Lumped constant for [18F]fluorodeoxyglucose in skeletal muscles of obese and nonobese humans

Pauliina Peltoniemi1, Peter Lönnroth2, Hanna Laine1, Vesa Oikonen1, Tuula Tolvanen1, Tove Grönroos1, Lena Strindberg2, Juhani Knuuti1, and Pirjo Nuutila1,3

1 Turku Positron Emission Tomography Center and 2 Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, University of Gothenburg, 41345 Gothenburg, Sweden; and 3 Department of Medicine, University of Turku, F-20520 Turku, Finland


    ABSTRACT
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quantitative 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) positron emission tomography (PET) has been widely used to calculate glucose utilization in skeletal muscle. FDG-PET results depend partly on the lumped constant (LC), which accounts for the differences in the transport and phosphorylation between [18F]FDG and glucose. In this study, we estimated the LC for [18F]FDG directly in normal and in insulin-resistant obese subjects by combining FDG PET with the microdialysis technique. Eight obese [age 29.4 ± 1.0 yr, body mass index (BMI) 33.6 ± 1.0 kg/m2] and eight nonobese (age 25.0 ± 1.0 yr, BMI 23.1 ± 1.0 kg/m2) males were studied during euglycemic hyperinsulinemia (1 mU · kg-1 · min-1 for 150 min). Muscle blood flow was measured using 15O-labeled water and PET. Muscle [18F]FDG uptake (rGUFDG) was calculated with Patlak graphic analysis. Interstitial glucose concentration of the quadriceps femoris muscle was measured simultaneously with [18F]FDG scanning using microdialysis. Muscle glucose uptake (by microdialysis, rGUMD) was calculated by multiplying glucose extraction by regional muscle blood flow. A significant correlation was found between rGUMD and rGUFDG (r = 0.78, P < 0.01). The LC was determined as the ratio of the rGUFDG to the rGUMD. The LC averaged 1.16 ± 0.16 and was similar in the obese and nonobese subjects (1.15 ± 0.11 vs. 1.16 ± 0.07, respectively, not significant). In conclusion, the microdialysis technique can be reliably combined with FDG PET to measure glucose uptake in skeletal muscle. Direct measurements with these two independent techniques suggest an LC value of 1.2 for [18F]FDG in human skeletal muscle during insulin stimulation, and the LC appears not to be sensitive to insulin resistance.

glucose transport; glucose phosphorylation; insulin resistance; microdialysis; positron emission tomography


    INTRODUCTION
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE IS QUANTITATIVELY the most important glucose-utilizing tissue, accounting for 70-80% of glucose utilized during hyperinsulinemia (11). Use of positron emission tomography (PET) and 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) as a glucose tracer enables noninvasive regional measurements of muscle glucose uptake in vivo in humans. This method has been widely used to study healthy subjects (36) and, recently, also insulin-resistant subjects (23, 27, 28, 37, 48, 54).

The tracer [18F]FDG is a labeled glucose analog in which the natural D-glucose's other hydrogen in the 2-position is replaced by fluorine-18. It is an ideal positron-emitting radionuclide, having low positron energy (0.64 MeV) and a half-life of 109.8 min, which allows kinetic studies (56). [18F]FDG is transported into the muscle cell by the same carriers as glucose, and inside the cell, it is phosphorylated to [18F]FDG-6-phosphate by hexokinase II. However, affinities of deoxyglucose for transport and phosphorylation differ from those of glucose, because glucose transporters favor deoxyglucose over glucose, whereas the affinity of hexokinase is higher for glucose than for deoxyglucose (6, 20). Therefore, a correction factor called the lumped constant (LC) has been experimentally derived and used to correct for differences in transport and phosphorylation between FDG and glucose. After phosphorylation, [18F]FDG-6-phosphate can be incorporated into glycogen (55), but it cannot enter glycolysis (18). Consequently, [18F]FDG-6-phosphate is irreversibly trapped in the muscle cell.

The microdialysis technique has been successfully employed to directly measure regional metabolic balance in human subcutaneous and muscle tissue (21). The technique has been previously validated to estimate interstitial glucose concentration (16, 30). Microdialysis samples the interstitial fluid and, provided that in situ calibration is performed, enables the estimation of glucose concentration in the interstitial fluid (29). The extraction of glucose can be calculated, and, if the regional tissue blood flow is quantitated, the glucose uptake can be estimated by use of Fick's equation (21). Thus PET techniques and microdialysis both open unique possibilities for measurement of local metabolic balance rates. Moreover, the combination of techniques seems ideal, because PET provides more accurate blood flow measurements than plethysmography (38), which is more commonly used with microdialysis. Use of PET and radiowater allows direct and regional assessment of skeletal muscle blood flow without interference from other leg tissues and without invasive catheterizations.

In skeletal muscle studies, an LC of 1.0 has been commonly used (31, 36) despite the differences in the transport and phosphorylation of FDG relative to that of glucose (6, 20). Recently, Kelley et al. (24) determined the LC for [18F]FDG in human skeletal muscle using FDG PET, [3H]glucose, and venous occlusion strain-gauge plethysmography. Their data suggested an LC value of 1.2 in healthy lean volunteers, and insulin did not seem to influence the LC. However, we have previously shown that whole leg perfusion is not identical to skeletal muscle perfusion (24); furthermore, in the study of Kelley et al., only normal subjects, not insulin-resistant subjects, were included. In a number of previous studies, a close relationship was demonstrated between whole body glucose uptake measured with the euglycemic clamp technique and femoral muscle glucose uptake measured with FDG PET (34, 36, 37). When an LC of 1.0 was used, FDG PET-derived glucose uptake was similar, as measured with the forearm balance technique, and was 79% of the whole body glucose uptake (36) measured with the insulin glucose clamp technique. However, in insulin-resistant states such as obesity, where impaired cellular glucose uptake is the major cause of insulin resistance (5, 26) and changes in rates of glucose transport have been demonstrated (13), the LC might be altered.

PET combined with the microdialysis technique enables direct, relatively noninvasive measurement of the LC in vivo in human skeletal muscle. In the present study, we used these techniques to estimate the LC for [18F]FDG in healthy humans. We also tested whether the LC is altered in insulin-resistant obese subjects.


    RESEARCH DESIGN AND METHODS
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Eight nonobese [age 25 ± 1 yr, body mass index (BMI) 23.1 ± 1.0 kg/m2] and eight obese (age 29 ± 1 yr, BMI 33.6 ± 1.0 kg/m2) males were studied. The characteristics of the study subjects are shown in Table 1. The subjects were healthy, as judged by history, physical examination, and routine laboratory tests, and they were not taking any medications. The nature, purpose, and potential risks of the study were explained to all subjects before they gave their written informed consent to participate. The study was approved by the Joint Commission of the Ethics Committee of the University of Turku and Turku University Central Hospital.

                              
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Table 1.   Subject characteristics

Study design. The design of this study is shown in Fig. 1. Studies were performed after an overnight fast. Alcohol and caffeine were prohibited 24 h before the study, and the subjects were instructed to avoid strenuous physical activity 1 day before the study. The subjects were supine during the study. Two catheters were inserted, one in an antecubital vein of the left hand for the infusion of glucose and insulin and for injections of [15O]H2O and [18F]FDG and one in the radial artery for blood sampling. The microdialysis catheter was inserted into the left vastus medialis muscle. The study for each subject consisted of a 150-min normoglycemic hyperinsulinemic (1 mU · kg-1 · min-1) period. During hyperinsulinemia, normoglycemia was maintained using a variable rate of infusion of 20% glucose (12). The steady state for serum insulin and arterial plasma glucose concentrations was kept during the clamp for 150 min. During the period of FDG scanning, between 60 and 150 min, the coefficient of variation (CV) for arterial glucose concentrations was 3.2 ± 0.8% without any trend and was similar between the study groups.


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Fig. 1.   Study design. Arrows denote the time of intravenous injections of positron emitting tracers [15O]H2O and 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG). Hatched rectangles denote the time period of dynamic scanning.

At 0 min, an intravenous infusion of insulin (1 mU · kg-1 · min-1) was started. At 40 min from the start of the clamp, muscle blood flow was measured in the femoral region using [15O]H2O. Thereafter, at 60 min, [18F]FDG was injected for the measurement of muscle [18F]FDG uptake. Microdialysis samples were collected during the [18F]FDG scanning. Blood pressure and heart rate were determined basally and every 30 min throughout the study by means of an automatic oscillometric blood pressure analyzer (model HEM-705C, Omron, Tokyo, Japan). Blood samples for the measurement of radioactivity, serum insulin, and plasma glucose concentrations were taken as detailed below.

Microdialysis. The microdialysis procedure was performed as previously described (29, 33). The microdialysis catheter (dialysis membrane 16 × 0.5 mm, 20 kDa mol mass cut-off; CMA Microdialysis, Stockholm, Sweden) was inserted into the left vastus medialis muscle 10 cm above the knee joint (Fig. 2), without any local anesthesia, by vertical puncture of the surface of disinfected skin with a 20-gauge intravenous plastic cannula, which was then inserted into the muscle. The steel mandrin was removed, and the microdialysis catheter was inserted subsequently, after which the plastic cannula was also removed, leaving the catheter in the muscle. The inlet of the microdialysis catheter was connected to a microinjection pump (CMA/100, CMA Microdialysis), which perfused the catheter at a flow rate of 2.5 µl/min. Isotonic saline with an addition of 2.0 mmol/l glucose was used as the perfusion fluid. After 60 min of equilibration, the first four dialysates were collected at 5-min intervals and thereafter at 10-min intervals until the end of the study. After calibration of the microdialysis catheter, interstitial glucose concentrations were determined. The CV for interstitial glucose concentrations was 9.5 ± 1.5% between 60 and 150 min and was similar between the study groups.


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Fig. 2.   Example of [18F]FDG image as determined with positron emission tomography (PET) from the femoral region. To demonstrate the location of the microdialysis probe in the quadriceps femoris muscle (arrow), [18F]FDG (2% of study dose) was injected into the probe after the study.

Calibration procedure. The internal reference technique was applied to assess the in vivo probe recovery in each probe (30). For internal reference calibration, 5 µCi/ml of [3H]glucose (Amersham) were added to the perfusates, and the percentage of loss over the membrane was taken as an estimate of recovery (32). The mean in vivo recovery for glucose was 20 ± 0.1%.

Production of PET tracers. For production of 15O (t1/2 = 123 s), a low-energy deuteron accelerator, Cyclone 3, was used (Ion Beam Application, Louvain-la-Neuve, Belgium). [15O]H2O was produced by means of a dialysis technique in a continuously working water module (10). Sterility and pyrogenity tests were performed daily to verify the purity of the product. [18F]FDG (t1/2 = 109 min) was synthesized with an automatic apparatus as described by Hamacher et al. (15). The specific radioactivity at the end of the synthesis was 76 GBq/µmol, and the radiochemical purity exceeded 98%.

Image acquisition and processing. An eight-ring ECAT 931/08-tomograph (Siemens/CTI, Knoxville, TN) was used for image acquisition. The scanner has an axial resolution of 6.7 mm and an in-plane resolution of 6.5 mm (50). The subject was positioned in the tomograph with the femoral region within the gantry. Before the emission scannings, a transmission scan for correction of photon attenuation was performed for 20 min with a removable ring source containing 68Ge. All data were corrected for dead time, decay, and measured photon attenuation. For image processing, a recently developed Bayesian iterative reconstruction algorithm using median root prior with 150 iterations and the Bayesian coefficient of 0.3 was applied (1).

Regions of interest. Regions of interest (ROIs) were drawn in the anteromedial muscle compartments of both femoral regions in four cross-sectional slices, carefully avoiding large blood vessels (44). Localization of the muscle compartments was verified by comparing the flow images with the transmission image, which provides a topographical distribution of tissue density. The ROIs outlined in the flow images were copied to the [18F]FDG images to obtain quantitative data from identical regions.

Measurement of muscle blood flow. For measurement of blood flow, 1.1-1.5 GBq [15O]H2O was injected intravenously, and dynamic scanning was performed for 6 min (6 × 5-s, 6 × 15-s, and 8 × 30-s frames). To determine the input function, blood from the radial artery was continuously withdrawn using a pump at a speed of 6 ml/min. The radioactivity concentration was measured with a two-channel detector system (Scanditronix, Uppsala, Sweden) calibrated to the well counter (Wizard 1480; Wallac, Turku, Finland) and the PET scanner, as previously described (46). The arterial input curve was corrected for dispersion and delay as previously described (38). The autoradiographic method and a 250-s integration time were applied to calculate blood flow pixel by pixel. This method has been previously validated against the steady-state method (46) and blood flow measured with strain-gauge plethysmography (38).

Measurement of muscle glucose uptake with [18F]FDG (rGUFDG). For measurement of [18F]FDG uptake, 0.18-0.25 GBq of [18F]FDG was injected intravenously over 2 min, and a dynamic scan for 90 min was started (1 × 60-s, 6 × 30-s, 1 × 60-s, 3 × 300-s, and 7 × 600-s frames). Arterial blood samples for measurement of plasma radioactivity were withdrawn as previously described (36).

The three-compartment model of [18F]FDG kinetics was used as described previously (19, 35, 36, 47). Plasma and tissue time-activity curves for the anteromedial muscle compartments were analyzed graphically to quantitate the fractional rate of tracer uptake (Ki) (41). The time-activity points 10-90 min after the [18F]FDG injection were used to determine the slope by linear regression. The rGUFDG was calculated by multiplying Ki by the plasma glucose concentration ([Glc]p).

Measurement of rGUMD with microdialysis. After calibration, interstitial fluid glucose concentration was measured using microdialysis. Glucose uptake was calculated on the basis of Fick's equation by multiplying glucose extraction fraction with blood flow (21). Blood flow was measured using [15O]H2O and PET in the same muscle compartment where the interstitial glucose concentration was measured, i.e., in the vicinity of the microdialysis probe. The muscle volume in which blood flow was measured was ~15 cm3. The extraction fraction for glucose was calculated as previously described (16, 21) using the glucose permeability surface area product (PS)~4 ml · min-1 · 100 g-1 (7, 39, 52, 53) and the measured arterial (A) and interstitial (I) concentrations. Thereafter, the rGUMD was calculated by multiplying glucose extraction by blood flow (BF)
rGU<SUB>MD</SUB><IT>=</IT>[A<IT>−</IT>I]<IT>×</IT>[<IT>1−</IT>e<SUP>−PS/BF</SUP>]<IT>×</IT>BF

Calculation of LC. The LC was calculated by dividing rGUFDG measured using PET by the rGUMD measured using microdialysis
LC<IT>=</IT>rGU<SUB>FDG</SUB>/rGU<SUB>MD</SUB><IT>=K</IT><SUB>i</SUB><IT>×</IT>[Glc]<SUB>p</SUB>/rGU<SUB>MD</SUB>

Whole body glucose uptake. Whole body glucose uptake was determined independently of the PET measurements by use of the euglycemic hyperinsulinemic clamp technique as previously described (12). During hyperinsulinemia, normoglycemia was maintained by use of a variable rate of infusion of 20% glucose based on arterial plasma glucose measurements (22). The rate of whole body glucose uptake was calculated from the same time period that the measurements of muscle blood flow and glucose uptake were performed.

Other measurements. Arterial and plasma glucose were determined in duplicate by the glucose oxidase method (Analox GM7 Analyzer, Analox Instruments, London, UK). Serum insulin concentrations were measured basally and at 30-min intervals during the insulin infusion using a double-antibody radioimmunoassay (Pharmacia Insulin RIA kit, Pharmacia). Body fat content was estimated from four skinfolds (subscapular, triceps brachii, biceps brachii, and crista iliaca), as measured with calipers.

Statistical methods. All results are expressed as means ± SE. The differences between the two groups were compared using Student's unpaired t-test or the Wilcoxon matched pairs test, where appropriate. Correlations were calculated using Pearson's correlation analysis. The statistical calculations were performed with the Statistica (StatSoft, Tulsa, OK) statistical program. Significance was set at the P < 0.05 level.


    RESULTS
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INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
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Metabolic characteristics during the studies. Characteristics of the study subjects are shown in Table 1. During hyperinsulinemia, plasma glucose concentrations were similar [5.3 ± 0.03 vs. 5.3 ± 0.02 mmol/l, obese vs. nonobese subjects, respectively, not significant (NS)], but serum insulin concentrations were slightly higher in the obese than in the nonobese subjects (67 ± 5.0 vs. 49 ± 4.0 mU/l, respectively, P < 0.05).

Whole body glucose uptake. Insulin-stimulated whole body glucose uptake expressed per body weight was 123% higher in the nonobese than in the obese subjects (32.3 ± 2.6 vs. 13.4 ± 1.0 µmol · kg body wt-1 · min-1, nonobese vs. obese, P < 0.001; Fig. 3). When expressed per lean body mass (LBM), insulin-stimulated whole body glucose uptake was 70% higher in the nonobese than in the obese subjects (38.9 ± 3.3 vs. 23.4 ± 2.0 µmol · kg body wt-1 · min-1, respectively, P < 0.001).


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Fig. 3.   Femoral muscle glucose uptake (rGU), measured with PET (rGUPET) and with microdialysis (rGUMD), and whole body glucose uptake rates in the nonobese (open bars) and obese (filled bars) subjects. ***P < 0.001.

Muscle blood flow. Rates of insulin-stimulated muscle blood flow were slightly higher in the nonobese than in the obese subjects, but this difference was statistically nonsignificant (4.2 ± 1.1 vs. 1.9 ± 0.3 ml · 100 g-1 · min-1, P = 0.07; Table 2).

                              
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Table 2.   Results of PET and microdialysis measurements

Muscle glucose uptake measured using [18F]FDG and PET. During insulin stimulation, fractional [18F]FDG uptake values (Ki) were higher in femoral muscles of the nonobese than in the obese subjects (0.009 ± 0.002 vs. 0.004 ± 0.003 min-1, respectively, P < 0.01; Table 2 and Fig. 4). Consequently, muscle glucose uptake values measured with [18F]FDG (rGUFDG) and calculated by multiplying Ki by the plasma glucose concentration were significantly higher in the nonobese than in the obese subjects (46.3 ± 8.2 vs. 18.8 ± 1.4 µmol · kg muscle-1 · min-1, respectively, P < 0.001; Table 2). Muscle glucose uptake values measured with [18F]FDG correlated with rates of insulin-stimulated muscle blood flow in the pooled data (r = 0.60, P < 0.05; data not shown). Muscle glucose uptake values measured using [18F]FDG correlated with whole body glucose uptake values expressed per LBM in the pooled data (r = 0.84, P < 0.01; Fig. 5).


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Fig. 4.   Graphic analysis (Patlak) of femoral muscle [18F]FDG uptake showed a linear increase between the distribution volume (y-axis) and the normalized plasma time in minutes (x-axis), indicating metabolic trapping.



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Fig. 5.   Relationships between whole body glucose uptake expressed per lean body mass (LBM) and femoral muscle glucose uptake measured with [18F]FDG (rGUFDG) and microdialysis (rGUMD) in obese () and nonobese (open circle ) subjects.

Muscle glucose uptake measured by microdialysis. Insulin-stimulated femoral muscle rGUMD was significantly higher in the nonobese than in the obese subjects (41.4 ± 5.9 vs. 17.0 ± 1.6 µmol · kg muscle-1 · min-1, nonobese vs. obese, P < 0.005; Table 2 and Fig. 3). During hyperinsulinemia, interstitial glucose concentrations were higher in the obese (4.13 ± 0.17 mmol/l) than in the nonobese subjects (3.39 ± 0.16 mmol/l, P < 0.01; Table 2). There was an inverse correlation between interstitial glucose concentration and muscle glucose uptake values measured with [18F]FDG in the pooled data (r = -0.50, P < 0.05). Femoral muscle glucose uptake correlated with whole body glucose uptake expressed per LBM in the pooled data (r = 0.75, P < 0.01; Fig. 5).

Lumped constant. The LC, calculated by dividing rGUFDG by rGUMD, averaged 1.16 ± 0.07 for the entire group, 1.16 ± 0.16 for the nonobese, and 1.15 ± 0.11 and for the obese subjects (P = NS; Table 2). Regional PET-derived glucose uptake (rGUPET) correlated with rGUMD (r = 0.78, P < 0.01; Fig. 6). No association was found between the LC and whole body glucose uptake (P = 0.15, r = 0.43) or between the LC and serum insulin concentrations (P = 0.76, r = 0.14) in the pooled data or when the groups were analyzed separately (data not shown).


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Fig. 6.   Relationship between rGUPET and rGUMD in obese () and nonobese (open circle ) subjects.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was undertaken to estimate directly the LC for [18F]FDG in human skeletal muscle during insulin stimulation. Furthermore, we tested whether insulin resistance alters the LC. We found that the LC for [18F]FDG averaged 1.2 and was unaltered in insulin-resistant obese subjects. No correlation was found between the LC and serum insulin concentrations (range 37-85 mU/l) during the insulin clamp.

The model for quantitation rates of glucose utilization using labeled 2-deoxyglucose as a tracer was first introduced by Sokoloff et al. (49) to quantitate rates of cerebral glucose utilization in rats. This model has been subsequently applied in PET studies and used to quantitate glucose uptake rates with [18F]FDG in skeletal muscle in humans (27, 28, 36, 43, 54). This model is applicable only in homogenous tissues under steady-state conditions when [18F]FDG and [18F]FDG-6-phosphate are present in tracer amounts (42). In addition, the model assumes that dephosphorylation of [18F]FDG-6-phosphate is nonsignificant (41). The Ki can be calculated by use of graphic analysis of plasma and tissue time-activity curves (41). Thereafter, the rate of glucose uptake can be calculated by multiplying Ki by the [GLC]p divided by an LC, which accounts for differences in the transport and phosphorylation of [18F]FDG and glucose. In the present study, the steady-state conditions required by the model were attained by the insulin clamp technique, [18F]FDG was used in tracer amounts, and the graphic analysis of the plasma and tissue time-activity curves showed linear slopes (Fig. 4), supporting the assumption that [18F]FDG was irreversibly trapped within the muscle cells and not dephosphorylated.

From experimental animal studies, the LC for [18F]FDG in skeletal muscles has been estimated to be 1.0 (31), and this value has previously been commonly used in human skeletal muscle studies. Recently, Kelley et al. (24) determined the LC for [18F]FDG in human skeletal muscle using FDG PET, [3H]glucose, and venous occlusion strain-gauge plethysmography. Their data suggested an LC value of 1.2 in healthy lean volunteers, and insulin did not seem to influence the LC. However, we have previously shown that whole leg perfusion is not identical with skeletal muscle perfusion (45); furthermore, in the study of Kelley et al., only normal subjects, not insulin-resistant subjects, were included. Skeletal muscle LC for other 2-deoxyglucoses, i.e., for 2-deoxy[1-3H]glucose and 2-deoxy[1-14C]glucose, has been previously estimated in rats and found to range from 0.75 to 1.20 (4, 14). However, those results cannot be used directly for human skeletal muscle or for [18F]FDG, which is structurally more similar to glucose than either 2-deoxy[1-3H]glucose or 2-deoxy[1-14C]glucose. In the present study, by combining the microdialysis technique with PET, the LC for [18F]FDG was estimated directly in human skeletal muscle and averaged 1.2 in both the nonobese and the obese subjects during insulin stimulation. This finding is consistent with our previous studies (34, 36) and with the study of Kelley et al., suggesting that LC is close to unity.

Microdialysis has not previously been combined with the FDG-PET technique; it has been used mainly in the assessment of interstitial glucose concentration in human subcutaneous and muscle tissue. The interstitial glucose concentrations measured in the present study are comparable with those found in previous studies (16, 30, 32). All microdialysis catheters used in the present study were calibrated in steady-state conditions using the internal reference technique, which has been previously shown to give similar recovery coefficients to the equilibration technique in humans (16). After the calibrated assessment of interstitial glucose concentration, the glucose uptake was calculated using Fick's principle by multiplying the glucose extraction fraction with blood flow. The direct validation to measure glucose uptake using microdialysis against arteriovenous methods during insulin stimulation is lacking. The recent study of Stallknecht et al. (51) was designed to evaluate the reliability of the microdialysis technique for the assessment of glucose uptake in adipose tissue. They found that calculated and measured venous plasma glucose concentrations were identical in adipose tissue, but that glucose uptake rates calculated from microdialysis and 133Xe washout measurements and by Fick's principle only tended toward correlation. However, any small errors in absolute values of fasting plasma glucose calculated from microdialysis had significant influence on the calculated arteriovenous differences. Furthermore, when arteriovenous difference was multiplied by an inaccurate blood flow estimated from 133Xe washout, their finding of nonsignificant association between glucose uptake rates was not considered to be surprising (51). Challenging their results, we found a significant correlation between glucose uptake measured with microdialysis and FDG PET (r = 0.78, P < 0.01). Our study design differed from the study of Stallknecht et al. in that the present study was performed after an overnight fast during steady-state hyperinsulinemia leading to higher arteriovenous glucose differences. Furthermore, skeletal muscle perfusion was measured directly with radiowater in the vicinity of the microdialysis probe, in the same region of interest where the glucose and [18F]FDG uptake were measured. Use of PET and radiowater allows direct and regional assessment of skeletal muscle blood flow and metabolism without interference from other leg tissues and without invasive catheterizations. Regional glucose and [18F]FDG uptake were calculated in the same region with two different and independent methods, and from these measurements the LC was estimated. Thus, in the present study, the finding that muscle glucose uptake assessed by PET correlated closely with the uptake rates estimated by microdialysis suggests that these two methods can be reliably combined.

In the calculation of glucose uptake measured with microdialysis, the value of permeability surface area product (PS) is needed. Only preliminary data of the PS value for glucose in human skeletal muscle during physiological insulin stimulation are available, and its influence on the results must be carefully taken into account. Trap-Jensen and Lassen (52) determined the PS for fructose and found it to be 5.5 ml · min-1 · 100 g-1 in human forearm during exercise. Crone (8) reported the PS value for D-glucose to be 4.6 ml · min-1 · 100 g-1 in cat gastrocnemius muscle. For larger molecules such as 51Cr-EDTA and sucrose, PS values between 3.0 and 5.5 ml · min-1 · 100 g-1 in skeletal muscle have been reported (7, 39, 52, 53). These previously reported PS values for hexose molecule are in accordance with the applied PS value of 4 ml · min-1 · 100 g-1 (Fig. 7). Moreover, when it is taken into account that glucose is a smaller molecule than sucrose and 51Cr-EDTA and that the permeability has been demonstrated to be 60% higher for hexose than for sucrose in human forearm (53), there is strong evidence that an applied PS value of 4 ml · min-1 · 100 g-1 is adequate within the range of blood flow rates presently registered (9, 39, 40). This is also in agreement with the data from direct measurements of PS for glucose in the human forearm during insulin stimulation, which suggested that PS for glucose averaged 4 ml · min-1 · 100 g-1 and was not dependent on plasma insulin concentration (S. Gudbjornsdottir, J. Wahren, and P. Lönnroth, personal communication).


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Fig. 7.   Influence of previously reported permeability surface area product (PS) values (Refs. 7, 8, 39, 52, 53) on the calculated lumped constant value with blood flow rate of 3.1 ml · 100 g muscle-1 · min-1 (see Table 2).

The effect of the PS value on the LC is illustrated by the following example. If mean blood flow is 3.1 ml · 100 g-1 · min-1 (Table 2), and the PS value varies from 3 to 5 ml · min-1 · 100 g-1, the LC varies from 1.39 to 1.08 (Fig. 7). Hence, PS values >3 ml · min-1 · 100 g-1 have only a minor effect on the value of the LC. However, it should be noted that blood flow has been found to correlate with PS value, but the PS-to-blood flow ratio has been shown to be constant at such low blood flow rates as presented in this study (9). Thus the nonsignificant difference in blood flow rates between the groups in the present study was not influencing the LC value, which was found to be similar in the obese and nonobese subjects. Also, the recent data by Kelley et al. (24) give indirect support for the applied PS value; namely, if the PS is calculated on the basis of their data, using a value of 1.2 for LC, the PS value will be ~4 ml · min-1 · 100 g-1.

In the present study, blood flow and glucose uptake rates correlated, and, in accord with previous observations in obese subjects (25), the obese subjects of this study tended to have a lower blood flow rate. It has been suggested that the lower blood flow rate demonstrated in insulin-resistant muscle per se could mediate the insulin resistance by means of failure to deliver hormones and/or nutrients to the cells (2, 25). However, some other studies have shown that the impact of variations in blood flow in normal (32) and insulin-resistant muscle is minor or absent (16). The present study further underlines this conclusion, because obese subjects had a higher muscle interstitial glucose concentration compared with lean subjects. Thus, despite lower blood flow rates, insulin-resistant muscles have higher interstitial glucose levels due to a low elimination rate of glucose (16). Recently, it has been demonstrated that induction of insulin resistance in muscle reduces blood flow rate (17). Thus one could speculate that the attenuated action of insulin on glucose metabolism might be a causal factor behind the reduced blood flow of insulin-resistant muscle.

Glucose transporters favor [18F]FDG over glucose, whereas the affinity of hexokinase for glucose is higher than that for 2-deoxyglucose (6); thus one could hypothesize that the LC might be altered in states such as obesity, where rates of either glucose transport or phosphorylation are changed. Obesity is a major cause of insulin resistance in humans (3), and it is characterized by an inability of insulin to normally increase glucose uptake in skeletal muscles. Previous in vivo studies have demonstrated impaired cellular glucose uptake to be the major cause of insulin resistance in obesity (5, 26). Furthermore, in a study of intact muscle strips, decreased rates of glucose transport have been found in muscles of morbidly obese subjects (13). Yet, in the present study, we found that the LC was unchanged in the insulin-resistant obese subjects, who (consistent with previous data) exhibited a 55% reduction in whole body and a 59% reduction in femoral muscle glucose uptakes compared with the nonobese subjects. Interstitial glucose concentrations measured with the microdialysis technique were higher in the obese than in the nonobese subjects. From the present data, we are unable to conclude whether glucose transport or hexokinase activity is altered in the obese subjects, but the present finding of higher interstitial glucose concentration of the obese subjects seems to support the hypothesis that glucose transport is decreased in obesity. This is in accord with a previous study by Kelley et al. (23), which demonstrated a reduction in the insulin response of the transport rate constant in obese nondiabetic subjects.

In summary, it is feasible to combine the microdialysis technique with PET for the assessment of glucose uptake in skeletal muscle in vivo. By use of these two independent methods, direct measurements of the LC in human skeletal muscle demonstrate the LC value of 1.2 for [18F]FDG. Furthermore, the LC appears not to be sensitive to insulin resistance.


    ACKNOWLEDGEMENTS

We thank all the personnel in the Turku PET Centre for skillful technical assistance.


    FOOTNOTES

This work was supported by grants from the Research Foundation of the Orion Corporation (P. Peltoniemi), the Novo Nordisk Foundation (P. Nuutila), the Academy of Finland (P. Nuutila), the Turku University Foundation (P. Peltoniemi), and by the Yrjö Jahnsson Foundation (P. Peltoniemi), the Swedish Researcher Council (Project 10864), the Swedish Diabetes Association, and Novo Pharma.

Address for reprint requests and other correspondence: P. Peltoniemi, Turku PET Center, Univ. of Turku, PO Box 52, 20520 Turku, Finland (E-mail: papelto{at}utu.fi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 March 2000; accepted in final form 6 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
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