The regulation and importance of glucose uptake in the isolated Atlantic cod heart: rate-limiting steps and effects of hypoxia
1 Ocean Sciences Centre, Memorial University of Newfoundland, St John's,
Newfoundland, Canada, A1C 5S7
2 Department of Biological Sciences, Idaho State University, Pocatello, ID
83209-8007, USA
* Author for correspondence (e-mail: wdriedzic{at}mun.ca)
Accepted 8 March 2004
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Summary |
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Key words: glucose uptake, glucose transport, cytochalasin B, 2-deoxyglucose, hypoxia, heart, cardiac performance, cod, Gadus morhua
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Introduction |
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Myocardial glucose metabolism in vertebrates is primarily dependent on the
uptake of extracellular glucose. Glucose enters the cell and is phosphorylated
by hexokinase to glucose-6-phosphate (G-6-P), which in turn enters the
glycolytic pathway common to both glucose breakdown and glycogenolysis. In the
rat heart, the maximal in vitro activity of hexokinase is about
5-fold greater than the maximal rate of glucose transport
(Randle and Tubbs, 1979).
Glucose uptake is the rate-limiting step for the mammalian heart under anoxia;
however, under normoxic conditions with insulin present, phosphorylation of
glucose to G-6-P becomes rate limiting
(Morgan et al., 1961
;
Cheung et al., 1978
) despite
high levels of hexokinase. In the mammalian heart, most of the extracellular
glucose enters the cell via two specific Na+-independent
proteins, GLUT-1 and GLUT-4. GLUT-1 is thought to account for glucose entry
under basal conditions, while GLUT-4 is responsive to insulin. Under
oxygen-limiting conditions there is translocation of both these transporters
from intracellular sites to the sarcolemma, where facilitative diffusion of
glucose is realized (Montessuit et al.,
1998
; Behrooz and Ismail-Beigi,
1999
).
The fish heart, similar to the mammalian heart, has the capacity to utilize
glucose as a metabolic fuel under aerobic conditions
(Lanctin et al., 1980;
Driedzic and Hart, 1984
;
Sidell et al., 1984
; West et
al., 1993
,
1994
) and is dependent upon
the uptake of extracellular glucose to maintain performance under conditions
of oxygen deprivation (Driedzic et al.,
1978
; Bailey et al.,
2000
) or elevated work (West
et al., 1993
). Unlike mammal hearts, how glucose uptake is
regulated in fish remains unresolved. Blasco et al.
(1996
) found that the in
vivo uptake of 2-deoxyglucose (2-DG) in the heart and brain of the brown
trout exceeded the accumulation of 2-deoxyglucose-6-phosphate (2-DG-6-P) by
1.72.6 times, although in six other tissues there was a 1:1 ratio.
Assuming that 2-DG and glucose are handled in the same fashion, this finding
implies that the phosphorylation of glucose, and not glucose transport, is
rate limiting in the fish heart even though the in vitro activity of
hexokinase in fish heart is very high
(Driedzic and Gesser, 1994
)
and greatly exceeds maximal rates of glucose uptake and utilization. This
latter characteristic would suggest that transport, and not phosphorylation,
is a limiting factor.
The recent cloning and sequencing of glucose transporter (GLUT) proteins in
several fish species suggest that facilitative diffusion of glucose into
myocytes is the first step for glucose utilization and an important site for
metabolic regulation in fish (Planas et
al., 2000; Teerijoki et al.,
2001
). In non-contracting eel ventricle strips, glucose uptake is
stimulated under anoxia by about 50%. Treatment of these strips with
cytochalasin B (a general inhibitor of glucose transporters) eliminated the
anoxia-stimulated component but had no effect on basal transport
(Rodnick et al., 1997
). These
results raise the possibility that another glucose transport mechanism, such
as Na+-dependent glucose uptake, is important in the fish heart.
This mechanism has been demonstrated in frog skeletal muscle
(Kitasato and Marunaka, 1985
)
and occurs in other fish tissues such as the kidney
(Kanli and Terreros, 1997
) and
intestine (Reshkin and Ahearn,
1987
).
In the current study, we utilized the isolated cod heart and cardiac tissue to: (1) evaluate the impact of GLUT transporter inhibition on performance and glucose uptake under hypoxia; (2) measure glycogen and free glucose levels in hearts subjected to two different oxygen levels and the presence/absence of glucose; (3) test the hypothesis that there is an Na+-sensitive component to glucose uptake; and (4) examine the relationship between 2-DG uptake and 2-DG-6-P accumulation to assess if uptake or phosphorylation is rate limiting. We conclude that the limiting step in glucose uptake is glucose transport and there is an obligatory requirement for enhanced GLUT activity for hypoxic performance. Na+-coupled glucose transport is absent and, by default, we propose that a significant component of glucose entry in the teleost heart is based on simple diffusion.
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Materials and methods |
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Preparations
Ventricle strips
Animals were netted individually, killed by a sharp blow to the head,
doubly pithed and weighed. The heart was quickly excised and placed in
ice-cold medium for marine teleosts (in mmol l-1): 150 NaCl, 5.0
KCl, 1.5 CaCl2, 0.17 MgSO4, 0.17
NaH2PO4, 2.33 Na2HPO4, 11.0
NaHCO3, 5.0 D-glucose, gassed with 99.5% air:0.5%
CO2, and with pH set to 7.8 at 8°C. After cutting through the
pericardium, the entire heart was excised, and the ventricle was dissected
free of the atrium and bulbus arteriosus. The ventricle was bisected and four
strips (11.5 mm wide and 810 mm long) were cut by slicing
parallel to the long axis of the ventricle with a razor blade. To permit
recovery from the effects of tissue slicing, ventricle strips were
pre-incubated for 60 min in stoppered 25 mlErlenmeyer flasks containing 2 ml
of basic medium. Medium was supplemented with 35 mmol l-1 mannitol
and 0.1% bovine serum albumin (BSA) (Cohn Fraction V, essentially fatty acid
free). Flasks were gassed continuously with 99.5% air:0.5% CO2 and
incubated at 8°C in a reciprocating incubator (Haake, model SWB 20) at 60
cycles min-1 throughout the experiment. Osmolarity was kept
constant in all incubations (340 mosmol l-1) by varying the
concentration of mannitol, such that the sum of D-glucose,
mannitol, pyruvate and 2-deoxyglucose (2-DG) concentrations was 40 mosmol
l-1. Mannitol was also used as a marker of the extracellular space
because of its similar diffusion properties to those of glucose; however,
unlike glucose, it does not enter the myocyte. To determine whether there is
an Na+-sensitive component of 2-DG uptake in the cod heart, we
compared uptake rates in basic medium with those in Na+-free medium
containing equimolar amounts of choline Cl (150) and choline HCO3
(11). We also tested whether phloridzin inhibits glucose uptake at a
concentration (1 mmol l-1) that significantly inhibits facilitative
glucose transport in fish cells (Teerijoki
et al., 2001
) and mammalian skeletal muscle
(Cheng et al., 1978
). The
water content of ventricle strips (ml g-1) was determined by drying
tissue to a constant mass in a vacuum oven set at 70°C.
Perfused hearts
The perfusion medium was identical to the medium used for ventricle strips
except for the presence or absence of D-glucose. Temperature of the
circulating perfusion medium was maintained at 10°C by connecting the
water-jacketed condensers and other chambers to a circulating water bath.
D-glucose (5 mmol l-1) was added to the media of several
groups including the control groups. Also, one group from each study received
25 µmol l-1 cytochalasin B, an inhibitor of facilitative glucose
transport proteins (Rodnick et al.,
1997). Cytochalasin B was dissolved in DMSO and added to the
perfusion medium just prior to the experiment. The final amount of DMSO was
0.025%. Under normoxic conditions, the perfusate was gassed with 99.5%
air:0.5% CO2, whereas severely hypoxic conditions were achieved by
gassing with 99.5% N2:0.5% CO2. Perfusate oxygen partial
pressure (PO2) was measured by collecting
perfusion medium from the input chamber in a glass (Hamilton) syringe and
injecting it into a temperature-controlled chamber housing an E101 oxygen
electrode (Analytical Sensors Inc., Sugar Land, TX, USA). The oxygen electrode
was connected to an OM-200 oxygen meter (Cameron Instrument Company, Port
Aransas, TX, USA). PO2 during the stabilization
period was 17.8±0.08 kPa. At 5, 15 and 30 min into the perfusion with
hypoxic medium, PO2 was 5.87±0.27 kPa,
5.20±0.27 kPa and 4.27±0.27 kPa, respectively.
Performance studies
Cod were killed and their hearts were placed in ice-cold perfusion medium.
Hearts were quickly mounted onto the perfusion apparatus as described
previously (Driedzic and Bailey,
1994). The apparatus had two water-jacketed condensers. The first
condenser was filled with perfusion medium containing 5 mmol l-1
glucose and gassed under normoxic conditions. The second condenser was used
for experimental treatments and could be gassed with either air or nitrogen
mixtures. Gassing of these condensers began approximately 30 min prior to the
heart being mounted. The condensers were connected to a water-jacketed filling
chamber with side arm, which provided perfusate to the input cannula. The
input and output cannula were made of stainless steel tubing and had side arms
to allow for pressure measurements. The atrium of the heart was tied onto the
input cannula, and the bulbous arteriosus tied onto the output cannula, both
by a 2-0 silk ligature. The output cannula was connected to another
water-jacketed chamber (output) with side arm set 20 cmabove the heart.
Overflow from both the input and output chambers was re-circulated back into
one of the two condensers.
Once the heart was mounted, perfusion medium from the first condenser
(normoxic with glucose) was pumped to the input chamber at a rate that allowed
a constant overflow from the side arm and thus a constant input pressure
(pre-load, 0.29 kPa). Hearts were electrically stimulated with a Grass Model
SD9 square-wave generator set at 5 V and 200 msduration. Contraction rate was
set to 36 beats min-1 based on physiological heart rates of 39
beats min-1 seen in resting cod subjected to normoxic conditions
(Axelsson, 1988). All hearts
were forced to work against an imposed after-load of 1.96 kPa. Hearts were
kept under these conditions for 1530 min until flow rates stabilized.
After this cardiac parameter had stabilized, perfusion with this medium
continued for an additional 10 min before doing any experimental
manipulations. This was considered time `0'. The control group (normoxic with
glucose) did not have to be switched over to the second condenser. However,
the other four experimental conditions (normoxic medium without glucose,
hypoxic medium with glucose, hypoxic medium without glucose, and hypoxic
medium with added glucose and 25 µmol l-1 cytochalasin B) were
switched to the second condenser at this time.
All hearts were perfused for an additional 2 h or until hearts failed, as determined by flow less than 1 ml min-1. Once the experiment was terminated, the atrium and bulbous arteriosus were cut away from the ventricle, and the ventricle was cut in half, rinsed with ice-cold glucose-free perfusion medium, blotted and weighed. The two ventricle pieces were frozen in liquid nitrogen and kept in a 80°C freezer for biochemical analysis.
Measurement of 2-DG uptake and extracellular space in ventricle strips
2-DG uptake is defined as transport into the cell and intracellular
phosphorylation by hexokinase. Phosphorylated 2-DG is trapped in the myocyte
and this makes it possible to measure sugar transport. Uptake measurements
were conducted as described previously
(Rodnick et al., 1997). After
the 10 min rinse step to remove glucose from the extracellular space,
ventricle strips were incubated in 1.5 ml of medium containing 2 mmol
l-1 pyruvate, 1 mmol l-1
2-deoxy-D-[3H(G)]glucose (37 kBq ml-1;
specific activity 222 kBq nmol-1; New England Nuclear, Guelph, ON,
Canada), 37 mmol l-1 [U-14C]mannitol (3.7 kBq
ml-1; specific activity 2 kBq nmol-1; New England
Nuclear) and 0.1% BSA for 1060 min. Radioisotopes were dried under
gaseous nitrogen to remove the ethanol and water. Preliminary studies
indicated that it takes between 10 and 20 min for full equilibration of the
radiolabeled mannitol in the incubation medium with the muscle extracellular
space. To terminate experiments, ventricle strips were blotted briefly on
filter paper moistened with ice-cold medium, freeze-clamped and stored at the
temperature of liquid nitrogen. Strips were weighed and processed by boiling
for 10 min in 1 ml of water followed by centrifugation (1000 g
for 10 min at room temperature). After centrifugation, a portion of the
supernatant was counted. The 2-DG and 2-DG-6-P of the muscle extract were
separated by ion-exchange chromatography using a small column (0.5 ml bed
volume) of DEAE-Sephacel (Jacobs et al.,
1990
). 2-DG and 2-DG-6-P were eluted with successive washes of
distilled water and 0.2 mol l-1 HCl, respectively. Duplicate
samples of crude muscle extracts, both column fractions and diluted incubation
medium were place in scintillation vials containing Ecolume (ICN Biochemicals,
Costa Mesa, CA, USA) and counted in a Packard 2500TR liquid scintillation
counter with channels preset for dual-label counting. Extracellular space and
intracellular concentrations of 2-DG and 2-DG-6-P were determined as described
previously for mammalian skeletal muscle
(Hansen et al., 1994
). The
intracellular space was obtained by subtracting the volume of the
extracellular fluid from the total tissue water space.
Measurement of 2-DG uptake and extracellular space in perfused hearts
For these experiments, the perfusion system was redesigned in order to
decrease the total volume of the perfusion medium and hence reduce the
radioisotope requirement. A total volume of 50 ml was achieved by redirecting
the overflow from the output chamber directly to the input chamber. This
eliminated the need for the two water-jacketed condensers.
Similar to the performance experiments, hearts were mounted and initially subjected to normoxic media containing 5 mmol l-1 D-glucose, and flow and output pressure were recorded in order to determine when the preparation was stable. Most preparations stabilized within 30 min. Once stable, the perfusion medium was quickly replaced by a medium containing 5 mmol l-1 D-glucose, 2-[3H(G)]-deoxy-D-glucose (18.5 kBq ml-1) and D-[1-14C]-mannitol (5.55 kBq ml-1). [14C]mannitol was added to calculate the extracellular fluid. Once the medium was replaced, it was gassed with either 99.5% air:0.5% CO2 (normoxic) or 99.5% N2:0.5% CO2 (hypoxic). One additional group received a hypoxic medium containing 25 µmol l-1 cytochalasin B. PO2 was measured for 20 and 25 min into the stabilization period, 10 min after the switch to radioactive normoxic medium and every 5 min during perfusion with the radioactive hypoxic medium. PO2 during the stabilization period was 17.7±0.08 kPa. 5, 10 and 15 min into the perfusion with hypoxic medium, PO2 was 4.93±0.04 kPa, 2.9±0.27 kPa and 2.13±0.13 kPa, respectively.
After 15 min of perfusing with the radioisotopes, the atrium and bulbous
arteriosus were cut away from the ventricle; the ventricle was cut in half,
rinsed in ice-cold glucose-free perfusion medium, weighed and frozen in liquid
nitrogen. Ventricle samples (150 mg) were homogenized in nine volumes of
6% perchloric acid, then duplicate samples (200 µl) of homogenate and 10 ml
Ecolume were counted for both 3H and 14C.
Extracellular space (ml g-1 ventricle) and the intracellular
concentration of 2-DG (µmol g-1 ventricle) were calculated as
previously described (Rodnick et al.,
1997). Although it is possible that perfusion times of 15 min
would be insufficient for full equilibration of the extracellular marker
(mannitol), 15 min was chosen because it was important that the hearts were
still functioning at a high level. In the performance experiments, two out of
eight hypoxic hearts perfused with medium containing glucose and cytochalasin
B failed by 30 min, and half had failed after 40 min. Even if our estimates of
glucose uptake in the perfused heart are low, this should not negate the
positive effect of hypoxia on glucose uptake and the negative impact of the
cytochalasin B treatment.
Instrumentation and data analysis
Input (pre-load) and output (after-load) pressures were read using a
Biotronix BL 630 pressure transducer connected to a Biotronix meter. During
the performance study, flows were measured using a Biotronix pulsed logic
flowmeter (model BL610). However, a Transonic flowmeter (model T206) with a 2N
in-line flow probe (Transonic Systems, Inc., Ithaca, NY, USA) was used to
measure flows in the glucose uptake studies. Both the pressure transducer and
flowmeter were interfaced to a PowerLab computerized system, and data were
collected online every 5 min for 30 s using the accompanying software program.
Power was calculated using the formula
(Driedzic, 1992):
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Assays
Glycogen
Approximately 50 mg of ventricle was homogenized in 0.5 ml 30% KOH and
boiled for 10 min. Glycogen was precipitated by adding 0.3 ml of 2% aqueous
Na2SO4 and 2.0 ml absolute ethanol. After the mixture
was centrifuged for 10 min at 1500 g, the supernatant was
decanted, the pellet washed with 2 ml 66% ethanol and dissolved in 1 ml warm
distilled water. An equal volume of 1.2 mol l-1 HCl was then added,
and the sample heated in a boiling water bath for 2 h. Hydrolysates were
frozen in liquid nitrogen and stored at 80°C until analyzed for
glucose content.
Glucose assay conditions were based on a procedure modified from Bergmeyer
et al. (1974). Briefly, an
assay medium was prepared containing 250 mmol l-1 imidazole, 5 mmol
l-1 MgSO4, 10 mmol l-1 ATP and 0.8 mmol
l-1 NADP+. A 100 µl aliquot of the sample was added
to a spectrophotometer cuvette and diluted 1:10 with the assay media. 10 µl
of glucose-6-phosphate dehydrogenase (G-6-PDH) was added to remove any
endogenous G-6-P. Absorbance was read at 340 nm on a DU640 spectrophotometer
(Beckman Coulter, Mississauga, ON, Canada) after 10 min. Hexokinase was then
added and the absorbance read after 2530 min.
Free glucose and lactate
Approximately 50 mg of the ventricle was homogenized in nine volumes of 6%
perchloric acid. This homogenate was spun at 10 000 g for 10
min and the supernatant was collected. Free glucose was measured using the
modified procedure from Bergmeyer et al.
(1974) mentioned above and
lactate was assayed using a commercial kit (Sigma # 826-UV).
Statistical analysis
For the performance studies, analyses of variance (ANOVAs) were done at
each time interval, and decisions were made with regard to statistical
differences between groups using pairwise comparisons, after performing a
Bonferroni adjustment. Statistical analysis of the glycogen, glucose and
lactate data and glucose uptake measurements was performed using one-way ANOVA
followed by Tukey's post-hoc test. P0.05 was considered
to be statistically significant for all studies.
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Results |
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Isolated heart performance under different conditions is shown in Fig. 1. Normoxic hearts perfused with glucose continued to perform for at least 120 min. After 120 min,power output g-1 was 88.7±11.1% of the original value. When glucose was excluded from the perfusate, six of seven normoxic hearts also contracted for 120 min and power output was 76.5±9.8% of the original value. This value was not significantly different from the normoxic group receiving glucose. One heart failed after 110 min. Under hypoxic conditions, seven of eight hearts receiving glucose maintained 69.9±8.9% of the initial power for 30 min. After 120 min, however, half the hearts failed and the power output of the remaining four hearts was just 38.3±9.2% of initial values. After 35 min, performance of oxygen-deprived hearts receiving glucose was significantly lower than hearts that were well oxygenated.
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Although the exclusion of glucose did not significantly affect the performance of hearts that were well oxygenated, there was a profound effect when glucose was excluded from the hypoxic medium. Hearts performed at a significantly lower level after 20 minwhen glucose was excluded from the hypoxic medium. At 20 min, one heart had failed and the mean power output g-1 of the other five hearts was just 38.0±10.3% of initial performance. With glucose in the hypoxic medium, hearts were still performing 73.4±7.6% of the original values after 20 min. Half of these hearts were also still performing after 120 min whereas all hearts perfused with no glucose in the medium failed by 40 min. These observations promote extracellular glucose as an important, if not essential, energy substrate in the working cod heart during hypoxia.
Adding cytochalasin B to the hypoxic medium with glucose caused a significant decrease in heart performance after 40 min when compared to the hypoxic with glucose group (P<0.01). Hearts performed at 69.4±8.7% of basal values after 40 min when perfused with the hypoxic medium (with glucose) but only 28.2±13.6% when cytochalasin B was added. At 40 min, only 50% of these hearts were still functioning, and at 60 min only two of eight hearts continued to develop power at 9.2±4.4% of the initial value. The fact that cytochalasin B binds to the facilitative glucose transporter and inhibits glucose transport activity provides further evidence that extracellular glucose is necessary to maintain heart performance under oxygen-limiting conditions.
Glucose uptake in the perfused heart
Fig. 2 shows the changes in
2-DG uptake in isolated hearts induced by hypoxia or hypoxia and cytochalasin
B. Hypoxia stimulated glucose uptake from 1.64±0.12 µmol
g-1 15 min-1 (normoxic values) to 5.69±0.09
µmol g-1 15 min-1 (P<0.01). The addition
of 25 µmol l-1 cytochalasin B to the hypoxia medium partially
inhibited (4.70±0.32 µmol g-1 15 min-1) the
stimulation of 2-DG uptake induced by anoxia (P<0.01), but these
levels were still higher than the normoxic conditions.
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Intracellular concentrations of glycogen, free glucose and lactate
Mean heart glycogen levels were 10.98±1.78 µmol glucose
g-1 tissue in hearts perfused under normoxic conditions (with
glucose). These levels were significantly higher (P0.05) than in
the hearts perfused under any other condition
(Fig. 3A). When hearts were
perfused with the hypoxic medium containing glucose, glycogen levels were less
than half (4.94±1.57 µmol glucose g-1 tissue) the
corresponding normoxic values. Similar heart glycogen levels were also seen
when glucose was omitted from the normoxic medium (5.23±1.30 µmol
glucose g-1 tissue). The exclusion of glucose from the hypoxic
medium or the addition of cytochalasin B caused heart glycogen levels to drop
even lower to 1.27±0.15 µmol glucose g-1 tissue and
1.42±0.34 µmol glucose g-1 tissue, respectively. These
last two groups hypoxia with no glucose and hypoxia with glucose and
cytochalasin B were not significantly lower than the hypoxic with
glucose group or the normoxic without glucose group. Clearly, the absence of
exogenous glucose and/or oxygen, or the presence of cytochalasin B, promotes
glycogenolysis and the use of endogenous glucose for energy production.
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Free glucose in the heart showed a very similar pattern to that of glycogen
(Fig. 3B). The highest levels
(0.86±0.15 µmol glucose g-1 tissue) of free glucose were
found in hearts perfused with the normoxic medium with glucose. These levels
were significantly higher (P0.05) than all other conditions
except for the hearts perfused with normoxic medium without glucose
(0.48±0.0.12 µmol glucose g-1 tissue; P=0.054).
Glucose levels were 0.35±0.06 µmol glucose g-1 tissue in
hearts perfused with a hypoxic with glucose medium. Again, the exclusion of
glucose from the hypoxic medium or the addition of cytochalasin B caused the
lowest glucose levels found in the heart (0.12±0.03 µmol glucose
g-1 tissue and 0.20±0.06 µmol glucose g-1
tissue, respectively). Lactate levels in the heart did not change under any
condition and the overall value was 10.85±0.33 µmol g-1
tissue (N=41).
Effects of Na+-free medium or phloridzin on 2-DG uptake
To define whether there is an Na+-dependent component of glucose
transport in cod cardiac tissue, ventricle strips were incubated in
Na+-free medium. Iso-osmotic replacement of Na+ with
choline had no effect on 2-DG uptake, suggesting that no
Na+-coupled sugar transporters were present
(Fig. 4). By contrast, when
ventricle strips were exposed to phloridzin (1 mmol l-1), 2-DG
uptake was inhibited by 50% (P<0.01). Based on these measurements,
facilitative transport of glucose appears to represent a major physiological
component of cardiac glucose uptake.
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Accumulation of 2-DG and 2-DG-6-P in ventricle strips
Our measures of the extracellular space after 20 minequilibration in
ventricle strips from cod (0.219±0.037 ml g-1 wet mass;
N=12) are comparable to studies of the rat heart using mannitol as
the marker (0.236±0.012 g g-1 wet mass;
Dobson and Cieslar, 1997) and
suggest that damage due to cutting the tissue or muscle incubation was
minimal. Uptake of 2-DG was linear for 60 min in non-contracting ventricle
strips exposed to 1 mmol l-1 2-DG
(Fig. 5A), and phosphorylated
2-DG represented the majority of total intracellular 2-DG
(Fig. 5B). After a 20 min
incubation, free 2-DG was detected in only six out of 12 strips, and the
concentration (0.036±0.009 mmol l-1 intracellular fluid) was
much lower than the extracellular value (1 mmol l-1). After 60 min,
free 2-DG was detectable in three of six strips at the concentration of
0.07±0.03 mmol l-1. This concentration represents <10% of
the total intracellular 2-DG. These findings provide evidence that, under
these conditions, 2-DG uptake rates reflect transport activity, transport is
rate-limiting and the accumulation of 2-DG-6-P provides an accurate index of
glucose transport activity.
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Discussion |
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Heart performance under hypoxia requires facilitated glucose transport
The initial power output (1.14 mW g-1) of perfused cod hearts
was slightly lower than reported in vivo values (1.77 mW
g-1; Axelsson and Nilsson,
1986) and approximately 17% of maximum pumping capacity (6.6 mW
g-1) as measured in situ (P. C. Mendoça, A. G.
Genge, E. J. Deitch and K. Gamperl, unpublished data). Furthermore, the
cardiac output of our in vitro hearts (21.5±1.2 ml
min-1 kg-1 fish) is well within the range (1729.1
ml min-1 kg-1 fish) of those reported for resting cod at
10°C (Jones et al., 1974
;
Petersson and Nilsson, 1980
;
Axelsson, 1988
). Thus, both
these parameters indicate that the perfused hearts were working at similar
levels to those of resting cod.
The cod heart does not require extracellular glucose to maintain power
under aerobic conditions. Hearts perfused with or without exogenous glucose
performed close to 100% of their initial value during the first 60 min.
Furthermore, hearts perfused with glucose in the medium performed only
slightly better during the next 60 min than those hearts perfused without
glucose. This confirms previous results indicating that the teleost heart can
maintain performance levels with endogenous fuels
(Driedzic and Hart, 1984).
Under severe hypoxic conditions, with glucose in the medium, cod hearts
were able to maintain a reduced level of performance for periods ranging from
30 to 120 min. This ability to sustain a lower level of performance during
severe hypoxia or anoxia has been seen in other fish such as the American eel,
bullhead, yellow perch and two species of armored catfish (Tamoatá and
Acari-bodó; Bailey et al.,
1999). These fish, including the cod, show excellent resistance to
the impairment of oxidative phosphorylation, which is indicative of a high
level of anaerobic metabolism. With the omission of glucose in the hypoxic
medium, cod hearts in this study failed within 40 min. Again, these results
are consistent with earlier findings, which showed that an exogenous glucose
supply was necessary to maintain force/pressure development in the eel heart
under anoxia (Bailey et al.,
2000
).
At 40 min, the power output of hypoxic hearts perfused with medium
containing glucose and cytochalasin B was significantly lower than hearts
perfused under hypoxia with glucose in the medium. Since cytochalasin B
impairs facilitated glucose transport into cells by binding to glucose
transporter proteins (Silverman,
1991), this result provides the first direct evidence that
facilitated glucose transport is essential to maintain the contractile
performance of the fish heart under oxygen-limiting conditions.
Hypoxia increases glucose uptake but only partly by an increase in facilitated transport
We can only estimate glucose utilization rates in the cod heart because it
is unclear if 15 min was sufficient time for the equilibration of the
extracellular marker, and a lumped constant that is the ratio of 2-DG uptake
to glucose uptake was not determined to correct for differences between tissue
uptake of these two compounds. Nevertheless, the rate of glucose uptake under
normoxia was similar to that reported for contracting eel ventricle strips
(Rodnick et al., 1997) and
rainbow trout hearts in vitro
(West et al., 1993
). Hypoxia
resulted in a 3-fold increase in glucose uptake compared with normoxic
conditions. This increase was much larger than the increase seen in glucose
uptake by eel ventricle strips, where the magnitude of the increase was less
than 50% of basal rates. It is probable that the difference in the impact of
hypoxia/anoxia relates to differences in energy demand by the contracting cod
heart versus the non-contracting eel ventricle strips. The addition
of cytochalasin B to the hypoxic medium in the perfused hearts led to a
decrease in the hypoxia-stimulated component of glucose uptake; however,
approximately 80% of glucose uptake was insensitive to cytochalasin B. A
cytochalasin B-insensitive component to glucose uptake was also observed in
the eel heart and was suggested by the authors
(Rodnick et al., 1997
) to be a
result of either membrane damage, a non-transporter-mediated process or
reduced affinity of the fish heart glucose transporter for cytochalasin B
relative to better-studied mammalian systems. The use of perfused hearts in
the current study suggests that tissue damage is not a major problem, leaving
the latter two issues still unresolved.
Extracellular glucose provides most of the energy under hypoxia
Severe hypoxia resulted in a significant decrease in glycogen levels in the
cod and eel hearts (Bailey et al.,
2000). Our data allow a calculation of the contribution of both
extracellular glucose and on-board glycogen to energy production. The rate of
2-DG uptake under hypoxia was
0.38 µmol g-1
min-1 (Fig. 2), which on the basis of a net ATP yield of two per glucose molecule equates to
0.75 µmol ATP g-1 min-1. This value though will be an
underestimate of glucose uptake if 2-DG accumulation underestimates uptake
(i.e. a lumped constant less than 1 see below). The rate of production
of glucosyl units from glycogen, under hypoxia with glucose in the medium, can
be calculated by the difference in the amount of glycogen between the normoxic
with glucose and the hypoxic with glucose groups divided by the average
perfusion time. This value was
0.06 µmol glucosyl unit g-1
min-1 (Figs 2,
3), which on the basis of a net
ATP yield of three per glucose equates to 0.18 µmol ATP g-1
min-1. These data reveal for the first time the magnitude of the
dependence of the fish heart upon extracellular glucose for the energy
production under hypoxia. ATP production from extracellular glucose is
minimally about 4-fold greater than that from glycogenolysis. These
independent biochemical measures are consistent with the finding of
glucose-dependent performance, including the current work with cod and studies
with other species (Driedzic et al.,
1978
; Bailey et al.,
2000
).
Despite the enhanced utilization of glycogen and myocardial glucose, the
amount of lactate (µmol g-1 tissue) in the heart did not change
under the various conditions. Although not measured, it is presumed that the
lactate was released from the heart into the perfusate, the same situation
reported for anoxic hearts from the sea raven and ocean pout, where anaerobic
metabolism was utilized to support ATP production as indicated by the
increased perfusate lactate levels leaving the heart
(Turner and Driedzic,
1982).
The absence of an Na+-sensitive component to glucose uptake
Hexose uptake by teleost cardiac muscle, to our knowledge, has not been
examined in the absence of extracellular Na+. An implication of the
present experiments is that Na+-sensitive glucose transport does
not exist in fish cardiac muscle. This contrasts with frog skeletal muscle,
where a minor (26%) Na+-sensitive component of sugar transport has
been reported (Kitasato and Marunaka,
1985). The fact that both phloridzin (current study) and
cytochalasin B (current study; Rodnick et
al., 1997
) were effective at inhibiting glucose uptake in fish
cardiac muscle promotes facilitative transport of glucose as an important
pathway by which sugars are transported across the myocyte sarcolemma.
However, the inability to completely block glucose uptake with either of these
inhibitors raises new questions about the contribution of other transmembrane
pathways and will require further investigation.
Glucose transport and phosphorylation may share control of glucose utilization
Based on our measurements of 2-DG uptake in cod ventricle strips and
perfused hearts, we provide new evidence that, in these preparations, membrane
transport is rate-limiting for glucose utilization. First, in non-working
ventricle strips, the majority of 2-DG was recovered as intracellular
2-DG-6-P, not free 2-DG. Second, the intracellular concentration of 2-DG was
much lower than the corresponding extracellular value. Third, concentrations
of free glucose in the working perfused heart decreased, not increased, when
glucose uptake was increased 3.5-fold by hypoxia. These findings suggest that
the rate of glucose phosphorylation is greater than the rate of transport into
the cell and that the rate-limiting step in the utilization of extracellular
glucose is transmembrane transport. Glucose utilization over a full range of
conditions though is possibly more complex than the current point of departure
studies reveal.
It was first shown by Morgan et al.
(1961) that the major control
of glucose uptake in the rat heart shifts between glucose transport and
phosphorylation, depending on the experimental conditions. These authors noted
an absence of intracellular glucose in isolated rat heart perfused with
glucose but no insulin. However, significant amounts of intracellular glucose
accumulated in hearts perfused with insulin and glucose above 2 mmol
l-1 or in hearts under anaerobic conditions receiving at least 4
mmol l-l glucose. Since then, others
(Cheung et al., 1978
;
Manchester et al., 1994
;
Kashiwaya et al., 1994
) have
confirmed that glucose transport and phosphorylation alternatively share the
control of glucose utilization in the rat heart dependent upon the
physiological conditions.
A switching of the prime control site for glucose utilization probably
exists in the fish heart as well. Although the current study presents evidence
that glucose transport in the cod heart is rate-limiting under both aerobic
and hypoxic conditions, experiments with other species suggest that glucose
phosphorylation is rate-limiting. For instance, in a study of brown trout
in vivo, held under normoxic conditions, radiolabeled 2-DG
accumulates to a much greater extent than 2-DG-6-P
(Blasco et al., 1996).
Similarly, increases in heart glucose during hypoxia have been reported for
diving African lungfish (Protopterus aethiopicus;
Dunn et al., 1983
), goldfish
(Carassius auratus; Shoubridge
and Hochachka, 1983
) and a small Amazon cichlid
(Cichlasoma sp.; Almeida-Val and
Farias, 1996
). The contrast between our findings with cod heart
and other studies is probably related to species differences and in vivo
versus in vitro models. Our isolated preparations lack hormonal signals
that may activate glucose uptake, in the same way as insulin and epinephrine
do in the mammalian heart (Doenst and
Taegtmeyer, 1998
). In addition, the level of glucose in the medium
was maintained at normoxic levels typical for cod. In some species, for
example sole (Via et al.,
1997
), goldfish (Walker and
Johansen, 1977
) and a species of armored catfish
(MacCormack et al., 2003
),
blood glucose may increase many-fold under hypoxia, which in turn could shift
the site of regulation from transport to phosphorylation. The range of blood
glucose levels in cod as a function of oxygen availability and other factors
has yet to be determined. Additional efforts, both in vivo and in
vitro, will be necessary to better define the conditions that regulate
cardiac glucose uptake in fishes that exhibit extreme species variability in
blood glucose (from zero to >50 mmol l-1) both within and
amongst species (Chavin and Young,
1970
; MacCormack et al.,
2003
).
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