Delayed Transcapillary Delivery of Insulin to Muscle Interstitial Fluid After Oral Glucose Load in Obese Subjects
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糖尿病学杂志 2005年第1期
the Lundberg Laboratory for Diabetes Research, University Hospital, Gteborg, Sweden
ABSTRACT
Obese subjects exhibit a delay in insulin action and delivery of insulin to muscle interstitial fluid during glucose/insulin infusion. The aim of the present study was to follow the distribution of insulin to skeletal muscle after an oral glucose load in obese subjects. We conducted an oral glucose tolerance test (OGTT) in 10 lean and 10 obese subjects (BMI 23 ± 0.6 vs. 33 ± 1.2 kg/m2; P < 0.001). Insulin measurements in muscle interstitial fluid were combined with forearm arteriovenous catheterization and blood flow measurements. In the obese group, interstitial insulin was significantly (35eC55%) lower than plasma insulin (P < 0.05) during the 1st h after the OGTT, whereas in lean subjects, no significant difference was found between interstitial and plasma insulin levels during the same time period. The permeability surface area product for glucose, representing capillary recruitment, increased in the lean group (P < 0.05) but not in the obese group (NS). Obese subjects had a significantly higher plasma insulin level at 90eC120 min after oral glucose (398 ± 57 vs. 224 ± 37 pmol/l in control subjects; P < 0.05). The significant gradient between plasma insulin and muscle interstitial insulin during the first hour after OGTT suggests a slow delivery of insulin in obese subjects. The hindered transcapillary transport of insulin may be attributable to a defect in insulin-mediated capillary recruitment.
Insulin has to traverse the capillary endothelium to reach the target cell in peripheral tissue. At steady-state insulin infusion, interstitial insulin is 40% lower than plasma insulin (1,2). Insulin-resistant obese subjects, however, have supranormal interstitial insulin levels as a result of the insulin resistance in the target cell (3).
In vivo studies have shown that insulin action or the glucose disposal rate lags after an increase in plasma insulin (4,5). As well, during insulin infusion, most studies, but not all (6), have shown that interstitial insulin lags after plasma insulin (1,4,7), thereby demonstrating that the endothelial wall may delay the transcapillary transport of insulin. Furthermore, the dynamics of glucose uptake correlate closely with changes in interstitial insulin rather than plasma insulin, suggesting that interstitial insulin levels determine the uptake rate of glucose (3,8). A further delay in insulin action has been demonstrated in insulin-resistant obese and type 2 diabetic subjects (9,10), 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 is markedly delayed in obese, insulin-resistant animals (11,12) as well as in obese humans (13) during insulin/glucose infusion. It has been suggested that the slow rate of insulin delivery in insulin-resistant states might be due to reduced capillary density (14) or capillary recruitment (15,16) or the diminished vasodilative response of insulin (17,18). This study was designed to study the distribution of insulin from plasma to the interstitial fluid in skeletal muscle in obese subjects after a physiological increase of insulin during an oral glucose load.
The time course of changes in plasma and muscle interstitial insulin was assessed by means of microdialysis during an oral glucose tolerance test (OGTT). Measurements of blood flow and arteriovenous concentration gradients were conducted in parallel to evaluate the permeability surface area product (PS) and glucose uptake rates.
RESEARCH DESIGN AND METHODS
The study subjects were 10 obese and 10 lean control subjects, none of whom were taking any regular medication. Subjects’ clinical characteristics are listed in Table 1. All subjects gave informed consent, and the study was approved by the ethical committee of the University of Gteborg.
The investigations started at 8:00 A.M. after an overnight fast in a room kept at 25°C. The subjects were investigated in a semisupine position. Glucose (75 mg) was dissolved in 250 ml of chilled lemon-flavored water. Measurements were performed after the OGTT.
Three microdialysis catheters (CMA, Microdialysis AB, Stockholm, Sweden) were inserted through the steel mandarin of a 20-gauge cannula into the brachioradialis muscle in the forearm. For insulin and inulin measurements, two catheters with a 12 x 0.5 mm dialysis membrane and a 100-kDA molecular cutoff were used, and for glucose and lactate measurements, a 16 x 0.5 mm catheter and 20-kDA molecular cutoff was applied. Microdialysate sampling for insulin, glucose, and lactate measurements was done at 15-min intervals for 30 min in the postabsorptive state and continued after the start of the OGTT for 120 min, in parallel with forearm blood flow measurements and arterial and deep venous blood samples for insulin, glucose, and lactate. Each sample was stored at eC18°C before analysis. The principle of muscle microdialysis has been previously described in detail (2,19). The inlets of the catheters were connected to a microinjection pump (CMA 100; CMA). The perfusion fluid consisted of isotonic saline to which was added 2.0 mmol/l glucose, 0.25 mmol/l lactate, and 0.5 mmol/l urea in catheters applied for glucose and lactate measurements and 1.5 mmol/l glucose and 1% albumin in catheters used for insulin and inulin measurements. The perfusion rate was 2.5 e蘬/min (in catheters measuring glucose and lactate) and 1.5 e蘬/min (in catheters measuring insulin and inulin). After a 45-min equilibration period, the dialysates for glucose, lactate, and insulin measurements were collected at 15-min intervals. Inulin was infused at a constant rate (24 ml/h) for 300 min, and dialysates for inulin were collected during the last 60 min. The study protocol is shown in Fig. 1.
Measurements of interstitial insulin were taken according to the external reference calibration technique (2). This technique is premised on the fact that the relation between the relative recoveries of two substances in vitro is the same as the relation between the recoveries for the same two substances in vivo. Inulin was used as a reference substance, as its molecular size is similar to that of insulin. The relation between the recovery of inulin and that of insulin in vitro was 2. The mean relative recovery of inulin (dialysate inulin/plasma inulin) in vivo was 6.1 ± 0.4%. The in vivo recovery of insulin was calculated for each subject; the mean calculated in vivo recovery of insulin was 3.0 ± 0.4%. The in vivo recovery factor was used for recalculating the ratio of steady-state dialysate insulin content to the interstitial insulin concentration. Urea was used as the endogenous reference when calculating the recovery of glucose and lactate (20).
Forearm blood flow was measured by venous occlusion plethysmography using a Whitney strain gauge (21).
Calculations.
The PS was estimated using the method for estimating the extraction fraction: V eC A = (I eC A) x (1 eC eeCPS/Q), where V is the venous plasma concentration, A is the arterial plasma concentration, I is the interstitial concentration, e is the base of the natural logarithm (e 2.71828), and Q is the plasma flow rate (22).
Glucose and insulin uptake were estimated using Fick’s principle: insulin uptake = plasma flow x A eC V (plasma concentrations), and glucose uptake = blood flow x A eC V (blood concentrations).
Analytic methods.
Glucose, lactate, and urea concentrations in the dialysate and plasma fractions were determined with a colorimetric method (glucose and lactate) and an ultraviolet method (urea) on a CMA 600 microdialysis analyzer (CMA Microdialysis). Plasma insulin was determined with an enzymatic immunoassay (Insulin ELISA; Mercodia, Uppsala, Sweden) and the concentration of insulin in the microdialysates with an ultrasensitive essay (Ultra-Sensitive Insulin ELISA; Mercodia; coefficient of variation 5eC6%). Inulin concentrations in plasma and dialysates were determined photometrically (23).
Statistics.
Results are expressed as means ± SE. The significance of differences was tested with Student’s t test and ANOVA, when appropriate. When data were nonparametrically distributed (as for insulin), the Mann-Whitney U test was applied for unpaired data; when comparing nonparametric data from different compartments (arterial, venous, and interstitial muscle), Wilcoxon’s signed rank test for paired observations was used. P < 0.05 was considered statistically significant. The StatView program for personal computers was applied.
In vitro, it takes 15eC20 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, 15 min were subtracted when presenting the time curves for interstitial insulin (Fig. 2A and B).
RESULTS
Insulin.
In the obese group, interstitial insulin was significantly lower than plasma insulin at 15, 30, 45, and 60 min after the OGTT (Fig. 2B), whereas in lean subjects, no significant difference between interstitial and plasma insulin levels was found during the first hour after the oral glucose load (Fig. 2A). The ratio of interstitial to plasma insulin was therefore higher in lean compared with obese subjects 60 min after glucose ingestion (Fig. 2A and B). At the end of the OGTT (90eC120 min), the interstitial insulin was 60% of the plasma insulin level in both lean (P < 0.05) and obese (P < 0.05) subjects (Fig. 2A and B). Thus, at the last part of the OGTT, the interstitial to plasma insulin ratio was similar in the two groups.
The obese subjects had a significantly higher plasma insulin level at 90eC120 min after oral glucose (398 ± 57 vs. 224 ± 37 pmol/l in control subjects; P < 0.05) (Fig. 3). In addition, interstitial insulin was higher in obese subjects (230 ± 35 vs. 146 ± 21 in lean control subjects; P < 0.05) at 90eC120 min. Insulin uptake rates in both groups are presented in Fig. 4. The insulin uptake increased significantly (P < 0.05) in both groups compared with basal values: 96 ± 27 in the obese group and 41 ± 6.5 fmol · 100 geC1 · mineC1 in the lean group (NS) at 60 min.
Glucose.
Arterial plasma glucose in the fasting state was 5.1 ± 0.1 vs. 5.2 ± 0.1 mmol/l, and venous glucose was 5.0 ± 0.1 vs. 5.1 ± 0.1 mmol/l in lean and obese subjects, respectively (NS) (Table 2). During OGTT, the arterial glucose level was 7.6 ± 0.7 vs. 8.3 ± 0.7 (NS) at 120 min, and venous glucose was 6.8 ± 0.4 vs. 7.8 ± 0.8 (NS) in lean and obese subjects, respectively. In the fasting state, interstitial glucose was 3.4 ± 0.4 vs. 4.1 ± 0.3 (NS) in lean and obese subjects, and at 120 min, it was 5.5 ± 0.6 vs. 6.3 ± 0.6 (NS), respectively. Glucose uptake increased significantly in both groups during the 1st h after oral glucose administration (Table 2).
Blood flow.
Forearm blood flow was similar in the lean and obese groups in both the postabsorptive state (1.6 ± 0.1 vs. 1.7 ± 0.2 ml · 100 geC1 · mineC1; NS) and at 90eC120 min (1.9 ± 0.2 vs. 1.6 ± 0.2 ml · 100 geC1 · mineC1; NS). Blood flow had a tendency to increase during the OGTT in lean subjects (P = 0.07), but not in obese ones.
DISCUSSION
This study demonstrated that obese subjects exhibit a significant gradient between plasma insulin and muscle interstitial insulin during the 1st h after an oral glucose load that was not found in lean control subjects. It is suggested that this significant gradient demonstrates a delayed transcapillary transport of insulin in obese subjects. Thus, the ratio of interstitial to plasma insulin was lower in obese than in lean subjects at the beginning of the OGTT. However, by the end of the OGTT, the interstitialeCtoeCplasma insulin ratio was similar in the two groups (60%), as was previously demonstrated (2,5).
Most earlier studies have investigated insulin action at steady-state insulin levels, and cellular studies from skeletal muscle have shown that intracellular insulin signaling is defective in obese subjects (24). However, another aspect of insulin resistance is a delay in insulin action. Previously, it has been reported that the time of onset of insulin action is delayed in obese subjects during insulin/glucose infusion (10,13). A delayed transcapillary insulin transport in insulin-resistant states has also been reported from in vitro studies (11) and some (1,12,25,26), but not all (6,27), in vivo studies. In a recent study from our laboratory (13), the delay in insulin action was accompanied by a slow delivery of insulin to the muscle interstitial fluid during insulin/glucose infusion in the obese state. However, the distribution of insulin from blood to muscle tissue during more physiological conditions, represented by an OGTT in the present study, has not previously been reported in obese subjects.
During high insulin levels in steady-state conditions, capillary delivery of insulin is sufficient in insulin-resistant muscle (2,3,28). In this study, interstitial muscle insulin was even higher in obese than in lean subjects at the end of the OGTT, confirming earlier data of interstitial insulin levels in obese rats (28) and humans (3) at the end of insulin infusion. The insulin resistance associated with obesity that is observed after prolonged insulin stimulation thus does not seem to be caused by a deficient transcapillary delivery of insulin and glucose but rather by cellular insulin resistance. However, during noneCsteady-state conditions, exemplified by an OGTT in the present study, a hindered insulin distribution is observed in obese, insulin-resistant subjects. The reason for the sluggish insulin distribution may be decreased capillary density, blood flow, capillary recruitment, or permeability in the muscle tissue of obese subjects. Capillary density is negatively correlated with obesity (14); further, it is proposed that low capillary density would prolong the distance along which insulin must be diffused and thus delay insulin action (14).
The issue of whether insulin-mediated vasodilation can modulate glucose uptake has been previously discussed. Muscle blood flow correlates positively with insulin-mediated glucose uptake during insulin infusion (17,29,30). However, the increase in glucose uptake is relatively higher and appears earlier than the increase in blood flow, which would suggest that increased blood flow is not the source of the augmented glucose uptake during the clamp procedure (31). Instead it has been suggested that an increased glucose uptake may stimulate blood flow. Supporting this view are the results of two studies in which insulin administered together with glucose locally in the forearm induced an increase in blood flow (32), whereas insulin infused with a metabolically inactive isomer of glucose did not (33). In a clamp study with lean, obese, and type 2 diabetic subjects, all three groups exhibited similar blood flow when a comparable glucose disposal rate was achieved in all groups (34).
In the present study, no clear difference in blood flow rate was detected between obese and lean subjects during the OGTT. In accordance with the above-mentioned studies (32,34), the higher insulin and glucose levels in the obese group may have compensated for the conjectured diminished blood flow in the obese group. As well, the physiological insulin levels achieved during the OGTT may not have been high enough to increase total limb flow.
Recent work by Vincent et al. (35) has suggested that physiological insulin levels can mediate capillary recruitment, even in the absence of a total blood flow increment. The increased capillary recruitment (35) may be of importance for the muscle glucose uptake. In addition, the PS for glucose was increased in healthy humans during an OGTT without a concomitant increase in blood flow (36). One interesting finding has been that in insulin-resistant states, insulin-mediated capillary recruitment (15,37) seems to be impaired. In the current study, there was a significant difference in interstitial versus plasma insulin in the first hour after the glucose load in the obese group. It may be speculated that decreased capillary recruitment in the obese group impaired the delivery of insulin in the beginning of the OGTT.
In the present study, the PS for glucose increased 1 h after glucose ingestion in the lean but not in the obese group, which may suggest a diminished capillary recruitment in obese subjects. It should be noted that the formula for determining the PS is not equivalent to measurements of capillary recruitment but rather a sum product of vasodilation/vasoconstriction and permeability over the tissue bed. However, a change in capillary recruitment influences the PS value. In addition, calculations of PS (which assumes homogeneity) may be weakened by the presence of heterogeneity of blood flow distribution in muscle (38). It may be assumed that this heterogeneity exists in both lean and obese subjects. Hence, it is reasonable to believe that the change in PS that is found in lean but not in obese subjects corresponds to a difference in change of permeability; that is, the capillary permeability area. Insulin, as well as inulin and glucose, pass through the capillary wall by means of passive diffusion; hence, the blunted increase in PS for glucose found in obese subjects may be relevant for the sluggish increase in interstitial insulin in these subjects.
The present data confirm the well-known observation that obese subjects have a higher insulin response after oral glucose, (39). Similarly, a brisk rise in plasma insulin level with an average peak at 30 min has been demonstrated in nondiabetic subjects in all weight classes (39).
Obese subjects have a high first-phase insulin response (40), which may be an attempt to compensate for the cellular insulin resistance in obesity (24). It has also been shown that a two-phase insulin infusion increases interstitial insulin levels more rapidly than a continuous insulin infusion, suggesting that a loss of the first insulin response may delay transcapillary delivery of insulin (41). In prediabetic states, the first insulin response is diminished (42), which may further deter the distribution of insulin to peripheral tissues. One previous interesting finding is that pulsatile insulin infusion in healthy lean individuals enhances glucose uptake in peripheral tissues more than a continuous insulin infusion (43).
In this study, the insulin response after OGTT showed considered variability in both groups (data not shown), and not all subjects had a biphasic insulin response. This finding concurs with earlier reports on a wide plasma insulin response after OGTT in healthy individuals (44). In the current study, the insulin uptake rate seemed to be delayed in the obese group, even though direct evidence for this cannot be provided from the present data as the variable (often two-phase) plasma insulin response after oral glucose does not allow calculations of the time to reach half-maximal concentrations. Instead, we suggest that the delayed insulin distribution in obese subjects was demonstrated by the significant concentration gradient between plasma and muscle interstitial insulin seen in the beginning of the OGTT. In the lean group, on the other hand, no significant gradient was found between plasma and interstitial insulin in the beginning of the OGTT, suggesting no major hindrance of insulin transport over the capillary wall. The presence of a minor gradient cannot be excluded in this relatively small group of lean subjects. However, the main finding is the major gradient found in the obese subjects as the result of a significant group difference. The present data agree with those from another recent microdialysis study of transcapillary insulin transport in muscle in lean subjects, where a significant gradient between plasma and interstitial insulin was observed at the peak but not in the beginning of the OGTT (45).
In summary, it appears that obese subjects need a higher plasma insulin response than lean subjects to achieve a similar time curve for muscle interstitial insulin. Also, an increased interstitial muscle insulin level at the end of the OGTT may be of pathogenetic importance for insulin resistance in muscle, as high ambient insulin levels may further desensitize the cells to insulin (46).
ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Research Council (project nos. 10864, 11330, and 12206), the Swedish Diabetes Association, Nordisk Insulinfond, Novo Nordisk Pharma AB, the Inga-Britt and Arne Lundberg Foundation, and Gteborgs Lkarsllskap.
We are grateful to Sren Sndergaard, Helena Odenstedt, and Margareta Landeen for their technical assistance and to Gerd Nilsen for administrative support.
OGTT, oral glucose tolerance test; PS, permeability surface area product
REFERENCES
Jansson PA, Fowelin JP, von Schenck HP, Smith UP, Lonnroth PN: Measurement by microdialysis of the insulin concentration in subcutaneous interstitial fluid: importance of the endothelial barrier for insulin. Diabetes42 :1469 eC1473,1993
Sjostrand M, Holmang A, Lonnroth P: Measurement of interstitial insulin in human muscle. Am J Physiol276 :E151 eCE154,1999
Castillo C, Bogardus C, Bergman R, Thuillez P, Lillioja S: Interstitial insulin concentrations determine glucose uptake rates but not insulin resistance in lean and obese men. J Clin Invest93 :10 eC16,1994
Yang YJ, Hope ID, Ader M, Bergman RN: Insulin transport across capillaries is rate limiting for insulin action in dogs. J Clin Invest84 :1620 eC1628,1989
Bergman RN, Yang YJ, Hope ID, Ader M: The role of the transcapillary insulin transport in the efficiency of insulin action: studies with glucose clamps and the minimal model. Horm Metab Res Suppl24 :49 eC56,1990
Mokshagundam SP, Peiris AN, Stagner JI, Gingerich RL, Samols E: Interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals. Metabolism45 :951 eC956,1996
Niklasson M, Holmang A, Lonnroth P: Induction of rat muscle insulin resistance by epinephrine is accompanied by increased interstitial glucose and lactate concentrations. Diabetologia41 :1467 eC1473,1998
Yang YJ, Hope ID, Ader M, Bergman RN: Importance of transcapillary insulin transport to dynamics of insulin action after intravenous glucose [erratum in Am J Physiol 173:Sect. E after table of contents, 1997]. Am J Physiol266 :E17 eCE25,1994
Prager R, Wallace P, Olefsky JM: In vivo kinetics of insulin action on peripheral glucose disposal and hepatic glucose output in normal and obese subjects. J Clin Invest78 :472 eC481,1986
Nolan JJ, Ludvik B, Baloga J, Reichart D, Olefsky JM: Mechanisms of the kinetic defect in insulin action in obesity and NIDDM. Diabetes46 :994 eC1000,1997
Wascher TC, Wolkart G, Russell JC, Brunner F: Delayed insulin transport across endothelium in insulin-resistant JCR:LA-cp rats. Diabetes49 :803 eC809,2000
Holmang A, Niklasson M, Rippe B, Lonnroth P: Insulin insensitivity and delayed transcapillary delivery of insulin in oophorectomized rats treated with testosterone. Acta Physiol Scand171 :427 eC438,2001
Sjostrand M, Gudbjornsdottir S, Holmang A, Lonn L, Strindberg L, Lonnroth P: Delayed transcapillary transport of insulin to muscle interstitial fluid in obese subjects. Diabetes51 :2742 eC2748,2002
Lillioja S, Young AA, Culter CL, Ivy JL, Abbott WG, Zawadzki JK, Yki-Jarvinen H, Christin L, Secomb TW, Bogardus C: Skeletal muscle capillary density and fiber type are possible determinants of in vivo insulin resistance in man. J Clin Invest80 :415 eC424,1987
Clerk LH, Rattigan S, Clark MG: Lipid infusion impairs physiologic insulin-mediated capillary recruitment and muscle glucose uptake in vivo. Diabetes51 :1138 eC1145,2002
Wallis MG, Wheatley CM, Rattigan S, Barrett EJ, Clark AD, Clark MG: Insulin-mediated hemodynamic changes are impaired in muscle of Zucker obese rats. Diabetes51 :3492 eC3498,2002
Laakso M, Edelman SV, Brechtel G, Baron AD: Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man: a novel mechanism for insulin resistance. J Clin Invest85 :1844 eC1852,1990
Laakso M, Edelman SV, Brechtel G, Baron AD: Impaired insulin-mediated skeletal muscle blood flow in patients with NIDDM. Diabetes41 :1076 eC1083,1992
Lonnroth P, Jansson PA, Smith U: A microdialysis method allowing characterization of intercellular water space in humans. Am J Physiol253 :E228 eCE231,1987
Strindberg L, Lonnroth P: Validation of an endogenous reference technique for the calibration of microdialysis catheters. Scand J Clin Lab Invest60 :205 eC211,2000
Siggaard-Andersen J: Venous occlusion plethysmography on the calf: evaluation of diagnosis and results in vascular surgery. Dan Med Bull17 (Suppl. I) :1 eC68,1970
Paaske WP: Capillary permeability in skeletal muscle. Acta Physiol Scand101 :1 eC14,1977
Waugh WH: Photometry of inulin and polyfructosan by use of a cysteine/tryptophan reaction. Clin Chem23 :639 eC645,1977
Goodyear LJ, Giorgino F, Sherman LA, Carey J, Smith RJ, Dohm GL: Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. J Clin Invest95 :2195 eC2204,1995
Niklasson M, Daneryd P, Lonnroth P, Holmang A: Effects of exercise on insulin distribution and action in testosterone-treated oophorectomized female rats. J Appl Physiol88 :2116 eC2122,2000
Miles PD, Li S, Hart M, Romeo O, Cheng J, Cohen A, Raafat K, Moossa AR, Olefsky JM: Mechanisms of insulin resistance in experimental hyperinsulinemic dogs. J Clin Invest101 :202 eC211,1998
Cline GW, Petersen KF, Krssak M, Shen J, Hundal RS, Trajanoski Z, Inzucchi S, Dresner A, Rothman DL, Shulman GI: Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N Engl J Med341 :240 eC246,1999
Holmang A, Mimura K, Bjorntorp P, Lonnroth P: Interstitial muscle insulin and glucose levels in normal and insulin-resistant Zucker rats. Diabetes46 :1799 eC1804,1997
Laakso M, Edelman SV, Olefsky JM, Brechtel G, Wallace P, Baron AD: Kinetics of in vivo muscle insulin-mediated glucose uptake in human obesity. Diabetes39 :965 eC974,1990
Baron AD, Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G: Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J Clin Invest96 :786 eC792,1995
Yki-Jarvinen H, Utriainen T: Insulin-induced vasodilatation: physiology or pharmacology Diabetologia41 :369 eC379,1998
Dela F, Larsen JJ, Mikines KJ, Galbo H: Normal effect of insulin to stimulate leg blood flow in NIDDM. Diabetes44 :221 eC226,1995
Ueda S, Petrie JR, Cleland SJ, Elliott HL, Connell JM: The vasodilating effect of insulin is dependent on local glucose uptake: a double blind, placebo-controlled study. J Clin Endocrinol Metab83 :2126 eC2131,1998
Mather KJ, Mirzamohammadi B, Lteif A, Steinberg HO, Baron AD: Endothelin contributes to basal vascular tone and endothelial dysfunction in human obesity and type 2 diabetes. Diabetes51 :3517 eC3523,2002
Vincent MA, Dawson D, Clark AD, Lindner JR, Rattigan S, Clark MG, Barrett EJ: Skeletal muscle microvascular recruitment by physiological hyperinsulinemia precedes increases in total blood flow. Diabetes51 :42 eC48,2002
Gudbjornsdottir S, Sjostrand M, Strindberg L, Wahren J, Lonnroth P: Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle. J Clin Endocrinol Metab88 :4559 eC4564,2003
Youd JM, Rattigan S, Clark MG: Acute impairment of insulin-mediated capillary recruitment and glucose uptake in rat skeletal muscle in vivo by TNF-. Diabetes49 :1904 eC1909,2000
Kalliokoski KK, Laaksonen MS, Knuuti J, Nuutila P: Perfusion distribution between and within muscles during intermittent static exercise in endurance-trained and untrained men. Int J Sports Med24 :400 eC403,2003
Yalow RS, Glick SM, Roth J, Berson SA: Plasma insulin and growth hormone levels in obesity and diabetes. Ann N Y Acad Sci131 :357 eC373,1965
Beard JC, Ward WK, Halter JB, Wallum BJ, Porte D Jr: Relationship of islet function to insulin action in human obesity. J Clin Endocrinol Metab65 :59 eC64,1987
Getty L, Hamilton-Wessler M, Ader M, Dea MK, Bergman RN: Biphasic insulin secretion during intravenous glucose tolerance test promotes optimal interstitial insulin profile. Diabetes47 :1941 eC1947,1998
Gautier JF, Wilson C, Weyer C, Mott D, Knowler WC, Cavaghan M, Polonsky KS, Bogardus C, Pratley RE: Low acute insulin secretory responses in adult offspring of people with early onset type 2 diabetes. Diabetes50 :1828 eC1833,2001
Schmitz O, Arnfred J, Nielsen OH, Beck-Nielsen H, Orskov H: Glucose uptake and pulsatile insulin infusion: euglycaemic clamp and [3eC3H]glucose studies in healthy subjects. Acta Endocrinol (Copenh)113 :559 eC563,1986
Yalow RS, Rose HG, Bauman WA: Hyperinsulinemia. Am J Med85 :22 eC30,1988
Herkner H, Klein N, Joukhadar C, Lackner E, Langenberger H, Frossard M, Bieglmayer C, Wagner O, Roden M, Muller M: Transcapillary insulin transfer in human skeletal muscle. Eur J Clin Invest33 :141 eC146,2003
Garvey WT, Olefsky JM, Marshall S: Insulin receptor down-regulation is linked to an insulin-induced postreceptor defect in the glucose transport system in rat adipocytes. J Clin Invest76 :22 eC30,1985(Mikaela Sjstrand, Soffia )
ABSTRACT
Obese subjects exhibit a delay in insulin action and delivery of insulin to muscle interstitial fluid during glucose/insulin infusion. The aim of the present study was to follow the distribution of insulin to skeletal muscle after an oral glucose load in obese subjects. We conducted an oral glucose tolerance test (OGTT) in 10 lean and 10 obese subjects (BMI 23 ± 0.6 vs. 33 ± 1.2 kg/m2; P < 0.001). Insulin measurements in muscle interstitial fluid were combined with forearm arteriovenous catheterization and blood flow measurements. In the obese group, interstitial insulin was significantly (35eC55%) lower than plasma insulin (P < 0.05) during the 1st h after the OGTT, whereas in lean subjects, no significant difference was found between interstitial and plasma insulin levels during the same time period. The permeability surface area product for glucose, representing capillary recruitment, increased in the lean group (P < 0.05) but not in the obese group (NS). Obese subjects had a significantly higher plasma insulin level at 90eC120 min after oral glucose (398 ± 57 vs. 224 ± 37 pmol/l in control subjects; P < 0.05). The significant gradient between plasma insulin and muscle interstitial insulin during the first hour after OGTT suggests a slow delivery of insulin in obese subjects. The hindered transcapillary transport of insulin may be attributable to a defect in insulin-mediated capillary recruitment.
Insulin has to traverse the capillary endothelium to reach the target cell in peripheral tissue. At steady-state insulin infusion, interstitial insulin is 40% lower than plasma insulin (1,2). Insulin-resistant obese subjects, however, have supranormal interstitial insulin levels as a result of the insulin resistance in the target cell (3).
In vivo studies have shown that insulin action or the glucose disposal rate lags after an increase in plasma insulin (4,5). As well, during insulin infusion, most studies, but not all (6), have shown that interstitial insulin lags after plasma insulin (1,4,7), thereby demonstrating that the endothelial wall may delay the transcapillary transport of insulin. Furthermore, the dynamics of glucose uptake correlate closely with changes in interstitial insulin rather than plasma insulin, suggesting that interstitial insulin levels determine the uptake rate of glucose (3,8). A further delay in insulin action has been demonstrated in insulin-resistant obese and type 2 diabetic subjects (9,10), 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 is markedly delayed in obese, insulin-resistant animals (11,12) as well as in obese humans (13) during insulin/glucose infusion. It has been suggested that the slow rate of insulin delivery in insulin-resistant states might be due to reduced capillary density (14) or capillary recruitment (15,16) or the diminished vasodilative response of insulin (17,18). This study was designed to study the distribution of insulin from plasma to the interstitial fluid in skeletal muscle in obese subjects after a physiological increase of insulin during an oral glucose load.
The time course of changes in plasma and muscle interstitial insulin was assessed by means of microdialysis during an oral glucose tolerance test (OGTT). Measurements of blood flow and arteriovenous concentration gradients were conducted in parallel to evaluate the permeability surface area product (PS) and glucose uptake rates.
RESEARCH DESIGN AND METHODS
The study subjects were 10 obese and 10 lean control subjects, none of whom were taking any regular medication. Subjects’ clinical characteristics are listed in Table 1. All subjects gave informed consent, and the study was approved by the ethical committee of the University of Gteborg.
The investigations started at 8:00 A.M. after an overnight fast in a room kept at 25°C. The subjects were investigated in a semisupine position. Glucose (75 mg) was dissolved in 250 ml of chilled lemon-flavored water. Measurements were performed after the OGTT.
Three microdialysis catheters (CMA, Microdialysis AB, Stockholm, Sweden) were inserted through the steel mandarin of a 20-gauge cannula into the brachioradialis muscle in the forearm. For insulin and inulin measurements, two catheters with a 12 x 0.5 mm dialysis membrane and a 100-kDA molecular cutoff were used, and for glucose and lactate measurements, a 16 x 0.5 mm catheter and 20-kDA molecular cutoff was applied. Microdialysate sampling for insulin, glucose, and lactate measurements was done at 15-min intervals for 30 min in the postabsorptive state and continued after the start of the OGTT for 120 min, in parallel with forearm blood flow measurements and arterial and deep venous blood samples for insulin, glucose, and lactate. Each sample was stored at eC18°C before analysis. The principle of muscle microdialysis has been previously described in detail (2,19). The inlets of the catheters were connected to a microinjection pump (CMA 100; CMA). The perfusion fluid consisted of isotonic saline to which was added 2.0 mmol/l glucose, 0.25 mmol/l lactate, and 0.5 mmol/l urea in catheters applied for glucose and lactate measurements and 1.5 mmol/l glucose and 1% albumin in catheters used for insulin and inulin measurements. The perfusion rate was 2.5 e蘬/min (in catheters measuring glucose and lactate) and 1.5 e蘬/min (in catheters measuring insulin and inulin). After a 45-min equilibration period, the dialysates for glucose, lactate, and insulin measurements were collected at 15-min intervals. Inulin was infused at a constant rate (24 ml/h) for 300 min, and dialysates for inulin were collected during the last 60 min. The study protocol is shown in Fig. 1.
Measurements of interstitial insulin were taken according to the external reference calibration technique (2). This technique is premised on the fact that the relation between the relative recoveries of two substances in vitro is the same as the relation between the recoveries for the same two substances in vivo. Inulin was used as a reference substance, as its molecular size is similar to that of insulin. The relation between the recovery of inulin and that of insulin in vitro was 2. The mean relative recovery of inulin (dialysate inulin/plasma inulin) in vivo was 6.1 ± 0.4%. The in vivo recovery of insulin was calculated for each subject; the mean calculated in vivo recovery of insulin was 3.0 ± 0.4%. The in vivo recovery factor was used for recalculating the ratio of steady-state dialysate insulin content to the interstitial insulin concentration. Urea was used as the endogenous reference when calculating the recovery of glucose and lactate (20).
Forearm blood flow was measured by venous occlusion plethysmography using a Whitney strain gauge (21).
Calculations.
The PS was estimated using the method for estimating the extraction fraction: V eC A = (I eC A) x (1 eC eeCPS/Q), where V is the venous plasma concentration, A is the arterial plasma concentration, I is the interstitial concentration, e is the base of the natural logarithm (e 2.71828), and Q is the plasma flow rate (22).
Glucose and insulin uptake were estimated using Fick’s principle: insulin uptake = plasma flow x A eC V (plasma concentrations), and glucose uptake = blood flow x A eC V (blood concentrations).
Analytic methods.
Glucose, lactate, and urea concentrations in the dialysate and plasma fractions were determined with a colorimetric method (glucose and lactate) and an ultraviolet method (urea) on a CMA 600 microdialysis analyzer (CMA Microdialysis). Plasma insulin was determined with an enzymatic immunoassay (Insulin ELISA; Mercodia, Uppsala, Sweden) and the concentration of insulin in the microdialysates with an ultrasensitive essay (Ultra-Sensitive Insulin ELISA; Mercodia; coefficient of variation 5eC6%). Inulin concentrations in plasma and dialysates were determined photometrically (23).
Statistics.
Results are expressed as means ± SE. The significance of differences was tested with Student’s t test and ANOVA, when appropriate. When data were nonparametrically distributed (as for insulin), the Mann-Whitney U test was applied for unpaired data; when comparing nonparametric data from different compartments (arterial, venous, and interstitial muscle), Wilcoxon’s signed rank test for paired observations was used. P < 0.05 was considered statistically significant. The StatView program for personal computers was applied.
In vitro, it takes 15eC20 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, 15 min were subtracted when presenting the time curves for interstitial insulin (Fig. 2A and B).
RESULTS
Insulin.
In the obese group, interstitial insulin was significantly lower than plasma insulin at 15, 30, 45, and 60 min after the OGTT (Fig. 2B), whereas in lean subjects, no significant difference between interstitial and plasma insulin levels was found during the first hour after the oral glucose load (Fig. 2A). The ratio of interstitial to plasma insulin was therefore higher in lean compared with obese subjects 60 min after glucose ingestion (Fig. 2A and B). At the end of the OGTT (90eC120 min), the interstitial insulin was 60% of the plasma insulin level in both lean (P < 0.05) and obese (P < 0.05) subjects (Fig. 2A and B). Thus, at the last part of the OGTT, the interstitial to plasma insulin ratio was similar in the two groups.
The obese subjects had a significantly higher plasma insulin level at 90eC120 min after oral glucose (398 ± 57 vs. 224 ± 37 pmol/l in control subjects; P < 0.05) (Fig. 3). In addition, interstitial insulin was higher in obese subjects (230 ± 35 vs. 146 ± 21 in lean control subjects; P < 0.05) at 90eC120 min. Insulin uptake rates in both groups are presented in Fig. 4. The insulin uptake increased significantly (P < 0.05) in both groups compared with basal values: 96 ± 27 in the obese group and 41 ± 6.5 fmol · 100 geC1 · mineC1 in the lean group (NS) at 60 min.
Glucose.
Arterial plasma glucose in the fasting state was 5.1 ± 0.1 vs. 5.2 ± 0.1 mmol/l, and venous glucose was 5.0 ± 0.1 vs. 5.1 ± 0.1 mmol/l in lean and obese subjects, respectively (NS) (Table 2). During OGTT, the arterial glucose level was 7.6 ± 0.7 vs. 8.3 ± 0.7 (NS) at 120 min, and venous glucose was 6.8 ± 0.4 vs. 7.8 ± 0.8 (NS) in lean and obese subjects, respectively. In the fasting state, interstitial glucose was 3.4 ± 0.4 vs. 4.1 ± 0.3 (NS) in lean and obese subjects, and at 120 min, it was 5.5 ± 0.6 vs. 6.3 ± 0.6 (NS), respectively. Glucose uptake increased significantly in both groups during the 1st h after oral glucose administration (Table 2).
Blood flow.
Forearm blood flow was similar in the lean and obese groups in both the postabsorptive state (1.6 ± 0.1 vs. 1.7 ± 0.2 ml · 100 geC1 · mineC1; NS) and at 90eC120 min (1.9 ± 0.2 vs. 1.6 ± 0.2 ml · 100 geC1 · mineC1; NS). Blood flow had a tendency to increase during the OGTT in lean subjects (P = 0.07), but not in obese ones.
DISCUSSION
This study demonstrated that obese subjects exhibit a significant gradient between plasma insulin and muscle interstitial insulin during the 1st h after an oral glucose load that was not found in lean control subjects. It is suggested that this significant gradient demonstrates a delayed transcapillary transport of insulin in obese subjects. Thus, the ratio of interstitial to plasma insulin was lower in obese than in lean subjects at the beginning of the OGTT. However, by the end of the OGTT, the interstitialeCtoeCplasma insulin ratio was similar in the two groups (60%), as was previously demonstrated (2,5).
Most earlier studies have investigated insulin action at steady-state insulin levels, and cellular studies from skeletal muscle have shown that intracellular insulin signaling is defective in obese subjects (24). However, another aspect of insulin resistance is a delay in insulin action. Previously, it has been reported that the time of onset of insulin action is delayed in obese subjects during insulin/glucose infusion (10,13). A delayed transcapillary insulin transport in insulin-resistant states has also been reported from in vitro studies (11) and some (1,12,25,26), but not all (6,27), in vivo studies. In a recent study from our laboratory (13), the delay in insulin action was accompanied by a slow delivery of insulin to the muscle interstitial fluid during insulin/glucose infusion in the obese state. However, the distribution of insulin from blood to muscle tissue during more physiological conditions, represented by an OGTT in the present study, has not previously been reported in obese subjects.
During high insulin levels in steady-state conditions, capillary delivery of insulin is sufficient in insulin-resistant muscle (2,3,28). In this study, interstitial muscle insulin was even higher in obese than in lean subjects at the end of the OGTT, confirming earlier data of interstitial insulin levels in obese rats (28) and humans (3) at the end of insulin infusion. The insulin resistance associated with obesity that is observed after prolonged insulin stimulation thus does not seem to be caused by a deficient transcapillary delivery of insulin and glucose but rather by cellular insulin resistance. However, during noneCsteady-state conditions, exemplified by an OGTT in the present study, a hindered insulin distribution is observed in obese, insulin-resistant subjects. The reason for the sluggish insulin distribution may be decreased capillary density, blood flow, capillary recruitment, or permeability in the muscle tissue of obese subjects. Capillary density is negatively correlated with obesity (14); further, it is proposed that low capillary density would prolong the distance along which insulin must be diffused and thus delay insulin action (14).
The issue of whether insulin-mediated vasodilation can modulate glucose uptake has been previously discussed. Muscle blood flow correlates positively with insulin-mediated glucose uptake during insulin infusion (17,29,30). However, the increase in glucose uptake is relatively higher and appears earlier than the increase in blood flow, which would suggest that increased blood flow is not the source of the augmented glucose uptake during the clamp procedure (31). Instead it has been suggested that an increased glucose uptake may stimulate blood flow. Supporting this view are the results of two studies in which insulin administered together with glucose locally in the forearm induced an increase in blood flow (32), whereas insulin infused with a metabolically inactive isomer of glucose did not (33). In a clamp study with lean, obese, and type 2 diabetic subjects, all three groups exhibited similar blood flow when a comparable glucose disposal rate was achieved in all groups (34).
In the present study, no clear difference in blood flow rate was detected between obese and lean subjects during the OGTT. In accordance with the above-mentioned studies (32,34), the higher insulin and glucose levels in the obese group may have compensated for the conjectured diminished blood flow in the obese group. As well, the physiological insulin levels achieved during the OGTT may not have been high enough to increase total limb flow.
Recent work by Vincent et al. (35) has suggested that physiological insulin levels can mediate capillary recruitment, even in the absence of a total blood flow increment. The increased capillary recruitment (35) may be of importance for the muscle glucose uptake. In addition, the PS for glucose was increased in healthy humans during an OGTT without a concomitant increase in blood flow (36). One interesting finding has been that in insulin-resistant states, insulin-mediated capillary recruitment (15,37) seems to be impaired. In the current study, there was a significant difference in interstitial versus plasma insulin in the first hour after the glucose load in the obese group. It may be speculated that decreased capillary recruitment in the obese group impaired the delivery of insulin in the beginning of the OGTT.
In the present study, the PS for glucose increased 1 h after glucose ingestion in the lean but not in the obese group, which may suggest a diminished capillary recruitment in obese subjects. It should be noted that the formula for determining the PS is not equivalent to measurements of capillary recruitment but rather a sum product of vasodilation/vasoconstriction and permeability over the tissue bed. However, a change in capillary recruitment influences the PS value. In addition, calculations of PS (which assumes homogeneity) may be weakened by the presence of heterogeneity of blood flow distribution in muscle (38). It may be assumed that this heterogeneity exists in both lean and obese subjects. Hence, it is reasonable to believe that the change in PS that is found in lean but not in obese subjects corresponds to a difference in change of permeability; that is, the capillary permeability area. Insulin, as well as inulin and glucose, pass through the capillary wall by means of passive diffusion; hence, the blunted increase in PS for glucose found in obese subjects may be relevant for the sluggish increase in interstitial insulin in these subjects.
The present data confirm the well-known observation that obese subjects have a higher insulin response after oral glucose, (39). Similarly, a brisk rise in plasma insulin level with an average peak at 30 min has been demonstrated in nondiabetic subjects in all weight classes (39).
Obese subjects have a high first-phase insulin response (40), which may be an attempt to compensate for the cellular insulin resistance in obesity (24). It has also been shown that a two-phase insulin infusion increases interstitial insulin levels more rapidly than a continuous insulin infusion, suggesting that a loss of the first insulin response may delay transcapillary delivery of insulin (41). In prediabetic states, the first insulin response is diminished (42), which may further deter the distribution of insulin to peripheral tissues. One previous interesting finding is that pulsatile insulin infusion in healthy lean individuals enhances glucose uptake in peripheral tissues more than a continuous insulin infusion (43).
In this study, the insulin response after OGTT showed considered variability in both groups (data not shown), and not all subjects had a biphasic insulin response. This finding concurs with earlier reports on a wide plasma insulin response after OGTT in healthy individuals (44). In the current study, the insulin uptake rate seemed to be delayed in the obese group, even though direct evidence for this cannot be provided from the present data as the variable (often two-phase) plasma insulin response after oral glucose does not allow calculations of the time to reach half-maximal concentrations. Instead, we suggest that the delayed insulin distribution in obese subjects was demonstrated by the significant concentration gradient between plasma and muscle interstitial insulin seen in the beginning of the OGTT. In the lean group, on the other hand, no significant gradient was found between plasma and interstitial insulin in the beginning of the OGTT, suggesting no major hindrance of insulin transport over the capillary wall. The presence of a minor gradient cannot be excluded in this relatively small group of lean subjects. However, the main finding is the major gradient found in the obese subjects as the result of a significant group difference. The present data agree with those from another recent microdialysis study of transcapillary insulin transport in muscle in lean subjects, where a significant gradient between plasma and interstitial insulin was observed at the peak but not in the beginning of the OGTT (45).
In summary, it appears that obese subjects need a higher plasma insulin response than lean subjects to achieve a similar time curve for muscle interstitial insulin. Also, an increased interstitial muscle insulin level at the end of the OGTT may be of pathogenetic importance for insulin resistance in muscle, as high ambient insulin levels may further desensitize the cells to insulin (46).
ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Research Council (project nos. 10864, 11330, and 12206), the Swedish Diabetes Association, Nordisk Insulinfond, Novo Nordisk Pharma AB, the Inga-Britt and Arne Lundberg Foundation, and Gteborgs Lkarsllskap.
We are grateful to Sren Sndergaard, Helena Odenstedt, and Margareta Landeen for their technical assistance and to Gerd Nilsen for administrative support.
OGTT, oral glucose tolerance test; PS, permeability surface area product
REFERENCES
Jansson PA, Fowelin JP, von Schenck HP, Smith UP, Lonnroth PN: Measurement by microdialysis of the insulin concentration in subcutaneous interstitial fluid: importance of the endothelial barrier for insulin. Diabetes42 :1469 eC1473,1993
Sjostrand M, Holmang A, Lonnroth P: Measurement of interstitial insulin in human muscle. Am J Physiol276 :E151 eCE154,1999
Castillo C, Bogardus C, Bergman R, Thuillez P, Lillioja S: Interstitial insulin concentrations determine glucose uptake rates but not insulin resistance in lean and obese men. J Clin Invest93 :10 eC16,1994
Yang YJ, Hope ID, Ader M, Bergman RN: Insulin transport across capillaries is rate limiting for insulin action in dogs. J Clin Invest84 :1620 eC1628,1989
Bergman RN, Yang YJ, Hope ID, Ader M: The role of the transcapillary insulin transport in the efficiency of insulin action: studies with glucose clamps and the minimal model. Horm Metab Res Suppl24 :49 eC56,1990
Mokshagundam SP, Peiris AN, Stagner JI, Gingerich RL, Samols E: Interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals. Metabolism45 :951 eC956,1996
Niklasson M, Holmang A, Lonnroth P: Induction of rat muscle insulin resistance by epinephrine is accompanied by increased interstitial glucose and lactate concentrations. Diabetologia41 :1467 eC1473,1998
Yang YJ, Hope ID, Ader M, Bergman RN: Importance of transcapillary insulin transport to dynamics of insulin action after intravenous glucose [erratum in Am J Physiol 173:Sect. E after table of contents, 1997]. Am J Physiol266 :E17 eCE25,1994
Prager R, Wallace P, Olefsky JM: In vivo kinetics of insulin action on peripheral glucose disposal and hepatic glucose output in normal and obese subjects. J Clin Invest78 :472 eC481,1986
Nolan JJ, Ludvik B, Baloga J, Reichart D, Olefsky JM: Mechanisms of the kinetic defect in insulin action in obesity and NIDDM. Diabetes46 :994 eC1000,1997
Wascher TC, Wolkart G, Russell JC, Brunner F: Delayed insulin transport across endothelium in insulin-resistant JCR:LA-cp rats. Diabetes49 :803 eC809,2000
Holmang A, Niklasson M, Rippe B, Lonnroth P: Insulin insensitivity and delayed transcapillary delivery of insulin in oophorectomized rats treated with testosterone. Acta Physiol Scand171 :427 eC438,2001
Sjostrand M, Gudbjornsdottir S, Holmang A, Lonn L, Strindberg L, Lonnroth P: Delayed transcapillary transport of insulin to muscle interstitial fluid in obese subjects. Diabetes51 :2742 eC2748,2002
Lillioja S, Young AA, Culter CL, Ivy JL, Abbott WG, Zawadzki JK, Yki-Jarvinen H, Christin L, Secomb TW, Bogardus C: Skeletal muscle capillary density and fiber type are possible determinants of in vivo insulin resistance in man. J Clin Invest80 :415 eC424,1987
Clerk LH, Rattigan S, Clark MG: Lipid infusion impairs physiologic insulin-mediated capillary recruitment and muscle glucose uptake in vivo. Diabetes51 :1138 eC1145,2002
Wallis MG, Wheatley CM, Rattigan S, Barrett EJ, Clark AD, Clark MG: Insulin-mediated hemodynamic changes are impaired in muscle of Zucker obese rats. Diabetes51 :3492 eC3498,2002
Laakso M, Edelman SV, Brechtel G, Baron AD: Decreased effect of insulin to stimulate skeletal muscle blood flow in obese man: a novel mechanism for insulin resistance. J Clin Invest85 :1844 eC1852,1990
Laakso M, Edelman SV, Brechtel G, Baron AD: Impaired insulin-mediated skeletal muscle blood flow in patients with NIDDM. Diabetes41 :1076 eC1083,1992
Lonnroth P, Jansson PA, Smith U: A microdialysis method allowing characterization of intercellular water space in humans. Am J Physiol253 :E228 eCE231,1987
Strindberg L, Lonnroth P: Validation of an endogenous reference technique for the calibration of microdialysis catheters. Scand J Clin Lab Invest60 :205 eC211,2000
Siggaard-Andersen J: Venous occlusion plethysmography on the calf: evaluation of diagnosis and results in vascular surgery. Dan Med Bull17 (Suppl. I) :1 eC68,1970
Paaske WP: Capillary permeability in skeletal muscle. Acta Physiol Scand101 :1 eC14,1977
Waugh WH: Photometry of inulin and polyfructosan by use of a cysteine/tryptophan reaction. Clin Chem23 :639 eC645,1977
Goodyear LJ, Giorgino F, Sherman LA, Carey J, Smith RJ, Dohm GL: Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. J Clin Invest95 :2195 eC2204,1995
Niklasson M, Daneryd P, Lonnroth P, Holmang A: Effects of exercise on insulin distribution and action in testosterone-treated oophorectomized female rats. J Appl Physiol88 :2116 eC2122,2000
Miles PD, Li S, Hart M, Romeo O, Cheng J, Cohen A, Raafat K, Moossa AR, Olefsky JM: Mechanisms of insulin resistance in experimental hyperinsulinemic dogs. J Clin Invest101 :202 eC211,1998
Cline GW, Petersen KF, Krssak M, Shen J, Hundal RS, Trajanoski Z, Inzucchi S, Dresner A, Rothman DL, Shulman GI: Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N Engl J Med341 :240 eC246,1999
Holmang A, Mimura K, Bjorntorp P, Lonnroth P: Interstitial muscle insulin and glucose levels in normal and insulin-resistant Zucker rats. Diabetes46 :1799 eC1804,1997
Laakso M, Edelman SV, Olefsky JM, Brechtel G, Wallace P, Baron AD: Kinetics of in vivo muscle insulin-mediated glucose uptake in human obesity. Diabetes39 :965 eC974,1990
Baron AD, Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G: Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J Clin Invest96 :786 eC792,1995
Yki-Jarvinen H, Utriainen T: Insulin-induced vasodilatation: physiology or pharmacology Diabetologia41 :369 eC379,1998
Dela F, Larsen JJ, Mikines KJ, Galbo H: Normal effect of insulin to stimulate leg blood flow in NIDDM. Diabetes44 :221 eC226,1995
Ueda S, Petrie JR, Cleland SJ, Elliott HL, Connell JM: The vasodilating effect of insulin is dependent on local glucose uptake: a double blind, placebo-controlled study. J Clin Endocrinol Metab83 :2126 eC2131,1998
Mather KJ, Mirzamohammadi B, Lteif A, Steinberg HO, Baron AD: Endothelin contributes to basal vascular tone and endothelial dysfunction in human obesity and type 2 diabetes. Diabetes51 :3517 eC3523,2002
Vincent MA, Dawson D, Clark AD, Lindner JR, Rattigan S, Clark MG, Barrett EJ: Skeletal muscle microvascular recruitment by physiological hyperinsulinemia precedes increases in total blood flow. Diabetes51 :42 eC48,2002
Gudbjornsdottir S, Sjostrand M, Strindberg L, Wahren J, Lonnroth P: Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle. J Clin Endocrinol Metab88 :4559 eC4564,2003
Youd JM, Rattigan S, Clark MG: Acute impairment of insulin-mediated capillary recruitment and glucose uptake in rat skeletal muscle in vivo by TNF-. Diabetes49 :1904 eC1909,2000
Kalliokoski KK, Laaksonen MS, Knuuti J, Nuutila P: Perfusion distribution between and within muscles during intermittent static exercise in endurance-trained and untrained men. Int J Sports Med24 :400 eC403,2003
Yalow RS, Glick SM, Roth J, Berson SA: Plasma insulin and growth hormone levels in obesity and diabetes. Ann N Y Acad Sci131 :357 eC373,1965
Beard JC, Ward WK, Halter JB, Wallum BJ, Porte D Jr: Relationship of islet function to insulin action in human obesity. J Clin Endocrinol Metab65 :59 eC64,1987
Getty L, Hamilton-Wessler M, Ader M, Dea MK, Bergman RN: Biphasic insulin secretion during intravenous glucose tolerance test promotes optimal interstitial insulin profile. Diabetes47 :1941 eC1947,1998
Gautier JF, Wilson C, Weyer C, Mott D, Knowler WC, Cavaghan M, Polonsky KS, Bogardus C, Pratley RE: Low acute insulin secretory responses in adult offspring of people with early onset type 2 diabetes. Diabetes50 :1828 eC1833,2001
Schmitz O, Arnfred J, Nielsen OH, Beck-Nielsen H, Orskov H: Glucose uptake and pulsatile insulin infusion: euglycaemic clamp and [3eC3H]glucose studies in healthy subjects. Acta Endocrinol (Copenh)113 :559 eC563,1986
Yalow RS, Rose HG, Bauman WA: Hyperinsulinemia. Am J Med85 :22 eC30,1988
Herkner H, Klein N, Joukhadar C, Lackner E, Langenberger H, Frossard M, Bieglmayer C, Wagner O, Roden M, Muller M: Transcapillary insulin transfer in human skeletal muscle. Eur J Clin Invest33 :141 eC146,2003
Garvey WT, Olefsky JM, Marshall S: Insulin receptor down-regulation is linked to an insulin-induced postreceptor defect in the glucose transport system in rat adipocytes. J Clin Invest76 :22 eC30,1985(Mikaela Sjstrand, Soffia )