominantly in endothelial and glial cells
(Gould and Seatter, 1997
;
Devaskar et al., 1991
). The
reason for differences in GLUT1 expression between Atlantic cod and rainbow
trout brain is unknown. Finally, we report here for the first time the
presence of a high level of GLUT1 in fish gill and kidney.
The tissue distribution and relative abundance of GLUT1 in Atlantic cod
differs from that of other GLUTs described for fish. Planas et al.
(2000) and Capilla et al.
(2002
) described a GLUT4 from
brown trout with abundant transcript in skeletal muscle, gill and kidney but
poor expression in heart, brain and liver. This GLUT4 occurs in much higher
levels than GLUT1 in red and white muscle of rainbow trout and, at least in
red muscle, is insulin sensitive (Capilla
et al., 2002
). Panserat et al.
(2001
) reported a
mammalian-like GLUT2 in rainbow trout that is strongly expressed in kidney and
liver but to a lesser extent in heart, brain and muscle. More recently, a
GLUT3 was cloned and sequenced from grass carp that is most abundant in kidney
with lower levels in heart, brain, gill, liver and muscle
(Zhang et al., 2003
). The
general picture for fish tissue that is emerging is that heart, brain and gill
have strong expression of GLUT1, skeletal muscle GLUT4, liver GLUT2, and
kidney GLUTs 1, 2, 3 and 4.
During development of the Atlantic cod embryo and early larval stages
nutrients from the yolk are mobilized to meet energetic needs. Endogenous
carbohydrates constitute less than 1% of the total energetic substances with
amino acids and lipids being the primary metabolic fuels
(Finn et al., 1995). GLUT1
expression was monitored at various stages of development from fertilization,
through hatching into larval fish, to 55 days of age when feeding on dry food
with a body mass of about 100 mg. GLUT 1 transcript was detected in Atlantic
cod from the very earliest stages of fertilized eggs. This finding is
consistent with that of rainbow trout embryos, in which GLUT1 is evident from
early gastrulation (Teerijoki et al.,
2001a
). In Atlantic cod, GLUT1 level became abundant at hatch and
continued through to day 55. There is no indication that developmental stage
and/or change in diet impact on the level of GLUT1 expression.
There was no difference in GLUT1 mRNA expression in heart, brain, kidney or
gill in Atlantic cod exposed to 40% DO2 for 6 h. This is in
contrast to a wide range of mammalian tissues that show increased expression
with hypoxia challenge (Behrooz and
Ismail-Beigi, 1999). Moreover, Zhang et al.
(2003
) noted that in grass
carp, a treatment of 7% DO2 for 4 h, resulted in an increase GLUT3
expression in kidney and gill with no change in heart, brain, liver or muscle.
It may be that the depth or length of the hypoxic challenge in the current
study was not sufficient to induce a change in GLUT1 expression, although the
treatment resulted in loss of equilibrium for about one-half of the fish. As
such, the possibility that this glucose transporter isoform is not hypoxia
sensitive in heart of Atlantic cod must be considered.
In hearts of a number of species there is a greater dependence on fatty
acid than carbohydrate metabolism for energy production at low temperature.
The corollary to this is that at high temperature, carbohydrates take on a
greater relative importance in terms of ATP supply. This contention is based
primarily upon performance challenges, enzyme activity levels and radioisotope
studies (Driedzic et al.,
1996). This is the first study that places a GLUT transporter into
this framework. Hearts from Atlantic cod, held at high temperature (8°C),
had marginally higher levels of normalized GLUT1 transcript than animals
acclimatized to winter conditions and sampled in the spring when water
temperature reached 4°C (0.51±0.06 vs 0.36±0.05;
P=0.071). It is possible that fish, at the lowest winter temperature
of approximately 1°C, would have even lower levels of GLUT1
expression; however, this remains to be assessed. In relation to this point,
however, the data are consistent with a decrease in the relative importance of
glucose as a metabolic fuel in fish hearts at low temperature.
There was no significant difference in the level of GLUT1 expression
between fed fish and those fasted for a 2-month period. If anything, there is
a suggestion of a small increase in GLUT1 level in the starved fish. This
finding, in Atlantic cod heart, differs from that of rat heart where GLUT1
mRNA level in 48 h fasted animals is only 14% of the level following a 5 h
fast (Kraegen et al., 1993).
The relatively constant level of GLUT1 mRNA in Atlantic cod heart during
starvation is similar to the situation in red and white skeletal muscle of
brown trout, in which a 45 day fast had no impact on GLUT1 expression
(Capilla et al., 2002
).
In summary, we have sequenced and cloned a putative glucose transporter from Atlantic cod that is homologous to a mammalian and trout GLUT1. This gene product is constitutive, has a wide tissue distribution, and is particularly abundant in heart, brain, gill and kidney. It is expressed upon fertilization and at all developmental stages. Levels of expression are not substantially altered under hypoxia in a number of tissues including heart, or in heart during low temperature challenge or starvation; however, the possibility that more severe hypoxia or larger temperature extremes would result in changes in gene expression cannot be ruled out. Overall, there is a ubiquitous tissue distribution of GLUT1 in Atlantic cod, consistent with other species, and the level of gene expression is relatively constant over a range of physiological conditions.
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Acknowledgments |
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References |
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