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Human Performance Laboratory and Department of Biochemistry, School of Medicine, East Carolina University, Greenville, North Carolina 27858-4353
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Hickey, Matthew S., Charles J. Tanner, D. Sean O'Neill,
Lydia J. Morgan, G. Lynis Dohm, and Joseph A. Houmard. Insulin activation of phosphatidylinositol 3-kinase in human skeletal muscle in
vivo. J. Appl. Physiol. 83(3):
718-722, 1997.
The purpose of this investigation was to determine
whether insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase)
activity is detectable in needle biopsies of human skeletal muscle.
Sixteen healthy nonobese males matched for age, percent fat, fasting
insulin, and fasting glucose participated in one of two experimental
protocols. During an intravenous glucose tolerance test (IVGTT)
protocol, insulin-stimulated PI3-kinase activity was determined from
percutaneous needle biopsies at 2, 5, and 15 min post-insulin
administration (0.025 U/kg). In the second group, a 2-h, 100 mU · m
2 · min1
euglycemic hyperinsulinemic clamp was performed, and biopsies were
obtained at 15, 60, and 120 min after insulin infusion was begun.
Insulin stimulated PI3-kinase activity by 1.6 ± 0.2-, 2.2 ± 0.3-, and 2.2 ± 0.4-fold at 2, 5, and 15 min, respectively, during
the IVGTT. During the clamp protocol, PI3-kinase was elevated by 5.3 ± 1.3-, 8.0 ± 2.6-, and 2.7 ± 1.4-fold above
basal at 15, 60, and 120 min, respectively. Insulin-stimulated
PI3-kinase activity at 15 min post-insulin administration was
significantly greater during the clamp protocol vs. the IVGTT
(P < 0.05). These observations suggest that insulin-stimulated PI3-kinase activity is detectable in
needle biopsies of human skeletal muscle, and furthermore, that the
euglycemic, hyperinsulinemic clamp protocol may be a useful tool to
assess insulin signaling in vivo.
insulin signaling; glucose; muscle biopsy
INTRODUCTION THE MOLECULAR ASPECTS of insulin signaling have been
the subject of intense scrutiny for many years (cf. Refs. 7, 8, 21).
Although the majority of progress in understanding the steps in the
intracellular signaling cascade have come from cell culture studies,
several investigations have suggested that insulin signaling is
defective in rodent models of obesity/insulin resistance (4, 5, 11, 12,
18). In particular, the dual-function lipid/serine protein kinase
phosphatidylinositol 3-kinase (PI3-kinase), which appears to be
involved in insulin-mediated glucose transport and glucose transporter
isoform GLUT-4 translocation (17, 23-25, 29), has been shown to be
downregulated in skeletal muscle from insulin-resistant rodents (5, 11,
18). Moreover, one member of our present group recently reported that
PI3-kinase activation by insulin in vitro is defective in human
skeletal muscle that is insulin resistant with respect to glucose
transport (15). Because insulin signaling may be modified by
alterations in the physiological milieu in intact organisms, this is a
question of considerable relevance with regard to establishing the
mechanisms that regulate insulin signaling. To our knowledge, only one
group has studied insulin-stimulated PI3-kinase activation in vivo (3). The purpose of this investigation was to determine the efficacy of
measuring insulin-stimulated PI3-kinase activity from needle biopsies
of human skeletal muscle by using either a bolus administration of
insulin during an intravenous glucose tolerance test or a euglycemic, hyperinsulinemic clamp, two common methods of measuring insulin action.
METHODS
Table 1.
Descriptive characteristics
Variable
Clamp
(n = 8)
IVGTT (n = 8)
Age, yr
23.8 ± 1.0
25.4 ± 1.9
(19.0-28.0)
(20.0-30.0)
%Fat
13.1 ± 1.4
14.0 ± 1.5
(8.0-20.8)
(7.0-19.0)
Fasting glucose, mM
5.2 ± 0.3
4.9 ± 0.1
(4.8-5.4)
(4.4-5.2)
Fasting insulin, pM
18.5 ± 1.1
18.7 ± 1.0
(13.5-29.5)
(14.2-31.0)
Values are means ± SE. Range is in parentheses. n, No.
of subjects. IVGTT, intravenous glucose tolerance test.
2 · min1)
was started. Blood for plasma glucose determination was obtained every
5 min throughout the test, and adjustments were made, as necessary, in
the rate of infusion of a 20% dextrose solution to maintain
euglycemia. Plasma insulin was determined every 10 min unless otherwise
noted. M value (glucose disposal rate) was determined in
six subjects as described by DeFronzo et al. (10). Data from two
subjects were not available because of an error in storing the glucose
infusion rate data.
Muscle biopsy.
Percutaneous muscle biopsies were obtained from the belly of the vastus
lateralis by using Bergstom needles with suction applied as described
previously (19). Biopsies were obtained basally (before IVGTT or clamp)
and at the following time points during each test: 2, 5, and 15 min
post-insulin administration in the IVGTT and at 15, 60, and 120 min
during the clamp. The time course during the IVGTT was chosen to mimic
bolus administration protocols reported in rodents (11, 12), in which
activation of PI3-kinase has a relatively short time course. The time
course of muscle biopsies during the clamp was on the basis of a
previous report regarding in vivo insulin-receptor tyrosine kinase
activation (13).
Baseline biopsies were obtained ~30 min before the start of each
test. During both procedures, subsequent biopsies were obtained from
alternating legs at each time point. Each of the biopsies was
~3-4 cm apart. During the clamp, the baseline and 60-min
biopsies were obtained from the same leg, as were the 15- and 120-min
biopsies. Thus during this procedure there was a minimum of 90 min
between sampling in the same leg. Because of the condensed time frame of the IVGTT protocol, samples were obtained from the same leg within
13 min (2- and 15-min samples). All biopsies obtained during the IVGTT
or clamp were rapidly frozen and stored in liquid nitrogen before
analysis for PI3-kinase activity.
PI3-kinase assay.
PI3-kinase was assayed according to the procedure of Goodyear et al.
(15) with minor modifications. Skeletal muscle tissue to be used for
determination of PI3-kinase activity was homogenized in (1:10, wt/vol)
ice-cold homogenization buffer containing 1% Nonidet P-40 in a
ground-glass mortar and pestle for 30 s. The homogenate was
subsequently solubilized for 60 min at 4°C on a rotator and then
centrifuged at 30,000 g for 1 h. The
supernatant (~500 µl) was incubated overnight at 4°C
with anti-phosphotyrosine conjugated to agarose beads (~40 µl,
Sigma A-1806). Immunoprecipitates were pelleted at 4°C for 30 s at
13,000 g and washed three times with
1% Nonidet P-40 in phosphate-buffered saline (pH 7.4) containing 100 µM
Na3VO4,
three times in ice-cold 100 mM tris(hydroxymethyl)aminomethane (Tris;
pH 7.5) with 100 mM LiCl and 100 µM
Na3VO4,
and two times with ice-cold 10 mM Tris (pH 7.5), containing 100 mM
NaCl, 1 mM EDTA, and 100 µM
Na3VO4.
The pellets were resuspended in 50 µl ice-cold 10 mM Tris (pH 7.5),
containing 100 mM NaCl, 1 mM EDTA, 10 µl of 100 mM
MgCl2, and 10 µl
phosphatidylinositol (2 µg/µl) and sonicated for 60 s in 10 mM Tris
(pH 7.5) with 1 mM EDTA. The reaction was started by the addition of 10 µl of 440 µM ATP containing 30 µCi
[32P]ATP. After 10 min, the reaction was stopped with the addition of 20 µl 8N HCl, and
labeled lipids were extracted with 160 µl methanol-chloroform
(1:1, vol/vol). After centrifugation for 60 s at 13,000 g, the lower phase (50 µl) was
separated by thin-layer chromatography on silica gel plates coated with
1% (wt/vol) potassium oxalate in a mobile phase of
chloroform-methanol-water-ammonium hydroxide (60:47:11.3:2,
vol/vol/vol/vol). The reaction products were visualized on a
phosphorimager and quantified by densitometry.
Statistics.
The magnitude of insulin stimulation of PI3-kinase activity was
determined by using repeated-measures analysis of variance. Where
applicable, post hoc analysis was performed by using Scheffé's procedure. Between-group differences were determined by using a
t-test. Relationships between selected
physiological variables were assessed by using simple linear regression
analysis. All data are presented as means ± SE. Statistical
significance was accepted as P < 0.05.
RESULTS
1 · min1
(n = 6). Mean fasting plasma insulin
was 18.7 ± 4.3 pM and increased to 783.2 ± 117.9, 1,029.4 ± 70.1, and 1,108.5 ± 95.3 pM by minutes 15, 60, and
120, respectively. Mean fasting plasma
glucose was 5.2 ± 0.3 mM and was maintained at 5.1 ± 0.2 mM
during the clamp. Peak PI3-kinase activity (60 min) was positively
related to M value (r = 0.73, P = 0.06, n = 6) (Fig.
2). In addition, peak PI3-kinase activity
was inversely related to body mass index
(r = 
0.65,
P = 0.08), percent body fat
(r = 0.54,
P = 0.16), and the percentage of type
IIb muscle fibers (r = 0.45,
P = 0.19), although these correlations
were not statistically significant.
IVGTT protocol. Insulin increased PI3-kinase activity over baseline levels by 1.6 ± 0.3-fold at 2 min (P = not significant), 2.2 ± 0.3-fold at 5 min (P < 0.05), and 2.2 ± 0.4-fold at 15 min. (P < 0.05). The mean Si was 6.2 ± 0.6 min/pM. Mean fasting plasma insulin was 18.5 ± 1.1 pM. Plasma insulin peaked at 2 min postbolus (207.2 ± 19.4 pM) and decreased to 92.3 ± 7.9 pM at 5 min postbolus. No corresponding insulin is available for the 15-min postbolus biopsy sample because this was not part of the IVGTT protocol. Mean fasting plasma glucose was 4.9 ± 0.1 mM. Peak PI3-kinase activity was not related to Si (r = 0.20, P = 0.45).
DISCUSSION
The molecular mediators of insulin action have been the subject of intense scrutiny for several years (cf. Refs. 6-8, 21). Because insulin resistance has been implicated in the pathophysiology of non-insulin-dependent diabetes (NIDDM), obesity, hypertension, and hyperlipidemia (9), the regulation of cellular insulin action is a topic of considerable relevance.
The results of this study complement a recent report by Bjornholm et al. (3), in which PI3-kinase activity was measured basally and 40 min after the onset of an insulin infusion in six control and six NIDDM subjects. These authors observed a twofold activation of PI3-kinase in the vastus lateralis in control subjects and no activation in the NIDDM patients. Importantly, the subjects studied by Bjornholm et al. were considerably older (~55 yr of age) than the subjects in the present study. Moreover, the peak plasma insulin was higher in the present study (1,029 vs. ~650 pM). Both of these factors may contribute to the differences in the magnitude of insulin stimulation in these studies. It should also be noted that Laville et al. (22) recently documented that acute hyperinsulinemia (3 h) increases PI3-kinase mRNA in human skeletal muscle. Taken together, these studies provide evidence that selected cellular aspects of insulin signaling in skeletal muscle are amenable to investigation in vivo in humans.
PI3-kinase is a dual-function lipid/serine protein kinase that is activated in response to insulin binding in several tissues, including skeletal muscle (5, 11, 12, 14-16). Studies on the role of PI3-kinase in insulin signaling have provided considerable evidence that the activation of this enzyme is an integral component of the cascade involved in GLUT-4 translocation and glucose transport (17, 23-25, 29). Thus the study of insulin-stimulated PI3-kinase activation in the context of measurement of whole body insulin action provides the opportunity to examine the integrity of a proximal step in the insulin-signaling cascade with respect to a functional end point, i.e., glucose uptake.
In the euglycemic clamp protocol, M value is assumed to primarily reflect skeletal muscle glucose disposal (10). In this context, it is noteworthy that M value was positively associated with peak PI3-kinase activation (r = 0.73, P = 0.06). Although the small number of subjects precludes any definitive statement, this relationship suggests that the assessment of PI3-kinase as described represents a physiologically relevant measurement. Further support for the use of the clamp comes from prior observations by Freidenberg et al. (13) that activation of the insulin receptor tyrosine kinase during a euglycemic clamp does not occur until ~60 min after the start of the insulin infusion. Our observation that PI3-kinase activation peaks at 60 min agrees closely with this observation. The reduction in activity at 120 min may relate to a downregulation of enzymatic activity. This phenomenon has been observed during short-term incubation of mouse solei (18), although Freidenberg et al. (13) observed a maintenance of insulin receptor tyrosine kinase activity in human skeletal muscle for up to 240 min during a clamp. The nature of the cellular events that are responsible for mediating insulin-stimulated glucose uptake, as well as the factors that regulate PI3-kinase activity, is just beginning to be understood.
In this context, we cannot exclude the possibility that PI3-kinase activity may have peaked at some time other than when biopsies were obtained. The ethical issues related to multiple biopsies in humans preclude an exhaustive time-course study. Moreover, there is some concern that the trauma associated with repeated biopsies may artifactually elevate PI3-kinase activity. Although we are not aware of any studies that have carefully examined the effect of tissue sampling on PI3-kinase activation in either rodents or humans, it should be noted that the process of obtaining rectus abdominus strips for muscle incubation, which involves the surgical removal of tissue similar to a needle biopsy, does not prevent a substantial insulin-stimulated activation of PI3-kinase (15). We took care during the clamp protocol to leave ~90 min between same-leg biopsies to avoid any confounding effects of the previous sample. Moreover, if a "biopsy effect" were evident, it would be expected to have a greater effect during the IVGTT protocol, when the time course of muscle sampling was condensed. In fact, PI3-kinase activation during this procedure was substantially lower than during the clamp.
The IVGTT protocol is, in many ways, a corollary of the bolus procedure used for several in vivo studies in rodents (5, 11, 12), with the obvious exception that the administered insulin dose is substantially lower in the human model (0.025 vs. 10 U/kg). In this respect, the relatively minor activation of PI3-kinase during this procedure is not surprising. After a bolus intravenous insulin administration, the peak interstitial concentration of insulin has been reported to be temporally delayed relative to the plasma by 13 min (1). Thus, whereas peak plasma concentration occurred at 2 min postbolus in the present study, interstitial concentration should have peaked at or near the time of the 15-min biopsy.
In both euglycemic clamp (20) and IVGTT protocols (1), interstitial insulin concentration has been reported to be ~50% of the plasma concentration at any given time point during each procedure. If we assume the same relationship applies to the present data, interstitial insulin would have been in the range of 500 pM at 60 min of the clamp protocol and ~50 pM at 15 min of the IVGTT. This difference can obviously be considered to contribute to the differences in the magnitude of PI3-kinase activation in each protocol in the present study. The physiological relevance of PI3-kinase activity is a point of emphasis for future studies. The plasma insulin levels during the clamp protocol are considerably higher than typical postprandial values. The extent to which PI3-kinase is meaningful with respect to insulin action is clearly dependent on the ability of physiological alterations in systemic insulin to activate the enzyme. Nevertheless, the limited data available in humans suggest that activation may be physiologically meaningful. Thus Bjornholm et al. (3) have documented a blunted activation in skeletal muscle of NIDDM vs. control patients, whereas we have observed a significant correlation between PI3-kinase activation and M value during the clamp. Because both studies involved small numbers of subjects, more work must be done to confirm and expand on these preliminary observations.
In conclusion, we have reported that PI3-kinase activity is detectable in needle biopsy samples of human vastus lateralis during a euglycemic, hyperinsulinemic clamp, and, to a lesser extent, during an IVGTT. The data suggest that peak activation of PI3-kinase occurs at ~60 min during the clamp, which is in good agreement with prior reports of the time course of insulin receptor tyrosine kinase activation in vivo (13). These preliminary observations are intended to provide a framework within which the cellular mechanisms of insulin signaling in human skeletal muscle can be investigated.
ACKNOWLEDGEMENTS
The authors thank the volunteers for participation in this study. The support of the Ambulatory Medical Unit of Pitt County Memorial Hospital is gratefully acknowledged. The assistance of Edward Tapscott is also appreciated.
FOOTNOTES
Received 4 November 1996; accepted in final form 22 April 1997.
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