Delayed Transcapillary Transport of Insulin to Muscle
Interstitial Fluid in Obese Subjects
Mikaela Sjöstrand,1 Soffia Gudbjörnsdottir,1 Agneta Holmäng,2 Lars Lönn,3 Lena Strindberg,1 and
Peter Lönnroth1
Insulin-resistant subjects have a slow onset of insulin
action, and the underlying mechanism has not been
determined. To evaluate whether a delayed transcapillary transport is part of the peripheral insulin resistance, we followed the kinetics of infused insulin and
inulin in plasma and muscle interstitial fluid in obese
insulin-resistant patients and control subjects. A total
of 10 lean and 10 obese men (BMI 24 ⴞ 0.8 vs. 32 ⴞ 0.8
kg/m2, P < 0.001) was evaluated during a hyperinsulinemic-euglycemic clamp (insulin infusion rate 120 mU 䡠
mⴚ2 䡠 minⴚ1) combined with an inulin infusion. Measurements of insulin and inulin in plasma were taken by
means of arterial-venous catheterization of the forearm
and microdialysis in brachioradialis muscle combined
with forearm blood flow measurements with vein occlusion pletysmography. The obese subjects had a significantly lower steady-state glucose infusion rate and,
moreover, demonstrated a delayed appearance of insulin (time to achieve half-maximal concentration [T1/2]
72 ⴞ 6 vs. 46 ⴞ 6 min in control subjects, P < 0.05) as
well as inulin (T1/2 83 ⴞ 3 vs. 53 ⴞ 7 min, P < 0.01) in the
interstitial fluid. Also, the obese subjects had a delayed
onset of insulin action (T1/2 70 ⴞ 9 vs. 45 ⴞ 5 min in
control subjects, P < 0.05), and their forearm blood
flow rate was significantly lower. These results demonstrate a delayed transcapillary transport of insulin and
inulin from plasma to the muscle interstitial fluid and a
delayed onset of insulin action in insulin-resistant
obese subjects. Diabetes 51:2742–2748, 2002
A
t steady state, insulin concentrations in the
interstitial fluid and in the lymph are markedly
lower than in plasma, suggesting an endothelial
barrier for the transcapillary transport of insulin (1–3). Additionally, a bulk of data suggests that the
equilibration of insulin over the endothelium is a timeconsuming process and that, as a result, the time kinetics
of insulin are slower in the interstitial fluid than in plasma
(2,4,5). In a study of perfused rat hearts (6), the endothelial
From the 1Lundberg Laboratory for Diabetes Research, Sahlgrenska University Hospital, Göteborg, Sweden; the 2Wallenberg Laboratory, Sahlgrenska
University Hospital, Göteborg, Sweden; and the 3Department of Radiology,
Sahlgrenska University Hospital, Göteborg, Sweden.
Address correspondence and reprint requests to Mikaela Sjöstrand, MD,
Lundberg Laboratory for Diabetes Research, Sahlgrenska University Hospital,
S-413 45 Göteborg, Sweden. E-mail: [email protected].
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.
2742
wall seemed to delay the transcapillary transport of insulin. Also, when insulin is infused, levels of interstitial
insulin in lymph (1) and in adipose tissue (2), as well as in
rat skeletal muscle (7), lag behind plasma insulin.
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 insulinresistant 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.
DIABETES, VOL. 51, SEPTEMBER 2002
M. SJÖSTRAND AND ASSOCIATES
TABLE 1
Clinical characteristics
n
Age (years)
Fasting plasma glucose (mmol/l)
Fasting plasma insulin (pmol/l)
Weight (kg)
BMI (kg/m2)
Lean body mass (kg)
Total adipose tissue mass (kg)
Visceral adipose tissue mass (kg)
Lean
Obese
10
40 ⫾ 4
5.4 ⫾ 0.2
41 ⫾ 7
77 ⫾ 2
24 ⫾ 0.8
61 ⫾ 1.5
15 ⫾ 1
2.6 ⫾ 0.2
10
43 ⫾ 3
5.6 ⫾ 0.1
107 ⫾ 20*
104 ⫾ 4†
32 ⫾ 0.8†
70 ⫾ 2.9‡
34 ⫾ 3†
6.6 ⫾ 0.6†
Data are means ⫾ SE. *P ⬍ 0.01, †P ⬍ 0.001, and ‡P ⬍ 0.05 for lean
versus obese subjects.
RESEARCH DESIGN AND METHODS
Subjects. Ten obese and ten lean control subjects, none of whom were taking
any regular medication, were investigated. The clinical characteristics of the
participants are listed in Table 1. Two lean control subjects were smokers, and
two lean and four obese subjects used tobacco snuff. All subjects gave
informed consent, and the study was approved by the ethical committee of the
University of Göteborg.
Study protocol. The investigations started at 8:00 A.M. after an overnight fast.
The subjects were studied in the supine position in a room kept at 25°C. In the
left arm, blood sampling cannulas were inserted into an anticubital vein
draining deep forearm tissues (16) and into the radial artery for A-V measurements. A vein in the contralateral arm was cannulated and used for infusion
of insulin, glucose, and potassium chloride. The study protocol is shown in
Fig. 1.
Fasting plasma glucose and insulin were measured. A euglycemichyperinsulinemic clamp was started as previously described (17). The clamp
protocol was modified to not include any prime infusion of insulin. The insulin
infusion rate was 120 mU 䡠 m⫺2 䡠 min⫺1 for 240 min and was paralleled with
glucose infusion to maintain euglycemia. Potassium chloride (0.1 mmol/l) was
infused at a rate of 10 mmol/h during the clamp to prevent hypokalemia.
Paralleled to the insulin infusion, inulin was infused at a constant rate (24
ml/h) for 300 min. Arterial and venous blood samples were taken every 15 min
for inulin and insulin and every 30 min for glucose. Each sample was stored
at ⫺18°C before analysis.
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 ⫻ 0.5 mm 100-kDA molecular cutoff were used, and
for glucose, another catheter with 16 ⫻ 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 microin-
jection 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
␮l/min (glucose measurements) and 1.5 ␮l/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. Fick’s principle was used to estimate
the regional rate of glucose and insulin uptake. During steady state, the
formula (A-V) 䡠 flow was applied.
Permeability surface area produc for insulin and glucose. The permeability surface area product (PS) was derived from the following formula:
A-V ⫽ ( A ⫺ I) ⫻ (1 ⫺ e⫺ps/Q)
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).
FIG. 1. Study protocol.
DIABETES, VOL. 51, SEPTEMBER 2002
2743
INSULIN KINETICS IN OBESITY
TABLE 2
Time (in minutes) to achieve T1/2 and Tmax concentrations of
insulin and inulin in arterial plasma and in muscle microdialysates (minus 20 min to compensate for the delay in the microdialysis system) in obese subjects and lean control subjects during
infusion of inulin (24 ml/h) and insulin (120 mU 䡠 m⫺2 䡠 min⫺1)
Arterial insulin
Obese subjects
Control subjects
Dialysate insulin
Obese subjects
Control subjects
Arterial inulin
Obese subjects
Control subjects
Dialysate inulin
Obese subjects
Control subjects
T1/2
Tmax
8 ⫾ 0.4
9 ⫾ 0.7
54 ⫾ 7
56 ⫾ 2
72 ⫾ 6
46 ⫾ 6*
148 ⫾ 8
98 ⫾ 9†
35 ⫾ 2
39 ⫾ 3
188 ⫾ 10
177 ⫾ 7
83 ⫾ 3
53 ⫾ 7†
153 ⫾ 7
123 ⫾ 8*
Data are means ⫾ SE. *P ⬍ 0.05, †P ⬍ 0.01 for obese subjects versus
control subjects.
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 Student’s
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.
RESULTS
Glucose infusion rate. Glucose infusion rates (GIRs)
achieved at 180 –240 min of the clamp were reduced in
obese subjects as compared with control subjects when
calculated as either per body weight (8.9 ⫾ 0.5 vs. 12.5 ⫾
0.7 mg 䡠 kg⫺1 䡠 min⫺1, P ⬍ 0.05) or per lean body mass
(13.1 ⫾ 0.6 vs. 15.4 ⫾ 0.8, P ⬍ 0.05). The obese subjects
showed a marked delay in onset of the insulin-mediated
increase of glucose disposal rate. The initiation phase of
insulin activation of glucose metabolism was expressed as
T1/2, adapting the GIR during the last hour of the clamp as
100%. In addition, the slope of the tangent to the initial
activation curve activation was characterized as recently
suggested in a study by Nolan et al. (9). Figure 2A
illustrates the time curves for GIR in the two groups. T1/2,
calculated from the mean of each individual curve of GIR,
was 70 ⫾ 9 min in obese subjects and 45 ⫾ 5 min in control
subjects (P ⬍ 0.05). The initial activation slopes were
reduced by ⬃25% in the obese group as compared with the
control group (Fig. 2B).
Insulin. The steady-state arterial plasma concentration of
insulin was 1,960 ⫾ 153 and 2,025 ⫾ 236 pmol/l (NS) and
in deep venous plasma was 1,701 ⫾ 206 and 1,911 ⫾ 198
pmol/l (NS) in lean and obese subjects, respectively.
Arterial and deep venous levels of insulin are depicted in
Fig. 3.
The A-V concentration difference of insulin in the basal
state was 8.5 ⫾ 4 vs. 22 ⫾ 11 pmol/l (NS) and during
2744
FIG. 2. A: Time course of whole-body GIR in lean and obese subjects
during a euglycemic-hyperinsulinemic clamp (120 mU 䡠 mⴚ2 䡠 minⴚ1). B:
Whole-body GIR in lean and obese subjects, plotted as percent of
maximal GIR at the last hour of the clamp. Data are means ⴞ SE. F,
lean subjects; E, obese subjects.
steady-state hyperinsulinemia was 124 ⫾ 37 vs. 139 ⫾ 44
pmol/l (NS) in lean and obese subjects, respectively.
Interstitial insulin levels during steady state were 1,199 ⫾
140 and 916 ⫾ 100 pmol/l in obese and control subjects,
respectively (NS). No difference in T1/2 or Tmax for plasma
FIG. 3. Arterial insulin (E), venous insulin (Œ), arterial insulin (F),
and venous insulin (}) in forearm during insulin infusion (120
mU 䡠 mⴚ2 䡠 minⴚ1) and insulin infusion (24 ml/h) in all subjects. Data are
means ⴞ SE (n ⴝ 20).
DIABETES, VOL. 51, SEPTEMBER 2002
M. SJÖSTRAND AND ASSOCIATES
FIG. 4. Insulin in dialysate in lean and obese subjects during a
euglycemic-hyperinsulinemic clamp (120 mU 䡠 mⴚ2 䡠 minⴚ1). Data are
means ⴞ SE. F, lean subjects; E, obese subjects.
insulin was seen between obese and control subjects
(Table 2). T1/2 and Tmax of insulin in arterial plasma were
achieved at 16 ⫾ 1 and 55 ⫾ 3 min (n ⫽ 20), respectively
(Fig. 3). However, the T1/2 and Tmax of insulin in microdialysates was delayed in obese subjects (P ⬍ 0.05) (Table 2
and Fig. 4).
Calculated insulin uptake was 199 ⫾ 55 and 143 ⫾ 30
fmol 䡠 100 g⫺1 䡠 min⫺1 in lean and obese subjects (NS).
Estimated PS for insulin during steady-state clamp was
0.5 ⫾ 0.3 vs. 0.3 ⫾ 0.1 ml 䡠 100 g⫺1 䡠 min⫺1 in lean and obese
subjects, respectively (NS).
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).
Glucose. Arterial and venous plasma glucose in the fasting state were 5.4 ⫾ 0.2 and 5.3 ⫾ 0.2 vs. 5.6 ⫾ 0.2 and
FIG. 6. Forearm blood flow in lean and obese subjects during a
euglycemic-hyperinsulinemic clamp (120 mU 䡠 mⴚ2 䡠 minⴚ1). Data are
means ⴞ SE. F, lean subjects; E, obese subjects.
5.4 ⫾ 0.1 mmol/l in lean and obese subjects, respectively
(NS). During steady-state clamping at 210 –240 min, arterial and venous glucose levels were 6.4 ⫾ 0.2 and 5.0 ⫾ 0.4
vs. 6.4 ⫾ 0.1 and 4.9 ⫾ 0.3 mmol/l in the lean and obese
groups, respectively (NS). At the end of the clamp, A-V
concentration difference of glucose was significant in both
groups (P ⬍ 0.01). Interstitial glucose in the fasting state
was 3.5 ⫾ 0.2 vs. 3.7 ⫾ 0.3 mmol/l and during steady-state
clamp was 4.0 ⫾ 0.3 vs. 4.3 ⫾ 0.3 mmol/l in lean and obese
subjects, respectively (NS). The calculated glucose uptake
tended to be higher in the lean group in the fasting state
(0.6 ⫾ 0.2 vs. 0.3 ⫾ 0.1 ␮mol 䡠 100 g⫺1 䡠 min⫺1, P ⫽ 0.07)
and was significantly higher during steady-state clamping
(5.1 ⫾ 1.2 vs. 2.1 ⫾ 0.5, P ⬍ 0.05) in lean and obese
subjects, respectively. The glucose uptake rate correlated
positively with the GIR (r2 ⫽ 0.32, P ⬍ 0.05). Estimated PS
during steady-state clamp was 2 ⫾ 1 vs. 2 ⫾ 1 ml 䡠 100 g⫺1 䡠
min⫺1 in lean and obese subjects, respectively (NS).
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 䡠 100 g⫺1 䡠 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).
DISCUSSION
FIG. 5. Inulin in dialysate in lean and obese subjects during a euglycemic-hyperinsulinemic clamp (120 mU 䡠 mⴚ2 䡠 minⴚ1). Data are means ⴞ
SE. F, lean subjects; E, obese subjects.
DIABETES, VOL. 51, SEPTEMBER 2002
This study demonstrates that the time kinetics of insulin in
muscle tissue are slower than in plasma in both obese and
lean subjects. This is in accordance with previous studies
in lymph (1) and in subcutaneous adipose tissue in humans (2). Importantly, activation of glucose disposal is
also slower than changes in plasma insulin but fits kinetically with insulin in lymph (26). The present data, obtained in the interstitial fluid, confirm the earlier finding of
an endothelial barrier for insulin, with a 40 – 60% lower
interstitial insulin level and slower kinetics than in plasma
(2,4).
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-medi2745
INSULIN KINETICS IN OBESITY
ated 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 testosteronetreated 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
2746
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–receptormediated 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 (34 –36). 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 insulinmediated glucose uptake in muscle (33,37,38). However,
during an insulin infusion, the increase in blood flow
DIABETES, VOL. 51, SEPTEMBER 2002
M. SJÖSTRAND AND ASSOCIATES
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 ⬃15–30% of insulin’s
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 Fick’s 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 Fick’s 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
DIABETES, VOL. 51, SEPTEMBER 2002
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-tomuscle 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.
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 Göteborgs Läkarsällskap.
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.
REFERENCES
1. Yang YJ, Hope ID, Ader M, Bergman RN: Insulin transport across capillaries is rate limiting for insulin action in dogs. J Clin Invest 84:1620 –1628,
1989
2. 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.
Diabetes 42:1469 –1473, 1993
3. Sjostrand M, Holmang A, Lonnroth P: Measurement of interstitial insulin in
human muscle. Am J Physiol 276:E151–E154, 1999
4. Yang YJ, Hope ID, Ader M, Bergman RN: Importance of transcapillary
insulin transport to dynamics of insulin action after intravenous glucose
[published erratum appears in Am J Physiol 173:section E, 1997, following
table of contents]. Am J Physiol 266:E17–E25, 1994
5. Niklasson M, Daneryd P, Lonnroth P, Holmang A: Effects of exercise on
insulin distribution and action in testosterone-treated oophorectomized
female rats. J Appl Physiol 88:2116 –2122, 2000
6. Brunner F, Wascher TC: Contribution of the endothelium to transcapillary
insulin transport in rat isolated perfused hearts. Diabetes 47:1127–1134,
1998
7. Niklasson M, Holmang A, Lonnroth P: Induction of rat muscle insulin
resistance by epinephrine is accompanied by increased interstitial glucose
and lactate concentrations. Diabetologia 41:1467–1473, 1998
8. 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 Invest 93:10 –16, 1994
9. Nolan JJ, Ludvik B, Baloga J, Reichart D, Olefsky JM: Mechanisms of the
kinetic defect in insulin action in obesity and NIDDM. Diabetes 46:994 –
1000, 1997
10. Wascher TC, Wolkart G, Russell JC, Brunner F: Delayed insulin transport
across endothelium in insulin-resistant JCR:LA-cp rats. Diabetes 49:803–
809, 2000
11. Holmang A, Niklasson M, Rippe B, Lonnroth P: Insulin insensitivity and
delayed transcapillary delivery of insulin in oophorectomized rats treated
with testosterone. Acta Physiol Scand 171:427– 438, 2001
12. King GL, Johnson SM: Receptor-mediated transport of insulin across
endothelial cells. Science 227:1583–1586, 1985
13. Bar RS, DeRose A, Sandra A, Peacock ML, Owen WG: Insulin binding to
microvascular endothelium of intact heart: a kinetic and morphometric
analysis. Am J Physiol 244:E447–E452, 1983
14. Steil GM, Ader M, Moore DM, Rebrin K, Bergman RN: Transendothelial
insulin transport is not saturable in vivo: no evidence for a receptormediated process. J Clin Invest 97:1497–1503, 1996
15. Hamilton-Wessler M, Ader M, Dea MK, Moore D, Loftager M, Markussen J,
Bergman RN: Mode of transcapillary transport of insulin and insulin analog
NN304 in dog hindlimb: evidence for passive diffusion. Diabetes 51:574 –
582, 2002
16. Moller N, Butler PC, Antsiferov MA, Alberti KG: Effects of growth hormone
on insulin sensitivity and forearm metabolism in normal man. Diabetologia
32:105–110, 1989
17. DeFronzo RA, Tobin JD, Andres R: Glucose clamp technique: a method for
quantifying insulin secretion and resistance. Am J Physiol 237:E214 –E223,
1979
2747
INSULIN KINETICS IN OBESITY
18. Lonnroth P, Jansson PA, Smith U: A microdialysis method allowing
characterization of intercellular water space in humans. Am J Physiol
253:E228 –E231, 1987
19. Muller M, Holmang A, Andersson OK, Eichler HG, Lonnroth P: Measurement of interstitial muscle glucose and lactate concentrations during an
oral glucose tolerance test. Am J Physiol 271:E1003–1007, 1996
20. Strindberg L, Lonnroth P: Validation of an endogenous reference technique
for the calibration of microdialysis catheters. Scand J Clin Lab Invest
60:205–211, 2000
21. Siggaard-Andersen J: Venous occlusion plethysmography on the calf:
evaluation of diagnosis and results in vascular surgery. Dan Med Bull 17
(Suppl. 1):1– 68, 1970
22. Chowdhury B, Sjostrom L, Alpsten M, Kostanty J, Kvist H, Lofgren R: A
multicompartment body composition technique based on computerized
tomography. Int J Obes Relat Metab Disord 18:219 –234, 1994
23. Kvist H, Chowdhury B, Grangard U, Tylen U, Sjostrom L: Total and visceral
adipose-tissue volumes derived from measurements with computed tomography in adult men and women: predictive equations. Am J Clin Nutr
48:1351–1361, 1988
24. Lonn L, Starck G, Alpsten M, Ekholm S, Sjostrom L: Determination of
tissue volumes: a comparison between CT and MR imaging. Acta Radiol
40:314 –321, 1999
25. Waugh WH: Photometry of inulin and polyfructosan by use of a cysteine/
tryptophan reaction. Clin Chem 23:639 – 645, 1977
26. 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 (Suppl. 24):49 –56, 1990
27. 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 Invest 78:472– 481, 1986
28. Mokshagundam SP, Peiris AN, Stagner JI, Gingerich RL, Samols E:
Interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and
lean individuals. Metabolism 45:951–956, 1996
29. Sjostrand M, Holmang A, Strindberg L, Lonnroth P: Estimations of muscle
interstitial insulin, glucose, and lactate in type 2 diabetic subjects. Am J
Physiol Endocrinol Metab 279:E1097–E1103, 2000
30. Holmang A, Mimura K, Bjorntorp P, Lonnroth P: Interstitial muscle insulin
and glucose levels in normal and insulin-resistant Zucker rats. Diabetes
46:1799 –1804, 1997
31. Holmang A, Bjorntorp P, Rippe B: Tissue uptake of insulin and inulin in red
and white skeletal muscle in vivo. Am J Physiol 263:H1170 –H1176, 1992
32. Krotkiewski M, Bylund-Fallenius AC, Holm J, Bjorntorp P, Grimby G,
Mandroukas K: Relationship between muscle morphology and metabolism
in obese women: the effects of long-term physical training. Eur J Clin
Invest 13:5–12, 1983
33. 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 Invest 85:1844 –1852, 1990
34. 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 Invest 80:415– 424, 1987
35. Lithell H, Lindgarde F, Hellsing K, Lundqvist G, Nygaard E, Vessby B, Saltin
2748
B: Body weight, skeletal muscle morphology, and enzyme activities in
relation to fasting serum insulin concentration and glucose tolerance in
48-year-old men. Diabetes 30:19 –25, 1981
36. Hedman A, Berglund L, Essen-Gustavsson B, Reneland R, Lithell H:
Relationships between muscle morphology and insulin sensitivity are
improved after adjustment for intra-individual variability in 70-year-old
men. Acta Physiol Scand 169:125–132, 2000
37. 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 Invest 96:786 –792,
1995
38. Laakso M, Edelman SV, Brechtel G, Baron AD: Impaired insulin-mediated
skeletal muscle blood flow in patients with NIDDM. Diabetes 41:1076 –
1083, 1992
39. Utriainen T, Malmstrom R, Makimattila S, Yki-Jarvinen H: Methodological
aspects, dose-response characteristics and causes of interindividual variation in insulin stimulation of limb blood flow in normal subjects.
Diabetologia 38:555–564, 1995
40. Baron AD, Clark MG: Role of blood flow in the regulation of muscle
glucose uptake. Annu Rev Nutr 17:487– 499, 1997
41. Baron AD, Tarshoby M, Hook G, Lazaridis EN, Cronin J, Johnson A,
Steinberg HO: Interaction between insulin sensitivity and muscle perfusion
on glucose uptake in human skeletal muscle: evidence for capillary
recruitment. Diabetes 49:768 –774, 2000
42. Holmang A, Muller M, Andersson OK, Lonnroth P: Minimal influence of
blood flow on interstitial glucose and lactate- normal and insulin-resistant
muscle. Am J Physiol 274:E446 –E452, 1998
43. Natali A, Quinones Galvan A, Pecori N, Sanna G, Toschi E, Ferrannini E:
Vasodilation with sodium nitroprusside does not improve insulin action in
essential hypertension. Hypertension 31:632– 636, 1998
44. Zierler K: Theory of the use of arteriovenous concentration differences for
measuring metabolism in steady and non-steady states. J Clin Invest
40:2111–2125, 1961
45. Staehr P, Hother-Nielsen O, Levin K, Holst JJ, Beck-Nielsen H: Assessment
of hepatic insulin action in obese type 2 diabetic patients. Diabetes
50:1363–1370, 2001
46. Beard JC, Ward WK, Halter JB, Wallum BJ, Porte D Jr: Relationship of islet
function to insulin action in human obesity. J Clin Endocrinol Metab
65:59 – 64, 1987
47. Getty L, Hamilton-Wessler M, Ader M, Dea MK, Bergman RN: Biphasic
insulin secretion during intravenous glucose tolerance test promotes
optimal interstitial insulin profile. Diabetes 47:1941–1947, 1998
48. Cerasi E, Luft R: The plasma insulin response to glucose infusion in
healthy subjects and in diabetes mellitus. Acta Endocrinol (Copenh)
55:278 –304, 1967
49. 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.
Diabetes 50:1828 –1833, 2001
50. DECODE Study Group/European Diabetes Epidemiology Group: Glucose
tolerance and mortality: comparison of WHO and American Diabetes
Association diagnostic criteria: diabetes epidemiology: collaborative analysis of diagnostic criteria in Europe. Lancet 354:617– 621, 1999
DIABETES, VOL. 51, SEPTEMBER 2002