Minimal influence of blood flow on interstitial glucose and lactate-normal and insulin-resistant muscle

A. Holmäng1, M. Müller2, O. K. Andersson3, and P. Lönnroth3

1 The Wallenberg Laboratory, Department of Heart and Lung Diseases and 3 The Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Göteborg University, S-413 45 Gothenborg, Sweden; and 2 Department of Clinical Pharmacology, Vienna University Hospital, A-1090 Vienna, Austria

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To study the regulation of the interstitial glucose concentration in skeletal muscle, nine control subjects and nine older and overweight non-insulin-dependent diabetes mellitus (NIDDM) subjects with extreme insulin resistance were investigated with microdialysis in the medial femoral muscle before and during a euglycemic insulin clamp. After an overnight fast, arterial plasma glucose concentration was 4.9 ± 0.1 and 8.5 ± 0.6 mmol/l (P < 0.001), respectively. The arterial-interstitial concentration ([a-i]) differences of glucose and lactate were 0.43 ± 0.16 (P < 0.05) and -0.13 ± 0.05 mmol/l, respectively, in normal subjects. In NIDDM subjects, [a-i] differences for glucose and lactate were nonsignificant. Muscle blood flow was similar in controls and NIDDM subjects. During the glucose clamp, the glucose [a-i] differences increased and the lactate [a-i] differences decreased significantly in both groups. The glucose 170 infusion rate was 8.0 ± 0.77 vs. 3.2 ± 0.51 mg · kg-1 · min-1 (P < 0.001), and blood flow was 9.9 ± 1.6 vs. 6.7 ± 0.9 ml · 100 g-1 · min-1 (P < 0.05) in controls and NIDDM subjects, respectively. These results show that 1) the capillary wall is rate limiting for muscle glucose uptake and lactate release in control subjects but not in postabsorptive hyperglycemic insulin-resistant subjects, 2) vasodilation during insulin infusion does not prevent the increase in [a-i] difference of glucose in normal subjects, and 3) in severely insulin-resistant muscle, the [a-i] difference of glucose is not extended despite lack of vasodilation.

skeletal muscle; glucose metabolism; microdialysis; muscle blood flow

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

INSULIN RESISTANCE in the muscles may include the inability of insulin to stimulate muscle glucose uptake by the glucose transporter system as well as the oxidation and storage of glucose (10). Accordingly, regardless of the underlying cause, in vitro investigations have demonstrated multiple defects in muscle cells from insulin-resistant subjects, including perturbations of the insulin receptor signal (5) as well as the glucose transporter proteins (12) and the glycogen synthase activity (30). In addition to these cell-associated defects, increasing interest has recently been focused on the putative role of the blunted vasodilatory effect of insulin repeatedly demonstrated in peripheral tissue, i.e., skeletal muscle, in insulin resistant-subjects (3, 4, 15). A lacking vasodilatory effect of insulin could lead to decreased capillary delivery of insulin and glucose to the muscle cell. In favor of such a pathogenic mechanism, a number of studies have shown that the ability of insulin and glucose to induce vasodilation in skeletal muscles also correlates closely to the rate of glucose uptake (4, 15). In contrast to this theory, however, it has been shown that glucose uptake and vasodilation induced by insulin have different concentration-effect curves as well as time kinetics (32). Moreover, the extent to which vasodilation contributes to glucose uptake during pharmacological vasodilation (23, 31) or after ingestion of food (20) or glucose (21) seems to be only minor in normal healthy individuals. It may thus be concluded that the pathogenetic significance of the hampered insulin-mediated vasodilation found in insulin-resistant subjects is still not clear.

We recently employed the calibrated microdialysis technique (18) to investigate the interstitial muscle glucose concentration (22). In that study, it was demonstrated for the first time that the interstitial glucose concentration in muscle is less than that in arterial plasma after overnight fasting, indicating that the capillary wall is rate limiting for the muscle glucose uptake (22). The finding of a significant arterial-interstitial concentration ([a-i]) difference then makes the calculation of the extraction fraction of glucose in the muscle possible (17). When the interstitial muscle glucose concentration and the extraction fraction were studied in insulin-resistant Zucker rats, the interstitial fluid concentration of glucose was found to be higher than normal and the a-i concentration difference of glucose was diminished (13). To elucidate the relationship between glucose uptake and blood flow in insulin-resistant muscle further, we investigated a group of normal individuals and a group of older and overweight patients with non-insulin-dependent diabetes mellitus (NIDDM) having severe insulin resistance. The two groups were not age matched and, naturally, did not have identical fasting glucose levels. The two groups were studied separately, and the data from muscle microdialysis and blood flow measurement were compared qualitatively.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
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Subjects

Four male and five female subjects with NIDDM and four male and five female younger and nondiabetic subjects with normal glucose tolerance were studied. Table 1 lists the clinical characteristics of the subjects. Three of the nine subjects with NIDDM were treated with oral hypoglycemic agents, which were discontinued 24 h before the study. The NIDDM subjects did not show clinical signs of late diabetic complications and were normotensive. Glycosylated hemoglobin (HbA1c) was 7.1 ± 0.5% (means ± SE). The normal reference range for the current assay is 3.3-5.3%. All subjects gave their informed consent, and the study was approved by the Ethics Committee of Göteborg University.

                              
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Table 1.   Clinical characteristics of subjects

Study Protocol

The investigations were started at 8 AM after an overnight fast. The subjects were studied in the supine position in a room kept at 25°C. A polyethylene catheter was placed in a left forearm vein for blood sampling. The forearm was heated with electric pads (60-70°C) to arterialize the venous blood (17). Each study started with a calibration period of 180 min, followed by a euglycemic clamp performed as previously described by DeFronzo et al. (11). The clamp started with a primed infusion of insulin (Actrapid, Novo Nordisk, Copenhagen, Denmark) for 10 min followed by a constant infusion rate of 1 mU · kg-1 · min-1 for 120 min. Blood samples were drawn every 5 min. The rate of glucose infusion was adjusted to maintain the glucose concentration at euglycemia (5 mM). Potassium chloride (0.1 M) was infused at a rate of 10 mmol/h during the clamp to prevent hypokalemia.

Microdialysis

In the present experimental procedures, a commercially available custom-made microdialysis catheter (16 × 0.5 mm, 20 kDa molecular weight cutoff; CMA-10, CMA, Stockholm, Sweden) was used. The inlet of the microdialysis catheter was connected to a microinjection pump (CMA 100, CMA) and perfused at a flow rate of 2.0 µl/min. Isotonic saline with addition of 2.5 mmol/l glucose and 250 µmol/l lactate was used as the perfusion fluid. Two catheters were inserted without anesthetics into the ipsilateral quadriceps femoris ~10-15 cm above the knee joint by the following procedure: the surface of the disinfected skin was punctured vertically with a 20-gauge cannula. The steel mandrel was removed and the microdialysis catheter was inserted subsequently. After 30 min equilibration, the dialysate was collected at 15-min intervals. After calibration of the microdialysis catheters (see Calibration Procedure), the euglycemic clamp was started and the glucose and lactate levels were followed for 2 h.

Muscle blood flow to the calf was measured before and after 90 min of the euglycemic clamp on the contralateral leg using vein occlusion plethysmography (1).

Calibration Procedure

In vitro. Concentration-independent transfer rates for glucose and lactate were described in vitro in previous experiments (21). The mean in vitro recovery in the dialysate for the catheter type employed in the present study was ~60% for glucose and 81% for lactate (21).

In vivo. In vivo probe recovery was assessed in each probe according to the equilibrium (18) and internal reference calibration techniques (21, 19).

The equilibrium calibration was carried out as previously described in detail (18). Briefly, known concentrations of glucose and lactate were added to the perfusate in a nonconsecutive order, and the net increase of glucose and lactate concentration in the dialysate was measured. The linear relationship between perfusate glucose and increase in dialysate concentration was established, and the point of no net influx (indicating the interstitial glucose concentration) was estimated by means of linear regression. For internal reference calibration, 5 µCi/ml [3H]glucose and [14C]lactate were added to the perfusate, and the percentage loss over the membrane was taken as an estimate of recovery (21).

The experiments according to the equilibrium method revealed a mean in vivo recovery of 0.29 ± 0.02% (n = 18) for glucose and 0.39 ± 0.02% for lactate (n = 18). The corresponding mean recovery values obtained by means of internal reference calibration were 0.27 ± 0.03 and 0.41 ± 0.02% [not significant (NS)], respectively.

Analytical Methods

Glucose and lactate concentrations in plasma and in the dialysate fractions were determined enzymatically using 10-µl samples for simultaneous analyses of glucose and lactate in a YSI 2700 select biochemical analyzer (Yellow Springs Instruments, Yellow Springs, OH). Radioactivity was counted in a liquid scintillation counter using a quenched-corrected (external standards), double-isotope program (1217 Rackbeta, LKB, Uppsala, Sweden). Tissue samples were initially checked for quenching interferences using internal standards for 14C and 3H.

Statistics

All statistical calculations were made on absolute values. All results are expressed as means ± SE. Significance of difference was tested with Student's t-test for paired observations and, when applicable, with analysis of variance. Linear regression analyses of the in vivo recovery experiments were performed according to the least-square method.

    RESULTS
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Methods
Results
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Plasma Insulin and Rates of Total Glucose Disposal

The insulin values are shown in Table 2. The basal insulin values tended to be higher in the older NIDDM subjects (15.7 ± 4.8 µU/ml) than in controls (4.7 ± 0.5 µU/ml), but this difference was not significant (P < 0.06). The insulin values achieved during the glucose clamp did not differ significantly between the two groups. The glucose disposal rate in the NIDDM subjects was significantly lower than in controls (P < 0.001; Fig. 1).

                              
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Table 2.   Plasma insulin concentrations (µU/ml) during euglycemic-hyperinsulinemic clamp


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Fig. 1.   Total body glucose uptake during a hyperinsulinemic euglycemic clamp (1 mU · kg-1 · min-1) in control (open bars) and non-insulin-dependent diabetes mellitus (NIDDM) subjects (filled bars). * P < 0.01. Data are means ± SE [analysis of variance (ANOVA)].

Glucose Levels Before and During the Glucose Clamp

In postabsorptive control subjects, the glucose concentration in arterial plasma and in interstitial fluid was 4.9 ± 0.2 and 4.5 ± 0.2 mmol/l, respectively, and the a-i concentration difference was significant (0.43 ± 0.16 mmol/l, P < 0.05; Table 3). In NIDDM subjects, postabsorptive hyperglycemia prevailed. The fasting glucose level in arterial plasma and interstitial fluid was 8.9 ± 0.6 and 8.4 ± 0.9 mmol/l, respectively. In this subject group, five individuals had higher glucose concentrations in plasma compared with interstitial fluid, and the a-i glucose concentration difference was not significant (0.54 ± 0.73 mmol/l, NS). Ninety minutes after the initiation of the glucose clamp, the interstitial glucose concentrations in controls were still significantly lower (4.4 ± 0.2 mmol/l) compared with the plasma concentrations (5.6 ± 0.1 mmol/l; P < 0.001), and the NIDDM subjects now also had a lower interstitial glucose concentration than that in plasma (5.6 ± 0.2 vs. 4.5 ± 0.3 mmol/l; P < 0.05; Table 4). The [a-i] differences of glucose increased gradually after 90 min of glucose clamp from the fasting state to 1.22 ± 0.25 mmol/l (P < 0.05) in controls (Fig. 2A), and in the NIDDM subjects the a-i glucose concentration difference was significant: 1.11 ± 0.27 mmol/l (P < 0.05; Fig. 2B).

                              
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Table 3.   LBF measurements and a-i differences of glucose and lactate before (0 min) and after 90-min euglycemic glucose clamp in control subjects

                              
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Table 4.   LBF measurements and a-i differences of glucose and lactate before (0 min) and after 90-min euglycemic glucose clamp in NIDDM subjects


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Fig. 2.   Microdialysis in quadriceps femoris muscle during a euglycemic glucose clamp in control (A) and in NIDDM (B) subjects. Two dialysis catheters were placed ipsilaterally and perfused with isotonic saline with addition of 2.5 mmol/l glucose and 250 µmol/l lactate. Each catheter was calibrated separately in vivo for relative recovery of glucose and lactate. Mean interstitial glucose concentration (bullet ) was compared with that in arterial plasma (open circle ) at 0, 30, 60, 90, and 120 min. A: * P < 0.05, ** P < 0.001; B: * P < 0.05, ** P < 0.01. Data are means ± SE (ANOVA).

Lactate Levels Before and During the Glucose Clamp

The mean interstitial concentration of lactate in the postabsorptive state, estimated by regression analysis, was 0.73 ± 0.04 mmol/l for controls and 0.95 ± 0.06 mmol/l for NIDDM subjects. In controls, this concentration was significantly higher than that in arterial plasma (0.60 ± 0.03 mmol/l, P < 0.05). In the NIDDM subjects, plasma lactate was higher than in interstitial fluid (1.11 ± 0.27 mmol/l), but the concentration differences did not reach statistical significance (Table 3). After 90 min of the glucose clamp, the interstitial lactate concentrations increased to 1.38 ± 0.10 mmol/l compared with the plasma concentrations (1.13 ± 0.10 mmol/l; P < 0.05) in controls, whereas in the NIDDM subjects there was still no significant difference between plasma concentrations (1.09 ± 0.09 mmol/l) and the interstitial concentrations (1.15 ± 0.09 mmol/l; Table 4).

The [a-i] differences of lactate in the postabsorptive state were -0.13 ± 0.05 mmol/l, and, after 90 min of glucose ingestion (-0.25 ± 0.10 mmol/l) in controls (Fig. 3A) and in the NIDDM subjects, these differences decreased significantly from 0.17 ± 0.15 to -0.06 ± 0.12 mmol/l (P < 0.05; Fig. 3B).


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Fig. 3.   Microdialysis in quadriceps femoris muscle during a euglycemic glucose clamp in control (A) and in NIDDM (B) subjects. Two dialysis catheters were placed ipsilaterally and perfused with isotonic saline with addition of 2.5 mmol/l glucose and 250 µmol/l lactate. Mean interstitial lactate concentration (bullet ) was compared with that in arterial plasma (open circle ) at 0, 30, 60, 90, and 120 min. * P < 0.05, ** P < 0.01. Data are means ± SE (ANOVA).

Leg Blood Flow Measurements

In controls, mean leg blood flow was 6.4 ± 1.0 ml · 100 g-1 · min-1 in the postabsorptive state and increased to 9.9 ± 1.6 ml · 100 g-1 · min-1 90 min after the start of the glucose clamp (P < 0.05), whereas the increase in the NIDDM group from 6.0 ± 0.9 to 6.7 ± 0.9 ml · 100 g-1 · min-1 was not statistically significant (Tables 3 and 4).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present results clearly show that insulin-resistant muscle has qualitatively different interstitial glucose kinetic characteristics compared with muscle from normal insulin-sensitive subjects. The regulation of the interstitial glucose and lactate levels was different in the two subject groups both in the postabsorptive state and during insulin infusion. The results also suggest that the blunted vasodilatory effect of insulin in the older NIDDM subjects does not explain the markedly reduced glucose uptake during insulin clamping. The results do not permit conclusions as to the mechanism behind the insulin resistance or what mechanisms regulate insulin kinetics and action in NIDDM. The aim of the study was to compare qualitatively the kinetics of glucose and lactate in insulin-resistant muscle, regardless of the underlying mechanisms, with those in normals. Therefore, an extremely insulin-resistant subject group of NIDDM overweight patients were compared with younger lean controls.

Postabsorptive State

In the younger lean control group, it was confirmed (21) that the interstitial muscle glucose concentration was less than that in arterial plasma after an overnight fast. Given the blood flow, in this study estimated indirectly by assessment of leg blood flow, the extraction of glucose can be estimated by the formula: [a-i] · (1 - e-PS/Q), where PS is the permeability surface (set to ~5 ml · min-1 · 100 g-1 for glucose) and Q is the plasma flow rate (9, 17). Application of this formula would give an estimated basal glucose uptake in femoral muscle of ~3 µmol · min-1 · 100 g-1, which is in agreement with earlier results from microdialysis (21) or arteriovenous difference measurements in the leg (20). In analogy with the calculated glucose uptake rate, the lactate release from muscle cells can be estimated to ~0.4 µmol · min-l · g-1 in the normal subjects.

In insulin-resistant muscle, no significant a-i difference of glucose was demonstrated despite the fact that the leg blood flow rate in the fasting state was not different from that in normals. In these subjects, capillary delivery of glucose is thus not rate limiting for glucose uptake during fasting hyperglycemia. Glucose is taken up predominantly by passive diffusion in this situation, in agreement with the concept that non-insulin-dependent glucose uptake is increased during fasting hyperglycemia (2, 11). Furthermore, the finding that the capillary wall is not rate limiting for glucose delivery in these subjects convincingly demonstrates the pathogenic significance of an intracellular defect behind the insulin resistance. Thus, in fasting hyperglycemic NIDDM subjects, muscle blood flow does not regulate glucose uptake. In harmony with this, lactate diffuses passively over the capillary wall despite the increased plasma lactate concentration in these subjects. Because the a-i concentration difference of lactate was not significant, in contrast to what was registered in the normal subjects, the lactate release could not be estimated by microdialysis measurements. However, earlier studies have demonstrated an increased rate of nonoxidative glucose metabolism in fasting NIDDM subjects (7, 8). Thus, despite a putative increase of lactate formation by muscle cells in insulin-resistant subjects having a normal blood flow rate in the leg, lactate equilibrates over the capillary wall. The tissue lactate release is limited by cellular production in this situation and is not restricted by the endothelial barrier.

Insulin Infusion

In lean control subjects the insulin infusion caused a threefold increase in a-i glucose concentration difference and, concomitantly, leg blood flow increased ~50%. It may thus be concluded that the insulin-mediated increase in muscle blood flow does not compensate for the enhanced glucose utilization in muscle by preventing the decrease in interstitial glucose concentration. Again, because the increase in leg blood flow is representative of the vasodilation taking place in muscle (15), the glucose extraction can be assessed. It should be noted that increased blood flow could be mediated by both increased velocity of the capillary bloodstream and recruitment of new capillaries and that both these processes have been demonstrated after insulin (25). This means that the extraction of glucose will be enhanced by increase of both PS for glucose (~7 ml · min-1 · 100 g-1) (28) and Q, and the resulting muscle glucose uptake in the normal subjects would be ~9 µmol · min-1 · 100 g-1. The fractional extraction, however, would decrease from 0.80 to 0.75 in this situation. Given the 50% increase in blood flow, this alone would increase glucose extraction by ~45%, which would constitute ~15% of the overall increase in muscle glucose uptake during insulin infusion. Hence, the increase in muscle glucose uptake is mediated by the vasodilation only to a minor extent. This is also in agreement with recent results from microdialysis measurements in femoral muscle during an oral glucose tolerance test (21). Also, the i-a concentration difference for lactate increases during insulin infusion as a result of the enhanced glucose metabolism despite the concomitant vasodilation. It may thus be suggested that the capillary wall constitutes a functional diffusion barrier for glucose and lactate not only during fasting but also during insulin infusion in normal subjects. Moreover, insulin-mediated vasodilation does not overcome this barrier, and the enhanced diffusion is instead driven by the increased concentration difference over the capillary wall as a result of the enhanced cellular uptake and metabolism of glucose.

In NIDDM subjects, insulin infusion was introduced during fasting hyperglycemia and the glucose infusion was balanced to reach the same level as that received in the normal group. During normoglycemia, the NIDDM subjects appeared to be severely insulin resistant, as indicated by the low glucose infusion rate (60% lower than in the normal group) despite the considerably higher plasma insulin values. It is also notable that in insulin-resistant muscles insulin infusion induced a gradual increase in the glucose a-i concentration difference, which reached the same amplitude as in normals after 90 min. During the same period, no increase of leg blood flow was registered, confirming a blunted vasodilatory effect of insulin (4).

Interestingly, the late appearance of a significant a-i glucose concentration difference in NIDDM subjects indicates a delayed onset of the insulin effect, as recently discussed by others (26). Moreover, it implies a shift in a rate-limiting step(s) for muscle glucose uptake, in which the capillary wall becomes rate limiting as normoglycemia is reached (Fig. 2B). Also, this suggests that the glucose transport protein, which evidently is less rate limiting for glucose uptake during hyperglycemia (33), may become rate limiting again during normoglycemia maintained by insulin.

When the glucose uptake rate is assessed (~6 µmol · min-1 · 100 g-1) during normoglycemia in the insulin-resistant muscles, it appears that the reduction in assessed muscle glucose uptake corresponds to a significant reduction in glucose infusion rate (35 and 60% reduction, respectively). The severe reduction in insulin sensitivity and glucose uptake is further demonstrated by the i-a concentration difference for lactate, which did not reach statistical significance during the insulin infusion, indicating reduced conversion of glucose to lactate (Fig. 3B).

The present data also make it possible to evaluate the putative role of the lacking vasodilatory effect of insulin in the occurrence of insulin resistance in the subjects presently investigated. It might be argued that, because a-i glucose concentration differences were similar during insulin infusion in the two subject groups, the only factor that differentiated the insulin effect is the blood flow. However, the present and previous data (21) from normal individuals show that vasodilation induced by insulin and glucose is not critical for the increase in muscle glucose uptake. Furthermore, if limited access to glucose was the regulating factor behind the reduced glucose uptake in insulin-resistant subjects, the a-i concentration difference of glucose demonstrated here would have been increased and not nonexistent (fasting hyperglycemia) or normal (normoglycemic insulin clamp conditions). By contrast, it may be considered that, despite the low rate of glucose uptake in insulin-resistant muscle cells during insulin infusion, the a-i concentration difference of glucose appears to be normal, whereas muscle blood flow is reduced. We hypothesize that the a-i concentration difference is autoregulated in normal subjects by regulation of the blood flow through vasodilatory glucose metabolites such as lactate and CO2 (29) and that this "autoregulation" is diminished in severe insulin resistance.

Because reduction of cellular glucose uptake and not failing delivery of glucose is the factor determining the interstitial concentration, it may be presumed that pharmacological increase of the blood flow rate in insulin-resistant muscle would increase the interstitial glucose concentration and hence decrease the a-i concentration difference. Interestingly, we recently reported high interstitial glucose concentrations in normoglycemic insulin-resistant Zucker rats (13). In further support of the importance of cellular uptake of glucose for the interstitial concentration, we were recently able to demonstrate acute rapid reduction of muscular glucose uptake and concomitant increase of interstitial muscle glucose by beta -adrenergic stimulation in the rat (unpublished observation).

Although blood flow does not seem to regulate glucose uptake during steady-state insulin clamp conditions, it may be noted that the time course of onset of the insulin effect appeared to be severely delayed in the NIDDM subjects. It may be considered in this context that interstitial insulin levels are lower than in plasma (6) and that capillary delivery of insulin may thus be restricted and saturable (13, 27). A tentative reduction of muscle blood flow rate due to subnormal capillarization and vasodilatory response to insulin may thus delay the insulin effect. In support of this concept, we recently demonstrated a delayed distribution of insulin in femoral muscle of insulin-resistant testosterone-treated rats (24). These rats demonstrate a reduced muscular capillarization, which is normalized after long-term physical exercise (14). Physical exercise also normalizes the interstitial distribution time of insulin in these animals (24).

In summary, the capillary wall is rate limiting for glucose uptake and lactate release in normal muscle during euglycemic conditions but not in hyperglycemic elderly and overweight NIDDM subjects. The vasodilatory effect of insulin mediates only a small increase of glucose uptake in muscles, and a blunted vasodilation cannot explain the reduced glucose uptake. The putative role of vasodilation in the insulin distribution in the interstitial fluid must be further explored.

    ACKNOWLEDGEMENTS

The laboratory assistance provided by Lena Strindberg is gratefully acknowledged.

    FOOTNOTES

This study was supported by grants from the Swedish Research Council (project no. 10864, 11330, and 12206), the Inga-Britt and Arne Lundberg Foundation, the Swedish Diabetes Association, and the Nordisk Insulin Fond.

Address for reprint requests: A. Holmäng, Wallenberg Laboratory, Göteborg Univ., S-413 45 Göteborg, Sweden.

Received 14 July 1997; accepted in final form 12 November 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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