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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 11 5525-5533
Copyright © 2001 by The Endocrine Society

Other Original Articles

Evidence for Spatial Heterogeneity in Insulin- and Exercise-Induced Increases in Glucose Uptake: Studies in Normal Subjects and Patients with Type 1 Diabetes

Pauliina Peltoniemi, Hannele Yki-Järvinen, Hanna Laine, Vesa Oikonen, Tapani Rönnemaa, Kari Kalliokoski, Olli Raitakari, M. Juhani Knuuti and Pirjo Nuutila

Turku PET Centre (P.P., H.L., V.O., K.K., O.R., M.J.K., P.N.) and Department of Medicine (H.L., T.R., P.N.), University of Turku, FIN-20520 Turku, Finland; and Department of Medicine (H.Y.-J.), University of Helsinki, FIN-00014 Helsinki, Finland

Address all correspondence and requests for reprints to: Dr. Pauliina Peltoniemi, Turku PET Centre, University of Turku, P.O. Box 52, FIN-20520 Turku, Finland. E-mail: papelto{at}utu.fi

Abstract

It is unknown whether resistance to insulin- or exercise-stimulated glucose uptake reflects a spatially uniform or nonuniform decrease in glucose uptake within skeletal muscle. We compared the distributions of muscle glucose uptake and blood flow in eight patients with type 1 diabetes (age 24 ± 1 yr, body mass index 22.0 ± 0.8 kg/m2) and seven age- and weight-matched normal subjects using positron emission tomography, [18F]-fluoro-deoxy-glucose, and [15O]-water. Both groups were studied during euglycemic hyperinsulinemia and one-legged exercise. Heterogeneity was evaluated by calculating relative dispersion (SD divided by mean * 100%) of glucose uptake (RDg) and flow (RDf) in all pixels within a region of interest in femoral muscle. At rest insulin-stimulated glucose uptake was significantly lower in the type 1 diabetic patients (42 ± 7 µmol/kg per min) than in the normal subjects (78 ± 9 µmol/kg per min, P < 0.001), while muscle blood flows were similar (26 ± 1 vs. 31 ± 3 ml/kg muscle per min, respectively). The exercise-induced increment in glucose uptake but not in blood flow was also significantly lower in the type 1 diabetic patients than in the normal subjects. Heterogeneity of glucose uptake but not of blood flow was greater in the insulin-resistant type 1 diabetic patients both at rest (RDg 31 ± 1 vs. 25 ± 2%, patients with type 1 diabetes vs. normal subjects, P < 0.05) and during exercise, compared with normal subjects (27 ± 1 vs. 21 ± 2%, respectively, P < 0.05). Exercise increased both glucose uptake and blood flow several-fold and significantly decreased both RDg and RDf. Heterogeneity of RDg, was inversely associated with total glucose uptake (r = -0.54, P < 0.001, pooled data) and was highest in the most insulin-resistant patients. We concluded that both glucose uptake and blood flow are characterized by heterogeneity in human skeletal muscle, whose magnitude is inversely proportional to respective mean values. This implies that an increase in glucose uptake in human skeletal muscle is not a phenomenon, by which each unit increases its glucose uptake by a fixed amount but rather a spatially heterogeneous process.

INSULIN RESISTANCE OF glucose uptake is characterized by a decrease in the rate of glucose uptake per muscle mass. In addition, insulin resistance, at least in obese subjects, is characterized by a time-dependent or temporal defect—insulin activates glucose uptake more slowly in obese than nonobese subjects (1). Thus, previous studies have demonstrated variation in mean rates of insulin-stimulated glucose uptake as well as temporal heterogeneity. Regarding spatial variation in glucose uptake, Iversen et al. (2) used radioactive microspheres and 2-deoxy-glucose to quantitate rates of blood flow and glucose uptake in 25-mm3 sections of rabbit skeletal muscle. Considerable heterogeneity was observed in blood flow and glucose uptake both at rest and during exercise. Specific fiber-type distributions could not explain either the regional heterogeneity in blood flow or glucose uptake. Thus, the anatomical and physiological basis for the observed heterogeneity remained unclear.

There are no previous data on possible heterogeneity of glucose uptake in human skeletal muscle. Specifically, it is unknown whether stimuli such as exercise or insulin increase glucose uptake uniformly (i.e. by a fixed amount in each muscle fiber or larger muscle unit) or whether the increase is heterogeneous. At least theoretically, insulin or exercise could increase muscle glucose uptake by a fixed and similar amount in each pixel, or these stimuli could increase glucose uptake more in areas with high, compared with low, glucose uptake (i.e., by the same percentage in each pixel). Effects of these alternatives on relative heterogeneity of glucose uptake is simulated in the present study. Use of positron emission tomography (PET) and a median root prior (MRP)-based method for reconstruction reduces statistical noise and reconstruction artifacts sufficiently to allow pixel-by-pixel quantitation of glucose uptake also in human skeletal muscle (3). In the present study, we determined whether: (a) glucose uptake is heterogeneous in human skeletal muscle when measured in 65-mm3 volume fractions, (b) flow heterogeneity colocalizes with heterogeneity of glucose uptake, (c) relative heterogeneity of glucose uptake is higher in insulin-resistant than -sensitive individuals, and (d) relative heterogeneity of glucose uptake or flow depends on absolute rates of glucose uptake or flow or on the stimuli (insulin with or without exercise) used to increase glucose uptake.

Experimental Design and Methods

Subjects

Eight men with type 1 diabetes and seven normal subjects with no family history of diabetes or hypertension were studied (Table 1Go). All subjects were nonsmokers. Duration of type 1 diabetes averaged 7 ± 1 yr. The mean daily dose of insulin was 56 ± 5 U using multiple insulin injection regimens. The type 1 diabetic patients were normotensive and had no clinical or laboratory evidence of diseases other than type 1 diabetes and no signs of micro- or macrovascular disease, as determined by retinal photographs and autonomic nervous function tests. The normal men were healthy as judged by history, physical examination, and routine laboratory tests and were not taking any medications. Written informed consent was obtained after the nature, purpose, and potential risks of the study were explained to the subjects. The study was approved by the Joint Commission of Ethics of the University of Turku and Turku University Central Hospital.


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  		Table 1. Subject characteristics

 
Study design

Prior to the PET study, whole-body maximal oxygen consumption and maximal isometric contractile force of the quadriceps femoris muscle were determined as detailed below. The study design is shown in Fig. 1Go. The PET studies were performed after an overnight fast. Alcohol and caffeine were prohibited 1 d before the study, and the subjects were instructed to avoid strenuous physical activity for 1 d before the study. In the morning of the study, the usual dose of intermediate-acting insulin was reduced by half and no short-acting insulin was given. Two catheters were inserted, one in an antecubital vein of the left hand for infusion of glucose, insulin, and injections of [15O]H2O and [18F]FDG and another in the radial artery for blood sampling. The study for each subject consisted of a 150-min normoglycemic hyperinsulinemic (1mU/kg per min) period (0–150 min). During hyperinsulinemia, normoglycemia was maintained using a variable rate of infusion of 20% glucose. The subjects performed intermittent isometric exercise with one leg between 45 and 150 min (Fig. 1Go). After 60 min of hyperinsulinemia, muscle blood flow, and after 90 min muscle oxygen consumption were measured simultaneously in both femoral regions using [15O]H2O infusion and [15O]O2 inhalation techniques, and muscle glucose uptake was measured immediately thereafter using [18F]FDG and PET (Fig. 1Go). Blood samples for measurement of radioactivities and serum insulin concentrations were taken as detailed below.



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  		Figure 1. Study design. The arrows denote the time of iv injections/inhalation of positron-emitting tracers [15O]H2O, [15O]O2, and [18F]FDG. Shaded rectangles denote the time period of dynamic scanning.

 
Production of PET tracers and image acquisition

[18F]FDG (t1/2 = 109 min) was synthesized with an automatic apparatus as described by Hamacher et al. (4). The specific radioactivity at the end of the synthesis was 76 GBq/µmol and the radiochemical purity exceeded 98%. For production of [15O] (t1/2 = 123 sec), a low-energy deuteron accelerator Cyclone 3 was used (Ion Beam Application Inc., Louvain-la-Neuve, Belgium). [15O] was produced by the [14N](d,n) [15O] reaction on natural nitrogen gas (5). Radiochemical purity of [15O]O2 was 97%. [15O]H2O was produced using a dialysis technique in a continuously working water module (6). Sterility and pyrogenicity tests were performed daily to verify the purity of the product.

An ECAT 931/08 tomograph (Siemens/CTI Inc., Knoxville, TN) was used for image acquisition. The images were obtained from the femoral region. 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. Images were processed using an MRP reconstruction algorithm, which effectively reduces noisiness of PET images and enables analysis of pixels sized 9.6 mm2 (3).

Regions of interest

Regions of interest (ROIs, 40.3 ± 1.8 cm3) were drawn in the anteromedial muscle compartments (vastus medialis and vastus intermedius muscles) of both femoral regions in four consecutive cross-sectional slices in both legs, carefully avoiding large blood vessels. 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 [15O]O2 and [18F]FDG images to obtain quantitative data from identical regions.

Measurement of muscle glucose uptake

For measurement of glucose uptake, 0.19–0.25 GBq of [18F]FDG was injected iv over 2 min, and a dynamic scan for 30 min was started (8 x 15 sec, 2 x 30 sec, 2 x 120 sec, 1 x 180 sec, 4 x 300 sec). Arterial blood samples for measurement of plasma radioactivity were withdrawn, as previously described (7). The three-compartment model of [18F]FDG kinetics was used as described previously (7). Plasma and tissue time-activity curves for the anteromedial muscle compartments were analyzed graphically to quantitate the fractional rate of tracer uptake Ki (8). The rate of glucose uptake was obtained by multiplying Ki by the plasma glucose concentration divided by a lump constant term (LC): rate of glucose uptake = plasma glucose concentration /LC x Ki. The lumped constant accounts for differences in the transport and phosphorylation of [18F]FDG and glucose. A lumped constant value of 1.2 for skeletal muscle was used based on recent studies (9, 10, 11).

Regional heterogeneity of glucose uptake

Fractional rates of tracer uptake Ki were extracted from the pixel-by-pixel [18F]FDG images. A single large ROI (622 ± 24 pixels) outlined in the flow images was copied to [18F]FDG images and used for analysis of glucose uptake distribution. Data from four planes were pooled, and the average and SD of glucose uptake values were calculated. Relative dispersion of glucose uptake heterogeneity (RDg) was calculated by dividing the SD of glucose uptake by average glucose uptake value.

Measurement of muscle blood flow and oxygen consumption

For measurement of blood flow, 1.2–1.7 GBq of [15O]H2O was injected iv, and dynamic scanning for 6 min (6 x 5 sec, 6 x 15 sec, 8 x 30 sec frames) was performed (12, 13). The delay- and dispersion-corrected arterial radioactivity was used as an input function. The autoradiographic method and a 250-sec integration time were applied to calculate blood flow pixel by pixel (13, 14, 15, 16, 17, 18). For measurement of muscle oxygen consumption, [15O]O2 inhalation technique, recently validated in our laboratory, was used (19). The subjects inhaled the gas containing 1.23 ± 0.1 GBq of [15O]O2 via a mouthpiece and PET imaging of the femoral region was thereafter performed for 7 min with time frames of 6 x 5 sec, 6 x 15 sec, 6 x 30 sec, 2 x 60 sec, and the arterial radioactivity was measured (19).

Regional heterogeneity of blood flow

Blood flow values were extracted from the pixel-by-pixel flow image. A single large ROI drawn onto the anteromedial muscle compartment of the femoral region in four cross-sectional slices was used for analysis of flow distribution. On average this ROI consisted of 622 ± 24 pixels. Data from four planes were pooled and averaged, and the SD of blood flow values were calculated. Relative dispersion of blood flow heterogeneity (RDf) was calculated by dividing the SD of blood flow by mean flow (14, 15, 16, 20, 21).

Simulation study

To examine how an increase in mean glucose uptake/blood flow can influence relative heterogeneity of these parameters, a simplified mathematical model was used. Based on the in vivo glucose uptake/flow data, the average glucose uptake/flow was increased 5-fold using the following simulations (Fig. 2Go): fixed and similar increments in all pixels (A) and similar percentage increments in each pixel (B) (i.e., the absolute increments were higher in the active than in the less active areas). In both A and B, the absolute increase was kept constant.



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  		Figure 2. Simulation study. A, Effect of a fixed increment of glucose uptake in each pixel; relative dispersion (RD) decreases. B, Effect of similar percentile increment in each pixel; RD remains unchanged.

 
Exercise during the PET study and measurement of maximal isometric knee extension force

The subjects were lying supine in the PET scanner with the femoral regions in the gantry and the right leg, fixed at a knee ankle of 50 degrees, was fastened to a dynamometer (I-KON, Chattanooga Group Ltd., Oxfordshire, UK) (Fig. 1Go). During 45–150 min of hyperinsulinemia, intermittent isometric exercise was performed with one leg (19). Care was taken to fasten the subjects carefully to the imaging table to avoid any movements in the femoral region during the study. Exercise consisted of 2-sec isometric knee extension intermittent with 2 sec of rest. Exercise intensity was set at 10% of maximal isometric force measured before the PET study with a dynamometer (KinCom, Chattex Corp., Chattanooga, TN). Maximal oxygen uptake was determined using an electrically braked cycle ergometer (Ergoline 800 S, Bitz, Germany) with a continuous incremental protocol (19).

Whole-body glucose uptake and other measurements

Whole-body glucose uptake was quantitated, independent of PET measurements, using the euglycemic hyperinsulinemic clamp technique (22). During hyperinsulinemia, the rate of glucose infusion, corrected for changes in the glucose pool size, was used as a measure of whole-body glucose uptake (23). Whole-body glucose uptake was calculated from the same time period when the measurements of blood flow, oxygen consumption, and muscle glucose uptake were performed (60–150 min).

Arterial and plasma glucose were determined in duplicate by the glucose oxidase method (Analox GM7 analyzer; Analox Instruments Ltd., Hammersmith, London, UK). Serum-free insulin concentrations were measured using RIA (Pharmacia insulin RIA kit, Pharmacia Diagnostics AB, Uppsala, Sweden) after precipitation with polyethyleneglycol (24). Body fat content was estimated from four skinfolds (subscapular, triceps brachii, biceps brachii, and crista iliaca) as measured with caliper (25).

Noise contribution in flow and glucose uptake images

To examine how noise changes statistics of the measurements, we made empirical measurements of the pixel-by-pixel noise with varying integrated low activity levels in a uniform phantom (26). The methodological dispersion was decreased with increasing radioactivity but reached an almost constant level in the activity levels corresponding to the tissue activity during flow and FDG measurements in the present study (26). For the flow images reconstructed using MRP reconstruction method relative dispersion of the pixel-by-pixel noise at the activity levels of the resting muscles averaged 10% and of the exercising muscles 6% (26). For the glucose uptake images, pixel-by-pixel noise with varying integrated activity levels averaged approximately 6% at the activity levels of resting muscles and 5% at the activity levels of exercising muscles.

Statistical methods

All results are expressed as mean ± SEM. Baseline comparisons between the two groups were performed by using the t test. For correlation analysis, Pearson’s correlation coefficients were calculated. Statistical calculations were performed using the SAS statistical program package (SAS Institute, Inc., Cary, NC). Significance was inferred at two-tailed P < 0.05.

Results

Whole-body and femoral muscle glucose uptake, blood flow, and oxygen consumption

During hyperinsulinemia serum-free insulin concentrations (52 ± 1 vs. 55 ± 4 mU/liter, normal subjects vs. type 1 diabetic patients, NS) and plasma glucose concentrations (5.2 ± 0.1 vs. 5.4 ± 0.1 mmol/liter, respectively, NS) were comparable. Insulin-stimulated whole-body glucose uptake, expressed per body weight, was 42% lower in the type 1 diabetic patients (24 ± 3 µmol/kg body weight x min-1) than in the normal subjects (43 ± 2 µmol/kg body weight x min-1, P < 0.01). Insulin-stimulated glucose uptake in resting femoral muscle was 48% lower in the patients with diabetes than the normal subjects (42 ± 7 vs. 78 ± 9 µmol/kg muscle x min-1, P < 0.001, Fig. 3Go). During exercise insulin-stimulated glucose uptake increased significantly in both groups (P < 0.05), but the increment induced by exercise was significantly and 49% lower in the patients with type 1 diabetes than in the normal subjects (94 ± 21 vs. 186 ± 29 µmol/kg muscle x min-1, P < 0.05). The correlation coefficient between whole-body and femoral muscle glucose uptake was 0.83 in the normal subjects (P < 0.001) and 0.75 (P < 0.01) in the type 1 diabetic patients. During hyperinsulinemia resting rates of muscle blood flow were similar in both groups (Fig. 3Go). Exercise induced a significant (P < 0.001) and similar 4- to 5-fold increase of blood flow in both groups (Fig. 3Go). During hyperinsulinemia, resting rates of oxygen consumption were comparable in both groups (1.9 ± 0.3 vs. 1.5 ± 0.3 ml/kg muscle x min, normal subjects vs. patients with type 1 diabetes, NS). Exercise increased rates of oxygen consumption similarly and more than 10-fold in both groups (to 27 ± 2 vs. 23 ± 4 ml/kg muscle x min, respectively, NS).



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  		Figure 3. Rates of muscle glucose uptake and RDg (top) and rates of muscle blood flow and RDf (bottom) during hyperinsulinemia (INS) and exercise (INS+EX) of normal subjects (CONT) and patients with type 1 diabetes (DM). *, P < 0.05, ***, P < 0.001.

 
Simulation study

When glucose uptake was increased by a fixed amount in all pixels, RDg was decreased by 80% (Fig. 2AGo). When uptake was increased by a similar percentage (e.g., 5-fold in each pixel), RDg and the shape of histogram remained unchanged (Fig. 2BGo). In this case, the absolute increment of glucose uptake was higher in the active areas, compared with the less active areas.

Heterogeneity of glucose uptake (RDg) and blood flow (RDf)

Insulin-stimulated glucose uptake was heterogeneously distributed at rest and during exercise in both study groups. In the patients with type 1 diabetes, relative heterogeneity of glucose uptake RDg was higher both under resting conditions (31 ± 1 vs. 25 ± 2%, patients with type 1 diabetes vs. normal subjects, P < 0.05) and during exercise (27 ± 1 and 21 ± 2%, respectively, P < 0.05, Figs. 3Go, 4Go, and 5Go). Exercise decreased RDg significantly (-4 ± 1 and -4 ± 2%, respectively, P < 0.05) and similarly in both groups. In Fig. 5Go it can be seen that the 3-to 5-fold increases in glucose uptake by exercise changed relative heterogeneity relatively little, although small significant decreases were observed. This suggests that exercise increased glucose uptake predominantly by similar fold increases (i.e., areas already using glucose increased their absolute glucose uptake more than previously inactive areas). Figure 5Go also shows the effect of exercise on relative flow heterogeneity. The pattern of distribution is intermediate between stimulation models 2A and 2B.



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  		Figure 4. Individual examples of frequency histograms of muscle glucose uptake (left) and blood flow values (right) in a normal subject (CONT) and a patient with type 1 diabetes (DM) during hyperinsulinemia (INS) and exercise (INS + EX). Absolute glucose uptake/flow values (x-axis) are plotted against their frequency (y-axis).

 


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  		Figure 5. Frequency histograms of mean relative muscle glucose uptake (left) and blood flow (right) values in a group of normal subjects (CONT) and in a group of patients with type 1 diabetes (DM) during hyperinsulinemia (INS) and exercise (INS + EX). Relative glucose uptake/flow (x-axis) were calculated by dividing the SD of glucose uptake/flow by mean glucose uptake/flow, and these values were plotted against their frequency of all relative values of glucose uptake/flow (y-axis).

 
Muscle blood flow showed considerable relative heterogeneity both at rest (RDf 43 ± 6 vs. 43 ± 4%, normal subjects vs. patients with diabetes) and during exercise (29 ± 3 vs. 30 ± 2%, respectively, P = 0.94) in both groups (Figs. 3Go, 4Go, and 5Go). Exercise decreased RDf similarly and significantly (-14 ± 4 and -13 ± 3%, respectively; P < 0.002) in both groups.

Relationship between mean glucose uptake and flow and relative heterogeneity

The rate of femoral muscle glucose uptake was inversely correlated with relative heterogeneity of glucose uptake (r = -0.53, P < 0.01, Fig. 6Go) when all data were analyzed together. This relationship was particularly strong in the normal subjects in the exercising femoral region during hyperinsulinemia (r = -0.88, P < 0.01, Fig. 6Go). The rate of femoral muscle blood flow was also inversely correlated with relative heterogeneity of flow (r = -0.40, P < 0.05) when all data were analyzed together (data not shown).



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  		Figure 6. Relationship between glucose uptake and relative heterogeneity of glucose uptake (top) normal subjects (CONT) and in patients with type 1 diabetes (DM) during hyperinsulinemia (INS) and exercise (INS + EX).

 
Coupling between flow and metabolism and its relationship to insulin resistance

To examine, whether glucose uptake within an individual skeletal muscle region was correlated with blood flow, pixel-by-pixel analysis was performed in each subject under resting conditions and during exercise. An example of such an analysis in one patient with type 1 diabetes and a normal subject is shown in Fig. 7Go. Under resting hyperinsulinemic conditions, blood flow and glucose uptake did not seem to be correlated (mean ± SE of individual correlation coefficients 0.11 ± 0.07 and 0.11 ± 0.06, respectively, for normal subjects and type 1 diabetic patients). In contrast, during exercise flow and glucose uptake appeared to be better correlated with each other (mean ± SE of individual correlation coefficients 0.51 ± 0.03 and 0.46 ± 0.08, respectively). As shown above, there was no difference in average blood flow rates between the groups although glucose uptake was significantly lower in the patients with type 1 diabetes than the normal subjects. Figure 7Go illustrates that this was also evident when examined at the pixel-by-pixel level.



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  		Figure 7. Relationship between single pixel muscle blood flow and glucose uptake values during insulin and exercise in a normal subject (CONT) and a patient with type 1 diabetes (DM) extracted from pixel-by-pixel images.

 
Discussion

The present data provide some novel information regarding the distribution of glucose uptake and blood flow in human skeletal muscle. The type 1 diabetic patients had reduced rates of insulin-stimulated glucose uptake and a blunted increment in glucose uptake by exercise, compared with the normal subjects, and blood flows were similar between the groups under all conditions. The greater heterogeneity of glucose uptake in the type 1 diabetic patients than the normal subjects disappeared when adjusted for by the difference in glucose uptake between the groups. These data support the idea that spatial heterogeneity characterizes the abilities of both exercise and insulin to increase glucose uptake in human skeletal muscle.

Each method for quantitating heterogeneity of blood flow or glucose uptake in animal or human skeletal muscle has limitations such as those resulting from sampling errors. The within-region variation of flow and glucose uptake in the present study was markedly higher than could be expected if the variation was caused only by technical factors such as pixel-by-pixel inhomogeneity. The methodological variation with the PET and MRP reconstruction method is of the same magnitude (coefficient of variation, 5–10%) as has been previously reported using the microsphere method (coefficient of variation, 8–10%) to quantitate blood flow (27, 28). Our estimate of the RDgs under resting conditions and during exercise (21–25%) in normal subjects is comparable with that previously reported for the lateral gastrocnemius and flexor digitorum muscle of the rabbit (21–24%) (2, 29). The finding that RDg both under resting conditions and during exercise is lower than RDf is also consistent with previous animal data (2, 29). Our estimate of RDf at rest (43% both in the normal subjects and the type 1 diabetic patients) is within the range of values reported in studies in which heterogeneity has been estimated in animals using microspheres (20, 29) and in our previous human studies (14, 16, 17, 18). The magnitude of relative dispersion of blood flow during exercise (29–30%) is similar to that previously observed in animal (30, 2) and human (18) studies.

The present methodology has some advantages, compared with some previous approaches. First, the method is suitable for use in humans, and second, it involves neither invasive manipulation of tissue nor its innervation, factors that do profoundly affect at least flow heterogeneity (32). Limitations of the applied method relate to resolution and pixel size. Considering the diameter of a capillary (5–7 µm) relative to the dimensions of a voxel (3.1 x 3.1 x 6.75 mm = 65 mm3), the values of flow and glucose uptake heterogeneity do not measure heterogeneity at the capillary level. Methods such as the 1-methylxanthine method, which is applicable only in rats (33) and the microbubble method (33) are possibly more accurate indicators of capillary flow, but these methods do not allow quantitation of heterogeneity of glucose uptake. Measurement of heterogeneity at the level of capillaries does not necessarily provide physiologically the most relevant information because skeletal muscle blood flow is controlled by microvascular units rather than individual capillaries (20). Our recent study using fractal analysis of blood flow also suggests that relative heterogeneity of blood flow observed in 65-mm3 voxels is preserved also in microvascular units (26).

In the present study, we found a rather close inverse association between the mean rate of glucose uptake and its relative heterogeneity. This relationship was evident especially during exercise but could also be discerned under insulin-stimulated conditions in the type 1 diabetic patients (Fig. 6Go). The greater heterogeneity of glucose uptake in the type 1 diabetic patients could therefore be attributed to their lower rate of glucose uptake. On the other hand, because relative heterogeneity is calculated by dividing SD by the mean, the role of mean glucose uptake in determining its relative heterogeneity remains uncertain. This criticism is equally valid for calculation of relative flow heterogeneity. Because the relationship between mean glucose uptake and relative heterogeneity appeared similar regardless of the conditions (hyperinsulinemia alone or combined with exercise, Fig. 6Go), spatial heterogeneity could represent a more general mechanism via which various stimuli increase glucose uptake in skeletal muscle.

We have previously observed colocalization of mean flow with mean insulin-stimulated glucose uptake in skeletal muscle (14). However, this result was based on quantitation of glucose uptake in three ROIs drawn on a cross-section of the entire femoral region rather than on pixel-by-pixel comparison of flow and glucose uptake within one region of interest (Fig. 6Go). Thus, the present analysis is based on quantitation of rates of flow and glucose uptake in more than 2000 pixels in one ROI rather than comparison of three mean values of three ROIs within one femoral cross-section. When analyzed within individual skeletal muscles, flow in 65 mm3 voxels seemed to be correlated with glucose uptake under exercise- and insulin-stimulated conditions but not during insulin-stimulated conditions alone. Whether lack of a correlation under resting conditions is related to insensitivity of the methods used or a small range of variation, compared with the exercise-stimulated state, is uncertain. Other investigators have shown doses of insulin, which do not change total flow, to increase significantly the amount of muscle tissue drained by the forearm vein and that the amount of tissue newly recruited by insulin is closely correlated with glucose uptake (21). In the perfused rat hindlimb, studies using laser Doppler flowmetry and methylxanthine technique to detect changes in capillary (nutritive) flow have also demonstrated insulin to increase glucose uptake and nutritive flow without altering total flow (35, 36, 37).

Skeletal muscle is a heterogeneous tissue and consists of fibers with different metabolic and contractile characteristics (38). In rats a single fiber type predominates in several muscles (39, 40), whereas in humans all muscles consist of mixtures of various fiber types (41, 42). The proportion of slow, oxidative type I fibers averages 40–70% in most human muscles (41). Type I fibers have a greater capacity for aerobic activity than fast-twitched, type II fibers because they have more mitochondria and higher concentrations of oxidative enzymes. Insulin resistance has been shown to correlate with reduced proportions of slow-twitch, oxidative fibers and increased proportions of fast-twitch, glycolytic fibers (43). Also, the concentration of the insulin-sensitive glucose transporter GLUT4 is higher in type I than type IIa or IIb fibers (44, 45). However, insulin-resistant patients with type 1 diabetes have not been found to have a different fiber type from normal subjects (46), which makes it unlikely that differences in fiber type were responsible for the observed differences in the heterogeneity of glucose uptake.

As shown in Figs. 3Go and 5Go, the type 1 diabetic patients were insulin resistant even though blood flow and relative flow heterogeneity were similar to those in normal subjects. This finding is in keeping with previous studies showing that chronic hyperglycemia causes insulin resistance in type 1 diabetic patients (47, 48, 49) and that the defect induced by chronic hyperglycemia is localized to glucose extraction rather than blood flow (50). Thus, even if flow and glucose uptake are coupled, glucose uptake can be impaired even when glucose delivery is normal.

In conclusion, our in vivo data demonstrate that skeletal muscle glucose uptake is heterogeneously distributed, and this heterogeneity is inversely proportional to mean glucose uptake. This implies that an increase in glucose uptake in human skeletal muscle is not a phenomenon, in which each unit increases its glucose uptake by a fixed amount but rather a spatially heterogeneous process.

Acknowledgments

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

Footnotes

This work was supported by grants from the Academy of Finland (to P.N. and H.Y.-J.), Novo Nordisk Foundation (to P.N. and H.Y.-J.), the Foundation for Diabetes Research (to P.P.), the Turku University Foundation (to P.P.), the Yrjö Jahnsson Foundation (to P.P.), the Aarne Koskelo Foundation (to P.P.), and the Research Foundation of Orion Corporation (to P.P.).

Abbreviations: LC, Lumped constant term; MRP, median root prior; PET, positron emission tomography; RDf , relative dispersion of blood flow; RDg, relative dispersion of glucose uptake; ROI, region of interest.

Received February 14, 2001.

Accepted July 23, 2001.

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