Measurement of interstitial insulin in human muscle

Mikaela Sjöstrand, Agneta Holmäng, and Peter Lönnroth

Lundberg Laboratory for Diabetes Research and the Wallenberg Laboratory, Department of Internal Medicine and Heart and Lung Diseases, Sahlgrenska University Hospital, Gothenburg University, S-413 45 Gothenburg, Sweden

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous measurements in lymph and adipose tissue have indicated that interstitial insulin concentrations are ~40% lower than in plasma. Measurements of insulin in human muscle interstitial fluid have not been performed yet. We developed a new external reference technique for calibration of microdialysis catheters in situ. This technique allows correct assessments of interstitial peptide concentrations and was employed to estimate the insulin concentration in medial quadriceps femoris muscle in 11 individuals (age: 37 ± 3 yr; body mass index: 25.2 ± 1.2 kg/m2) during a two-step euglycemic hyperinsulinemic clamp. At steady-state insulin and glucose infusion, plasma glucose was 5.9 ± 0.2 mmol/l, plasma insulin was 155 ± 17 mU/l, and interstitial muscle insulin was 67 ± 19 mU/l (n = 9; P < 0.01). At a higher insulin infusion rate, the steady-state plasma insulin concentration was 379 ± 58 mU/l, and interstitial insulin concentration was 180 ± 40 mU/l (P < 0.01). The data show for the first time that high physiological and supraphysiological plasma insulin levels give 30-50% lower interstitial concentrations of insulin in the muscle. The importance of capillary delivery as a rate-limiting step for the insulin effect is suggested.

insulin sensitivity; muscle tissue; microdialysis

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

EVALUATION OF THE sensitivity of insulin in muscle tissue involves the relationship between the effect and the concentration of insulin in the ambience of the muscle cells. Recently, investigations on the mechanisms regulating the delivery of insulin to the interstitial fluid have generated a bulk of data suggesting that the concentration of insulin in plasma may differ from that in the interstitial fluid. First, insulin sensitivity in muscle seems to be related to the capacity to increase blood flow and capillary recruitment (4, 10). Second, results from cultured cells in vitro suggest the active transport of insulin to the interstitial fluid by transendocytosis of the insulin receptor-hormone complex through the capillary endothelium (13). Third, the significance of a saturable pathway for insulin transport has been indicated in different studies of the kinetics of plasma insulin (5, 16). Furthermore, direct measurements of insulin in lymph (1) and in the subcutaneous interstitial fluid (11) suggest that the interstitial concentration of insulin is ~50% lower than in arterial plasma under steady-state euglycemic insulin clamp conditions. However, direct evidence for the existence of a significant arterial-interstitial concentration difference of insulin in the muscles is not provided.

The present study was performed to further explore the hypothesis that the capillary wall may be rate limiting for insulin delivery to the interstitial fluid in the muscle. Microdialysis measurements of interstitial fluid concentrations of insulin were done in the medial quadriceps femoris muscle (14) under euglycemic hyperinsulinemic conditions in healthy volunteers. The data demonstrate for the first time a significant arterial-interstitial concentration difference of insulin in the muscle.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Subjects

Four male and seven female healthy volunteers with normal glucose tolerance and no regular medication were studied. Table 1 lists the clinical characteristics of the subjects. All subjects gave their informed consent, and the study was approved by the Ethics Committee of Gothenburg University.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Clinical characteristics

Study Protocol

The investigations were started at 0800 after an overnight fast. The subjects were studied in the supine position in a room kept at 25°C. A polyethylene catheter was placed in a left forearm vein for blood sampling. The forearm was heated with electric pads (~70°C) to arterialize the venous blood (12). Each study started with a bolus injection of inulin (Inutest; Kemiflor, Stockholm, Sweden) followed by a constant intravenous infusion (24 ml/h) for 360 min. With this protocol, steady-state plasma inulin levels were achieved within 240 min (18). A euglycemic clamp performed as previously described by DeFronzo et al. (6) started 30 min after the initiation of the inulin infusion. The clamp started with a primed infusion of insulin (Actrapid; Novo Nordisk, Copenhagen, Denmark) for 10 min followed by a constant infusion rate of 120 mU · m-2 · min-1 for 120 min. Thereafter, the insulin infusion was increased to 240 mU · m-2 · min-1 and continued for 150 min. Blood samples were drawn every 5 min. The rate of glucose infusion was adjusted to maintain euglycemic plasma glucose concentrations. Potassium chloride (0.1 M) was infused at a rate of 10 mmol/h during the clamp to prevent hypokalemia.

Microdialysis

In the present experimental procedures, commercially available custom-made microdialysis catheters (12 × 0.5 mm, 100-kDa molecular mass cutoff; CMA-10; CMA, Stockholm, Sweden) were used. The inlet of the microdialysis catheter was connected to a microinjection pump (CMA 100; CMA) and was perfused with isotonic saline with the addition of 1.5 mmol/l glucose and 1% human albumin at a perfusion speed 1 µl/min. Catheters were inserted without anesthetics in the medial quadriceps femoris muscle ~10-15 cm above the knee joint by the following procedure. The surface of the disinfected skin was punctured vertically with a 20-gauge cannula. The steel mandrin was removed, and the microdialysis catheter was inserted. Microdialysates were collected at 30-min intervals.

Muscle blood flow to the calf was measured after 90 min of the euglycemic clamp at the lower level of insulin infusion and after 90 min at the higher level of insulin infusion in the contralateral leg by the vein occlusion plethysmography method (3).

Calibration Procedure

Validation of an external reference calibration technique. In a previous study, the significant binding of insulin to microdialysis catheter material was demonstrated (11). In the same study, it was shown that this binding phenomenon made conventional calibration procedures such as the equilibration technique less applicable in insulin measurements. It was also demonstrated that, after correction for unspecific binding, the relationship between insulin and inulin in vivo recovery (recovery = dialysate concentration/interstitial concentration) was similar to the relationship of the recoveries of the same two substances in microdialysis experiments conducted in vitro in plasma (11). This may lead to the assumption that, if the ratio of microdialysis recovery of two substances is similar irrespective of the ambient medium, the in vivo recovery of any substance could be estimated by the knowledge of the relationship between in vitro recoveries of the substance of interest and a reference substance provided that the in vivo recovery of the reference could be accurately measured. Such a calibration technique should be convenient in measurements of multiple substances and, also, would be time saving compared with equilibration calibration techniques (14). Indirectly, this calibration technique was previously suggested and even validated in a study in rat muscle where the ratios of microdialysis recoveries in vivo and in vitro were demonstrated to be similar for a number of different exogenous substances (7). In the present study, an exogenous reference calibration technique was validated for insulin measurements in the rat muscle. After validation, the same technique was applied for measurements in human muscle.

Calibration of microdialysis in rat muscle. Male Sprague-Dawley rats (n = 20) weighing 200-300 g were used. Twenty-four hours before study, an osmotic minipump was implanted subcutaneously (Alzet 2001 B; Alza, Palo Alto, CA) containing [14C]inulin (Amersham) given as a constant infusion (0.1 mCi/rat). After 24 h, the rats were anesthetized (Inactin; RBI, Natick, MA), and catheters were inserted in the right jugular vein (infusions) and in the left carotid artery (blood sampling). Under euglycemic conditions, ascertained by a glucose infusion, insulin (Actrapid) was infused at 5 mU · kg-1 · min-1 for 140 min. Muscle microdialysis was performed in both medial femoral muscles by two 10- to 15-mm microdialysis tubings (BAS, Indianapolis, IN) perfused with isotonic saline containing 1% bovine albumin and (in experiments including retrodialysis calibration of glucose) [3H]glucose (0.5 mM, 0.1 mCi/ml; Amersham) at a rate of 1 µl/min.

Relative microdialysis recovery in vivo for inulin was calculated as dialysate/plasma concentration of [14C]inulin. Recovery of glucose was calculated as the percentage efflux of [3H]glucose according to the internal reference calibration technique previously evaluated (14). In vivo recovery of insulin was calculated according to the exogenous reference technique using the formula R1 = R2, where R1 = recovery of insulin/recovery of inulin in vitro, and R2 is the corresponding recovery ratio obtained in vivo.

In experiments made in vitro in plasma, microdialysis was performed with the same catheter material and perfusate without labeled glucose (12). Briefly, the microdialysis catheters were placed in a jar containing human plasma with added [14C]inulin (0.025 mCi/ml) and insulin (1,000 mU/l; Actrapid). The jar was placed on a water bath at 37°C (250 strokes/min). Dialysate perfusion speed was 1 µl/min. After 60 min, microdialysate was collected hourly for 4 h and was analyzed for inulin, insulin, and glucose.

The data in Table 2 show that all three substances had ~50% lower recoveries in vivo compared with in vitro. This permitted us to conclude that, first, the previous finding (7) that the relationship between in vitro/in vivo recoveries is similar for any substance also is relevant for insulin measurements, since the insulin concentration in interstitial fluid could be calculated from the knowledge of the in vitro recovery ratio of either reference (i.e., inulin or glucose) and the reference concentration in the interstitial fluid. Second, by the exogenous reference calibration technique, a high-precision estimate of the interstitial insulin concentration was achieved since insulin, if used as a reference in turn, could make accurate calculations of interstitial glucose possible.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Relative recovery in vivo of inulin, insulin, and glucose

Calibration of microdialysis in human muscle. To characterize the different diffusion properties and to correct for differences in binding of the compounds to the catheter, experiments were performed in which inulin and insulin were dialyzed in plasma at 37°C in vitro using the double-cannulated probes for measurements in human muscle. The in vitro experiments were done according to the same protocol described above. Results from nine such experiments demonstrated that the relative recovery of inulin in microdialysates was 47 ± 7%. Relative recovery of insulin in vitro was 21 ± 1, 23 ± 2, 20 ± 2, and 22 ± 1% in the four hourly sampled dialysates.

The mean inulin recovery (dialysate inulin/plasma inulin) in experiments performed in vivo was 11 ± 3%. The in vivo recovery of insulin was then calculated in each subject according to the above formula. The mean calculated in vivo recovery of insulin was 5.0 ± 0.1%. The in vivo recovery factor was used for recalculating steady-state dialysate insulin content (2 ± 1, 3 ± 1, 10 ± 3, and 9 ± 3 mU/l in the four samples collected at 90-, 120-, 240-, and 270-min clamping times, respectively) to interstitial insulin concentrations.

Analytical Methods

Glucose concentrations in plasma were determined enzymatically using 10-µl samples on a YSI 2700 select biochemical analyzer (Yellow Springs Instruments, Yellow Springs, OH).

Plasma insulin was measured with a double-antibody radioimmunoassay (Pharmacia, Uppsala, Sweden). The concentration of insulin in microdialysates was determined with an enzymatic immunoassay (DAKO Diagnostics, Cambridge, UK). No difference in cross-reactivity for insulin was evident from measurements with two analysis techniques when identical plasma samples from rats, investigated before and during clamp, were examined. Inulin concentrations in plasma and in the dialysate fractions were determined photometrically using 20-µl samples according to the micromethod described by Waugh (20).

Statistics

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). All results are expressed as means ± SE. Plasma and dialysate inulin values were from the end of each experiment to ensure steady-state conditions. Significance of differences was tested with Student's t-test for paired observations, and, when several comparisons were performed, ANOVA was used. Fisher's least significant difference test was used for post hoc analyses.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Plasma glucose was kept constant during the clamp. When plasma insulin was increased during clamp step 2, the glucose infusion rate and leg blood flow increased significantly (Table 3). At both clamp steps, a significantly lower insulin concentration was measured in interstitial fluid compared with plasma (Fig. 1). The mean interstitial-to-plasma insulin concentration ratio was 0.43 and 0.47 at clamp steps 1 and 2, respectively.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   GIR for plasma and leg blood flow during a two-step hyperinsulinemic euglycemic clamp


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Plasma (filled bars) and interstitial (open bars) levels of insulin at steady state during a two-step euglycemic hyperinsulinemic clamp (120 and 240 mU · m-2 · min-1). *** P < 0.001, n = 11 subjects. Data are means ± SE.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present data show for the first time that the insulin concentration in human muscle interstitial fluid is significantly lower than in plasma during steady-state conditions. The data confirm the previous hypothesis, based on lymph measurements (5) and microdialysis in the subcutaneous tissue (11), that an arterial-interstitial concentration difference exists for insulin in the muscles when plasma insulin is kept at high physiological or supraphysiological levels. This allows us to draw the conclusion that, even at steady-state plasma insulin, the delivery of insulin to the interstitial fluid is not rapid enough to balance the elimination rate of muscle insulin. This, in turn, implies that the capillary delivery of insulin is partly rate limiting for the insulin consumption in the muscle. Also, the previous data from lymph (1) and subcutaneous microdialysis studies (11) have demonstrated an increase of the concentration difference over the capillary wall at increasing supraphysiological plasma insulin levels, harmonizing with the concept of a saturable transport system for insulin capillary delivery (13).

We recently demonstrated the existence of such a saturable transport system in quadriceps muscle in the rat (8). However, measurements in leg lymph did not give any evidence for saturability of the delivery of insulin to leg lymph (19). The present data do not allow conclusions as to the putative existence of a saturable system for transcapillary transport of insulin. Only indirect evidence was provided by the fact that the relative concentration gradient of insulin was identical at both clamp steps despite the significant increase of leg blood flow at clamp step 2 (Table 3). We cannot explain the difference in data obtained in different tissues and species concerning the putative existence of a saturable transport system for insulin. However, it can not be excluded that diffusion of insulin as well as active transport systems, such as the transendocytosis pathway, may be differently effective in different organs and, hence, might balance the interstitial fluid concentration of insulin with varying efficiency.

Furthermore, a saturable insulin transport system opens up the possibility that an arterial interstitial concentration gradient of insulin may be less extensive or even absent at a low physiological concentration range. Accordingly, data obtained from microdialysis studies in the rat demonstrated a significant insulin concentration difference over the capillary wall only at plasma concentrations beyond those exerting a half-maximum effect on glucose disposal (8). Due to the poor efficiency of the microdialysis sampling presently used, low physiological insulin concentrations were not detectable in the present study. However, it is clear from the present data that a significant concentration gradient over muscle capillary walls could be demonstrated in humans at submaximal insulin stimulation, indicating that the insulin concentration difference between the interstitial and plasma compartment may be physiologically significant.

The fact that the capillary wall is rate limiting for the uptake of both insulin (the present study) and glucose (14) suggests that the microcirculatory system and its capacity to recruit muscle capillaries is essential to optimize the time kinetics for the distribution of insulin in peripheral tissues. Interestingly, insulin-resistant human subjects not only have a decreased muscle capillary density (2) but also show a delay in peripheral insulin action (16). In experimental studies in the testosterone-treated rat, we have demonstrated reduction of capillary density as well as delayed insulin distribution and insulin resistance in muscle tissue (9, 15). It may be argued that the present insulin data might have been calculated less precisely due to the fact that the dialysis recovery of insulin was low and the multiplication factor based on insulin measurements thus was high. Furthermore, the variance in dialysate insulin was balanced by the same variance in inulin in vivo recovery in each experiment, since all catheters were calibrated in situ to ensure the accuracy of the measurements. We thus conclude that the concentration gradient of insulin over the capillary wall demonstrated here could not be artifactual, dependent on inaccurate calibration techniques.

In summary, microdialysis measurements in the human quadriceps muscle demonstrate for the first time a significant (~50%) concentration difference of insulin over the capillary wall. The pathophysiological significance of this finding obtained during hyperinsulinemic clamping conditions may be demonstrated further in future investigations including insulin-resistant subjects with a reduced muscle capillary density.

    ACKNOWLEDGEMENTS

The laboratory assistance provided by Lena Strindberg and Britt-Marie Larsson is gratefully acknowledged.

    FOOTNOTES

This study was supported by grants from the Swedish Research Council (project nos. 10864, 11330, and 12206), the Swedish Diabetes Association, Nordisk Insulin Fond, and Inga-Britt and Arne Lundberg Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. Sjöstrand, Lundberg Laboratory for Diabetes Research, Sahlgrenska Univ. Hospital, S-413 45 Gothenburg, Sweden.

Received 18 February 1998; accepted in final form 29 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ader, M., and R. N. Bergman. Importance of transcapillary insulin transport to dynamics of insulin action after intravenous glucose. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E17-E25, 1994[Abstract/Free Full Text].

2.   Allenberg, K., K. Johansen, and B. Saltin. Skeletal muscle adaptation to physical training in type II (non-insulin-dependent) diabetes mellitus. Acta Med. Scand. 223: 365-373, 1988[Medline].

3.   Andersson, D. K., L. Hansson, and R. Sivertsson. Primary hypertension refractory to triple drug teatment. A study on central and peripheral hemodynamics. Circulation 58: 615-621, 1978[Medline].

4.   Baron, A. D. Hemodynamic actions of insulin. Am. J. Physiol. 267 (Endocrinol. Metab. 30): E187-E202, 1994[Abstract/Free Full Text].

5.   Bergman, R N., J. Yang, I. D. Hope, and M. Ader. The role of the transcapillary insulin transport in the efficiency of insulin action: studies with glucose clamps and the minimal model. Horm. Metab. Res. Suppl. 24: 49-56, 1990[Medline].

6.   DeFronzo, R., R. Gunnarson, O. Björkman, M. Olsson, and J. Wahren. Effects of insulin on peripheral and splachnic glucose metabolism in non-insulin dependent (type 2) diabetes mellitus. J. Clin. Invest. 76: 149-155, 1985[Medline].

7.   Deguchi, Y., T. Terasaki, S. Kawasaki, and A. Tsuji. Muscle microdialysis as a model study to relate the drug concentration in tissue interstitial fluid and dialysate. J. Pharmacobio-Dyn. 14: 483-492, 1991[Medline].

8.   Holmäng, A., K. Mimura, P. Björntorp, and P. Lönnroth. Interstitial muscle insulin and glucose levels in normal and insulin-resistant Zucker rats. Diabetes 46: 1799-1804, 1997[Abstract].

9.   Holmäng, A., J. Svedberg, E. Jennische, and P. Björntorp. Effects of testosterone on muscle insulin sensitivity and morphology in female rats. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E555-E560, 1990[Abstract/Free Full Text].

10.   James, D. E., K. M. Burleigh, L. H. Storlien, S. P. Bennett, and E. W. Kraegen. Heterogeneity of insulin action in muscle: influence of blood flow. Am. J. Physiol. 251 (Endocrinol. Metab. 14): E422-E430, 1986[Abstract/Free Full Text].

11.   Jansson, P. A., J. Fowelin, H. von Schenck, U. Smith, and P. Lönnroth. Measurement by microdialysis of the insulin concentration in subcutaneous interstitial fluid. Diabetes 42: 1469-1473, 1993[Abstract].

12.   Jansson, P. A., A. Larsson, U. Smith, and P. Lönnroth. Glycerol production in subcutaneous adipose tissue in lean and obese man. J. Clin. Invest. 89: 1610-1617, 1992[Medline].

13.   King, G. L., S. M. Johnson, and I. Jialal. Processing and transport of insulin by vascular endothelial cells. Am. J. Med. 79: 43-47, 1985[Medline].

14.   Müller, M., A. Holmäng, O. K. Andersson, H. G. Eichler, and P. Lönnroth. Measurement of interstitial muscle glucose and lactate concentrations during an oral glucose tolerance test. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E1003-E1007, 1996[Abstract/Free Full Text].

15.  Niklasson, M., P. Daneryd, P. Björntorp, P. Lönnroth, and A. Holmäng. The effects of testosterone and exercise on insulin kinetics in rat (Abstract). Int. J. Obes. 20, Suppl. 4: 78, 1996.

16.   Olefsky, J., J. W. Farquhar, and G. Reaven. Relationship between fasting plasma insulin level and resistance to insulin-mediated glucose uptake in normal and diabetic subjects. Diabetes 22: 507-513, 1973[Medline].

17.   Prigeon, R. L., M. E. Roder, D. Porte, Jr., and S. E. Kahn. The effect of insulin dose on the measurement of insulin sensitivity by the minimal model technique. Evidence for saturable insulin transport in humans. J. Clin. Invest. 97: 501-507, 1996[Abstract/Free Full Text].

18.   Schachter, D., N. Freikel, and I. L. Schwartz. Movement of inulin between plasma and interstitial fluid. Am. J. Physiol. 160: 532-535, 1950.

19.   Steil, G. M., M. Ader, D. M. Moore, K. Rebrin, and R. N. Bergman. Transendothelial insulin transport is not saturable in vivo. No evidence for a receptor-mediated process. J. Clin. Invest. 97: 1497-1503, 1996[Abstract/Free Full Text].

20.   Waugh, W. H. Photometry of inulin and polyfructosan by use of a cysteine/tryptophan reaction. Clin. Chem. 23: 639-644, 1977[Medline].


Am J Physiol Endocrinol Metab 276(1):E151-E154
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society