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J Am Coll Cardiol, 2004; 43:1690-1697, doi:10.1016/j.jacc.2003.12.038
© 2004 by the American College of Cardiology Foundation
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Delayed response of insulin-stimulated fluorine-18 deoxyglucose uptake in glucose transporter-4-null mice hearts

Marcus V. Simões, MD, PhD*,*, Silvia Egert, PhD*, Sibylle Ziegler, PhD*, Masao Miyagawa, MD, PhD*, Sybille Reder, BS*, Terry Lehner, BS*, Ngoc Nguyen, BS*, Maureen J. Charron, PhD{dagger} and Markus Schwaiger, MD*

* Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Munich, Germany
{dagger} Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA

Manuscript received July 20, 2003; revised manuscript received November 27, 2003, accepted December 9, 2003.

* Reprint requests and correspondence: Dr. Marcus V. Simões, Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Str. 22, 81675 Munich, Germany.
msimoes{at}fmrp.usp.br


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
OBJECTIVES: We sought to evaluate the time course of insulin-stimulated myocardial glucose uptake (MGU) in mice that had undergone ablation of glucose transporter-4 (GLUT4).

BACKGROUND: The relative importance of GLUT4, the most abundant insulin-responsive glucose transporter, to modulate myocardial glucose metabolism is not well defined.

METHODS: Myocardial glucose uptake was assessed at various time points after glucose (1 mg/g) and insulin (8 mU/g) injection in GLUT4-null (G4N) (n = 48) and wild-type (WT) (n = 48) mice with 18F-2-deoxy-2-fluoro-D-glucose (FDG) using in vivo positron emission tomography (PET), in vitro gamma-counter biodistribution, and isolated, perfused hearts.

RESULTS: Baseline assessment with PET imaging showed comparable MGU in G4N (0.66 ± 0.12) and WT (0.67 ± 0.11, p = 0.70) mice. Early after insulin injection, WT mice demonstrated a 3.5-fold increase in MGU (2.45 ± 0.45, p = 0.03), whereas G4N mice presented no increase (1.11 ± 0.24, p = 0.28). At 60 min, MGU was comparable in G4N (3.19 ± 0.60) and WT (2.66 ± 0.47, p = 0.28) mice. In vitro gamma-counter biodistribution evaluation confirmed in G4N mice a lack of MGU increase early after insulin, but a slow response over 120 min. The isolated, perfused hearts of G4N mice during short-term (15 min) insulin stimulation displayed no increase in MGU (0.08 ± 0.01 ml/g/min), whereas WT mice presented a threefold increase (0.22 ± 0.01 ml/g/min, p < 0.01). With long-term (60 min) insulin stimulation, similar MGU was found in G4N (0.31 ± 0.02 ml/g/min) and WT (0.33 ± 0.04 ml/g per min, p = 0.04) mice.

CONCLUSIONS: The G4N mice displayed an increase of MGU in response to insulin similar to that of controls, but with a markedly delayed time response. Our findings underscore the important role of GLUT4 in the rapid adaptive response of myocardial glucose metabolism.

Abbreviations and Acronyms
  2-DG = 2-deoxyglycose
  FDG = 18F-2-deoxy-2-fluoro-D-glucose
  GLUT = glucose transporter
  G4N = GLUT4-null mice
  MGU = myocardial glucose uptake
  PET = positron emission tomography
  SUV = standard uptake value
  WT = wild-type mice

To meet its high-energy demand, the working heart has the ability to select the fuel among several exogenous substrates (1). The majority of energy is derived from long-chain fatty acids, but myocardial cells can rapidly change their energy source by stimulation of glycolysis in response to ischemia, insulin, increased workload, or catecholamine stimulation (2).

Recent observations have demonstrated that switching of substrates and increasing glycolytic flux are important compensatory mechanisms to cope with higher energy demand under pathologic conditions such as myocardial hypertrophy (3) and congestive heart failure (4). Furthermore, activation of glycolysis seems to play a critical role as a protective mechanism during acute ischemia and infarction (5,6). These observations underscore the importance of studying cardiac glucose metabolism, with direct implication for the development of new therapeutic strategies (7–9).

Several observations suggest that glucose transport through the sarcolemma is a rate-limiting step of myocardial glycolytic flux (10,11). Glucose enters cardiomyocytes through specific glucose transporter (GLUT) proteins—GLUT1 and GLUT4 (12). The most abundant glucose transporter in the heart is GLUT4, which is localized mainly in intra-cellular vesicles but is rapidly translocated to the plasma membrane, providing increased glucose transport in response to insulin (13), catecholamine stimulation (14), increased workload, ischemia, and hypoxia (15). A large amount of GLUT1 in the myocardium is localized to the sarcolemma and is considered to be responsible for basal glucose transport, even though its translocation can also be induced by stimuli that provoke GLUT4 accumulation in the sarcolemma (14,16,17).

Abnormalities in GLUTs have been demonstrated in association with changes in myocardial energy metabolism in cardiac hypertrophy and failure (18,19) and in the diabetic heart (20). In these conditions, a reduced sensitivity to insulin stimulation is associated with diminished expression or activity of GLUT4. Even though the role of GLUT1 in regulating glucose metabolism of the heart has been consistently demonstrated (9,21), the relative importance of GLUT4 remains unclear.

A murine model with ablation of the GLUT4 gene has been recently described (22). Besides retarded growth and reduced adipose tissue, these animals present myocardial hypertrophy with normal systemic arterial blood pressure and depressed systolic function with aging (23). Despite these cardiac abnormalities, the only study to date investigating myocardial metabolism in GLUT4-null (G4N) mice unexpectedly reported myocardial glucose uptake (MGU) comparable to that observed in wild-type (WT) control animals (24).

This study sought to investigate the changes in baseline and insulin-stimulated MGU in G4N and WT mice, using in vivo positron emission tomographic (PET) imaging, in vitro gamma-counter tissue biodistribution studies, and isolated heart preparations measuring myocardial accumulation of the positron-emitting glucose analogue 18F-2-deoxy-2-fluoro-D-glucose (FDG).


   Methods
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 Methods
Results
 Discussion
 References
 
Animals.   Mice with disruption of the GLUT4 gene were obtained from Albert Einstein College of Medicine, Bronx, New York (22). Age-matched B6CBAF1/JICO WT mice (Charles River Laboratories, Sulzfeld, Germany) were controls. The animals were fasted for 2 h before the studies. The descriptive characteristics of the investigated mice are listed in Table 1. The Bavarian Regional Ethic Committee for Animal Research approved the protocols of animal care and experimentation.


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  		Table 1 Descriptive Characteristics of the Investigated Animals

 
Materials.   Chemicals, unless otherwise specified, were purchased from Sigma (Merck), Deisenhofen, Germany.

Immunoblotting of GLUTs.   Membrane enrichment was performed with minor modifications (25). Briefly, samples were homogenized and submitted to centrifugation at 500 g (10 min) and 170,000 g (60 min). The resulting pellet was dissolved in homogenization buffer with 0.5% Triton X-100. The sample protein concentration was determined by a commercial assay (BCA Protein Assay, Pierce, Rockford, Illinois). Per sample, 50 µg protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel, as previously described (25).

FDG biodistribution experimental protocols.   For gamma-counter biodistribution studies and PET imaging, animals received intra-peritoneal injections of human insulin (8 mU/g body weight) and glucose (1 mg/g body weight). A dose of FDG (40 µCi) was injected in the tail vein under ether inhalation anesthesia in relation to the insulin/glucose application as follows: 1) simultaneous injection; 2) 30-min delay; 3) 60-min delay; and 4) 120-min delay. The baseline FDG uptake was assessed without glucose/insulin injection.

PET imaging.   Imaging by PET was performed at baseline, after simultaneous injection of the insulin/glucose solution and FDG, and after a 60-min delay. Animals were anesthetized with intra-muscular injection of midazolam (0.1 mg/kg), fentanyl (1 µg/kg), and medetomidin (10 µg/kg). Images were acquired in an EXACT HR+ scanner (CTI/Siemens, Erlangen, Germany) 40 min after intravenous FDG administration. Image acquisition protocols consisted of 10-min static emission and 10-min transmission. The images were reconstructed using an iterative ordered subsets expectation maximization reconstruction algorithm with eight subsets and four iterations. Three orthogonal plane images were generated for drawing regions of interest. Maximum FDG uptake values in the heart were obtained for the standard uptake value (SUV) calculations, applying corrections for body weight and injected activity.

Gamma-counter biodistribution studies.   Animals were euthanized 40 min after the FDG injection. The heart was rapidly harvested and rinsed in saline solution, and excess liquid was removed. After weighing the organ, the radioactivity was measured using a well gamma-counter (Cobra Quantum, Packard Instruments Company, Meriden). Cardiac FDG uptake was then expressed as the SUV obtained by calculating the ratio of myocardial FDG activity to injected dose normalized to the body and heart weights.

Isolated heart perfusion preparation.   Isolated heart perfusion was used to investigate whether the results obtained in the intact animal could also be obtained in an experimental setting where systemic factors potentially influencing glucose uptake are controlled (hormonal and metabolic states) by a standardized perfusate (14).

The preparation was performed as previously described (14). Briefly, the animals were euthanized under anesthesia (65 mg/kg intra-peritoneal sodium pentobarbital), and the hearts were harvested, placed in an ice-chilled buffer, and mounted on a non-recirculating retrograde perfusion system via ascending aorta cannulation at a constant flow rate of 3 ml/min (modified Langendorff preparation). The perfusate consisted of a modified Krebs-Henseleit-Bicarbonate buffer (pH 7.4), warmed at 37°C, oxygenated with 95% oxygen/5% carbon dioxide, and containing (in mmol/l): NaCl 117, KCl 4.7, MgSO4 1.1, KH2PO4 1.2, CaCl2 2.5, NaHCO3 20, and glucose 10.

A metal hook was inserted into the apex of the heart and connected to a tension transducer (GM3, Scaime Inc., Cedex, France), and the product of developed tension versus heart rate was continuously monitored (26). The hearts were allowed to stabilize for 10 min.

Then, FDG (10 µCi/ml of buffer) was added to the perfusate at various time points, as described in the perfusion protocols listed subsequently. The FDG accumulation was measured by using two external coincidence detectors interfaced with a computer (14). The acquired data were then corrected for heart weight and buffer radioactivity concentration. The FDG uptake rate was obtained from the slope of linear regression of the time-activity curve and expressed as ml/g/min.

Perfusate containing insulin (10 mU/ml) was used in an attempt to reproduce the time delays employed in the biodistribution studies for short-term insulin stimulation (15-min perfusion with FDG-containing buffer allowed the acquisition of baseline data; insulin was then added for an additional 15-min FDG uptake data acquisition) and long-term insulin stimulation (insulin-containing buffer was perfused for 60 min with the FDG uptake data acquisition performed during the last 15 min).

Statistical analysis.   Results are described as mean value ± SEM. To test the significance of mean differences, non-parametric distribution free tests were employed. The Mann-Whitney U test was used for non-paired samples, and the Wilcoxon matched-pairs test for paired samples. A p value <0.05 was considered significant.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
The characteristics of the animals are described in Table 1. Male and female animals were used, as gender did not influence any of the parameters assessed. The difference in body weight reflects the retarded growth and reduced fat tissue in animals with GLUT4 gene disruption. A 29% increase in heart weight and 50% increase in the heart/body weight ratio were observed, compatible with myocardial hypertrophy, as previously described in these genetically modified animals (24).

Immunoblot analysis of myocardial tissue confirmed a lack of GLUT4 protein in transgenic mice (Fig. 1). Protein quantification obtained from three different membrane preparations showed a 1.7 ± 0.2-fold increase in the expression of GLUT1 when compared with WT mice (p = 0.01).



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  		Figure 1 Western blot analysis of heart tissue for GLUT4 and GLUT1 proteins. As expected, there is an absence of the GLUT4 protein in homozygous (–/–) GLUT4-null mice. The GLUT1 protein displays a twofold increase in GLUT4-null mice (–/–) compared with wild-type mice (+/+). GLUT = glucose transporter.

 
PET imaging.   Representative examples of PET images are displayed in Figure 2, and the mean data of the myocardial FDG uptake obtained through the analysis of PET images are summarized in Figure 3A. A comparable radiotracer retention in hearts from G4N and WT mice was obtained in the non-stimulated baseline condition (p = 0.84).



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  		Figure 2 Representative positron emission tomography images obtained in wild-type and GLUT4-null mice under each of the experimental conditions. At baseline, no myocardial [18F]-2-deoxy-2-fluoro-D-glucose (FDG) activity was detected in the cardiac area of either animal. After simultaneous injection of insulin and FDG, the wild-type mouse presented with increased radiotracer uptake in the cardiac area (arrow), whereas no activity was identifiable in the GLUT4-null mouse. After 60 min, both animals had conspicuous myocardial FDG uptake of comparable intensity. GLUT = glucose transporter.

 


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  		Figure 3 Bar graphs summarizing the mean data of myocardial [18F]-2-deoxy-2-fluoro-D-glucose (FDG) uptake in GLUT4-null and wild-type mice (expressed as standard uptake values [SUVs], y-axis), using quantitative analysis of (A) in vivo positron emission tomography imaging and (B) in vitro gamma-counter tissue biodistribution studies. The positron emission tomography study was performed under the following conditions: baseline, simultaneous injection of insulin and FDG, and 60-min delay. The gamma-counter biodistribution study provides a more detailed time course of insulin-stimulated FDG uptake as 30-min and 120-min delay times points were also investigated. *p < 0.05 for comparisons with baseline values. {dagger}p < 0.05 for comparisons between GLUT4-null and wild-type animals in each experimental condition. GLUT = glucose transporter.

 
After simultaneous injection of insulin and FDG, WT mice presented a significant 3.5-fold increase in FDG uptake when compared with baseline (p = 0.0079), while maintaining similar levels of uptake after 60 min. On the other hand, the G4N mice presented no significant increase in FDG uptake after simultaneous injection of insulin and FDG when compared with baseline (p = 0.10). This uptake value was significantly reduced compared with the result obtained in the corresponding WT group (p = 0.032).

Under long-term insulin stimulation (60 min), G4N mice yielded a significant fivefold increase in FDG retention (p = 0.008) and were comparable to the WT animals investigated under the same experimental conditions (p = 0.55).

Gamma-counter biodistribution studies.   The results of biodistribution studies confirmed the results obtained by PET imaging that indicated a delayed response of FDG uptake in the G4N animals compared with the WT animals. The mean data are summarized in Figure 3B. In non-stimulated baseline conditions, comparable FDG uptake was detected in both animal groups (p = 0.6857) (Fig. 3B). However, there was a difference between G4N and WT mice regarding the time course of insulin stimulation in FDG uptake. Wild-type mice presented a significant 10-fold increase of FDG uptake after simultaneous injection, compared with the baseline condition (p = 0.0286). The maximum response of FDG uptake in WT animals occurred at a 30-min delay between insulin and radiotracer injection (about a 57-fold increase vs. baseline values, p = 0.0286), a response level that remained constant at 60- and 120-min delays (p = 0.0061 and 0.0159, respectively).

The G4N mice presented a delayed response in FDG uptake, confirming imaging data. After simultaneous injection, no significant increase in FDG uptake, compared with baseline, was detected (p = 0.889). In addition, there was a significant difference in FDG uptake between G4N and corresponding WT animals observed after simultaneous injection (p = 0.0286). Progressive augmentation of FDG uptake was observed at 30, 60, and 120 min. Even though a significant increase, compared with baseline, was demonstrated at 30 and 60 min (~10- and 20-fold increases, p = 0.0286 and 0.0061, respectively), the responses of G4N mice were reduced when compared with those obtained in WT mice (p = 0.0286 and 0.0175, respectively). The maximum response in G4N mice was found at 120 min, with FDG uptake values comparable in both animal types (Fig. 3B).

Isolated heart perfusion.   The product of heart rate (beats/min) times developed tension (g) was an index of cardiac function. At baseline, similar values were found in G4N (575.0 ± 93.2 beats/min·g) and WT mice (757.1 ± 80.3 beats/min·g, p = 0.16). No significant difference was found between baseline and end perfusion values in either group (569.7 ± 96.5 beats/min·g [p = 0.81] and 780 ± 91.5 beats/min·g [p = 0.38] for G4N and WT hearts, respectively).

Representative traces of time-activity curves are displayed in Figure 4, and the mean data are summarized in Figure 5. The FDG uptake rate assessed in the isolated heart perfusion preparation yielded comparable values in the G4N and WT hearts under baseline conditions (p > 0.99). With the short-term insulin perfusion protocol, the WT animals presented a significant threefold increase in the FDG uptake rate compared with control values (p = 0.0079), whereas G4N mice hearts presented no significant difference (p = 0.22).



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  		Figure 4 Examples of [18F]-2-deoxy-2-fluoro-D-glucose (FDG) time-activity curves obtained in isolated heart perfusion, using external coincidence detectors. This approach allows the evaluation of insulin-stimulated myocardial FDG uptake without the influence of systemic factors. (A) Short-term insulin effect: compared with the initial portion of the curve (baseline), an increase of the FDG uptake rate (slope of the curve segment) is seen in the wild-type mouse heart shortly after insulin is added to the perfusion buffer (arrow). Conversely, no change is observed in the GLUT4-null mouse heart after the addition of insulin (arrow). (B) Long-term insulin stimulation: hearts were perfused with insulin containing buffer 60 min after in vivo administration of insulin/glucose. In this condition, both GLUT4-null and wild-type hearts had an increased FDG uptake rate compared with the corresponding baseline value. GLUT = glucose transporter.

 


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  		Figure 5 Bar graph summarizing the mean data of the [18F]-2-deoxy-2-fluoro-D-glucose (FDG) uptake rate (expressed as ml/g/min, y-axis) obtained in isolated perfused hearts from GLUT4-null and wild-type mice in each of the experimental conditions (x-axis). *p < 0.05 for comparison with baseline. {dagger}p < 0.05 for comparisons between GLUT4-null and wild-type mice in each experimental condition. GLUT = glucose transporter.

 
Under long-term insulin stimulation perfusion, a significantly higher FDG uptake rate was observed, compared with baseline, in both WT (p = 0.04) and G4N mice (p = 0.04). No significant difference was observed between WT and G4N mice hearts in the long-term perfusion protocols.


   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
Our results, based on MGU obtained in vivo as well as in isolated heart preparation, demonstrated that G4N mice hearts elicit FDG uptake comparable to that observed in WT hearts in the baseline non-stimulated condition. As expected, G4N mice did not exhibit an early response in FDG uptake to insulin administration. However, GLUT4-deficient hearts displayed FDG uptake similar to that of WT animals after 2-h exposure to insulin.

Few previous studies have investigated the consequences of GLUT4 gene ablation on myocardial metabolism (24,27,28). To date, there has been only one study by Stenbit et al. (24), which investigated myocardial metabolism in the G4N mice model. Glucose uptake was assessed using the glucose analogue 2-deoxyglycose (2-DG) and phosphorous-31 spectroscopy in isolated hearts perfused over 75 min. In contrast to our results, Stenbit et al. (24) reported a higher 2-DG uptake rate in G4N mice than in WT mice with insulin stimulation, and no late increase in 2-DG uptake was found.

These discordances can be explained by methodologic differences of the isolated heart perfusion preparations. Firstly, Stenbit et al. (24) investigated hearts that were taken from non-fasted animals. In this condition, the MGU rate is augmented at the beginning of perfusion and is unlikely to show a further increase with the addition of insulin in the perfusate (27). Secondly, the perfusate used by Stenbit et al. (24) included free fatty acids (palmitate, hydroxybutyrate) as energy substrates in addition to glucose. The oxidation of competitive substrates can lead to inhibition of MGU (2). One can speculate that inhibition of glucose uptake by competitive substrates is less marked in the G4N mice, as they present decreased mRNA levels of enzymes of fatty acid oxidation, as demonstrated by the Stenbit et al. (24) group, resulting in a reduced capacity of fatty acid oxidation.

On the other hand, the results of the present investigation concerning the normal baseline and deficient short-term, insulin-stimulated FDG uptake are concordant with results obtained by Tian and Abel (27), who investigated a transgenic mice model with selective ablation of the GLUT4 gene in heart tissue. Glucose uptake was also assessed in isolated hearts by measuring accumulation of the glucose analogue 2-DG with phosphorous-31 spectroscopy. In the baseline condition, hearts from fasted GLUT4-deficient and control mice presented similar levels of glucose uptake. During a 16-min period of insulin administration, cardiac GLUT4-deficient mice did not present an increase in glucose uptake, in contrast to control animals, which demonstrated a marked increase.

One mechanism probably involved in the preserved baseline glucose uptake in G4N mice is a compensatory increase of myocardial GLUT1 content. Corroborating this hypothesis, we demonstrated a 1.7-fold increase in GLUT1 protein content in the myocardial tissue of G4N mice. This finding is in agreement with the previous observations reported by Katz et al. (22), who found a 1.5-fold increase in GLUT1 myocardial content. Abel et al. (28) also reported a threefold increase in myocardial GLUT1 content in mice with selective ablation of GLUT4 in the heart. However, compensatory increases of additional GLUTs recently described cannot be discarded (29).

Despite the increased myocardial content of GLUT1, the G4N mice presented no elevation of basal FDG uptake. This finding is in contrast to previous observations in which increased GLUT1 expression was associated with augmented baseline glucose uptake (21). Nonetheless, it is plausible that the increase in GLUT1 expression in our model occurs in an extent needed to compensate for the absence of GLUT4 and to re-establish a normal, but not increased, glucose transport level.

It is conceivable that the increase in FDG uptake after prolonged insulin stimulation involves increased activity of GLUT1 by increased protein expression or translocation to the sarcolemma and/or upregulation of its activity. This hypothesis is supported by the results of one previous study demonstrating that long-term insulin stimulation preferentially upregulates GLUT1 expression, but not GLUT4, in rat hearts (30). Further evidence obtained in other insulin-responsive tissues submitted to long-term insulin stimulation supports this hypothesis. Studies in rat adipocytes have demonstrated maintenance of GLUT4 protein expression and increased GLUT1 expression when submitted to prolonged insulin stimulation (31). In addition, the trafficking of GLUTs is also affected differentially by short- and long-term insulin stimulation. Cultured 3T3-L1 adipocytes submitted to short-term (30 min) treatment with insulin increased the surface labeling of GLUT1 and GLUT4 to 3.5- and 12-fold, respectively. The expression of GLUT1 in the cell membrane of cultured 3T3-L1 adipocytes submitted to 24 h of insulin stimulation has been shown to be markedly increased (four-fold), whereas the level of GLUT4 at the cell surface was down-regulated (reduction of 53%), suggesting that the increase in GLUT1 compensates for the decrease in GLUT4 (32).

A further mechanism to explain the late insulin-stimulated increase in glucose uptake could be the stimulation of glucose uptake secondary to the effect of a sharp decrease of plasma free fatty acid levels in G4N mice after insulin administration. However, this explanation is not supported by our findings of increased insulin-stimulated glucose uptake in isolated hearts, a more controlled condition in which the influences of a marked reduction of other substrates' availability for oxidation are absent. These results suggest that an intrinsic myocardial mechanism, rather than a systemic metabolic effect, is responsible for the observed changes.

Myocardial insulin resistance can be defined as a decreased response of glucose uptake to the stimulatory effect of insulin. This is a metabolic abnormality with a high prevalence and is associated with a number of pathologic conditions, such as diabetes (20), myocardial hypertrophy and heart failure (18), and ischemic heart disease (33). Evidence derived from animal models (20,34,35) and clinical studies (18) suggests that a reduced expression and/or activity of GLUT4 is involved in the mechanism of insulin resistance. Thus, our transgenic mouse model with absence of GLUT4 can be considered as a model of extreme myocardial insulin resistance. In this context, our results, showing reduced FDG uptake shortly after insulin stimulation, are concordant with previous observations in humans in whom PET imaging was used and demonstrating reduced insulin-stimulated FDG uptake in patients exhibiting insulin resistance associated with cardiac hypertrophy and ischemic heart disease (33,36).

Study limitations.   Myocardial glucose uptake was assessed by measuring FDG accumulation in this investigation. This glucose analogue enters the myocardial cell through the same facilitative transporters as glucose (37). However, it has been previously reported that different stimuli or hormonal conditions can produce differential hexokinase affinity for glucose as well as for the glucose analogue FDG, yielding different uptake rates for each of these compounds (38). It was suggested that the estimation of absolute glucose uptake rates using FDG is not reliable without applying specific correction constants for every metabolic condition. Although this can represent a limitation of the present investigation, FDG kinetics were used as a functional index of glucose transport, and not for measuring absolute myocardial glucose utilization. On the other hand, this potential limitation does not affect the results obtained from the direct comparison of G4N and WT animals submitted to the same experimental conditions.

This investigation used a PET scanner designed for human clinical studies. The inherent limited spatial resolution and the consequent partial volume effect were implicated in a reduced sensitivity of the PET images when compared with gamma-counter biodistribution studies. This can explain the differences between the two methods in evaluating the magnitude of FDG uptake increase after insulin (Figs. 3A and 3B).

Conclusions.   We demonstrated that the G4N mice lack early insulin-stimulated FDG uptake. Myocardial uptake of FDG in the G4N mice in response to insulin is delayed but reaches levels comparable to those of control animals. Our results confirm a significant role of GLUT4 in the modulation of glucose myocardial metabolism, as it allows for a rapid glucose transport adaptation. During prolonged or long-term stimulation, other mechanisms, probably involving regulation of GLUT1 activity/translocation, play an important role in determining the glucose transport rate.

The findings of this study have potential implications in pathologic states in which reduced GLUT4 expression and insulin resistance were demonstrated, such as cardiac hypertrophy and diabetes (18,20). Although these patients present with preserved basal cardiac glucose metabolism and function, they demonstrate a poor tolerance to ischemia/reperfusion injury, which can be related to the inability to rapidly adapt the rate of glycolysis (39,40).


   Acknowledgments
 
The authors gratefully acknowledge the technical assistance of the PET staff for performing imaging acquisition and the PET chemistry/cyclotron staff for the FDG production.


   Footnotes
 
This study was supported by a grant from the Deutsche Forschungs Gemeinschaft (DFG, Schw 236/3) and by grants HL58119 and DK47425 (to Dr. Charron) from the National Institutes of Health, Bethesda, Maryland. Dr. Simões is supported by FAPESP research grant 01/04868-8.


   References
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 Abstract
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 Discussion
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