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Department of Physiology, Midwestern University, Downers Grove, Illinois 60515
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Osborn, Brett A., June T. Daar, Richard A. Laddaga, Fred D. Romano, and Dennis J. Paulson. Exercise training increases sarcolemmal GLUT-4 protein and mRNA content in diabetic heart. J. Appl. Physiol. 82(3): 828-834, 1997.
This study determined whether dynamic exercise training of
diabetic rats would increase the expression of the GLUT-4 glucose
transport protein in prepared cardiac sarcolemmal membranes. Four
groups were compared: sedentary control, sedentary diabetic, trained
control, and trained diabetic. Diabetes was induced by intravenous
streptozotocin (60 mg/kg). Trained control and diabetic rats were run
on a treadmill for 60 min, 27 m/min, 10% grade, 6 days/wk for 10 wk.
Sarcolemmal membranes were isolated by using differential
centrifugation, and the activity of sarcolemmal
K+-p-nitrophenylphosphatase
( pNPPase; an indicator of
Na+-K+-adenosinetriphosphatase
activity) was quantified. Hearts from the sedentary diabetic group
exhibited a significant depression of sarcolemmal pNPPase
activity. Exercise training did not significantly alter
pNPPase activity. Sedentary diabetic rats exhibited an 84 and 58% decrease in GLUT-4 protein and mRNA, respectively, relative to
control rats. In the trained diabetic animals, sarcolemmal GLUT-4
protein levels were only reduced by 50% relative to control values,
whereas GLUT-4 mRNA were returned to control levels. The increase in
myocardial sarcolemmal GLUT-4 may be beneficial to the diabetic heart
by enhancing myocardial glucose oxidation and cardiac performance
myocardial glucose oxidation; diabetic cardiomyopathy
INTRODUCTION STUDIES IN EXPERIMENTAL animals and humans have shown
that diabetic subjects exhibit depressed myocardial contractile
performance, particularly under stress conditions such as enhanced
workload (5, 12). Clinically, the diabetic heart also exhibits enhanced sensitivity to ischemic-reperfusion injury (12, 24, 33). It has been
suggested that limited glucose availability during these stress
conditions may account for these alterations in myocardial performance
in the diabetic individual (6, 17, 25, 29). Because transport is the
rate-limiting step for myocardial glucose uptake (17), a defect in the
glucose transport system could be particularly detrimental. Studies
have also shown that diabetes is associated with a decrease in the
number of sarcolemmal glucose transport proteins (6, 25).
Exercise training of diabetic subjects has been shown to limit the
depression in myocardial pump function (18) and improve the recovery of
contractile function after a period of ischemia and reperfusion (16,
19). The incidence of reperfusion arrhythmias was also reduced by
exercise training of diabetic animals (1). One of the beneficial
effects of exercise training on myocardial performance may be mediated
through an enhancement of the myocardial glucose transport system. In
control nondiabetic animals, it has been shown that myocardial glucose
uptake during rest and exercise is significantly increased after
7-13 wk of exercise training (11). The enhanced ability of the
exercise trained heart to utilize glucose may exert a protective effect
in the event of an ischemic episode. This suggests that exercise exerts
an effect by modulating glucose transport in control animals. In fact,
it has been reported by us that exercise training of diabetic rats enhances glucose oxidation rates of diabetic hearts (20). In addition,
Hall et al. (8) showed that exercise training of diabetic rats
attenuated the reduction in whole heart total GLUT-4 glucose transport
protein levels. The purpose of this study was to ascertain whether
exercise training would affect sarcolemmal GLUT-4 levels as well as the
mRNA levels in the diabetic myocardium, hence providing a possible
mechanism for the beneficial effects of exercise training on the
diabetic heart.
METHODS
80°C.
Metabolic status.
For each experiment, the effects of the various treatment protocols on
the severity of the diabetic state were assessed by measuring blood
glycosylated hemoglobin (gHb) and plasma levels of glucose, free fatty
acids, triglycerides, and total cholesterol. Blood concentration of
total gHb was estimated by using 50 µl of blood in a microscale
ion-exchange column method. The ion-exchange columns were purchased
from Helena Laboratories (Beaumont, TX). Plasma glucose was measured by
using the two-step enzymatic method (hexokinase and glucose-6-phosphate
dehydrogenase, Sigma Chemical, St. Louis, MO). Plasma triglycerides and
total cholesterol levels were measured by using kits purchased from
Sigma Chemical. Free fatty acids were measured by using a kit from Wako
Pure Chemical Industries.
Cytochrome oxidase.
The plantaris muscle was homogenized by using a polytron for 2 s at a
speed setting of 6 [~18,000 revolutions/min (rpm)] with a
2-min wait on ice, and then the procedure was
repeated 2-3 times until a homogenous homogenate was
attained. An isolation medium containing 0.1 M KCl and (in mM) 50 3-(N-morpholino)propanesulfonic acid (MOPS), 10 MgSO4, and 1 ethylenediaminetetraacetic acid was used. Cytochrome oxidase
activity was measured spectrophotometrically at 25°C as described
by Wharton and Tzagoloff (30).
Isolation of sarcolemmal vesicles.
The method described by Pierce and Dhalla (21) was used. Three to four
hearts were pooled and used for each isolation procedure. Hearts were
then rapidly excised, trimmed of atria and extraneous tissues, and
weighed before being processed. The tissues were then minced with
scissors and homogenized in 20 ml of (in mM) 250 sucrose, 1 dithiothreitol (DTT), 20 tris(hydroxymethyl) aminomethane (Tris)-maleate (pH 7.6), and 25 sodium pyrophosphate, as well as 0.1 M
KCl by using a Sorvall Omnimixer (speed setting of 6) 2 times for 12 s
each. A 0.2-ml aliquot was removed and stored with 1.8 ml of (in mM)
250 sucrose, 1 DTT, and 20 Tris-maleate for next-day biochemical
assays. The remaining homogenate was mixed with 20 ml of (in mM) 250 sucrose, 1 DTT, 20 Tris-maleate, and 500 sodium pyrophosphate, as well
as 1.2 M KCl, and centrifuged at 48,400 g for 45 min by using a Sorvall RC5B
rotor (20,000 rpm). The pellet was resuspended by hand by using a
Dounce homogenizer in 13 ml of (in mM) 250 sucrose, 1 DTT, and 20 Tris-maleate and incubated with 30,000 U of purified precrystalline
deoxyribonuclease (DNase) I (>1,400 U/mg dry weight,
Worthington Biochemical, Freehold, NJ). The DNase I was previously
suspended in 2 ml of the above solution at 30°C for 45 min. The
suspension was polytroned at a speed setting of 6 (~18,000 rpm) 3 times for 7 s each and then diluted into 2 tubes of 15-20 ml of
(in mM) 250 sucrose, 1 DTT, and 20 Tris-maleate. These suspensions were
then centrifuged at 17,400 g (12,000 rpm) at 4°C for 15 min. The resultant supernatant was centrifuged
at 100,000 g (30,000 rpm) for 60 min
by using a Beckman 30 rotor. The pellet was resuspended in 6 ml of 40% sucrose. This suspension was layered on the bottom of a sucrose gradient consisting of 2 ml of 27% sucrose [containing 0.02 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N
,N-tetraacetic acid (EGTA)], 1 ml of 30% sucrose, 2 ml of 34% sucrose, 2 ml of 37% sucrose, and 3 ml of 40% sucrose. All sucrose solutions contained 2 mM NaCl. The gradient solution was centrifuged overnight (16-18 h) in a Beckman 40 rotor at 104,000 g
(40,000 rpm) at 4°C. The sarcolemmal vesicles were recovered as a
diffuse band above the 30:34% sucrose interface. The sarcolemmal
fraction was diluted and centrifuged at 30,000 rpm for 45 min. The
pelleted sarcolemmal membranes were resuspended in 140 mM NaCl and 20 mM MOPS (pH 7.4) and stored in liquid
N2 atmosphere for future analysis.
Enzyme activities.
To monitor the relative purity of the membrane fractions prepared by
using this fractionation procedure and to assess
Na+-K+-adenosinetriphosphatase
(ATPase) activity, total
K+-p-nitrophenylphosphatase
( pNPPase) activity was measured in sarcolemmal vesicle preparations. The reaction medium contained (in mM)
50 Tris, 5 MgCl2, 1 EGTA, 5 p-nitrophenylphosphate, and 20 KCl (pH 7.8) (21). Basal
pNPPase activity was measured in a similar medium without
KCl, and this value was subtracted from the total pNPPase
activity to obtain the
K+-pNPPase activity.
The reaction was started by the addition of 10 µg sarcolemmal protein
and stopped after 8 min by the addition of 2 ml of 1 N NaOH. The amount
of p-nitrophenyl formed was measured at an absorbance of 410 nm. A preliminary experiment showed that the
homogenate pNPPase activity of the control and diabetic
groups was 0.89 and 0.37 µmol
phenol · mg
1 · h1,
respectively. The activities of the sarcolemmal vesicles were increased
by at least 18-fold over those measured in the homogenates.
Immunoblot (Western blot) analyses.
Sarcolemmal vesicles containing ~75 µg protein (determined by
bicinchoninic assay protein kit from Pierce Chemical, Rockford, IL)
were mixed with Laemmli sample buffer (14) containing 2.5% dithiothreitol and 1% sodium dodecyl sulfate (SDS). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10%
resolving gel and then transferred from the gel to an Immobilon membrane (Sigma Chemical) by electrotransfer. The membrane was blocked
for 2 h with 5% Carnation low-fat instant milk in Tris-buffered saline
(TBS), followed by incubation at 25°C for 16 h in polyclonal antibody ECU4 (East Acres Biologicals, Southbridge, MA) specific for
the last 12 amino acids of the GLUT-4 COOH-terminal (9). The membranes
were washed alternatively in TBS and TBS-0.05% polyoxyhylenesorbitan monolaurate (Tween 20) and then probed for 4 h with
125I-labeled protein A, (New
England Nuclear, Wilmington, DE). Membranes were washed, dried, and
autoradiographed for 48 h at 70°C. To determine the amount
of 125I associated with GLUT-4,
immunolabeled bands from the nitrocellulose sheets (45 kDa) were
excised and counted on a Cobra-1 gamma counter (model 5003, Packard).
Each gel contained sarcolemmal membrane samples from all four groups.
Because of differences in the specific activity of the labeled probe
from one experiment to another and transfer efficiencies between gels,
the number of counts for the 125I
associated with GLUT-4-immunolabeled bands from each of the experimental groups was expressed as a percentage of the control value
of each respective gel. Periodic replicates of gels were performed to
confirm that the results being obtained were consistent.
In a separate set of control, diabetic, and exercise-trained diabetic
rats, the levels of GLUT-4 were assayed in heart homogenates. In this
experiment, tissue was homogenized in (in mM) 20 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 1 EDTA, and 250 sucrose, pH 7.4 (1:79 wt/vol). Protein content was determined in duplicate from aliquots of homogenate with the bicinchoninic assay method (Pierce Chemical). The homogenate was diluted with Laemmli sample buffer. Fifty micrograms of protein were
added to each lane of the gel. Samples were run in duplicate and
subjected to SDS-PAGE with a 10% resolving gel. Prestained molecular
weight standards were run in lanes adjacent to samples. Next, proteins
were electrophoretically transferred to a nitrocellulose membrane
(Immobilon-P, Millipore, Bedford, MA). Membranes were blocked for 1 h
at room temperature with a solution of 50 mM Tris, 200 mM NaCl, and
0.025% Tween 20 and 0.02% sodium azide [TBS + Tween
(TBST)], pH 7.5, containing 5% Carnation nonfat dry
milk. Nitrocellulose membranes were then incubated overnight in a
750-fold dilution of polyclonal antibody ECU4. Membranes were rinsed
twice briefly, followed by a single wash for 15 min and 2 final washes for 5 min at room temperature with TBST + 1% milk. Subsequently, a
60-min incubation with a 1,000-fold dilution of a rabbit horseradish peroxidase-conjugated antibody (Amersham, Arlington Heights, IL) in
TBST + 1% milk was performed. Membranes were then washed as described
above. Protein bands were detected by exposing the membranes to
enhanced chemiluminescence detection reagents (Amersham) in a dark
room, wrapping the membranes in Saran wrap, and finally exposing them
to Kodak X-OMAT AR film.
RNA blot analyses.
Myocardial tissue was homogenized in 1 ml of a guanidinium
thiocyanate-2-mercaptoethanol solution (50:0.36 vol/vol) for 2 × 10-s bursts at a setting of 6 (~18,000 rpm) by using a Polytron (Brinkmann, Westbury, NY). Total RNA was then isolated by extraction in
phenol-chloroform, followed by isopropanol precipitation from the
aqueous phase. The RNA was then pelleted by centrifugation at
10,000 g. The pellet was washed twice
with 70% ethanol. After centrifugation, the RNA was dissolved in
pyrocarbonic acid diethyl ester-treated water as described by
Chomczynski and Sacchi (4). The amount of RNA was estimated by
measuring the absorbance at 260 nm. The 260/280 ratio was used to check
for purity. For Northern blot analyses, 25 µg of total RNA were
electrophoresed on a 1.2% agarose-formaldehyde gel and, by the use of
the Rapid Downward Transfer System (Turboblotter, Schleicher & Schuell,
Keene, NH), transferred onto Nytran membranes (Schleicher & Schuell).
RNA integrity and efficiency were verified by staining gels with
ethidium bromide. The RNA was visualized and photographed by
ultraviolet transillumination to ensure that the RNA was intact. The
intensity of the 18S and 28S ribosomal bands were used to ensure that
the amount of total RNA loaded onto each lane of the gel was constant. An RNA ladder (GIBCO-BRL, Grand Island, NY) was utilized to calculate size or length of the mRNA. Nearly full-length cDNAs for the rat GLUT-4
glucose transporter and glycerol-3-phosphate dehydrogenase (G3PDH) were
32P labeled to a specific activity
of 1.5-3.5 × 109
counts · min1 · µg1
by the Random Primers Labeling System (GIBCO-BRL). The GLUT-4 probe was
kindly provided by Dr. G. Bell (2) and purified by using a Qiagen
column kit. The G3PDH probe was obtained from Clontech, Palo Alto, CA.
Blots were then prehybridized for 3 h at 51°C in a solution of 40%
deionized formamide, 5× standard sodium citrate (SSC), 5×
Denhardt's solution, 0.1 mg/ml salmon sperm DNA, 0.5 mM
NaPO4, 10% Dextran sulfate, and
0.5% SDS. Hybridization was carried out at 51°C for 18-24 h
in 40% deionized formamide and 5× SSC by using the
32P-labeled cDNA probe. The
membranes were then washed at high stringency (55°C, 0.1% SSC,
0.1% SDS, 20 min) and then autoradiographed at 70°C by
using Kodak XAR-5 film. The resulting autoradiograms were quantified by
the amount of 32P associated with
GLUT-4. Labeled bands from the nitrocellulose sheets (2.8 kb for GLUT-4
and 1.1 kb for G3PDH) were excised and counted on a TRICARB CA liquid
scintillation counter (model 1900, Packard). Results were corrected for
background determined in apparently unlabeled areas of the membranes.
Data analysis.
Data obtained for each group were compared to statistically determine
significant differences by using a one-way analysis of variance test
and a Newman-Keuls multiple-comparison test for post hoc comparisons
among treatment groups. A value of P < 0.05 is considered significant.
RESULTS
Sedentary diabetic rats exhibited an elevation in blood levels of gHb (%) as well as levels of plasma glucose, triacylglycerol, and cholesterol relative to those of the control group (Table 1). Exercise training of diabetic rats significantly lowered blood gHb as well as plasma glucose, cholesterol, and triglyceride levels. Previously, we have shown that exercise training of diabetic rats did not alter levels of blood gHb (20). The explanation for this inconsistency is uncertain but may relate to the level of training produced in diabetic rats by the different trainers used in each study. Exercise training of control rats had no effect on blood gHb and plasma glucose, but plasma cholesterol and triglycerides were decreased. Plasma free fatty acids were decreased in both exercise-trained groups relative to the respective sedentary groups.
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Cytochrome oxidase activity of the plantaris muscle was assayed to determine whether the exercise protocol produced a significant training effect (Table 1). The cytochrome oxidase activity of the sedentary diabetic rats was decreased relative to the sedentary control group. Exercise training significantly increased skeletal muscle activity of this enzyme in both control and diabetic rats by 24 and 22%, respectively, relative to their sedentary counterparts. However, because body weights of the control and diabetic rats were different, this similar percent increase in plantaris cytochrome oxidase activity does not necessarily indicate that both groups received the same degree of training but, rather, that the exercise program produce a significant training effect in each group.
Sedentary control rats exhibited a significant increase in body weight over the 10-wk protocol. Exercise training significantly attenuated this increase in body weight. Sedentary diabetic rats had significantly lowered body weights relative to sedentary control rats. However, exercise training of diabetic rats did not significantly affect body weight.
Table 2 shows the effects of exercise training on sarcolemmal pNPPase activity. The activity of this enzyme was significantly depressed in the sedentary diabetic animals relative to that in the sedentary control. This depression in K+-pNPPase activity was similar to that reported by others (21). The activity of the K+-pNPPase in the trained diabetic rats was comparable to that of the sedentary diabetic group.
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In the present study, whole heart homogenates from sedentary control,
sedentary diabetic, and exercise-trained diabetic rats were analyzed
for total GLUT-4 levels (Fig. 1). The
diabetic total heart GLUT-4 level was decreased relative to that of the
control heart. In two exercise-trained diabetic hearts, the levels of total GLUT-4 were increased toward control levels but did not completely normalize levels. This finding is consistent with that of
Hall et al. (8).
Cardiac sarcolemmal GLUT-4 levels are shown in Fig.
2. A typical Western blot of
sarcolemmal proteins probed with polyclonal antibody ECU (GLUT-4) is
shown (Fig. 2A).
Sedentary diabetic rats exhibited a significant decrease in sarcolemmal
GLUT-4 concentration compared with those of the sedentary control
group. The mean value for the diabetic group was only 16% of the
control value. Exercise training of diabetic rats improved the levels
of GLUT-4 but did not produce a complete normalization. GLUT-4 levels
were increased to 50% of the control value. Although this difference
was significantly less than the control level, it was also
significantly higher than the sedentary diabetic group. Exercise
training of control rats had no effect on sarcolemmal GLUT-4.
Significantly different from D,
P < 0.05.
The effects of diabetes and exercise training on myocardial GLUT-4 mRNA
levels are shown in Fig. 3.
Sedentary diabetic rats exhibited a significant 60% decrease in GLUT-4
mRNA levels relative to sedentary control rats. Exercise training of
diabetic rats increased significantly the levels of GLUT-4 mRNA to
control levels such that there were no significant differences between
these two groups. Exercise training of control rats had no effect on GLUT-4 mRNA. To ensure that these differences in GLUT4 mRNA were not
due to nonspecific changes in total mRNA levels, the housekeeping gene
G3PDH was also measured. No differences in mRNA for G3PDH were found
among the four groups (Fig. 4).
Significantly
different from D, P < 0.05.
DISCUSSION
Streptozotocin-induced diabetes has been shown to produce a 53% decrease in glucose transport activity in cardiac sarcolemmal vesicles compared with that in controls (6). This effect was attributed to a decrease in the number of cell-surface GLUT-4 transporters. The sedentary diabetic rats in this study exhibited a similar reduction in whole heart and sarcolemmal GLUT-4 levels. This decline in the number of sarcolemmal glucose transport proteins may limit the availability of glucose, which is an important source of ATP for the myocardium during periods of increased work or ischemia (6, 17, 25). It has been hypothesized that these metabolic impairments may contribute, in part, to the depression seen in cardiac contractile performance of diabetic hearts in response to increased work (5, 6, 12, 18). It may also explain the clinical findings that the diabetic heart is at a greater risk for myocardial ischemia and its associated complications (1, 12, 24, 33).
Clinical reports have indicated that exercise training reduces the risk of cardiac complications associated with diabetes mellitus (15, 26). The beneficial effects of exercise training on the diabetic heart have been confirmed in experimental studies (1, 16, 18-20). In the present study, exercise-trained diabetic rats exhibited a partial normalization of whole heart and sarcolemmal GLUT-4 protein levels as well as a complete return to control levels for the mRNA levels of this protein. Previously, Hall et al. (8) showed that exercise training of diabetic rats produced a partial enhancement in the levels of total GLUT-4 protein in the whole heart. These findings suggest that exercise training of diabetic rats enhances the synthesis of new GLUT-4 protein and its incorporation into the sarcolemmal membrane. The exercise-training protocol used in this study had no effect on sarcolemmal GLUT-4 protein and mRNA levels of control (nondiabetic) rats. The lack of effect in this case may be due to the fact that myocardial levels of GLUT-4 were already at optimal levels. In addition, because the control rats were larger than the diabetic rats, the level of cardiovascular stress in both groups elicited by the training sessions may not have been identical.
It has been suggested that a correlation exists between GLUT-4 protein concentration and oxidative enzyme activity in skeletal muscle, suggesting that the training-induced increase in muscle GLUT-4 is coregulated with the increased expression of enzymes associated that control glucose utilization (3). In the present study, it is interesting to note that the diabetic rats exhibited a decrease in plantaris muscle cytochrome oxidase activity that was partially reversed by exercise training of diabetic rats. Kainulainen et al. (10) also found that the activity of cytochrome oxidase was decreased in diabetic skeletal muscle and that exercise training of diabetic rats prevented this decrease. However, in this study the increase in plantaris muscle cytochrome oxidase activity was not associated with an increase in plantaris GLUT-4 content. Although we did not measure levels of plantaris muscle GLUT-4, we did show that decrease in myocardial GLUT-4 levels in the diabetic heart was partially prevented in the exercise-trained diabetic rats, suggesting that the regulation of skeletal muscle and heart GLUT-4 levels may be different.
The metabolic signal(s) responsible for the effect of exercise training on myocardial GLUT-4 expression has yet to be identified. Although it has been shown previously that the exercise-training protocol used in this study does not alter plasma insulin concentration, it is possible that the sensitivity of the heart to insulin could be enhanced (20). Increased production of adenosine during periods of exercise may also be a possible metabolic mediator for enhanced GLUT-4 expression. Adenosine and conditions that stimulate the release of adenosine (such as exercise, ischemia, increased workloads, and enhanced contractile activity) have been shown to enhance GLUT-4 translocation (13, 23, 27, 28). Moreover, the diabetic heart has been shown to exhibit decreased sensitivity to adenosine (22), which may further contribute to reduced sarcolemmal GLUT-4 content. Alterations in the intracellular levels of fatty acid and/or glucose metabolites may also provide the metabolic signal (31).
The present study did not assess directly whether this increase in myocardial GLUT-4 in the exercise-trained diabetic hearts resulted in enhanced glucose uptake, glycolysis, or glucose oxidation. However, previously, it has been shown that exercise training of diabetic rats enhances the ability of the diabetic heart to oxidize exogenous glucose (20). Interestingly, the effects of exercise training on glucose oxidation and myocardial sarcolemmal GLUT-4 content were of similar magnitude. In both cases, exercise training produced only a partial return of these parameters toward control levels; i.e., the values for the exercise-trained diabetic rats were about halfway between the sedentary diabetic and control values. Other studies have shown that exercise training will enhance the enzymatic pathways associated with glucose metabolism. Exercise training of control rats has been shown to increase the maximal activity of glycolytic enzymes in rat myocardium, including hexokinase, pyruvate kinase, and lactate dehydrogenase (7, 32).
The present study confirmed that sarcolemmal pNPPase activity is diminished in the diabetic animal relative to that of the control group (21). However, exercise training did not attenuate this depression. Although protein levels for the Na+-K+ pump do not appear to be directly altered by exercise training, the enhancement of glucose metabolism in the exercise-trained diabetic heart may also enhance activity of Na+-K+ pump (17). It has been suggested that the ATP for sarcolemmal Na+-K+-ATPase is derived predominantly from glycolysis. Exercise training, by enhancing glycolytic capacity of the heart, may help maintain adequate fuel for the sarcolemmal Na+-K+-ATPase activity.
In summary, the results of the present study provide evidence for the effectiveness of exercise training in reducing some of the cardiac complications associated with diabetes mellitus. Exercise training of diabetic rats resulted in an upregulation of sarcolemmal GLUT-4 protein and mRNA. This increase in sarcolemmal GLUT-4 may enhance myocardial glucose oxidation (20) and improve cardiac pump performance (18). The increase in myocardial sarcolemmal GLUT-4 may be beneficial to the diabetic heart by enhancing myocardial glucose oxidation and cardiac performance.
ACKNOWLEDGEMENTS
This study was supported by a Feasibility Grant from the American Diabetes Association (D. J. Paulson) and a Grant-in-Aid from the American Heart Association of Metropolitan Chicago (F. D. Romano).
FOOTNOTES
Address for reprint requests: D. J. Paulson, Dept. of Physiology, Midwestern University, 555 31st St., Downers Grove, IL 60515.
Received 7 May 1996; accepted in final form 28 October 1996.
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