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Lundberg Laboratory for Diabetes Research (S.G., M.S., L.S., P.L.), Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden; and Department of Clinical Physiology (J.W.), Karolinska Hospital, S-17176 Stockholm, Sweden
Address all correspondence and requests for reprints to: Soffia Gudbjörnsdóttir, Lundberg Laboratory for Diabetes Research, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail: soffia. gudbjornsdottir{at}medic.gu.se.
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Abstract |
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Intramuscular microdialysis in combination with the forearm model and blood flow measurements was performed in healthy males, studied during an oral glucose tolerance test or during a one-step or two-step euglycemic hyperinsulinemic clamp.
PS for glucose increased significantly from 0.29 ± 0.1 to 0.64 ± 0.2 ml/min·100 g after oral glucose tolerance test, and glucose uptake increased from 1.2 ± 0.4 to 2.6 ± 0.6 µmol/min·100 g (P < 0.05). During one-step hyperinsulinemic clamp (plasma insulin, 1.962 pmol/liter), PS for glucose increased from 0.2 ± 0.1 to 2.3 ± 0.9 ml/min·100 g (P < 0.05), and glucose uptake increased from 0.6 ± 0.2 to 5.0 ± 1.4 µmol/min·100 g (P < 0.05). During the two-step clamp (plasma insulin, 1380 ± 408 and 3846 ± 348 pmol/liter), the arterial-interstitial difference and PS for insulin were constant. The PS for glucose tended to increase (P = not significant), whereas skeletal muscle blood flow increased from 4.4 ± 0.7 to 6.2 ± 0.8 ml/min·100 ml (P < 0.05).
The present data show that PS for glucose is markedly increased by oral glucose, whereas a further vasodilation exerted by high insulin concentrations may not be physiologically relevant for capillary delivery of either glucose or insulin in resting muscle.
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Introduction |
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High plasma concentrations of insulin lead to vasodilation and recruitment of capillaries in skeletal muscle (12, 13, 14, 15). A small vasodilatory effect exerted by physiological insulin concentrations is reported in some but not all studies (16), and the importance of the vasodilatory effect of insulin has been debated (17). Furthermore, the vasodilation registered as an increase in blood flow exerted by insulin occurs later than does the effect of insulin on glucose uptake, and also, the dose dependency is different (17). On the other hand, indirect measurements of capillary recruitment as a phenomenon have yielded data indicating that insulin may indeed stimulate capillary recruitment and that this may occur before, and independently of, any increase in the blood flow rate (18).
To more precisely assess the importance of the vasodilatory effect of insulin, direct measurements of the permeability surface (PS) area of insulin and glucose should be carried out. We have previously (8) shown that PS area can be calculated by forearm arteriovenous cannulation in combination with microdialysis and blood flow measurements, according to Renkin (19). Evidence for the fact that the microdialysis approach enables high-precision estimates of the glucose uptake rate was given in a recent study including comparative data obtained concomitantly by means of positron emission tomography assessment of [18F]deoxyglucose uptake in human skeletal muscle (20).
Combining the forearm model with im microdialysis and blood flow measurements provides, for the first time, a unique opportunity to directly estimate PS area for glucose and insulin. In the present study, the effect of oral glucose and high insulin concentrations on PS was studied. Due to the poor permeability and low concentrations of insulin in muscle during physiological conditions, PS of insulin was measured only at high plasma levels. The aim of the study was to obtain relevant and direct data on the capillary permeability and to test the hypothesis that this is regulated under physiological conditions and, if so, whether it is related to changes in blood flow.
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Subjects and Methods |
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Twenty-eight subjects were investigated in three different studies, as described below. The clinical characteristics of the subjects are given in Table 1
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Study 2. To investigate the effect of insulin on PS for glucose, we studied 10 healthy, nonsmoking male volunteers (age, 40 ± 4 yr; BMI, 24 ± 0.8 kg/m2) on no regular medication, applying a one-step euglycemic hyperinsulinemic clamp after an overnight fast. Some of the subjects in study 2 delivered some additional data that were reported in another study from our laboratory (8).
Study 3. To further study the effect of insulin and blood flow on PS for glucose as well as for insulin, we used a two-step euglycemic hyperinsulinemic clamp in 10 healthy, nonsmoking male volunteers (age, 26.5 ± 5 yr; BMI, 23 ± 2.8 kg/m2) with no regular medication.
The subjects were recruited through advertising in a local newspaper. All subjects gave their informed consent, and the study was approved by the Ethics Committee of Göteborg University.
Study protocol
The investigations were started at 0800 h after an overnight fast. The subjects were studied in the supine position in a room kept at 25 C. Under local anesthesia, catheters were inserted into the right antecubital vein for inulin, glucose, and insulin infusions, into a deep vein in the left antecubital fossa, and into the left radial artery for blood sampling. Forearm blood flow was measured every 30 min in the right arm by venous occlusion plethysmography using Whitney strain gauge. The hand blood circulation was occluded by inflation to 200 mm Hg of a cuff at the wrist. In study 1, an OGTT was then performed. A constant iv insulin infusion (infusion rate, 24 ml/h) was given for 360 min to achieve steady-state plasma inulin levels (studies 2 and 3) (21). In study 3, a bolus injection of inulin (Inutest, Kemiflor, Stockholm, Sweden) was given before the infusion. A euglycemic clamp, as previously described by DeFronzo et al. (22), was started 30 min after initiation of the inulin infusion. In study 2, the clamp protocol was modified to exclude any prime infusion of insulin (insulin infusion rate, 120 mU x m2 x min-1). In study 3, the clamp was started with a primed infusion of insulin (Actrapid, Novo Nordisk, Copenhagen, Denmark) for 10 min, followed by a constant infusion rate of 120 mU/m2·min for 120 min. Thereafter, the insulin infusion rate was increased to 240 mU/m2·min and continued for another 120 min. Blood samples for glucose analyses were drawn every 5 min. In both studies 2 and 3, the rate of glucose infusion was adjusted to maintain the blood glucose concentration at about 5.5 mM/liter. Potassium chloride (0.1 M) was infused at a rate of 10 mmol/h during the clamp to prevent hypokalemia.
Microdialysis catheters were inserted without anesthetics into the left brachioradialis muscle using the following procedure: the surface of the disinfected skin was punctured vertically with a 20-gauge cannula. The steel mandrin was removed, and the microdialysis catheter was inserted. Two commercially available custom-made microdialysis catheters (16 x 0.5 mm, 20 kDa molecular mass cutoff, CMA-10; and 12 x 0.5 mm, 100 kDa cutoff, CMA-10; CMA Microdialysis, Stockholm, Sweden) were used. The inlet of the microdialysis catheter was connected to a microinjection pump (CMA 100, CMA Microdialysis) and perfused with isotonic saline with addition of 1.5 mmol/liter glucose. For insulin and inulin measurements, 1% human albumin was added. The flow rate was 2.5 µl/min in catheters for glucose measurements, 1.5 µl/min in study 2, and 1 µl/min in study 3 (in the catheters measuring insulin and inulin). Microdialysates were collected at 30-min intervals.
To characterize the different diffusion properties and to correct for differences in binding of the compounds to the catheters, two different calibration techniques were used. For calibration of the catheters measuring glucose, the internal reference technique was applied, and for catheters measuring insulin, the external reference technique was used. The latter technique includes experiments in which inulin and insulin are dialyzed in plasma at 37 C in vitro. The relation between recovery of inulin and insulin in vitro was 2. The mean inulin recovery (dialysate inulin/plasma inulin) in experiments performed in vivo was 6.7 ± 0.4% in study 2 and 11 ± 3% in study 3. The relation between recovery of inulin and insulin in vitro should be identical to that estimated in vivo (23). The in vivo recovery of insulin was then calculated in each subject according to the formula R1 = R2, where R1 is recovery of insulin/recovery of inulin in vitro and R2 is recovery of insulin/recovery of inulin in vivo.
The mean calculated in vivo recovery of insulin was 3.4 ± 0.2% in study 2 and 5.0 ± 0.1% in study 3. The in vivo recovery factor was used for recalculating steady-state dialysate insulin content to interstitial insulin concentrations.
PS product area was estimated according to the equation for estimating the extraction fraction: V - A = (I - A) x (1 - e -PS/Q), where V is the venous plasma concentration, I is the interstitial concentration, A is the arterial plasma concentration, PS is the permeability surface product area, and Q is the plasma flow rate (24, 25).
Uptake was estimated by Fick’s principle: Insulin uptake = plasma flow x A - V (plasma concentrations) and glucose uptake = blood flow x A - V (blood concentrations).
Analytical methods
Glucose concentrations in plasma were determined enzymatically using 10-µl samples on a YSI 2700 select biochemical analyzer (Yellow Springs Instrument Co., Inc., Yellow Springs, OH) in study 3 and on a CMA 600 analyzer (CMA Microdialysis) in studies 1 and 2.
Plasma insulin was measured with a double antibody RIA (Pharmacia, Uppsala, Sweden) in studies 1 and 3, and with an enzymatic immunoassay (Mercodia, Insulin ELISA, Uppsala, Sweden) in study 2. Concentration of insulin in microdialysates was determined with two enzymatic immunoassays (Mercodia, Insulin ELISA, study 2; and Dako Diagnostics Ltd., Cambridgeshire, UK, study 3). Inulin concentrations in plasma and in the dialysate fractions were determined photometrically, according to the micromethod described by Waugh (26).
Statistics
The results are expressed as means ± SE of the mean (SEM). Significance was tested with Student’s t test and, for nonparametric data, with Wilcoxon’s signed-rank test for paired observations.
Statistical calculations of mean insulin and glucose levels as well as blood flow were made on absolute values obtained during the last 60 min at every clamp step (steady-state). Plasma and dialysate inulin values were from the end of each experiment to ensure steady-state conditions.
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Results |
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)During the OGTT in healthy subjects after one night’s fasting, arterial plasma glucose was 5.4 ± 0.1, 9.5 ± 0.5, and 6.9 ± 0.3 mmol/liter at 0, 90, and 150 min, respectively, and arterial insulin was 30 ± 2.4, 270 ± 30, and 162 ± 18 pmol/liter at 0, 90, and 150 min, respectively. The AI difference for glucose increased significantly from 2.6 ± 0.3 mmol/liter at 0 min to 4.7 ± 0.6 mmol/liter at 90 min. The PS for glucose after the overnight fast was 0.29 ± 0.08 ml/min·100 g and increased significantly to 0.64 ± 0.2 ml/min·100 g (P < 0.05) at 120 min during the OGTT. Glucose uptake was increased from 1.2 ± 0.4 µmol/min·100 g at 0 min to 2.6 ± 0.6 µmol/min·100 g at 150 min (P < 0.05). Due to low concentrations, it was not possible to measure interstitial insulin. Forearm skeletal muscle blood flow was unchanged during the OGTT [1.8 ± 0.2 vs. 1.9 ± 0.2 ml/min·100 ml; P = not significant (ns)].
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)After an overnight fast, arterial insulin in healthy subjects was 47 ± 7 pmol/liter; at steady state, during the one-step clamp, arterial insulin was 2270 ± 174 pmol/liter. Fasting plasma glucose was 5.4 ± 0.1 mmol/liter and at steady state, during the clamp, arterial plasma glucose was 6.4 mmol/liter. The AI glucose difference did not change significantly (1.9 vs. 2.3 mmol/liter at 0 and 240 min, respectively). The PS for glucose did increase significantly, from 0.2 ± 0.1 to 2.3 ± 0.9 ml/min·100 g (P < 0.05), and glucose uptake increased from 0.6 ± 0.2 to 5.0 ± 1.4 µmol/min·100 g (P < 0.05).
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and Fig. 1)
During the two-step clamp, plasma insulin levels during steady state were 1597 ± 478 and 3979 ± 402 pmol/liter at an insulin infusion rate of 120 mU/min and 240 mU/min, respectively (P < 0.0001), and plasma glucose was 6.02 ± 0.1 and 6.1 ± 0.2 mmol/liter (ns) [blood glucose, 5.5 ± 0.14 and 5.8 ± 0.13 mmol/liter (ns)]. The glucose infusion rate (GIR) at steady state was 11.2 ± 0.5 and 13.1 ± 0.8 at insulin infusion 120 mU/min and 240 mU/min, respectively (P < 0.0001). The AI difference for insulin was constant during the two different clamp steps, namely 46% vs. 53% (ns), as can be seen in Fig. 1. The PS for insulin was the same during the two different clamp steps [0.6 ± 0.2 and 0.4 ± 0.1 ml/min·100 g (ns)], and PS for glucose was 2.2 ± 0.8 and 4.5 ± 1.6 ml/min·100 g (ns) at the insulin infusion rate of 120 mU/min and 240 mU/min, respectively. Glucose uptake was 8.1 ± 2.1 and 13.5 ± 4.4 µmol/min·100 g at an insulin infusion of 120 mU/min and 240 mU/min, respectively (ns). Skeletal muscle blood flow increased from 4.4 ± 0.7 ml/min·100 ml to 6.2 ± 0.8 ml/min·100 ml at an insulin infusion of 120 mU/min and 240 mU/min, respectively (P < 0.05).
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Discussion |
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Previous measurements of PS for glucose or substances of a similar molecular size as glucose have been performed indirectly in human skin (27) and in perfused limbs in experimental animals (19, 28, 29). The results from such studies have in general shown PS values in the range of 1.7–6.0 ml/min·100 g. The present data on PS for glucose obtained during insulin stimulation are confirmatory of previous results. By contrast, the PS for glucose presented here in the postabsorptive state in resting muscle appears to be only 0.2–0.3 ml/min·100 ml, indicating that PS assessed in a more physiological situation may differ from PS during nonphysiological conditions, such as maximal vasodilation. In support for this concept, the PS for lactate and glycerol appeared to be approximately 1 ml/min·100 g in the perfused original fat pad in dogs during rest (29). Direct measurements of PS for glucose in the human skeletal muscle under the conditions prevailing in the present study have, to our knowledge, not been reported previously in the literature.
A significant 2-fold increase in the PS for glucose was registered after the OGTT. As previously observed, the AI of glucose increased despite the increased tissue perfusion (30). It may, therefore, be concluded that the capillary recruitment exerted by an oral glucose load does not fully counterbalance the increased elimination rate of glucose in the skeletal muscle. The importance of the small but significant increase in PS area was, however, evident with use of the formula for calculating the extraction fraction of glucose (see Study protocol). At an elimination rate of 3.1 ± 0.8 µmol/min·100 g at 90 min (Table 2
), a tentative maintenance of PS at 0.3 ml/min·100 g would result in total deprival of interstitial glucose. It may, therefore, be concluded that the 2-fold increase in PS is essential for the glucose uptake after an OGTT.
It should also be noted that the calculations for glucose uptake ought to be done during steady state, which is usually not the case during an OGTT. In this study, the blood flow was constant throughout the OGTT, and the arterial, venous, and interstitial concentrations of glucose did not change more than 15% at 60–120 min why it may be considered reasonable to estimate the glucose uptake.
In the present study 2, the PS for glucose was measured during an insulin infusion. The data show a marked increase in PS for glucose and a concomitant increase in forearm blood flow. The PS value of approximately 2 ml/min·100 g obtained after 240 min of insulin infusion is comparable to the value previously achieved in human skin (27) and in perfused animal hind limbs (19, 29). Moreover, the parallel increase in PS and blood flow confirms earlier observations in skeletal muscle investigations (31). Consequently, the data suggest that capillary recruitment and the resulting increase in PS may be related to the vasodilatory effect of insulin. The small but significant increase in PS after OGTT in the present study was, however, observed without any concomitant change in forearm blood flow. It should be noted here that the relationship between PS and blood flow previously reported was observed in perfused muscle with dilated arteries and high blood flow rates (31). The data previously reported on the relationship between PS for small hydrophilic compounds and blood flow show a positive and direct (i.e. linear) correlation with blood flow rates considerably higher than those presently investigated (31). However, the relationship between low ranges of PS and blood flow has not been thoroughly investigated. Therefore, it may be suggested that the increase in PS for glucose presently observed was not paralleled by an increase in skeletal muscle blood flow, or else that a tentative increase in blood flow was beyond the limit of detection. However, the present data do not give direct support to the recent hypothesis that capillary recruitment exerted by insulin is not related to any increase in blood flow (18), because small changes in blood flow may have been overlooked due to the variability in the plethysmographic method. All together, the present finding of nonparallelity in changes of PS vs. flow when measured postabsorptively suggests marked heterogeneity of flow under these conditions.
To measure capillary permeability of both glucose and insulin, high rates of insulin infusion were used in a two-step fashion in the present study 3. Insulin is markedly less permeable than glucose, and to obtain detectable insulin concentrations in microdialysates, plasma insulin was kept unphysiologically high at two different levels. The PS value for insulin estimated in our study 3 was similar to previous data on substances of similar molecular size, reporting on vasodilated skeletal muscle in the rat (19, 29), and to values previously described for inulin (32). At clamp step 2, a significant increase in forearm blood flow was observed in the absence of significant changes in PS for either glucose or insulin (Table 4). It should be noted that the relationship between PS and blood flow is nonlinear when the blood flow is high, because the capillary permeability is less dependent on the blood flow rate (31). Furthermore, substances of a larger molecular size, such as insulin, show only minute changes in capillary permeability when the blood flow is altered (31). Therefore, it should be anticipated that PS for insulin in skeletal muscle is kept at approximately 0.5 ml/min·100 g in vasodilated beds and during two different unphysiologically high plasma levels of insulin, even if blood flow changes.
The present data do not allow us to speculate on the permeability of insulin during more physiological conditions. It is unclear how much influence permeability after OGTT has on the insulin effect because the elimination rate of insulin could not be calculated during the OGTT. However, a time lag in the insulin effect recently observed in insulin-resistant subjects (8) would be the result of a tentative lack of capillary recruitment after oral glucose intake. The importance of capillary delivery of insulin to the interstitial fluid for the time of onset of insulin action was also reported in that study (8).
In general, insulin-resistant obese and/or type II diabetes subjects do not show any vasodilatory effect of insulin (33, 34), and the insulin effect on peripheral glucose uptake correlates well with the skeletal muscle blood flow (35). However, it has been shown that in insulin-resistant muscle, capillary delivery of glucose does not fail when the elimination rate of glucose from the interstitial fluid is impaired (30). In this situation, the insulin resistance in the muscle cells is rate limiting for the glucose uptake and the interstitial fluid concentration of glucose is normal or even increased (8, 36). At steady-state conditions, capillary delivery of insulin is sufficient in insulin-resistant muscle (36), but when plasma insulin increases, the following increase in interstitial insulin as well as the time of onset of insulin action is slow (8, 37). In this context, it should be emphasized that the present data, which show that insulin regulates the PS and capillary delivery of glucose, were obtained in healthy subjects. The present results do not imply that a failing vasodilatory effect of insulin in insulin-resistant subjects may be an underlying mechanism of the insulin resistance per se.
It is also important to note that the present data were obtained in resting muscle and that they may not be comparable to those obtained during muscle contraction. Previous investigations have shown that passive contraction results in high blood flow rates and diminished AI differences of glucose but not of insulin (38).
The present data show that PS area product for glucose is lower than previously anticipated in human muscle during fasting. An increase was seen after oral glucose intake, contributing significantly to the increased glucose uptake. By contrast, the marked further increase exerted by high insulin concentrations may not be physiologically relevant in resting muscle. Previous investigations have suggested that glucose metabolism itself may increase peripheral blood flow, theoretically through enhancement of lactate production in the muscle (39). Furthermore, it has been demonstrated that insulin-mediated activation of nitric oxide exerts vasodilation in muscle (40). Because OGTT was more effective in increasing the PS than was insulin alone, the present data suggest that vasodilatory effects of glucose metabolism and lactate production (39) may be more important than those of insulin and nitric oxide (40) under physiological conditions.
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Footnotes |
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Abbreviations: AI, Arterial-interstitial; BMI, body mass index; GIR, glucose infusion rate; ns, not significant; OGTT, oral glucose tolerance test; PS, permeability surface.
Received March 12, 2003.
Accepted July 1, 2003.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, and interstitial 
glucose (A) and insulin (B) during a two-step clamp (study 3). Mean ± seam.