1 Lundberg Laboratory for Diabetes Research, Sahlgrenska University Hospital, Göteborg, Sweden
2 Wallenberg Laboratory, Sahlgrenska University Hospital, Göteborg, Sweden
3 Department of Radiology, Sahlgrenska University Hospital, Göteborg, Sweden
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ABSTRACT |
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INTRODUCTION |
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Furthermore, dynamics of glucose uptake correlate closely with the changes in interstitial insulin rather than with plasma insulin, suggesting that interstitial insulin levels determine the uptake rate of glucose (4,8). In subjects with obesity and type 2 diabetes, insulin-mediated glucose disposal and leg glucose uptake are delayed (9), suggesting that a delayed transcapillary transport of insulin could contribute to the insulin resistance in these subjects. Moreover, it has recently been demonstrated that insulin transport was markedly delayed in obese insulin-resistant animals (10,11).
In vitro studies in cultured cells have demonstrated that transendothelial insulin transport is saturable (12), which is compatible with a receptor-mediated transport process. Also, insulin receptors have been identified on endothelial cells (13). On the other hand, in vivo investigation in dogs demonstrated a reduced plasma/lymph gradient of insulin during an insulin infusion, indicating that diffusion, rather than a receptor-mediated saturable mechanism, regulates the transcapillary delivery of insulin (14). A nonsaturable transfer of insulin through the endothelium was indicated in another study in which the transport of insulin from the vascular to the interstitial fluid was shown to be concentration dependent (6). Further support for a nonsaturable process, such as diffusion as a mode of insulin transport over the capillary wall, has been demonstrated in a study in dogs in which the transport of an insulin analog with a low receptor affinity was not affected by the presence of insulin levels sufficiently high to saturate endothelial insulin receptors (15). The dynamics of infused insulin and inulin in human muscle interstitial fluid has, to our knowledge, not been explored. The aim of this study was 1) to investigate whether a delay in transcapillary insulin transport could contribute to the insulin resistance in obese subjects and 2) to compare the transcapillary transport of inulin and insulin. The study was therefore designed to measure the time of distribution of insulin and inulin from plasma to the interstitial fluid in human skeletal muscle in obese subjects and age-matched lean control subjects.
The time course of changes in plasma and muscle interstitial insulin and inulin was assessed by means of microdialysis during a constant infusion of insulin and inulin. Measurements of blood flow and arterial-venous (A-V) concentration gradients were conducted in parallel to evaluate capillary permeability.
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RESEARCH DESIGN AND METHODS |
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Three microdialysis catheters (CMA Microdialysis AB; CMA, Stockholm) were inserted at a 45° angle (through the steel mandarin of a 20-gauge cannula) into the right brachioradialis muscle. Control experiments in our laboratory, including computed tomography (CT) examinations or electromyogram, have shown that catheter position is satisfactorily ascertained by this procedure. For inulin and insulin measurements, two catheters with a dialysis membrane of 12 x 0.5 mm 100-kDA molecular cutoff were used, and for glucose, another catheter with 16 x 0.5 mm 20-kDa molecular cutoff was applied. The principle of muscle microdialysis has been described previously in detail (3,18, 19). The inlets of the catheters were connected to a microinjection pump (CMA 100). The perfusion fluid consisted of isotonic saline with addition of 1.5 mmol/l glucose and 1% albumin. The perfusion rate was 2.5 µl/min (glucose measurements) and 1.5 µl/min (in catheter measuring insulin and inulin). After 45 min equilibration, the dialysates for insulin, inulin, and glucose measurements were collected at 15-min intervals.
Calibration of the catheters for glucose measurements was performed using urea as an internal reference (20). In this study, the mean in vivo recovery obtained was 18 ± 1% for glucose. Measurements of interstitial insulin were taken according to the external reference calibration technique (3) at steady state. The relation between recovery of inulin and insulin in vitro was 1.7. The mean relative recovery of inulin (dialysate inulin/plasma inulin) in experiments performed in vivo was 6.7 ± 0.4%. The in vivo recovery of insulin was then calculated for each subject. The mean calculated in vivo recovery of insulin was 3.4 ± 0.2%. The in vivo recovery factor was used for recalculating steady-state dialysate insulin contents to interstitial insulin concentrations. Forearm blood flow was measured by venous occlusion pletysmography using a Whitney strain gauge (21).
CT was conducted to determine body composition. Examinations were made with a General Electric HiSpeed Advantage CT system (HSA, release RP2; GE Medical Systems, Milwaukee, WI) with the following settings: 120 kV, 5-mm slice thickness, and fixed filtration. One scan was obtained at the trunk, i.e., the fourth lumbar vertebra level (L4), and one scan at the forearm, i.e., the midpart of the m. brachioradialis level. The images were transferred to a separate UNIX-based analyzing unit. Tissue areas were determined as previously described (22), with the following precision errors calculated from double determinations: 0.5% subcutaneous adipose tissue, 1.2% visceral adipose tissue, 0.3% muscle tissue, and 3.4% bone tissue. The total adipose tissue and visceral adipose tissue volumes were calculated from predictive equations according to Kvist et al. (23). Estimated mass is obtained by multiplying tissue volumes by the density of adipose tissue, 0.923 g/cm3. Lean body mass can then be determined as body weight - adipose tissue (24).
Calculations
Insulin and glucose uptake rates.
Ficks principle was used to estimate the regional rate of glucose and insulin uptake. During steady state, the formula (A-V) · flow was applied.
Permeability surface area produc for insulin and glucose.
The permeability surface area product (PS) was derived from the following formula:
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where A is the arterial plasma concentration, V is the deep venous plasma concentration, I is the interstitial concentration, ln is the natural logarithm, and Q is the plasma flow.
Analytical methods.
Glucose and urea concentrations in plasma and in the dialysate fractions were determined with a colorimetric method (glucose) and a UV method (urea), on a CMA 600 microdialysis analyzer. Blood glucose during the clamp was analyzed enzymatically on a YSI 2700 select biochemical analyzer (Yellow Springs Instrument, Yellow Springs, OH). Plasma insulin was determined with an enzymatic immunoassay (Insulin ELISA; Mercodia, Uppsala, Sweden), and the concentration of insulin in the microdialysates was determined with an ultrasensitive essay (Mercodia Ultra sensitive Insulin ELISA). Inulin concentrations in plasma and dialysates were determined photometrically (25).
Statistics.
T1/2 and Tmax for the increase of dialysate insulin and inulin indicate the time to achieve half-maximal and maximal concentrations, respectively. In vitro, it takes 20 min to detect a stable change in dialysate levels of insulin and inulin after the concentration in the surrounding medium has been changed (data not shown). Thus, 20 min were subtracted when presenting T1/ 2 and Tmax of interstitial insulin and inulin, as shown in Table 2. Kinetic analyses were done individually, and the means of T1/2 and Tmax are presented. For calculation of interstitial concentrations, the mean of four samples collected during the last clamp hour was used. The results are expressed as means ± SE. Significance of difference was tested with Students t test for paired and unpaired observations. When data were nonparametrically distributed, the Mann-Whitney U test was applied for unpaired data. P < 0.05 was considered statistically significant. Simple regression analysis was also used, and the Stat-View program was applied.
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RESULTS |
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Inulin.
Steady-state concentrations of inulin in arterial plasma were 221 ± 16 and 215 ± 8 mg/l and in deep venous plasma were 222 ± 15 and 209 ± 8 mg/l in lean and obese subjects, respectively (NS). Arterial and deep venous levels of inulin for all subjects are shown in Fig. 3. The T1/2 of inulin in dialysate was significantly longer than in plasma (68 ± 5 vs. 34 ± 2 min; P < 0.01, n = 20) and was further extended in obese as compared with lean subjects (P < 0.05) (Table 2 and Fig. 5).
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Blood flow.
Forearm blood flow was lower in the obese group than in the control group, both in the postabsorptive state (1.6 ± 0.2 vs. 2.2 ± 0.2 ml · 100 g-1 · min-1, P < 0.05) and during steady-state hyperinsulinemia (1.9 ± 0.2 vs. 3.1 ± 0.3, P < 0.05). Blood flow rate increased significantly during the hyperinsulinemic-euglycemic clamp in lean versus obese subjects (P < 0.05) (Fig. 6).
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DISCUSSION |
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The main finding in this study, reported for the first time, is the marked delay of insulin delivery to the muscle interstitial fluid demonstrated in obese subjects. Earlier studies have shown a kinetic defect in the insulin-mediated activation of glucose uptake in obese subjects (9,27). Insulin-stimulated glucose uptake is the result of the insulin signal, including insulin receptor binding and activation of tyrosine kinase, propagation of the intracellular signal, and translocation and activation of glucose transporters. The relative contribution of a delayed transcapillary transport of insulin in addition to the insulin resistance produced by tentative defects in the insulin signaling system is not clear. Mostly, insulin sensitivity is measured at steady state, and a kinetic defect of insulin action is not taken into account. However, delays in insulin action, as produced by rightward shifts of the dose-effect curve of insulin and by slow transcapillary delivery of insulin, should both make important contributions to the postprandial hyperglycemia in insulin-resistant subjects.
In contrast to a present study by Mokshagundam et al. (28), no delay of transcapillary insulin transport in adipose tissue has been found in either obese or lean individuals. Apart from the fact that different tissues were studied, a possible explanation for the diverging results might be that Mokshagundam et al. administered a bolus infusion of insulin that raised the interstitial insulin more rapidly, whereas we used a continuous infusion. Nolan et al. (9) found no delay in the activation of insulin receptor tyrosine kinase in either obese or type 2 diabetic subjects. This would indicate that the distribution of insulin and binding to the receptor were not delayed and that the kinetic defect in insulin action was located posterior to the receptor. However, because the muscle biopsies for analyzing insulin receptor tyrosine kinase activation were taken in 30-min intervals, a kinetic defect in activation might have been difficult to detect. In contrast, delayed insulin transport was shown in perfused heart from insulin-resistant rats (10) as well as in vivo in rat muscle (5). In the latter study, exercise was shown to reverse the slow onset of insulin action in oophorectomized testosterone-treated rats. This finding is in harmony with another microdialysis study of muscle in insulin-resistant rats in which a slow insulin distribution in muscle was combined with a delayed onset of insulin action (11). Our present data demonstrate a delayed appearance of insulin to the muscle interstitial fluid in obese subjects. A reduced clearance of insulin in the skeletal muscle may also influence the appearance time of insulin in the muscle interstitial fluid. However, since insulin and the inert substance inulin are equally delayed in obese subjects, we do not believe the elimination of insulin is altered. Also, a change in distribution space would not affect time of appearance but rather the maximum concentration. Therefore, we suggest a delayed transcapillary transport of insulin in the obese group.
In the present study, we cannot rule out that the delay in GIR is affected by a decreased inhibition of hepatic glucose production (HGP) in the obese group. However, the rate of suppression of HGP is reported to be similar in lean and obese subjects during insulin infusion rates comparable with those used in the present study (27). Also, the delay in GIR activation is of the same magnitude and in the same time interval as the delay in increase of muscle insulin.
Thus, we suggest that the rightward shift of the insulin time-effect curve found in obesity might not only be explained by receptor or postreceptor defects and the resulting high EC50 (half-maximal effective concentration) values, but also by a delayed transport of insulin over the capillary wall.
The muscle interstitial insulin concentrations measured during steady-state hyperinsulinemia did not differ between obese and lean subjects. In a previous study of type 2 diabetic patients, we reported levels of muscle interstitial insulin similar to those in control subjects (29). Increased interstitial insulin levels were also reported in obese Zucker rats (30) and in lymph in obese humans (8). In addition, in this as well as a previous report (8), both insulin uptake and capillary permeability for insulin were normal in obese subjects. Therefore, it may be concluded that insulin levels in the interstitial fluid are normal at steady state in obese subjects and that the reduced GIR presently demonstrated at steady state is not due to failing capillary delivery of insulin.
Inulin is an inert water-soluble substance transported from plasma to peripheral tissue by passive diffusion and is eliminated through glomerular filtration. In this study, time of appearance of inulin in the muscle interstitial fluid was of the same magnitude as insulin. Because insulin and inulin have similar molecular weights, this indirectly supports the theory that insulin and inulin are transported by the same mechanism (diffusion) over the capillary wall. Also, in rat skeletal muscle time kinetics of insulin did not seem to differ markedly from those of inulin (31). In vitro, a transendothelial receptor-mediated pathway for insulin transport has been demonstrated. Labeled insulin can be transported through the endothelial cells, and this transport route can be inhibited by unlabeled insulin and by antibody against the insulin receptor (12). In contrast, in vivo studies of lymph indicate a nonsaturable non-receptor-mediated transport, such as diffusion (14). In muscle of lean rats, the plasma/interstitial concentration ratio increases in higher physiological concentration ranges that might be compatible with a saturable transport system (30). However, this was not evident in obese Zucker rats (30) or in human muscle (29). The present data, indicating that insulin and inulin are transported by the same mode of action (according to their similar kinetics in muscle microdialysates), further suggest that a non-receptor-mediated mechanism, such as diffusion, constitutes a major transport route of insulin.
The delay of transcapillary delivery of both inulin and insulin to the muscle interstitial fluid in obese subjects may depend on their having a lower capillary density (32) or a defective vasodilating response to insulin (33). A low capillary density is believed to constitute a mechanism behind insulin resistance in the muscle (3436). Furthermore, a longer diffusion distance between capillaries and muscle cells in insulin-resistant subjects has been suggested (34).
The blunted increase in forearm blood flow in the obese subjects during hyperinsulinemia in this study is consistent with earlier reports (33) that also demonstrate an impaired insulin-induced vasodilation in obesity.
Muscle perfusion correlates positively with insulin-mediated glucose uptake in muscle (33, 37,38). However, during an insulin infusion, the increase in blood flow appears later and is relatively lower than the increase of GIR, which is why a casual relationship between blood flow and glucose uptake may not be clear (39). The importance of this late vasodilatory effect of insulin relative to the activation of the insulin-mediated glucose uptake in the muscle has not been entirely defined, but earlier data have suggested that the insulin-mediated increase in blood flow accounts for 1530% of insulins overall action to stimulate muscle glucose uptake (19,40). Additionally, an increase in muscle perfusion seems to be more important for the insulin effect as insulin sensitivity and A-V difference of glucose increase (41).
It should be noted in this context, however, that in insulin-resistant muscle, blood flow is not rate limiting and vasodilation leads to increased interstitial glucose levels without increasing the glucose transport rate (42,43). The present study, in contrast, shows that insulin-induced vasodilation might be important for the time of onset of insulin action under non-steady-state conditions.
Estimation of the glucose uptake rate in forearm by application of Ficks principle may be hazardous when the blood flow is not stable (44). Therefore, kinetic analyses of forearm glucose uptake rates were not performed. As both activation of GIR and delivery of insulin to muscle interstitial fluid were delayed in the obese group, this delay should also be expected for muscle glucose uptake. In this context, it is important to note that forearm glucose uptake calculated by Ficks formula correlates closely with GIR under steady-state but not non-steady-state conditions. It is reasonable to believe that forearm muscle glucose uptake correlates with GIR, even under non-steady-state conditions, and that a lack of correlation might be due to the above difficulties involved with calculations when blood flow changes.
In this study, interstitial glucose was 35% lower than in plasma at steady-state hyperinsulinemia, confirming earlier reports (29,42). The estimated PS values were similar in obese and lean subjects during stable hyperinsulinemia. Nevertheless, a difference in PS between obese and lean subjects in the fasting state cannot be excluded. However, it seems unlikely because forearm blood flow did not change dramatically in either group after insulin infusion.
The HGP is expected to be shut off at the insulin levels presently used (45), which is why we believe that the GIRs observed in this study mainly represent muscle glucose uptake. This proposal was further supported by the finding that GIR and muscle glucose uptake were correlated.
Obese humans may have an increased first phase of insulin secretion (46), presumably to compensate for peripheral insulin resistance. This increase could also serve to compensate for a tentatively delayed delivery of insulin to the target tissue. In dogs, the interstitial insulin level increased more quickly after a biphasic insulin infusion than after a continuous infusion (47). In patients with early stages of type 2 diabetes, first-phase insulin release is lost despite the concomitant enhancement of second-phase secretion, which may lead to high postprandial glucose levels (48,49). It is possible that a delayed peripheral insulin action in type 2 diabetic subjects could further prolong the hyperglycemia after meals. Epidemiological studies have indicated that plasma glucose levels 2 h after a glucose load are strongly associated with all-cause and cardiovascular relative mortality risk (50).
In summary, the present data show for the first time that obese humans have a delayed distribution of insulin-to-muscle interstitial fluid as well as a slower activation of the glucose uptake rate. These data suggest that a delayed delivery of insulin may contribute to muscle insulin resistance in obesity.
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ACKNOWLEDGMENTS |
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The authors are grateful to Margareta Landén for technical assistance, Eva Bergelin and Lena Strid for help with CT examinations, and Gerd Nilsen for secretarial aid.
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FOOTNOTES |
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Received for publication 13 February 2002 and accepted in revised form 23 May 2002.
A-V, arterial-venous; CT, computed tomography; GIR, glucose infusion rate; HGP, hepatic glucose production; PS, permeability surface area product; T1/2, time to achieve half-maximal concentration; Tmax, time to achieve maximal concentration.
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REFERENCES |
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