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(Circulation. 1997;95:415-422.)
© 1997 American Heart Association, Inc.

Articles

Low-Flow Ischemia Leads to Translocation of Canine Heart GLUT-4 and GLUT-1 Glucose Transporters to the Sarcolemma In Vivo

Lawrence H. Young, MD; Yin Renfu, MD; Raymond Russell, MD, PhD; Xiaoyue Hu, MD; Michael Caplan, MD, PhD; Jianming Ren, PhD; Gerald I. Shulman, MD, PhD; Albert J. Sinusas, MD

the Department of Internal Medicine, Yale University School of Medicine, New Haven; the Department of Physiology, Yale University School of Medicine (M.C.), New Haven; and Bayer Metabolic Disorders Research Division (J.R.), West Haven, Conn.

Correspondence to Lawrence H. Young, MD, Section of Cardiovascular Medicine, Yale University School of Medicine, 333 Cedar St, FMP 323, New Haven, CT 06520-8017. E-mail lawrence.young{at}quickmail.yale.edu

	Abstract

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Background Myocardial ischemia increases heart glucose utilization in vivo. However, whether low-flow ischemia leads to the translocation of glucose transporter (GLUT)-4 and/or GLUT-1 to the sarcolemma in vivo is unknown.

Methods and Results In a canine model, we evaluated myocardial glucose metabolism in vivo and the distribution of GLUT-4 and GLUT-1 by use of immunoblotting of sarcolemma and intracellular membranes and immunofluorescence localization with confocal microscopy. In vivo glucose extraction increased fivefold (P<.001) and was associated with net lactate release in the ischemic region. Ischemia led to an increase in the sarcolemma content of both GLUT-4 (15±2% to 30±3%, P<.02) and GLUT-1 (41±4% to 58±3%, P<.03) compared with the nonischemic region and to a parallel decrease in their intracellular contents. Immunofluorescence demonstrated the presence of both GLUT-4 and GLUT-1 on cardiac myocytes. GLUT-1 had a more prominent cell surface pattern than GLUT-4, which was primarily intracellular in the nonischemic region. However, significant GLUT-4 surface labeling was found in the ischemic region.

Conclusions Translocation of the insulin-responsive GLUT-4 transporter from an intracellular storage pool to the sarcolemma occurs in vivo during acute low-flow ischemia. GLUT-1 is also present in an intracellular storage pool from which it undergoes translocation to the sarcolemma in response to ischemia. These results indicate that both GLUT-1 and GLUT-4 are important in ischemia-mediated myocardial glucose uptake in vivo.

Key Words: glucose • ischemia • metabolism • immunohistochemistry • endocardium

	Introduction

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Ischemia induces many changes in heart metabolism, including shifts from aerobic fatty acid metabolism to anaerobic glycolysis, which provides energy for critical myocardial cellular function.^{1 2} The ischemia-mediated increase in glucose utilization is characterized by enhanced rates of exogenous glucose uptake in vivo,^{3 4} which requires greater rates of transport across the plasma membrane. However, the mechanisms responsible in vivo for increased heart glucose transport with ischemia are not yet defined.

Of the seven reported members of the facilitative glucose transporter family, GLUT-4 and GLUT-1 are the primary forms expressed in adult mammalian heart muscle.⁵ Insulin-stimulated heart glucose uptake involves translocation of GLUT-4 from an intracellular membrane pool to the sarcolemma,^{6 7} where it is thought to account for insulin-mediated increases in glucose transport rates.^{8 9} Early in vitro studies demonstrated that hypoxia increases the number of glucose transporters present in the sarcolemma in both heart¹⁰ and skeletal muscle,¹¹ as assessed by cytochalasin B binding, but did not clearly identify the specific transporters involved. More recent studies have shown that hypoxia causes the translocation of the GLUT-4 transporter to the sarcolemma in L6 muscle cells¹² and perfused rat hearts.⁷ The latter study also found an increased percentage of GLUT-4 in sarcolemma prepared from isolated rat hearts after 15 minutes without perfusion (total global ischemia).⁷ However, no studies to date have assessed GLUT-4 translocation with myocardial ischemia in vivo.

GLUT-1 is also present in heart muscle^{13 14 15} and is found primarily on cardiac myocytes in the rat ventricle.¹⁶ GLUT-1 is generally considered to be responsible for "basal" glucose utilization in the setting of low fasting insulin concentrations and normal workload.⁵ However, previous studies have shown GLUT-1 translocation to the plasma membrane in L6 muscle cells and 3T3-L1 adipocytes during short-term hypoxia,¹² and there is evidence for a decrease in the cardiac intracellular GLUT-1 pool with anoxia.¹⁷ However, it is not known whether heart GLUT-1 is translocated to the sarcolemma during myocardial ischemia.

Thus, the present study was undertaken to assess the distribution of the heart GLUT-4 and GLUT-1 glucose transporters and their translocation in response to acute myocardial ischemia. We used a clinically relevant canine model of regional low-flow ischemia^{18 19} that is suitable for assessing both the acute in vivo metabolic and physiological responses to ischemia. Glucose transporter distribution was examined with both the cell fractionation technique and immunofluorescence with confocal microscopy.

	Methods

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Animal Preparation
Experiments were performed on fasting adult mongrel dogs with approval of the Yale Animal Care and Use Committee, in compliance with the guiding principles of the American Physiological Society. Animals (n=9) were anesthetized with sodium thiamyl (20 mg/kg IV), intubated, and ventilated on a respirator with halothane (1% to 2%), nitrous oxide (70%), and oxygen (30%) to maintain adequate anesthesia throughout the protocol.

A left lateral thoracotomy was performed for instrumentation of the heart as previously described.^{18 19} This instrumentation included a hydraulic occluder placed on the proximal LAD to create low-flow myocardial ischemia in the anterior region of the left ventricle. The degree of LAD stenosis was adjusted to achieve a 50% reduction in the distal LAD pressure (postoccluder) as monitored by a small catheter (25-gauge needle connected to polyethylene tubing).¹⁹ Similar catheters were placed for selective blood sampling from the cardiac veins draining the ischemic myocardium in the central LAD region and the nonischemic myocardium in the LCx region of the left ventricle.¹⁹ Doppler transducers (10 MHz) were sutured to the epicardial surface (adjacent to the venous sampling catheters) to measure transmural myocardial thickening within the ischemic and nonischemic regions.²⁰ A micromanometer catheter was placed to measure left ventricular pressure and dP/dt. Hemodynamic parameters were recorded with data acquisition software (Dataflow, Crystal Biotech). Core temperature was maintained throughout the procedure with heating pads and covering sheets.

Experimental Protocol
After instrumentation, baseline metabolic and hemodynamic measurements were made over a 30-minute period before low-flow ischemia. Mean aortic and distal LAD pressures were recorded before and during a 10-second total occlusion of the LAD (with a snare suture) to determine occlusion pressure. The hydraulic occluder was then adjusted until distal LAD pressure was midway between basal and occlusion pressures. This approach results in a 30% to 40% reduction in transmural myocardial blood flow within the ischemic LAD region, as previously reported.¹⁹ A stable reduction in distal LAD pressure and myocardial thickening was maintained for 80 minutes. Metabolic and hemodynamic measurements were repeated during the last 30 minutes (50 to 80 minutes) of stenosis. The animal was then killed with an overdose of halothane, and the heart was rapidly excised and immediately immersed in ice-cold saline. Myocardial tissue was directly processed for membrane preparation or immunofluorescence as described below.

Physiological Measurements of Heart Metabolism In Vivo
Quadruplicate samples of arterial and selected anterior and posterior cardiac venous blood (3 to 5 mL) were withdrawn during the 30-minute baseline and low-flow ischemia periods. The myocardial AV extractions for each substrate were determined from the mean values of the four samples drawn during the 30-minute sampling periods. Automated analyzers were used to measure the plasma concentrations of glucose (Beckman Instruments) and lactate (Yellow Springs Instruments). An automatic analyzer (OSM 3, Radiometer America Inc) was used to measure oxygen content in whole blood. Plasma was frozen and stored at -30°C for subsequent measurement of plasma free fatty acid concentrations with a microfluorometric assay.²¹ Plasma insulin was measured by the double-antibody radioimmunoassay technique (Diagnostic Systems). All concentrations were measured in duplicate.

Subcellular Membrane Fractionation
Ischemic left ventricular myocardium was selected from an anatomically defined region, bordered medially by the LAD and superiorly by the diagonal artery below the hydraulic occluder. Specific care was taken to avoid the border zone areas of myocardium perfused apically and laterally by distal circumflex branches. Nonischemic myocardium was taken from the posterior left ventricular wall. Both ischemic and nonischemic myocardium were divided into inner and outer halves. Approximately 10 g endocardium from the central ischemic region, corresponding to the site of the selective venous catheter, and a similar amount from the central nonischemic region were used for subsequent homogenization. The endocardium was studied because of the marked heterogeneity in the degree of flow reduction in the epicardium that occurs during partial LAD occlusion.^{18 19}

Membrane fractions were prepared according to a modification of a previously described procedure of differential and sucrose gradient centrifugation.^{11 22} In brief, the outer fibrous epicardial and endocardial surfaces were removed with a tissue slicer (Thomas Scientific) to avoid interference with the subsequent homogenization by fibrous strands. The myocardium was minced into 2-mm pieces. Crude homogenates (20% wt/vol) were prepared in a sucrose (250 mmol/L)/NaHCO₃ (10 mmol/L)/NaN₃ (5 mmol/L) buffer with an Ultra-Turrax T-25 (Kika-Works, Inc) polytron at setting 2 for 15 seconds and centrifuged at 1200g for 10 minutes. The pellets were rehomogenized for 15 seconds and again centrifuged at 1200g for 10 minutes. This supernatant constituted the crude membrane fraction, which was centrifuged at 190 000g for 1 hour. The membrane pellets were resuspended in a 25% sucrose solution with a glass homogenizer loaded onto a discontinuous sucrose gradient (25%, 30%, 35% wt/vol) and centrifuged for 20 hours at 150 000g. All procedures were performed at 4°C.

The sarcolemma fraction was collected from the upper half of the 25% sucrose layer and the intracellular membranes from the interface of the 30% and 35% sucrose layers. In addition, there was an intermediary distinct membrane band at the interface of the 25% and 30% fractions that was enriched for both sarcolemma and intracellular markers and thus was not used for assessment of transporter translocation. The membrane fractions were harvested and diluted fivefold in NaHCO₃ (10 mmol/L)/NaN₃ (5 mmol/L) solution and then spun at 190 000g for 1 hour. The resulting membrane pellets were resuspended in sucrose (250 mmol/L)/Tris (50 mmol/L), pH 7.4, and frozen at -80°C for subsequent use. The relative yields of sarcolemma and intracellular membranes were similar for ischemic and nonischemic myocardium.

Membrane Characterization
Membrane protein concentration was measured with a spectrophotometric assay (Bio-Rad Laboratories) using BSA as a standard. Enrichment of the membrane fractions for the sarcolemma and sarcoplasmic reticulum proteins, Na⁺,K⁺-ATPase and Ca²⁺-ATPase, respectively, was assessed by measurement of their enzymatic activity with a spectrophotometric assay as previously described.²³ In addition, qualitative enrichment for these marker proteins was assessed by immunoblot analysis with monoclonal antibodies to the ₁-subunit of the Na⁺,K⁺-ATPase and the SERCA2 calcium pump (Affinity Bioreagents, Inc). Immunoreactive protein was detected with peroxidase–goat anti-mouse IgG (Zymed Laboratories Inc) by chemiluminescence (ECL, Amersham Life Sciences).

GLUT-4 and GLUT-1 Immunoblot Analysis
SDS-PAGE was performed on sarcolemma and intracellular membranes (20 µg protein) with 10% gels under reducing conditions. The protein samples were diluted with Laemmli sample buffer containing 2% SDS with 3% dithiothreitol added to prevent protein aggregation. The gels were run at 200 V for 45 minutes in a minigel electrophoresis apparatus. Proteins were transferred to PVDF membranes (Trans-Blot membranes, Bio-Rad Laboratories) at 200 mA for 1 hour. Membranes were blocked initially for 1 hour with 5% milk in PBS at 37°C and then with 1% milk in PBS overnight at 4°C. Membranes were washed with (in mmol/L) NaCl 136, KCl 2.7, KH₂PO₄ 1.5, Na₂HPO₄ 8, and NaN₃ 3 and Triton X-100 (1%) and then incubated with primary antibodies with 1% milk in PBS at 37°C. The membranes were then washed and incubated with 2 µCi of ¹²⁵I-protein A (Amersham Co) with 1% milk in PBS at 25°C for 1 hour. They were washed, air-dried, and autoradiographed with XAR-5 film (Eastman Kodak Co) for 12 to 20 hours at -80°C with double intensifying screens (Sigma Chemical Co). On the basis of their locations identified on the autoradiograph, the bands were excised from the PVDF membrane and counted in a gamma well counter (Packard Instruments). The counts were corrected for background activity, which was measured from portions of adjacent membrane of similar size.

Partially purified serum from rabbits immunized with a synthetic peptide corresponding to the 15 C-terminal amino acids of the human/rat GLUT-4 transporter was used at a dilution of 1:8000. Crude serum from rabbits immunized with a synthetic peptide corresponding to the 18 C-terminal amino acids of the human/rat GLUT-1 transporter was used at a dilution of l:500 to 1000. Both anti–GLUT-4 and anti–GLUT-1 antibodies demonstrated specific immunoreactivity with canine heart, producing single distinct bands on the autoradiographs (Fig 1). The canine GLUT-4 band had a slightly higher molecular weight (just above 50 kD) than the GLUT-1 band (directly at 50 kD). In addition, specific immunoreactivity was further demonstrated by the elimination of antibody binding when antibody was preincubated with specific immunizing antigen before immunoblotting (Fig 1).

View larger version (25K): [in this window] [in a new window]   Figure 1. Autoradiograph of immunoblots of canine intracellular membranes with anti–GLUT-4 (1:8000) and/or anti–GLUT-1 (1:1000) antibodies indicating distinct GLUT-4 and GLUT-1 bands. Preincubation of antibodies with no competing antigen, synthetic GLUT-4 antigen (6 µg/mL final volume), or irrelevant antigen (6 µg/mL final volume) demonstrates specific inhibition of anti–GLUT-4 binding.

GLUT-4 and GLUT-1 Immunofluorescence
Immediately after the heart was removed, transmural sections of myocardial tissue (5 mm) from the center of the ischemic and nonischemic regions were placed in 3% paraformaldehyde for 2 hours and then transferred to 0.5% paraformaldehyde and stored at 4°C. Before sectioning, transmural slices of myocardium were immersed overnight for cryoprotection with 30% sucrose and 0.2% NaN₃ in PBS at 4°C. The tissue was then frozen in OCT Compound (Miles Inc) and stored at -80°C. Transmural sections (5 µm) were cut on a Cryostat microtome (Leica Instruments) at -30°C. The frozen sections were fixed onto gelatin-coated slides (0.5% gelatin with 0.05% CrKSO₄) by incubation at 60°C for 15 minutes.

The slide sections were hydrated with PBS, blocked with PBS with 1% BSA for 15 minutes, and then incubated with primary antibody for 2 hours at 25°C. Polyclonal rabbit GLUT-4 antibody was used at a dilution of 1:50 to 1:250 and GLUT-1 antibody at a dilution of 1:20 to 1:50. The slides were washed with PBS and then incubated with rhodamine-labeled goat anti-rabbit IgG (Boehringer Mannheim Co) at a dilution of 1:100 in PBS with 0.1% BSA for 1 hour. The slides were washed, Vectashield mounting solution (Vector Laboratories Inc) was added to prevent fading, and the slides were sealed with a cover slip. The slides were examined by confocal light microscopy (Zeiss Instruments).

Calculations
The content of glucose transporters in either the sarcolemma or intracellular membrane fraction was calculated as the product of the disintegrations per minute of ¹²⁵I-protein A binding per microgram membrane protein (density) and the measured protein yield of that fraction (micrograms membrane protein per gram tissue).⁷ The relative content of each fraction was then expressed as a percentage of the total sarcolemma plus intracellular transporters, eg, percent sarcolemma GLUT-4.⁷ This calculation indicates the distribution of transporters present in the two membrane fractions, which demonstrated high enrichments of sarcolemma and intracellular membrane markers, respectively, with little cross-contamination (see below). It also corrects for the interassay variations attributable to changes in the amount and specific activity of ¹²⁵I-protein A used for immunoblot quantification.

Statistical Analysis
All values are expressed as mean±SEM. Paired Student's t test was used to compare ischemic and nonischemic values. A value of P<.05 was considered significant.

	Results

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Cardiac Function and Myocardial Thickening
Inflation of the hydraulic occluder around the proximal LAD, which reduced distal LAD pressure by 50%, resulted in moderately severe hypokinesis of the ischemic anterior left ventricular region, as reflected by a 70% reduction in myocardial thickening (Table 1). In contrast, myocardial thickening within the nonischemic LCx posterolateral region remained constant (Table 1). Despite the regional anterior dysfunction, arterial blood pressure, left ventricular end-diastolic pressure and dP/dt, and cardiac output did not change significantly during the ischemic period (Table 1).

View this table: [in this window] [in a new window]   Table 1. Myocardial Contractile Function and Hemodynamics During Low-Flow Ischemia

Physiological Measurements of Myocardial Metabolism in Low-Flow Ischemia
Arterial substrate concentrations remained relatively constant during the experimental protocol (Table 2). Free fatty acid levels were mildly elevated, reflecting the effects of low fasting insulin concentrations (5±1 µU/mL), the administration of heparin (essential to maintain catheter patency), and anesthesia. Consequently, myocardial glucose extraction was relatively low in both the LAD and LCx regions before ischemia (Fig 2, Table 2). However, during the ischemic period, glucose extraction increased fivefold in the ischemic LAD region while remaining constant in the nonischemic region (Fig 2, Table 2). In keeping with enhanced rates of anaerobic glycolysis, low-flow ischemia resulted in significant lactate production within the LAD region (Fig 2, Table 2). In contrast, net lactate extraction was maintained in the nonischemic LCx region. The oxygen and free fatty acid extractions both increased in the ischemic region, reflecting more efficient uptake in response to reduced blood flow (Table 2). However, since transmural blood flow in the ischemic region is 30% to 40% less than that in the nonischemic zone in this model,¹⁹ the net glucose uptake would be approximately threefold higher and the net free fatty acid and oxygen uptake 30% lower in the ischemic region.

View this table: [in this window] [in a new window]   Table 2. Myocardial Substrate Extraction: Effect of Low-Flow Ischemia

View larger version (25K): [in this window] [in a new window]   Figure 2. Myocardial extraction (% of arterial) of glucose (A) and lactate (B) in the LAD and LCx regions during the 30 minutes before ischemia (Baseline) and during the last 30 minutes of low-flow ischemia (Low Flow). AV extractions were calculated from quadruplicate time points. *P<.01 vs both baseline LAD extraction and LCx extraction during low-flow ischemia. Values represent mean±SEM (n=9).

Characterization of Sarcolemma and Intracellular Membrane Vesicles
Sarcolemma membranes (25% sucrose fraction) were highly enriched for plasma membrane Na⁺,K⁺-ATPase enzyme activity compared with the crude membrane fraction, with minimal Ca²⁺-ATPase activity (Table 3). Conversely, intracellular membranes were enriched for Ca²⁺-ATPase activity, with minimal Na⁺,K⁺-ATPase activity (Table 3). Marker enzyme enrichments in each of the membrane fractions were comparable in myocardium from ischemic and nonischemic regions (Table 3). Similar results were observed by immunoblotting with antibodies to the ₁-subunit of the Na⁺,K⁺-ATPase and the sarcoplasmic reticulum SERCA2 Ca²⁺-ATPase (Fig 3).

View this table: [in this window] [in a new window]   Table 3. Membrane Fraction Marker Enzyme Activities

View larger version (36K): [in this window] [in a new window]   Figure 3. Representative autoradiograph showing relative enrichment (compared with a crude membrane [CM] fraction) of 1-Na+,K+-ATPase in the sarcolemma (SL) and SERCA-2 Ca2+-ATPase in the intracellular (IC) membrane fractions. Comparable results were found in membranes from ischemic and nonischemic regions.

Subcellular Membrane Distribution of GLUT-4 and GLUT-1
In nonischemic myocardium from these fasted animals, immunoblots demonstrated that GLUT-4 was located predominantly in the intracellular membrane fraction, whereas GLUT-1 was found in both the sarcolemma and intracellular fractions (Fig 4). When glucose transporter content was quantified by counting the activity of the bands (¹²⁵I-protein A binding/µg membrane protein) and multiplication by the yield of each fraction (see "Methods"), the relative sarcolemma distribution of GLUT-4 was 15±2%, whereas that of GLUT-1 was 41±4% (Fig 5).

View larger version (31K): [in this window] [in a new window]   Figure 4. Representative autoradiograph of GLUT-4 and GLUT-1 immunoblots of the sarcolemma and intracellular membrane fractions prepared from the nonischemic (LCx) and ischemic (LAD) regions. Duplicate bands were run for each membrane fraction and exposed with 125I-protein A binding. Antibody dilution was 1:8000 for GLUT-4 and 1:1000 for GLUT-1.

View larger version (24K): [in this window] [in a new window]   Figure 5. Sarcolemma and intracellular membrane content of GLUT-4 and GLUT-1 in myocardium from nonischemic and ischemic regions of the left ventricle. Glucose transporter content was quantified by 125I-protein A binding (cpm/µg membrane protein) multiplied by yield of membrane fraction. Data for each membrane fraction are expressed as a percentage of total GLUT-4 or GLUT-1 content in sarcolemma and intracellular membrane fractions. Values are mean±SEM (n=9). *P<.05 vs nonischemic myocardium.

In the ischemic myocardium, there was evidence of translocation of both GLUT-4 and GLUT-1 from their intracellular membrane pools to the sarcolemma (Fig 4). The relative sarcolemma distribution of GLUT-4 increased twofold, from 15±2% to 30±3% (P<.02), in the ischemic region, whereas the sarcolemma content of GLUT-1 increased modestly, from 41±4% to 58±3% (P<.03). A parallel decrease in the intracellular content of both GLUT-4 (from 85±2% to 70±3%, P<.02) and GLUT-1 (from 59±4% to 42±3%, P<.02) was found in the ischemic compared with the nonischemic region.

Ischemia also increased the sarcolemma glucose transporter density (¹²⁵I-protein A binding/µg sarcolemma protein) for both GLUT-4 (73±24% increase, P<.02) and GLUT-1 (39±15% increase, P<.05). There were parallel decreases in the intracellular membrane GLUT-4 and GLUT-1 density in the ischemic myocardium, but these were somewhat more variable and did not reach statistical significance. Ischemia also significantly increased the ratio of sarcolemma to intracellular transporter densities: the ratio for GLUT-4 increased from 0.22±0.06 to 0.58±0.38 (P<.02), and that for GLUT-1 increased from 0.9±0.3 to 1.9±1.2 (P<.04).

Immunofluorescence Localization of GLUT-4 and GLUT-1
Immunofluorescence studies of myocardial sections were performed with confocal light microscopy to (1) establish whether both GLUT-1 and GLUT-4 were present primarily on cardiac myocytes within the canine left ventricle and (2) determine whether either GLUT-4 or GLUT-1 translocation was demonstrable by this technique, which complements the isolation of subcellular membranes. In the nonischemic region, GLUT-4 demonstrated a predominantly intracellular pattern, with a minor degree of cell surface (sarcolemma) fluorescence detected on cardiac myocytes (Fig 6A and 6B). In contrast, GLUT-1 was present primarily on the sarcolemma, although some intracellular fluorescence could also be detected within cardiac myocytes (Fig 7A and 7B). Both antibodies primarily labeled cardiac myocytes, and there was little autofluorescence or nonspecific secondary antibody binding. In the nonischemic region, there were no transmural differences in overall GLUT-4 or GLUT-1 immunofluorescence.

View larger version (135K): [in this window] [in a new window]   Figure 6. Immunofluorescence of GLUT-4 in sections from nonischemic (low power in A, high power in B) and ischemic (low power in C, high power in D) regions of left ventricle by confocal microscopy.

View larger version (122K): [in this window] [in a new window]   Figure 7. Immunofluorescence of GLUT-1 in sections from nonischemic (low power in A, high power in B) and ischemic (low power in C, high power in D) regions of left ventricle by confocal microscopy.

In the ischemic region, more prominent cell surface GLUT-4 labeling was evident, with associated decreases in the intracellular fluorescence (Fig 6C and 6D). In these sections, which were taken from the center of the ischemic region, there was no evident transmural gradient in the subcellular distribution of GLUT-4. In addition, it was difficult to determine any increase in GLUT-1 surface fluorescence in the ischemic region, given the high degree of surface labeling in nonischemic cardiac myocytes (Fig 7C and 7D).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These results demonstrate changes in the subcellular distribution of both the GLUT-4 and GLUT-1 transporters, together with physiological evidence of increased glucose utilization, during acute regional low-flow myocardial ischemia in a clinically relevant in vivo model. Four significant findings in this study help to elucidate the mechanisms regulating glucose transport in the ischemic heart. First, it demonstrates translocation of the "insulin-regulatable" GLUT-4 transporter from an intracellular membrane fraction to the sarcolemma with an approximately twofold increase in sarcolemma GLUT-4 content during in vivo ischemia. Second, it provides evidence that GLUT-1, like GLUT-4, is found primarily on cardiac myocytes in canine left ventricle. Third, it indicates that GLUT-1 is present both in the sarcolemma (the physiologically active site for transport into the myocardium) and in an intracellular membrane storage pool. Finally, it demonstrates a moderate degree of translocation of GLUT-1 from this intracellular membrane pool to the sarcolemma (40% increase) as a mechanism contributing to the enhancement of glucose transport into the myocardium during acute ischemia.

The present evidence for GLUT-4 translocation in a model of regional low-flow ischemia in vivo is of interest in the context of previous in vitro studies that have demonstrated, either directly or indirectly, translocation of GLUT-4 in both heart^{7 17} and skeletal muscle^{11 24} in response to anoxia or chemical inhibition of oxidative phosphorylation. Our results are also consistent with those of the one prior study to examine ischemia-mediated GLUT-4 translocation, which subjected buffer-perfused rat hearts to total global ischemia for 15 minutes.⁷ The relative content of GLUT-4 in the sarcolemma membrane fraction in our model increased from 15% to 30% with ischemia, which was similar to that seen in the ischemic isolated rat heart (18% to 41%).⁷

The cell fractionation technique using sucrose gradient centrifugation provided canine cardiac membranes with high enrichments and little cross-contamination of the fractions and is well suited for translocation studies. Canine membranes prepared with this approach typically have higher enrichments than those isolated from rat heart.²⁵ In this study, the sarcolemma fraction was 25-fold enriched for Na⁺,K⁺-ATPase activity, compared with a 6-fold enrichment found in the previous study of the ischemic isolated rat heart.⁷ The highly enriched fractions make it unlikely that cross-contamination of membrane fractions contributed significantly to the results of the immunoblot experiments. Furthermore, the GLUT-4 and GLUT-1 antibodies used in these experiments provided high-quality immunoblot and immunofluorescence results in canine heart. The specific immunoreactivity of these antibodies was evaluated in preliminary experiments in which the synthetic peptide antigen eliminated antibody binding to canine cardiac membranes in a specific manner. In addition, the canine GLUT-4 protein consistently had a slightly higher apparent molecular weight by electrophoresis than the GLUT-1 protein, and immunoblots for each showed distinct single bands, further demonstrating the specificity of the antibody.

We initially performed immunofluorescence studies of the glucose transporters with confocal microscopy to establish that both glucose transporters were located predominantly on myocytes in the left ventricular myocardium. We were also able to demonstrate GLUT-4 translocation with this technique because of the very minor degree of GLUT-4 surface labeling in the comparison nonischemic region and the significant (twofold) increase in sarcolemma GLUT-4 content with ischemia (as indicated by the cell fractionation technique). However, it is not surprising that immunofluorescence was unable to demonstrate convincing changes in GLUT-1, given the predominant surface labeling pattern in the control region and the relatively modest (40%) increase in sarcolemma content by the cell fractionation technique. Although immunofluorescence provides important complementary information on transporter localization, the results are somewhat difficult to quantify, and the technique does not have the sensitivity of cell fractionation and immunoblot analysis using ¹²⁵I-protein A quantification.

The ischemia-mediated GLUT-4 translocation observed in the present study occurred in the setting of low fasting insulin levels and myocardial glucose uptake in vivo, when there was little GLUT-4 present in the sarcolemma in the nonischemic region. There is conflicting evidence as to whether ischemia and insulin are additive stimuli to GLUT-4 translocation. Global ischemia did not appear to increase GLUT-4 translocation in isolated rat hearts previously perfused with supraphysiological concentrations of insulin.⁷ In contrast, both hypoxia and contraction augment the stimulation of glucose transport associated with insulin in isolated skeletal muscle,^{11 26} suggesting that different mechanisms may be responsible for translocation with these stimuli and/or that different pools of GLUT-4–containing vesicles may be recruited (see below). Thus, further studies will be required to determine whether ischemia and physiological increases in insulin have additive effects on heart GLUT-4 translocation in vivo.

In addition, ischemia-mediated GLUT-4 translocation was observed in this anesthetized canine model, in which catecholamines are likely to be elevated. Increased catecholamines may have a direct effect to stimulate glucose uptake through GLUT-4 translocation²⁷ and an indirect effect to inhibit glucose utilization by increasing plasma free fatty acid concentrations. The effects of free fatty acids on translocation of heart glucose transporters are currently unknown. Nonetheless, both sarcolemma GLUT-4 and glucose uptake were relatively low in the nonischemic region, reflecting the predominant effect of the low fasting insulin concentrations on glucose transporter distribution in this study.

GLUT-1 is the primary transporter in brain and other tissues and is thought to be responsible for the "basal" glucose uptake in skeletal and cardiac muscle in the presence of low insulin concentrations when sarcolemma GLUT-4 content is low.⁵ The present study is consistent with the previous findings of the presence of GLUT-1 protein in rat heart^{14 15 16} and GLUT-1 mRNA in canine²⁸ and human²⁹ left ventricle. However, in contrast to the rat heart, in which GLUT-1 was located predominantly on myocyte intercalated disks,¹⁶ our immunofluorescence results suggest that GLUT-1 has a more uniform myocyte surface labeling pattern in the canine heart. Furthermore, in the rat, GLUT-1 is present in greater amounts in heart than in skeletal muscle.¹⁵ These observations are of interest with regard to the potential metabolic importance of GLUT-1 in heart compared with skeletal muscle, in which GLUT-1 localizes mainly to neurofilaments rather than to myocytes.^{30 31} Thus, GLUT-1 may have a greater role in myocyte glucose metabolism in heart than in skeletal muscle, perhaps reflecting the higher levels of basal contractile and metabolic activity in the heart.

The present results are the first to demonstrate in vivo translocation of GLUT-1 to the sarcolemma and indicate an additional important role for GLUT-1 in the heart during ischemia. However, they are consistent with in vitro studies that have indicated that inhibition of oxidative phosphorylation can acutely increase translocation of GLUT-1 to the plasma membrane in isolated hepatocytes³² and skeletal muscle L6 cells.^{12 33} In the perfused rat heart, rotenone decreases the GLUT-1 content of intracellular membranes, also suggesting translocation, although the sarcolemma content of GLUT-1 was not assessed.¹⁷ The translocation of GLUT-1 is of particular interest in view of recent reports indicating an increase in GLUT-1 expression as an additional adaptive response to hypoxia. GLUT-1 synthesis is stimulated by hypoxia in L6 skeletal muscle cells,^{12 33} and long-term hypoxia increases heart GLUT-1 in rats.¹³ In a preliminary report, patients with chronic ischemic heart disease also have an increase in the ratio of GLUT-1 to GLUT-4 mRNA in myocardial biopsies.²⁹ Thus, the GLUT-1 response to myocardial ischemia may involve initial translocation of preexistent transporters to the sarcolemma and subsequent expression of new transporters during longer periods of ischemia.

Although the present results demonstrate translocation of both GLUT-4 and GLUT-1 during regional low-flow ischemia, they do not directly address the mechanisms involved in the trafficking of either GLUT-4 or GLUT-1 vesicles between cytoplasmic and sarcolemmal pools.⁵ Of interest, both exocytosis and endocytosis of GLUT-4 vesicles in adipocytes involve a low-molecular-weight G protein,^{34 35} raising the possibility that changes in high-energy phosphates during myocardial ischemia might influence trafficking through changes in cytosolic GTP concentrations. In addition, the signaling pathways responsible for ischemia-mediated translocation of GLUT-4 and GLUT-1 to the sarcolemma are currently unknown. Insulin-mediated GLUT-4 translocation in skeletal muscle typically involves activation of phosphatidylinositol 3-kinase and rearrangement of the actin cytoskeleton.^{5 36} However, hypoxia-mediated GLUT-4 translocation in skeletal muscle is not affected by inhibition of these pathways,²⁴ raising the possibility that transporter translocation during myocardial ischemia might also involve a different mechanism.

The cofractionation of the intracellular vesicles containing glucose transporters and sarcoplasmic reticulum Ca²⁺-ATPase was helpful in terms of assessing the enrichment of this membrane fraction but does not necessarily indicate identity of these two membrane populations. Currently, little is known about the subcellular location or protein composition of intracellular GLUT-4 and GLUT-1 vesicles in heart muscle or the extent to which they might share the same storage site. In adipocytes, some data suggest that the two transporters appear to reside in similar but separate pools of intracellular vesicles,³⁷ whereas other studies suggest that there is a subpopulation of vesicles enriched with GLUT-4 that is highly insulin-sensitive.³⁸ In contrast, in rat heart, GLUT-1 has been shown to coprecipitate with intracellular GLUT-4 vesicles.¹⁵ Whether the associated translocation of GLUT-4 and GLUT-1 observed during ischemia reflects either a subpopulation of GLUT-4 vesicles containing GLUT-1 or simply parallel translocation of distinct vesicles will need to be clarified in further studies using immunoisolation or immunohistochemical techniques.

In the present study, we observed physiological evidence for increased glucose uptake in vivo and transporter translocation during moderate low-flow ischemia in a clinically relevant model. Despite transporter translocation, more severe ischemia may limit myocardial glucose uptake in vivo by decreasing delivery³⁹ and inhibit glycolysis by decreasing intracellular pH.⁴⁰ Thus, the degree of residual blood flow is no doubt critical in determining the physiological impact of transporter translocation on glucose utilization during myocardial ischemia in vivo.

	Selected Abbreviations and Acronyms

 

GLUT = glucose transporter LAD = left anterior descending coronary artery LCx = left circumflex artery PVDF = polyvinylidene difluoride

	Acknowledgments

 
This work was supported by grants from the American Heart Association (Florida Affiliate) and the US Public Health Service (P30-DK-45735, RO1-DK-40936, 5T32-DK-07058-20). The valuable assistance of Donald Dione, Syed Hasan, Laurie Finta, RN, and Rosemarie Teodosio was appreciated.

	Footnotes

 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.

Received May 13, 1996; revision received August 5, 1996; accepted August 28, 1996.

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