Download Impaired Glucose Transporter Activity in Pressure

Impaired Glucose Transporter Activity in
Pressure-Overload Hypertrophy Is an Early Indicator of
Progression to Failure
Ingeborg Friehs, MD; Adrian M. Moran, MD; Christof Stamm, MD; Steven D. Colan, MD;
Koh Takeuchi, MD; Hung Cao-Danh, PhD; Christine M. Rader, BA;
Francis X. McGowan, MD; Pedro J. del Nido, MD
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Background—Severe hypertrophy and heart failure are important risk factors in cardiac surgery. Early adaptive changes
in hypertrophy include increased ventricular mass-to-cavity volume ratio (M/V ratio) and increased dependence on
glucose for energy metabolism. However, glucose uptake is decreased in the late stages of hypertrophy when ventricular
dilatation and failure are present. We hypothesized that impaired glucose uptake would be evident early in the
progression of hypertrophy and associated with the onset of ventricular dilatation.
Methods and Results—Ten-day-old rabbits underwent banding of the descending aorta. Development of hypertrophy was
followed by transthoracic echocardiography to measure left ventricular M/V ratio. Glucose uptake rate, as determined
by 31P-nuclear magnetic resonance spectroscopy measuring 2-deoxyglucose conversion to 2-deoxyglucose-6-phosphate,
was measured in isolated perfused hearts obtained from banded rabbits when M/V ratio had increased by 15% from
baseline (compensated hypertrophy) and by 30% from baseline (early-decompensated hypertrophy). In age-matched
control animals, the rate of glucose uptake was 0.6160.08 mmol z g of wet weight21 z 30 min21 (mean6SEM). With a
15% M/V ratio increase, glucose uptake rate remained at control levels (0.660.05 mmol z g of wet weight21 z 30 min21),
compared with hearts with 30% increased M/V ratios, where glucose uptake was significantly lower (0.4260.05 mmol z
g of wet weight21 z 30 min21; P#0.05). Glucose transporter protein expression was the same in all groups.
Conclusions—Glucose uptake rate is maintained during compensated hypertrophy. However, coinciding with severe
hypertrophy, preceding ventricular dilatation, and glucose transporter protein downregulation, glucose uptake is
significantly decreased. Because of the increased dependence of the hypertrophied hearts on glucose use, we speculate
that this impairment may be a contributing factor in the progression to failure. (Circulation. 1999;100[suppl II]:
II-187–II-193.)
Key Words: hypertrophy n echocardiography n glucose n metabolism
O
ne of the earliest events during adaptive remodeling in
pressure-overload hypertrophy is an increase in ventricular muscle mass; this is considered an initial compensatory
response to maintain normal wall stress. If pressure overload
persists, progressive ventricular dilatation ensues, which then
leads to increased wall stress, afterload mismatch, and decreased cardiac output.1 Several adaptive mechanisms occur
during the development of hypertrophy; these include the
multiplication of sarcomeres,2 a switch to immature isoforms
of contractile proteins,3 and greater dependence on transsarcolemmal calcium influx for excitation-contraction coupling.4
In addition, an increase occurs in glucose use as a substrate
for energy production.5,6 The transition from compensated to
decompensated hypertrophy with ventricular dysfunction is
poorly understood.
In mammalian cells, glucose is not freely permeable across
the lipid bilayer; it enters the cells by facilitated diffusion.
Specific membrane proteins that passively transport glucose
down a concentration gradient achieve this process. In cardiac
myocytes, 2 types of glucose transporters have been described (GLUT-1 and GLUT-4); they are responsible for
most of the uptake of glucose under basal conditions and in
response to insulin stimulation, respectively.7–14 We previously showed that the glucose uptake rate is impaired in
hearts with decompensated pressure-overload hypertrophy
when ventricular dilatation and contractile dysfunction have
occurred in response to thoracic aortic banding.15
In light of the potential role of glucose metabolism in
maintaining contractile function during the progression of
pressure-overload hypertrophy, we hypothesized that im-
From the Departments of Cardiac Surgery (I.F., C.S., K.T., H.C.-D., C.M.R., P.J.d.N.), Pediatric Cardiology (A.M.M., S.D.C.), and Anesthesiology
(F.X.M.), The Children’s Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Pedro J. del Nido, MD, Department of Cardiac Surgery, Children‘s Hospital, Harvard Medical School, 300 Longwood Ave., Boston,
MA 02115. E-mail [email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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Circulation
November 9, 1999
paired glucose transport occurs early in the progression to
ventricular dilatation. In a rabbit model of thoracic aortic
banding, we monitored the progression of hypertrophy using
transthoracic echocardiography. In previous work with this
model, we observed that after aortic banding, an initial
increase occurs in the left ventricular (LV) mass to LV
volume ratio (M/V ratio), which reaches a plateau '30%
above prebanding levels. At this point, the rate of LV cavity
volume increases faster than the rate of LV mass production,
resulting in a fall in M/V ratio.16 This progression mimics the
progression seen clinically with pressure overload.17 To
determine the relationship between glucose uptake and progression of hypertrophy, we measured the glucose uptake rate
and glucose transporter expression in control and aorticbanded rabbits during the early rise and the plateau phase of
LV M/V ratio after aortic banding.
modified KH solution containing 10 IU/L bovine insulin, a reduced
glucose content (1 mmol/L), and a concentration of 3 mmol/L
2-deoxyglucose (2-DG; Sigma). 2-DG acts as a competitive inhibitor
of glucose, and it is transported into the cell via the same mechanism
(facilitative diffusion). Phosphorylation of 2-DG by hexokinase to
2-deoxyglucose-6-phosphate (2-DG-6-P) results in a distinct resonance peak. The rate of rise of this peak is proportional to glucose
uptake by the cell and serves to quantify the rate of glucose uptake.
The rate of degradation of 2-DG-6-P was determined in a separate
group of hearts by perfusion with a KH buffer with 10 mmol/L
glucose and 10 U/L insulin after the infusion of 2-DG and obtaining
spectra for an additional 30 minutes. The rate of 2-DG accumulation
(proportional to the rate of glucose uptake) was quantified by a
second-order polynomial function.
Glucose Uptake With [2-3H]Glucose
Pressure-overload hypertrophy was achieved by placing a 2-0 silk
suture around the descending aorta just distal to the ligamentum
arteriosum in 10-day-old New Zealand White rabbits, as previously
described.15 Implanting a fixed constriction in an immature animal
and allowing it to grow induced pressure-overload hypertrophy by 2
to 3 weeks of age in this model. The progression of LV hypertrophy
was determined by weekly transthoracic echocardiography (HewlettPackard Sonos 1500 Cardiac Imager) with a 7.5-MHz transducer.
During these procedures, the animals remained unsedated to avoid
the influences of anesthetics on the results.
Glucose utilization was determined using the [2-3H]glucose method
of Katz and Dunn.21 This method is based on the measurement of
tritiated water (3H2O) production from [2-3H]glucose. 3H2O was
separated from [2-3H]glucose by ion-exchange chromatography on a
borate form of AG-1X8 resin (Bio-Rad Laboratories).22 Initially, the
hearts were perfused with tracer-free perfusate for a stabilization
period of 20 minutes; they were subsequently perfused in a nonrecirculating mode with KH buffer containing 50 mCi of [2-3H]glucose
over a period of 30 minutes. The rate of 3H2O release was measured
in the effluent withdrawn at 5-minute intervals, and 1 mL was
dissolved in Insta-Gel Plus/XF solution (Packard Bioscience). Isotope counting was performed on a Beckman LS 6500 liquid
scintillation counter (Beckman Instruments Inc). Rates of glucose
uptake are reported as micromoles per grams of wet weight per
minute (mmol z g of wet weight21 z min21).
Echocardiography
Hexokinase Activity
Two-dimensional cross-sectional images and M-modes of the left
ventricle were obtained by echocardiography. Simultaneous measurements of ECG and LV short-axis dimensions by M-mode were
recorded on hard copy at a paper speed of 100 mm/s. These
examinations were started at 3 weeks of age and performed at
1-week intervals. LV epicardial and endocardial surfaces were traced
by computer-aided offline analysis on a bit-mapped digitizing tablet.
End-diastolic (maximum dimension) and end-systolic (minimum
dimension) LV wall thicknesses were determined using previously
described methods.18 –20 The M/V ratio was calculated, and the serial
evaluation of M/V ratio was expressed as percent change
from baseline.
Hypertrophied and control hearts from separate groups of animals
were rapidly excised and perfused with modified KH solution for 5
minutes. The left ventricle was weighed, frozen in liquid nitrogen,
and homogenized in a buffer containing (in mmol/L): TrisHCl 20,
KCl 900, MgCl2 10, EDTA 2, and glucose 10 and 0.5% Triton
(Sigma). The assay was performed on the homogenate in a buffer
containing (in mmol/L): TrisHCl 40, KCl 100, MgCl2 20, EDTA 4,
ATP 2, NADP1 0.25, and glucose 10 and 0.03 U of glucose-6phosphate dehydrogenase (Sigma). Control assays were performed
on a buffer solution in which glucose or homogenate was omitted.
The rate of reduction of NADP1 with glucose-6-phosphate dehydrogenase was measured spectrophotometrically at a wavelength of 340
nm. One milliunit of hexokinase represents the amount of enzyme
activity that forms in 1 nmol of glucose-6-phosphate in 1 minute.23,24
Methods
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2-Deoxyglucose Uptake
Glucose uptake rate was determined using 31P-nuclear magnetic
resonance (NMR) spectroscopy. Spectra were acquired in an 8.45
Tesla vertical Bruker spectrometer operating at a proton frequency of
145 MHz. The isolated heart in its perfusion chamber was positioned
within a 20-mm solenoid radiofrequency coil. During the stabilization period of 30 minutes, the magnetic field was optimized by
shimming on the free-induction decay of the proton signal from the
heart and the surrounding perfusate. Spectra were obtained by
signal-averaging 120 scans with a 2-s delay. Each spectrum took 4
minutes. Spectral peak areas were quantified by integration after
baseline correction with software provided by Bruker. The determined areas were normalized to an external standard (using 500
mmol/L of dimethylene phosphonic acid) contained in a balloon
adjacent to the heart.
The animals were euthanized by intravenous injection of an
overdose of ketamine (50 to 100 mg/kg); heparin (500 U/kg) was
also given intravenously. The hearts were rapidly excised and placed
in cold Krebs-Henseleit (KH) solution. After aortic cannulation, the
hearts were perfused in the nonworking, nonrecirculating Langendorff mode at a perfusion pressure of 80 mm Hg with oxygenated
KH solution containing (in mmol/L): NaCl 117, KCl 4.7, MgSO4 1.2,
CaCl2 1.8, NaHCO3 23.7, and glucose 10 (37°C, pH 7.4). After a
30-minute stabilization period, the perfusate was switched to a
Western Immunoblotting
Ventricular tissue from a separate set of hypertrophied and agematched littermates (control animals) was homogenized in ice-cold
buffer containing (in mmol/L): TrisHCl 20, EDTA 2, EGTA 0.5, and
PMSF 1, and 25 mg/mL leupeptin and 0.3 mol/L sucrose (pH 7.4); it
was then centrifuged at 1000g for 20 minutes. The crude supernatant
fraction was stored at 280°C for later analysis. Gel electrophoresis
with 10% SDS-PAGE gels was performed on samples of 25 mg of
protein from total homogenates in accordance with the method of
Laemmli.25 Proteins were then electrophoretically transferred to
nitrocellulose membranes. After transfer, the membranes were incubated in 5% nonfat dry milk in 10 mmol/L TrisHCl (pH 7.5),
100 mmol/L NaCl, and 0.1% Tween 20 for 1 hour at room
temperature to block unspecific binding sites; they were then
incubated with antibodies.
GLUT-1 and GLUT-4 (Genzyme Diagnostics) were used as
primary antibodies at a dilution of 1:1000; samples were then
incubated with horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research Labs) at a dilution of 1:10000. The
bound antibody was detected by the enhanced chemoluminescence
method according to the manufacturer’s instructions (Amersham
Life Science). This method depends on the production of light after
Friehs et al
Glucose Transport in Pressure-Overload Hypertrophy
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the oxidation of luminol by horseradish peroxidase in the presence of
H2O2. After exposure on films, quantitative protein analysis was
undertaken by laser densitometry.
Statistical Analysis
Data are expressed as mean6SEM and were analyzed using SigmaStat software (Jandel Scientific). Comparisons between groups
were made with 1-way ANOVA. Probability values were corrected
by Bonferroni’s post hoc correction. If normality and equal variance
testing were passed, a standard t test was used. For all these tests,
P#0.05 was considered statistically significant.
Animal Care
All animals received humane care in compliance with the Principles
of Laboratory Animal Care formulated by the National Society for
Medical Research and the Guide for the Care and Use of Laboratory
Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86
to 23, revised 1985). The protocol was reviewed and approved by the
animal care committee at Children’s Hospital in Boston.
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Results
Weekly measurements of LV M/V ratios demonstrated that
after aortic banding, an initial rapid increase of LV wall
thickness occurred, resulting in a rise in LV M/V ratio. This
reached a plateau '30% above prebanding levels by 3 to 4
weeks of age. We termed the phase leading up to this plateau
“compensated hypertrophy.” By 4 to 5 weeks of age, a
gradual decline in M/V ratio occurred due to a more rapid
increase in LV cavity volume relative to LV mass, indicative
of LV dilatation (decompensated phase). Figure 1 shows
representative M-mode echocardiographic images of a heart
with compensated hypertrophy and an age-matched control
heart.
On the basis of the echocardiographic findings, animals
with a '15% increase in M/V ratio from baseline (4 weeks of
age, compensated hypertrophy) and with a '30% rise in M/V
ratio (5 weeks of age, early-decompensated phase) were
studied and compared with age-matched control animals.
Paralleling these echocardiographic findings, the banded
animals showed a significant degree of hypertrophy, as
estimated by LV weight to body weight ratios, which were
3.460.4 (compensated phase) and 3.360.4 (earlydecompensated phase) in the hypertrophied groups compared
with 2.360.2 in controls. Body weights were not different
between the groups, and LV weight was 3.0860.26 g in
hearts with compensated hypertrophy, 2.8560.06 g in hearts
with early-decompensated hypertrophy, and 2.1660.29 g in
control hearts (P#0.05).
31
P-NMR spectra before and after a 30-minute infusion of
2-DG are shown in Figures 2A and 2B. Figure 2C depicts a
representative curve of the 2-DG-6-P peak during 2-DG
infusion and wash-out, indicating that 2-DG-6-P is not further
metabolized during the observation period, even after subsequent wash-out with glucose-containing buffer.
Figure 3A shows the rate of 2-DG-6-P accumulation over
a period of 30 minutes in the aortic-banded groups and
control animals. 2-DG accumulation was significantly lower
in hearts with early-decompensated hypertrophy ('30% increase of M/V ratio) than in control hearts (P#0.05), with a
slower rate of rise and lower total accumulation after 30
minutes. Figure 3B shows the rate of glucose uptake mea-
Figure 1. Representative M-mode echocardiographic pictures
from normal rabbit hearts (A) and those with compensated hypertrophy (B). Scale is not indicative of blood-pressure curve.
sured by cumulative tritiated water production from
[2-3H]glucose over a period of 30 minutes. In aortic-banded
animals at a '30% rise of M/V ratio, the glucose-uptake rate
was significantly lower compared with age-matched control
animals (P#0.05).
In a separate group of hearts (n54 per group), myocardial
protein was obtained from the left ventricle of rabbits with
compensated, early-decompensated, and late-decompensated
hypertrophy (6 weeks of age, M/V ratio beginning to fall).
The GLUT-1 and GLUT-4 protein content from the myocardium of aortic-banded groups and age-matched littermates, as
determined by immunoblotting, was not significantly different between any of the groups. A representative immunoblot
of GLUT-4 is depicted in Figure 4A, and a summary of the
densitometry results is shown in Figure 4B.
In a separate group of hearts perfused with KH solution
containing insulin and glucose, total hexokinase activity was
the same in hypertrophied and control hearts at a M/V ratio
increase of '30% (38.260.47 mU/mg protein compared with
42.861.62 mU/mg protein, respectively).
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Figure 2. Glucose uptake rate and
phosphorylation were measured as
accumulation of 2-DG-6-P by 31P-NMR
spectroscopy over 30 minutes. A, Representative NMR-spectra (PCr indicates
creatine phosphate assigned to 0 ppm;
standard, dimethyl phosphoric acid; Pi,
inorganic phosphate; and PME, phosphomonoester). B, Representative NMRspectra with typical 2-DG-6-P peak after
addition of 2-DG to the perfusate. C,
Representative 2-DG uptake curve: perfusion of hearts (n53; mean6SEM) with
2-DG over 40 minutes followed by 35
minutes of wash-out with glucose- and
insulin-containing buffer (change of solutions indicated by arrows).
Discussion
In this study, echocardiography was used to monitor the
progression of LV hypertrophy and to determine the timing
for the study of glucose uptake and glucose transporter
protein content. The observations derived from this investigation reveal that a decrease in glucose uptake rate is evident
early in the transition from compensated to decompensated
pressure-overload hypertrophy, as determined by an index of
LV M/V ratio. The defect in insulin-stimulated glucose
uptake precedes the downregulation of sarcoplasmic reticu-
lum Ca-ATPase (SERCA-2) and glucose transporter expression (GLUT-4 and GLUT-1).15,26
Under pathophysiological conditions such as hypertrophy
or during ischemia and early reperfusion, a high rate of
cardiac glucose metabolism may be crucial.27–29 Glucose
transport is thought to be rate-limiting for glucose use. In the
heart, 2 distinctive glucose transporters are responsible for
glucose uptake across the plasma membrane. The GLUT-1
transporter, which is present in low levels in most tissues, is
non-insulin–stimulated and is responsible for basal glucose
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Glucose Transport in Pressure-Overload Hypertrophy
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Figure 3. A, Glucose uptake rate and
phosphorylation were measured as
accumulation of 2-DG-6-P by 31P-NMR
spectroscopy over 30 minutes in aorticbanded animals showing '15% increase
in M/V ratio (3 to 4 weeks of age) and
'30% rise of M/V ratio (4 to 5 weeks of
age) and in age-matched littermates
(controls; n56/group). *P#0.05 between
groups. B, Glucose uptake rate determined by measuring cumulative tritiated
water production from [2-3H]glucose in
aortic-banded animals showing '30%
rise of M/V ratio compared with agematched littermates (n55/group).
*P#0.05 versus control.
use.11,12 GLUT-4 activity is regulated by insulin and is
expressed in tissues in which glucose transport needs to be
rapidly and markedly enhanced (adipocytes, skeletal and
cardiac muscle).13,14 In the basal state, GLUT-1 is evenly
distributed between the plasma membrane and low-density
microsomal pools, whereas GLUT-4 is almost entirely stored
in an intracellular pool.14,30,31 Insulin interacts with its receptor on the plasma membrane and stimulates the redistribution
of GLUT-4 to the sarcolemma.32,33 When insulin levels fall,
GLUT-4 is resequestered in intracellular vesicles.34 A decrease in GLUT-4 mRNA and protein is thought to be a
mechanism for insulin resistance in various models of diabetes, and it has been associated with lower myocardial glucose
uptake.14,35–38 In the present study, we did not find altered
GLUT-4 protein expression, as determined by immunoblotting of LV myocardial tissue, which indicates that insulin
signal transduction and/or glucose transporter activity is
altered in hypertrophied hearts.
In the present study, we used the distinct 31P-NMR peak of
2-DG-6-P to investigate the glucose transport capacity of
hypertrophied hearts. 2-Deoxy-D-glucose, a competitive inhibitor of D-glucose, is transported into the cell via the same
mechanism as glucose, is phosphorylated by hexokinase, and
accumulates as 2-DG-6-P, which is metabolized very slowly.
The rate of 2-DG-6-P accumulation, therefore, reflects both
transport across the plasma membrane and phosphorylation
of glucose, and it is a useful tracer of glucose transport and
phosphorylation in the isolated perfused heart.15,39 To confirm our findings with 2-DG uptake in hypertrophied hearts,
we used a radioactive tracer technique; the same results were
obtained.
In some models, 2-DG-6-P is further metabolized by
incorporation to glycogen at low rates.40 The rate of 2-DG6-P degradation is slow with respect to the rate of uptake;
degradation rates ranging from 70 to 110 minutes have been
previously reported.39,41,42 In the present study, 2-DG uptake
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November 9, 1999
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Figure 4. A, Representative immunoblot
of total homogenates from hearts with
compensated ('15% rise of M/V ratio),
early-decompensated ('30% increase of
M/V ratio from baseline), and latedecompensated hypertrophy (decline of
M/V ratio indicating dilatation) compared
with age-matched controls. B, Densitometry values (arbitrary densitometry
units) for total homogenates of hearts
with compensated, earlydecompensated, and latedecompensated hypertrophy and agematched controls (n54/group).
reached a plateau after 30 minutes of perfusion, and little, if
any, 2-DG-6-P was further metabolized during a wash-out
period. Similar findings have been reported when hypertrophied and normal hearts were compared in a spontaneously
hypertensive rat heart model.43 Therefore, we think that the
use of 2-DG is a reliable method of glucose uptake determination in our model.
In the current study, in hearts with compensated hypertrophy ('15% rise in M/V ratio), glucose uptake in response to
insulin was still maintained at near-normal levels; impaired
glucose uptake was detected before progression from compensated to decompensated hypertrophy at the peak M/V
ratio ('30% above baseline). It is conceivable that the
impaired response of GLUT-4 to insulin stimulation (eg,
defects in insulin receptor signaling or alterations in GLUT4-containing vesicle trafficking) could contribute to the development of decompensated cardiac hypertrophy. Similar
defects in the insulin-stimulation of glucose use and transport
in the heart have been demonstrated in animal models of
diabetes and insulin resistance.37,44 – 46 In the absence of
insulin, glucose transport is rate-limiting for glycolysis, as
reported for isolated rat cardiomyocytes.47 Therefore, an
important relationship may exist between the progression of
hypertrophy to failure and decreased glucose uptake because
a diminished rate of cardiac glucose use is thought to be
detrimental to heart function.48 We previously showed that
severe hypertrophy with impaired glucose transport has
functional significance with regard to impaired recovery after
experimental myocardial ischemia. Normalizing glucose
transport with vanadate significantly improved postischemic
recovery in the hypertrophied heart.15
Our data indicate that a decline of glucose uptake rate
occurs in the hypertrophied heart and, concomitantly with the
lack of glucose available for the myocardium, hypertrophy
progresses to myocardial failure. Insulin insensitivity of the
GLUT-4 transport system, a potential underlying mechanism,
may play a significant role. Because GLUT-4 protein levels
remain unchanged at this stage, we speculate that impaired
insulin signaling is responsible for this defect. Strategies to
overcome this defect by either direct bypass of insulindependent GLUT-4 activation or augmenting glucose transport to the myocytes by other means may improve myocardial
function and prevent the progression of hypertrophied hearts
to failure.
Acknowledgements
This work was supported in part by grants from the American Heart
Association (to P.J.d.N.) and the National Institutes of Health (HL
46207 to P.J.d.N. and HL 52589 to F.X.M.).
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Circulation. 1999;100:II-187-Ii-193
doi: 10.1161/01.CIR.100.suppl_2.II-187
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