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First published online November 1, 2006
Journal of Experimental Biology 209, 4490-4502 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02532
Sequence of Atlantic cod (Gadus morhua) GLUT4, GLUT2 and GPDH: developmental stage expression, tissue expression and relationship to starvation-induced changes in blood glucose
Ocean Sciences Centre, Memorial University of Newfoundland, St John's, Newfoundland, A1C 5S7, Canada
* Author for correspondence (e-mail: wdriedzic{at}mun.ca)
Accepted 7 September 2006
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Summary |
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Food deprivation for 2 months was used as a vehicle to monitor GLUT expression at different blood glucose levels. Starvation resulted in a decrease in blood glucose and liver glycogen that recovered following 20 days of re-feeding. GLUT4 expression in heart was decreased with starvation and increased with re-feeding. GLUT4 mRNA level in heart correlated with blood glucose. It is suggested that this relationship is related to insulin responsiveness. GLUT4 expression in white muscle increased with starvation and decreased with re-feeding. It is proposed that this is due to the necessity to maintain high levels of the glucose transporter protein in the face of starvation-associated proteolysis. GLUT2 expression in liver correlated with blood glucose, consistent with higher rates of glucose transport from liver to blood in the fed state than in the food-deprived state.
Glycerol-3-phosphate dehydrogenase (GPDH) cDNA was also cloned. It encodes a 351 amino acid protein, which is 73-90% identical to GPDH from numerous other fish species. GPDH is ubiquitously expressed. Expression in heart decreased with starvation and increased with refeeding, whereas expression in liver did not change with starvation.
In other studies, gene expression was monitored at nine time points from fertilization of eggs to larval development. GLUT4 is detectable in fertilized eggs and is fully expressed by the halfway to hatching point. GLUT2 is not evident at fertilization, is detectable at halfway to hatching, and fully expressed at hatching. GPDH expression was evident from fertilization.
Key words: Atlantic cod, Gadus morhua, glucose transporter, GLUT4, GLUT2, insulin responsiveness, starvation, GPDH
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The study of glucose transport in fish is receiving
considerable attention for a variety of reasons including species-specific
differences in blood glucose levels that range from less than 0.5 mmol
l-1 to in excess of 50 mmol l-1 (e.g.
Chavin and Young, 1970;
MacCormack et al., 2003), up
to sixfold changes in blood glucose level within species as a function of
hypoxia or osmotic challenge (Shoubridge
and Hochachka, 1983;
Sangiao-Alvarellos et al.,
2005), use of dietary carbohydrate in an aquaculture context
(Hemre et al., 2001), and the
phenomenon of glucose intolerance (Moon,
2001). GLUT1 has been characterized in common carp
(Teerijoki et al., 2001b),
rainbow trout (Teerijoki et al.,
2000; Teerijoki et al.,
2001a) and Atlantic cod (Hall
et al., 2004). GLUT3 has been characterized in grass carp
(Zhang et al., 2003) and
Atlantic cod (Hall et al.,
2005). Here we describe the sequence, developmental pattern,
tissue expression, and relationship to plasma glucose levels of GLUTs 4 and 2
in Atlantic cod. Food deprivation in Atlantic cod leads to mobilization of
liver lipid and glycogen, in association with decreases in plasma glucose and
insulin (Black and Love, 1986;
Hemre et al., 1990;
Sundby et al., 1991). We
capitalize upon this to assess the expression of GLUTs in association with
altered plasma glucose.
GLUT4 in mammals is expressed primarily in heart, skeletal muscle and
adipose tissue and is the only insulin-sensitive transporter within the class
I GLUTs (Wood and Trayhurn,
2003). A mammalian-like GLUT4 was cloned and sequenced from brown
trout (Planas et al., 2000)
and coho salmon (Capilla et al.,
2002). In brown trout GLUT4 was expressed primarily in red and
white skeletal muscle, gill, kidney and adipose tissue but only to a limited
extent in heart (Planas et al.,
2000). Also, GLUT4 is insulin sensitive in adipocytes from coho
salmon (Capilla et al., 2004).
In salmonids, red muscle shows the hallmarks of insulin responsiveness but
white muscle does not. Starvation of brown trout, leading to decreases in
plasma insulin and glucose, was associated with decreased GLUT4 mRNA levels in
red muscle but not in white muscle, and rainbow trout injected with porcine
insulin showed an increase in GLUT4 mRNA in red but not white muscle
(Capilla et al., 2002). Given
the weak expression of GLUT4 mRNA in brown trout heart and the apparent lack
of responsiveness in white muscle, despite presumed changes in glucose usage,
it was considered of interest to extend these findings to other species.
GLUT2 in mammals occurs primarily in liver, pancreas, intestine and kidney.
In liver, this glucose transporter serves in the bi-directional movement of
glucose, which is dependent upon dietary/hormonal status
(Wood and Trayhurn, 2003). In
rainbow trout, GLUT2 is expressed in liver, kidney and intestine
(Krasnov et al., 2001;
Panserat et al., 2001). There
was no change in expression found following 4 days of starvation
(Panserat et al., 2001).
The current study also provides an analysis of glycerol-3-phosphate
dehydrogenase (GPDH). The expression of the gene is regulated in liver of
rainbow smelt in accordance with rates of glycerol 3-phosphate (glycerol
3-P) and subsequent glycerol production
(Ewart et al., 2001;
Liebscher et al., 2006).
Glycerol 3-P is a precursor of triglyceride synthesis and glycerol is
produced from the breakdown of triglycerides. As such, we considered this
protein to be a potential candidate for change during alterations in
triglyceride management.
Here we report the complete sequence for GLUT4, GLUT2 and GPDH cDNAs, the timing of expression of the genes from fertilization to the larval stage, tissue distribution in juvenile fish, and the response to starvation. The most important finding is that heart GLUT4 and liver GLUT2 expression correlate with plasma glucose, whereas, white muscle GLUT4 does not.
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Materials and methods |
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cDNA cloning
Full length cDNAs for Atlantic cod GLUT4, GLUT2 and GPDH were cloned using
a combination of RT-PCR, RNA ligase-mediated rapid amplification of 5'
and 3' cDNA ends (RLM-RACE) and genome walking. The sequences of all
primers used in cDNA cloning are presented in
Table 1.
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The three partial cDNAs were cloned using the following RT-PCR methodology.
Total RNA was extracted using Trizol Reagent (Invitrogen, Burlington, ON,
Canada) from either red muscle (GLUT4) or liver (GLUT2, GPDH) and then treated
with amplification grade DnaseI (Invitrogen). RNA was reverse-transcribed with
an oligo(dT) primer using M-MLV reverse transcriptase (Invitrogen). PCR
amplification was performed using DyNAzyme EXT (MJ Research, Waltham, MA,
USA). Touchdown PCR was used with 40 cycles of 94°C for 30 s,
65°C
0.5°C per cycle for 30 s and 72°C for 1-2 min. PCR
products were subcloned into pGEM-T Easy (Promega, Madison, WI, USA) and
triplicate clones sequenced on both strands at MOBIX, McMaster University.
The 5' and 3' ends of the three partial cDNAs were cloned using
a commercial kit for RLM-RACE, GeneRacer Kit (Invitrogen) and
poly(A)+ RNA isolated from total RNA using the Oligotex mRNA Mini
Kit (Qiagen Inc.). PCR amplification was performed at 94°C at 30 s,
70°C0.3°C per cycle for 30 s and 72°C for 1-2 min for 40
cycles using DyNAzyme EXT (MJ Research) with the exception of GLUT2 3'
RACE, which was performed at 94°C for 10 s, 72°C for 1.5 min for 7
cycles followed by 94°C for 10 s, 67°C for 30 s, 68°C for 1 min
for 32 cycles, using Elongase Enzyme Mix (Invitrogen).
Where genome walking was performed, genomic DNA was extracted from liver
using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA)
according to the manufacturer's protocol. GenomeWalker libraries were
constructed using the Universal GenomeWalker Kit (BD Biosciences Clontech,
Palo Alto, CA, USA) according to the manufacturer's protocol. PCR
amplification was performed at 94°C for 10 s, 70°C0.3°C per
cycle for 30 s and 68°C for 3 min for 40 cycles using DyNAzyme EXT (MJ
Research).
GLUT4
For GLUT4, a partial cDNA was cloned using RT-PCR. Primers were designed
based upon consensus sequences from conserved areas of aligned vertebrate
GLUT4s. Primers 1 and 2 were used for the initial PCR reaction and 1 µl of
this was used as template for a semi-nested PCR using Primer 1 and Primer 3.
The 1180-nucleotide partial cDNA sequence was aligned with sequences from
three other glucose transporters cloned from Atlantic cod and gene-specific
PCR primers were designed in areas to specifically amplify GLUT4. 5'
RACE was performed using Primer 4 and Primer 5 (nested) with the GeneRacer
5' Primer and the GeneRacer 5' Nested Primer, respectively. The
461-nucleotide PCR product contained the 72-nucleotide 5' UTR and the
20-nucleotides of unverified 5' coding sequence. The 3' end of
GLUT4 was obtained using a combination of genome walking and 3'
RLM-RACE. Genome walking was initially chosen over 3' RACE to break up
the remaining 3' sequence into smaller fragments based upon the presence
of restriction enzymes within. The first walk was performed using Primer 6 and
Primer 7 (nested) with the GenomeWalker Adaptor Primer 1 (AP1) and the
GenomeWalker Nested Adaptor Primer 2 (AP2), respectively. A 575 bp PCR product
was amplified from the SspI library, which contained an additional
110 bp of ORF sequence. To obtain additional 3' sequence, a second
genome walk was performed using the intronic primers, Primers 8 and 9
(nested). A 676 bp PCR product was amplified from the MslI library,
which contained the final 222 nucleotides of the ORF and 181 nucleotides of
the 3' UTR. The remaining 3' sequence was obtained by RLM-RACE.
Primer 10 and Primer 11 (nested) were used with the GeneRacer 3' Primer
and the GeneRacer 3' Nested Primer, respectively. A 806 bp band was
generated that contained the remaining 735 nucleotides of the 3'
UTR.
GLUT2
For GLUT2, a partial cDNA was cloned by RT-PCR. Primer 12 was used with
Primer 13 for the primary PCR. 1 µl of this PCR reaction was used as
template for a semi-nested PCR using Primer 12 and Primer 14. A 993-nucleotide
PCR product was amplified. The 5' end of GLUT2 was cloned by RLM-RACE.
Primer 15 and Primer 16 (nested) were used to amplify a 469-nucleotide PCR
product that contained the 26-nucleotide 5' UTR and the remaining 231
nucleotides of 5' coding sequence. The 3' end of GLUT2 was also
cloned by RLM-RACE. Primer 17 and Primer 18 (nested) were used to amplify two
bands (475 bp and 539 bp) that contained the remaining 319 bp of the coding
sequence (CDS) and 3' UTR sequences of 106 bp and 170 bp, respectively.
The size difference is due to the presence of two polyadenylation signals at
position 1635 and 1695, respectively.
GPDH
For GPDH, a partial cDNA clone was amplified by RT-PCR. Primer sets 19 and
20 amplified a 363 bp product. The 5' end of GPDH was cloned by
RLM-RACE. Primers 21 and 22 (nested) were used to amplify an 800 bp PCR
product, which contained a 59 bp 5' UTR and an additional 711 bp of the
CDS. The 3' end of GPDH was also cloned by RLM-RACE. Primers 23 and 24
(nested) were used to amplify a 790 bp PCR product that contained the final 2
bp of the CDS and the 577 bp 3' UTR. It should be noted that a GPDH-like
clone the CDS of which shared 70% sequence identity with the GPDH described
here was also amplified during the cloning process. However, this clone
contained a 37 bp frameshift deletion and would therefore code for a nonsense
protein.
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.), which uses the CLUSTAL W algorithm
(Thompson et al., 1994). For
phylogenetic and molecular evolutionary analyses, alignments were imported
into MEGA version 2.1 (Kumar et
al., 2001). Phylogenetic trees were constructed using the
Neighbor-Joining (NJ) 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;
Tusnady and Simon, 2001).
Gene expression analysis by real-time reverse transcription PCR
Levels of GLUT4, GLUT2 and GPDH mRNA were quantified by real-time reverse
transcription PCR (qRT-PCR), using TaqMan® probe-based chemistry and the
7300 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The
sequences of the primers and probes used in gene expression analysis are
presented in Table 2. Primers
and probes for GLUT4 and GLUT2 were designed in areas to specifically amplify
their respective GLUT, when aligned with other GLUTs from Atlantic cod.
Primers and probes for GPDH were designed in areas to specifically amplify
GPDH and not the nonsense transcript that had been detected during the cloning
process. TaqMan® probes were designed to span intron-exon boundaries
(identified by genome walking) (data not shown) to eliminate amplification
from any contaminating genomic DNA that may remain following DNaseI
treatment.
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First-strand cDNA was synthesized from 1 µg of DnaseI-treated total RNA
using random primers and M-MLV Reverse Transcriptase (Invitrogen). PCR
amplification for the target genes was performed in a 25 µl reaction using
1 µl of cDNA, 900 nmol l-1 each of forward and reverse primer,
250 nmol l-1 TaqMan® probe and 1 x TaqMan® Universal
PCR Master Mix, with AmpErase® UNG (Applied Biosystems). Expression levels
of the target genes were normalized to 18S ribosomal RNA, using the Eukaryotic
18S rRNA Endogenous Control (VIC/MGB Probe, Primer Limited) (Applied
Biosystems). PCR amplification for the endogenous control was performed in a
separate 25 µl reaction using 1 µl of a 1:10 dilution of the cDNA, 1
x probe/primer mix and 1 x TaqMan® Universal PCR Master Mix,
with AmpErase® UNG (Applied Biosystems). The real-time analysis program
consisted of 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min and
40 cycles of 95°C for 15 s and 60°C for 1 min. On each plate, for
every sample, the target gene and endogenous control were tested in triplicate
along with no RT controls. The fluorescence threshold cycle (CT)
was determined using the 7300 PCR Detection System SDS Software Relative
Quantification Study Application (Version 1.2.3) (Applied Biosystems). The
relative starting quantity of each transcript was determined using the
comparative CT method for relative quantification
(Livak and Schmittgen,
2001).
In vivo experiments Developmental expression patterns
GLUT4, GLUT2 and GPDH expression was monitored throughout the developmental
period of Atlantic cod from egg to larval fish at intervals corresponding to
developmental stages and changes in diet. Temperature was maintained at
5-6°C. Eggs were sampled at fertilization (day 0), 45 degree days (HH -
halfway to hatching) and at 105 degree days (hatching). Post-hatch samples
were then taken at day 2 (feeding solely from yolk sac), day 19 (rotifers),
day 47 (rotifers/Artemia), day 52 (Artemia), day 54
[Artemia/dry food (GEMMA micro diet; Skretting, NB, Canada)], and day
60 (dry food). Typical body mass of day 60 larvae was about 100 mg. At each
interval, approximately 100 mg of biomass was used in the RNA extraction.
Individuals were pooled to achieve the required amount of biomass.
Fasting/re-feeding
Fasting/re-feeding was used as a means to alter blood glucose levels.
Atlantic cod, less than 1 year old, 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. Experiments were initiated on
March 1, 2005. Fed and starved fish were sampled after 1 and 2 months. The
food-deprived fish were then re-fed and sampled after 20 days. The Canadian
Council on Animal Care policy with respect to experiments requiring
withholding of food is that fish should not be permitted to lose more than 15%
of body mass during periods of food restriction. The current study falls
within these guidelines (see Results). Body mass, length, liver mass, blood
glucose and liver glycogen were determined for all fish. Total lipid level and
triglyceride levels were assessed in liver from fed and starved fish only.
Heart and white muscle GLUT4, liver GLUT2, and heart and liver GPDH expression
were assessed in fish starved for 2 months, fed fish sampled at the same time
and fish re-fed for 20 days following starvation.
Biochemical analysis
Glycogen and glucose were assayed as described elsewhere
(Clow et al., 2004) with the
exception that absorbance was determined with a DTX 880 microplate reader
(Beckman Coulter, Mississauga, ON, Canada). Lipids were extracted in
chloroform/methanol using a Folch procedure
(Folch et al., 1957) modified
according to Parrish (Parrish,
1998). Lipid classes were separated using a MARK V Iatroscan
(Iatroscan Laboratory, Tokyo, Japan) analyzer as described elsewhere
(Parrish, 1987).
Data analysis
For qRT-PCR analysis, normalized transcript levels are expressed relative
to a calibrator sample (assigned a value=1) in all studies. In the
developmental and tissue distribution experiments, the condition/tissue that
expressed the particular transcript at the lowest detectable level was the
calibrator. In the fasting/re-feeding experiments, the first sample analyzed
was the calibrator. Statistical analysis of data was performed using a one-way
ANOVA followed by Tukey's HSD post-hoc test. In all cases,
P<0.05 was considered to be statistically significant. Values are
expressed as means ± s.e.m.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Deduced amino acid sequence analysis
Atlantic cod GLUT4 encodes a 503 aa protein, which has a predicted
molecular mass of 54.7 kDa and an isoelectric point of 7.09. Atlantic cod
GLUT2 encodes a 506 aa protein, with a predicted molecular mass of 55.1 kDa
and an isoelectric point of 6.73.
Phylogenetic analysis (Fig. 1) was performed to determine the relatedness of Atlantic cod GLUT4 and GLUT2 to class I GLUTs (1-4) from other vertebrates, including all those reported in fish. Atlantic cod GLUT4 clusters with GLUT4s from coho salmon and brown trout, as well as, GLUT4s from other vertebrates. Atlantic cod GLUT2 clusters with the GLUT2 from rainbow trout and with GLUT2s from other vertebrates. They do not cluster with other fish class I GLUTs including GLUT1s from Atlantic cod, rainbow trout and common carp nor with GLUT3s from Atlantic cod and grass carp nor with a GLUT from pacific hagfish.
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Developmental and tissue expression patterns for Atlantic cod GLUT4, GLUT2 and GPDH
Atlantic cod were monitored from eggs to larval fish to determine at which
point expression of GLUT4, GLUT2 and GPDH becomes detectable. Gene expression
was analyzed in one sample from each time point by qRT-PCR. GLUT4 is weakly
expressed in the day 0 eggs (Fig.
3) but by halfway to hatching, is well expressed. GLUT2 is not
expressed at all in the day 0 eggs but is weakly expressed at halfway to
hatching. Upon hatching, GLUT2 is expressed at high levels. GPDH is expressed
in the day 0 eggs, with expression increasing threefold by halfway to
hatching.
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Tissues were examined for expression of GLUT4, GLUT2 and GPDH by qRT-PCR from a single juvenile Atlantic cod (body mass approximately 45 g) maintained at 8°C and fed a commercial diet (Shur Gain, Truro, NS, Canada) daily (Fig. 4). The calibrator sample for GLUT4 was intestine, for GLUT2, kidney and for GPDH, white muscle. GLUT4 is highly expressed in heart, red muscle and white muscle with lower levels in gill, gonad, kidney and intestine. GLUT2 is highly expressed in liver, intestine and kidney. GPDH is ubiquitously expressed, with highest levels in gonad, kidney, intestine, liver, brain and heart. Lower levels were detected in other tissues sampled.
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When values for GLUT expression, from all individuals, were plotted against blood glucose, there was a significant correlation between heart GLUT4 and blood glucose and between liver GLUT2 and blood glucose (Fig. 9). There was no correlation between white muscle GLUT4 and blood glucose.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Glucose transporters have several amino acid sequences and transmembrane
helices common to all classes, as well as class specific motifs
(Joost and Thorens, 2001;
Hruz and Mueckler, 2001). As
highlighted in Fig. 2A,
Atlantic cod GLUT4 and GLUT2 have all the features of glucose transporters in
general and of class I GLUTs, with the exception of a small number of amino
acid substitutions. However, the identification of structural features
important in the function of individual members of class I GLUTs may be
enhanced by comparison of species-specific sequences.
In the case of GLUT4, this is important when considering motifs that may
play a role in trafficking/targeting. Mammalian GLUT4 isoforms are
characterized by the presence of several unique motifs involved in protein
trafficking, including the FQQI motif at the amino terminus, and the LL motif
and the acidic cluster TELEYLGP located at the carboxyl terminus
(Holman and Sandoval, 2001;
Lalioti et al., 2001). In
mammalian cells, the FQQI domain is important in the sorting process that
separates GLUT4 from the endosome system
(Holman and Sandoval, 2001;
Lalioti et al., 2001). In
Atlantic cod and coho salmon (Capilla et
al., 2004) the FQQ motif is present, but the I is replaced with
another hydrophobic amino acid, L. In brown trout, the motif is FQHL
(Capilla et al., 2004). The LL
domain appears to be involved in sorting between the trans-Golgi
Network and GLUT4 storage vesicles; however, all three fish lack the LL motif,
being LG in Atlantic cod. It has been suggested that these domains may be
partially redundant in GLUT4 endocytosis
(Holman and Sandoval, 2001;
Lalioti et al., 2001), which
may be correct, based upon amino acid data from fish which have an FQQL domain
but are lacking the LL domain. The acidic cluster is present in fish, albeit
with minor amino acid substitutions.
In the case of GLUT2, all GLUT2s are characterized by the presence of an
elongated extracellular loop between transmembrane segments 1 and 2. However,
the size and sequence of this loop is highly variable, with an additional 32,
38, 14 and 15 amino acids in mammalian, chicken, rainbow trout and Atlantic
cod GLUT2s, respectively. Furthermore, transmembrane 7 of mammalian GLUT2s
contains an H(V/M)A motif that was thought to be needed for the transport of
D-fructose (Wu et al.,
1998). However, in chicken and both fish species, this motif is
not conserved, with sequences of QIS and HLS, respectively.
Expression of GLUTs during development and tissue distribution
The energy requirements of the developing Atlantic cod are met by lipids
and amino acids from fertilization to consumption of the yolk sac
(Finn et al., 1995).
Thereafter, carbohydrate becomes a component of the diet by virtue of the body
composition of the prey species, rotifers and Artemia. The final diet
of dry food contains 12% carbohydrate. GLUT4 is marginally detectable at
fertilization but shows about a 100-fold increase in expression level by
halfway to hatch. GLUT2 expression is not detectable at fertilization but is
so at the halfway to hatch point with a further fourfold increase at hatching.
Expression levels of GLUT 4 and 2 (reported here) and GLUT 1
(Hall et al., 2004) appear to
be relatively stable between hatching and the 100 mg larval stage, suggesting
that the fish are poised for carbohydrate transport and that the change in
metabolic fuel associated with the transition from yolk sac dependence to dry
food has little impact on relative gene expression of these glucose
transporters.
The tissue distribution of the GLUT 4 and 2 transporters in Atlantic cod is
similar to the mammalian paradigm (Wood
and Trayhurn, 2003). GLUT4 expression is most prevalent in heart,
followed by red muscle and white muscle, with lower levels in a number of
other tissues; whereas, GLUT2 mRNA is most highly expressed in liver, at lower
levels in intestine and kidney, and is undetectable in other tissues.
Tissue-specific expression of GLUTs 1, 2, and 3 in Atlantic cod is generally
consistent with that reported for GLUT1 and GLUT2 in rainbow trout
(Teerijoki et al., 2000;
Panserat et al., 2001) and
GLUT3 in grass carp (Zhang et al.,
2003). GLUT4 expression in heart appears to differ between
Atlantic cod, where it is highly expressed, and brown trout, where it is
expressed to only a limited extent (Planas
et al., 2000). The functional significance of this difference in
cardiac tissue remains to be resolved.
Impact of starvation on metabolic fuels and GLUT expression
The period of food deprivation was well within the tolerance limits of
Atlantic cod as evidenced by maintenance of condition factor and levels of
lipids in liver. However, the challenge resulted in decreases in blood glucose
and liver glycogen, as previously reported
(Black and Love, 1986;
Hemre et al., 1990). Such
starvation-induced decreases in blood glucose are associated with parallel
decreases in plasma insulin (Hemre et al.,
1990; Sunby et al., 1991). Although insulin was not measured in
the current experiments, we assume that it decreased with starvation, given
that the water temperature (5-8°C in previous studies; 8°C current
experiment) and the length of starvation (3-4 weeks in previous studies; 4-8
weeks in current experiment) were in the same range as in the earlier work of
Hemre et al. (Hemre et al.,
1990) and Sunby et al. (Sunby et al., 1991). Re-feeding led to a
recovery of blood glucose levels and an overshoot in liver glycogen, again as
reported by Black and Love (Black and Love,
1986). We note here for the first time a correlation between
glycogen content in liver and blood glucose, suggesting that the former sets
the glucose level available to other tissues.
Expression of GLUT4 in heart decreased during starvation, increased with
re-feeding and correlated with blood glucose. The most plausible explanation
for this is that high levels of blood glucose are associated with elevated
levels of insulin and this in turn results in activation of GLUT4
transcription. The scenario depicted for heart of Atlantic cod matches that in
red muscle of brown trout, in which starvation is associated with decreases in
blood insulin and glucose in association with decreases in red muscle GLUT4
(Capilla et al., 2002).
Expression of GLUT4 in white muscle was the mirror image of that in heart.
Food deprivation resulted in an increase in mRNA levels, which returned to
control values with re-feeding. GLUT4 expression in white muscle did not
correlate with blood glucose. Differences between red muscle and white muscle,
with respect to alterations in GLUT4 levels, have been previously noted in
trout although the significance of it may have gone unrecognized
(Capilla et al., 2002). GLUT4
mRNA levels decreased in red muscle of starved brown trout but in white
muscle, although the there was no significant difference in expression level,
the average value increased by about 25%. In rainbow trout injected with
porcine insulin, there was an increase in GLUT4 mRNA in red muscle but no
change in white muscle. It appears that the GLUT4 responsiveness to insulin
and/or high blood glucose levels noted in heart of Atlantic cod and red muscle
of trout does not carry over to white muscle. Indeed starvation is associated
with increases in GLUT4 mRNA. An explanation to account for this is that
during starvation there is proteolysis in white muscle of Atlantic cod
(Black and Love, 1986) that
probably includes GLUT4. The data presented here are for GLUT 4 mRNA and not
transporter protein. In order to maintain glucose transport there may need to
be an increase in synthesis of this protein and this is reflected in increased
GLUT4 expression. A response similar to that reported here for Atlantic cod
has been observed in rats, in which a 3-day fast led to a two- to threefold
increase in GLUT4 transcription and GLUT4 mRNA in white muscle but no change
in red muscle (Neufer et al.,
1993).
GLUT2 mRNA levels correlated with blood glucose. In mammals, GLUT2 serves
to facilitate glucose transport either into or out of liver cells, dependent
on dietary and hormonal status. One scenario to account for the data is that
as starvation proceeds, liver glycogen is depleted and delivery of glucose
from liver to blood is diminished therefore reducing the need to maintain high
levels of GLUT2 protein. In the only other study that we are aware of,
regarding GLUT2 in fish, the transporter was highly expressed in liver of
rainbow trout and the level was not influenced by 4 days of food deprivation
(Panserat et al., 2001).
Glycerol-3-phosphate dehydrogenase
GPDH from Atlantic cod encodes a deduced amino acid sequence that is very
similar to that found in a number of other fish species. The enzyme GPDH plays
a pivotal role in the synthesis of glycerol 3-phosphate required for
triglyceride/phospholipid synthesis and in the conversion of glycerol
3-phosphate to dihydroxyacetone in the breakdown of these components. GPDH
expression was apparent at fertilization, increased by about threefold by
halfway to hatching, and remained elevated throughout the remainder of the
developmental period. In juvenile fish, GPDH was detected in all tissues
sampled with the highest levels, typically occurring in those tissues with
high lipid turnover, such as brain, intestine and liver. The starvation
challenge was not substantive enough to induce changes in liver triglyceride
levels. In this context it is not surprising that GPDH mRNA levels did not
change in liver. GPDH expression in heart decreased with starvation and
recovered during re-feeding. The simplest explanation for this is a decrease
in phospholipid/triglyceride synthesis in heart during starvation associated
with decreased synthesis of glycerol 3-phosphate.
Conclusion
In summary, GLUT4, GLUT2 and GPDH cDNAs from Atlantic cod were cloned and
sequenced. In accordance with the mammalian model GLUT4 is expressed primarily
in heart and muscle, whereas GLUT2 is expressed at the highest levels in
liver. GPDH is expressed in all tissues assessed, with highest levels in
tissues that have high rates of lipid turnover. Starvation was associated with
decreases in blood glucose, liver glycogen and heart GLUT4 and GPDH. These
parameters recovered with re-feeding. By contrast, white muscle GLUT4
increased with starvation and returned to pre-starved levels with re-feeding.
The physiological significance of this is yet to be resolved. GLUT2 expression
in liver correlated with blood glucose levels, probably reflecting glycogen
depletion in liver during starvation with reduced movement of glucose from
liver to blood.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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