Division of Diabetes, Departments of 1 Medicine and Biochemistry,2 The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
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ABSTRACT |
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Although
chronic hyperinsulinemia has been shown to induce insulin resistance,
the basic cellular mechanisms responsible for this phenomenon are
unknown. The present study was performed 1) to determine the
time-related effect of physiological hyperinsulinemia on glycogen
synthase (GS) activity, hexokinase II (HKII) activity and mRNA content,
and GLUT-4 protein in muscle from healthy subjects, and 2)
to relate hyperinsulinemia-induced alterations in these parameters to
changes in glucose metabolism in vivo. Twenty healthy subjects had a
240-min euglycemic insulin clamp study with muscle biopsies and then
received a low-dose insulin infusion for 24 (n = 6) or
72 h (n = 14) (plasma insulin concentration = 121 ± 9 or 143 ± 25 pmol/l, respectively). During the
baseline insulin clamp, GS fractional velocity (0.075 ± 0.008 to
0.229 ± 0.02, P < 0.01), HKII mRNA content
(0.179 ± 0.034 to 0.354 ± 0.087, P < 0.05), and HKII activity (2.41 ± 0.63 to 3.35 ± 0.54 pmol · min1 · ng
1,
P < 0.05), as well as whole body glucose disposal and
nonoxidative glucose disposal, increased. During the insulin clamp
performed after 24 and 72 h of sustained physiological
hyperinsulinemia, the ability of insulin to increase muscle GS
fractional velocity, total body glucose disposal, and nonoxidative
glucose disposal was impaired (all P < 0.01), whereas
the effect of insulin on muscle HKII mRNA, HKII activity, GLUT-4
protein content, and whole body rates of glucose oxidation and
glycolysis remained unchanged. Muscle glycogen concentration did not
change [116 ± 28 vs. 126 ± 29 µmol/kg muscle,
P = nonsignificant (NS)] and was not correlated with
the change in nonoxidative glucose disposal (r = 0.074, P = NS). In summary, modest chronic hyperinsulinemia
may contribute directly (independent of change in muscle glycogen
concentration) to the development of insulin resistance by its impact
on the GS pathway.
hexokinase II; glucose transporter 4; skeletal muscle; insulin resistance
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INTRODUCTION |
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INSULIN RESISTANCE IS A CHARACTERISTIC finding in type 2 diabetes mellitus (8) and has been related to abnormalities in insulin receptor signal transduction (16, 31), glucose transport and phosphorylation (3, 21), and glycogen synthesis (2, 44). Although considerable evidence suggests that insulin resistance is genetically determined (23), other studies have shown that insulin resistance can be acquired secondary to hyperglycemia (18, 19, 50) or elevated plasma free fatty acid (FFA) levels (12, 32). Hyperinsulinemia is a constant finding in all insulin resistance states (8, 9, 24, 36). Most investigators have interpreted the hyperinsulinemia to represent a compensatory response to offset the impairment in insulin action (8, 36). However, in vivo (11, 40) and in vitro (13, 18) studies have shown that hyperinsulinemia can be a cause of insulin resistance. A physiological elevation in plasma insulin concentration (from 8 to 20 µU/ml) for as few as 3-5 days can induce insulin resistance, due to a reduction in glycogen formation (11). Consistent with this observation, exposure to high insulin concentrations inhibits glycogen synthase (GS) activity in cultured myocytes (18). However, no previous studies have examined the cellular mechanisms responsible for insulin-induced insulin resistance in skeletal muscle in humans. The present study was designed to examine the time-related effects of a physiological increment in plasma insulin concentration, similar to that observed in the fasting state in insulin-resistant individuals, on GS and hexokinase II (HKII) activity, HKII mRNA, and GLUT-4 protein in muscle tissue of human subjects.
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METHODS |
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Subjects.
Twenty healthy subjects [13 males, 7 females, age = 29 ± 1 yr, body mass index (BMI) = 23.7 ± 0.6 kg/m2,
%body fat = 22.6 ± 1.8%] participated in the low-dose
insulin infusion protocol. Two additional subjects (1 male, 1 female), matched for age and BMI, took part in a 72-h control saline infusion. All subjects had a normal oral glucose tolerance test, no subject had a
family history of diabetes, and none were taking any medications. Individuals participating in regular vigorous exercise were excluded. All subjects consumed an isocaloric diet containing 150-200 g of
carbohydrate for 3 days before each study, and body weight was stable
for
6 mo before study. Written voluntary consent was obtained from
each subject after an explanation of the purpose, nature, and potential
risks. The protocol was approved by the Institutional Review Board of
the University of Texas Health Science Center.
Euglycemic insulin clamp.
After a 10-h overnight fast, a 4-h euglycemic hyperinsulinemic clamp
study was performed as previously described (10). A 20-gauge Teflon catheter was inserted into an antecubital vein for
infusion of insulin, glucose, and
D-[3-3H]glucose. The tritiated glucose was
given as a primed (25 µCi) continuous (0.25 µCi/min) infusion
starting 2 h before the insulin clamp. A second catheter was
inserted retrogradely into a dorsal hand vein for blood sampling, and
the hand was kept in a heated box (60°C) throughout the study. After
a 120-min equilibration period, a percutaneous needle biopsy of the
vastus lateralis muscle was obtained (26), and the muscle
tissue was frozen immediately in liquid nitrogen and stored at 70°C
until analyzed. After the muscle biopsy, a 240-min insulin infusion was
started at 40 mU · m
2 · min
1.
Arterialized blood samples were collected every 5 min for plasma glucose determination, and a 20% glucose infusion was adjusted to
maintain euglycemia. Plasma tritiated glucose specific activity and
insulin concentrations were measured every 10-30 min during the
insulin clamp. All blood samples were promptly centrifuged and stored
at
20°C until analyses were performed. After 240 min, a second
biopsy was obtained from the vastus lateralis muscle of the
contralateral thigh. During the last 60 min of the basal insulin clamp
periods, oxygen consumption and carbon dioxide production rates were
measured continuously using indirect calorimetry (Deltatrac Metabolic
Monitor, SensorMedics, Savi Park, CA), and rates of glucose and lipid
oxidation were calculated as previously described (45).
Insulin-mediated nonoxidative glucose metabolism, which primarily
reflects glycogen synthesis (43), was calculated as the
difference between total body glucose disposal and glucose oxidation.
Lean body mass and FFM were determined in all subjects by use of
tritiated water (6).
Chronic insulin infusion.
Three to five days after the initial insulin clamp, subjects were
admitted to the General Clinical Research Center at 1800 and started on
a weight-maintaining diet (55% carbohydrate, 30% fat, 15% protein),
and calories were divided one-fifth for breakfast, two-fifths for
lunch, and two-fifths for dinner. This dietary regimen was kept
constant throughout the hospital stay. At 0800 on the day after
admission, a catheter was placed into an antecubital vein. After two
basal samples for insulin and glucose determination were collected, a
continuous insulin infusion (10 mU · m2 · min
1) was started
to approximately double the basal plasma insulin concentration while
maintaining euglycemia. The duration of insulin infusion was 24 (n = 6) or 72 h (n = 14). Plasma
glucose concentration was determined every 60-90 min from a second
catheter placed into a vein on the dorsum of the hand, and an exogenous
glucose infusion was adjusted to maintain the plasma glucose
concentration within 5% of the basal level. The plasma insulin
concentration was measured after 24 (n = 17), 48 (n = 11), and 72 h (n = 11). At
the end of 24 or 72 h, the insulin infusion was stopped, and the
glucose infusion was tapered off over 30-90 min. One hundred
twenty minutes after cessation of the continuous insulin infusion, a
repeat euglycemic insulin clamp was performed.
Analytical determinations. Plasma glucose was measured by the glucose oxidase method. Plasma insulin was determined by radioimmunoassay (Diagnostic Products, Los Angeles, CA). Tritiated glucose specific activity was determined on deproteinized plasma samples as previously described (11).
Muscle biopsies.
Muscle tissue was homogenized while still frozen in a buffer consisting
of 50 mM potassium phosphate, pH 7.4, 2 mM dithothreitol, 20 mM sodium
fluoride, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor,
20 µg/ml p-aminobenzamidine, 70 µg/ml
N-p-tosyl-L-lysine chlormethyl
ketone, and 170 µg/ml phenylmethysulfonyl fluoride. Homogenates were
centrifuged at 14,000 g, and the supernatant (cytosolic
fraction) was removed and kept on ice. The pellet (mitochondrial fraction) was resuspended in the extraction buffer containing 0.1%
Triton X-100. The soluble fraction contains >95% of the cytosolic enzymes, including GS, and the particulate fraction contains >98% of
the mitochondrial enzymes, such as pyruvate dehydrogenase
(27).
Enzyme activity assays.
The different temperature sensitivities of HKII and HKI were used to
separate the activities of the two enzymes (26). Aliquots of cytosolic and mitochondrial fractions were either heated at 45°C
for 1 h or kept on ice. Because HKII is denatured at 45°C, HK
activity assayed on the heated sample represents HKI activity, whereas
the activity assayed on the sample kept at 4°C represents total HK
(HKI + HKII) activity (26). HKII activity was
determined as the difference between these values. HK activity was
determined using a modification (15) of an enzyme-linked
fluorometric assay (34). The HK buffer consisted of (in
mM): 40 Tris · HCl, pH 7.5, 100 KCl, 20 MgCl2, 2 EDTA, 10 glucose, 2 ATP, 0.25 NADP+, and 0.01 U/ml glucose
6-phosphate dehydrogenase (Sigma Chemical, St. Louis, MO). Total assay
volume was 1.0 ml, and the assay was started by adding 10 µl of
muscle extract (30-60 µg protein). The increase in fluorescence
was monitored for 15 min by use of a fluorometer (Turner model 112, Sequoia-Turner, Mountain View, CA). Multiple assays performed on
separate portions of muscle biopsies from the same individuals
(n = 5) revealed the interassay coefficient of
variation (CV) to be 11.5 ± 1.6% over a range of activity of
1-4 pmol · min1 · µg protein
1.
GS activity was determined in cytosolic fractions using 0.1 mM
(GS0.1) and 10 mM (GS10) glucose 6-phosphate
(G-6-P), as previously described (27). GS
fractional velocity (GSFV) was defined as the ratio of GS
activities, determined as GS0.1/GS10. This
ratio is increased by insulin infusion in humans and represents
dephosphorylation and activation of glycogen synthase.
RNase protection assays for HKI and HKII mRNA. RNase protection assays for specific mRNA content of muscle biopsies were performed using Maxiscript and RNase Protection Assay Kits (Ambion, Austin, TX). A fragment of human HKII cDNA (15) was used as a template to produce an antisense RNA probe that protected a product of 231 nt, and a fragment of human HKI cDNA (33) was used to produced a probe that protected a product of 396 nt. Two micrograms of total RNA from each biopsy were used in the RNase protection assay. Protected products were separated by urea-polyacrylamide gel electrophoresis and quantified by PhosphorImage analysis (Molecular Dynamics, Sunnyvale, CA). A 28S rRNA probe was used as an internal standard to produce a control signal. In multiple experiments using aliquots of RNA from the same individuals, the intra-assay CV was <10%. Pre- and postinsulin RNA samples were always run in the same assay, and data were expressed in density units relative to the 28S rRNA signal.
GLUT-4 immunoblot analysis. Solubilized (Triton X-100) extracts (250 µg) of muscle biopsies were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with an antibody specific for GLUT-4 (Analytical Scientific, San Antonio, TX). Detection was accomplished with the enhanced chemiluminescence system (Amersham). Images were quantified by densitometry, and GLUT-4 content was expressed relative to a GLUT-4-positive control run on each gel.
Glycogen concentration. In 6 of the 14 subjects who received the 72-h insulin infusion, a small piece of muscle tissue (20-40 mg) was homogenized in 0.05 M sodium acetate, pH 4.55. Glycogen was digested with amyloglucosidase by a modification of the method of Chan and Exton (4), and glucose was assayed using a HK/glucose 6-phosphate dehydrogenase-coupled spectrophotometric assay.
Calculations and statistical analyses. During the last 30 min of tritiated glucose infusion, a steady-state plateau of tritiated glucose specific activity was achieved in all subjects, and the basal rate of hepatic glucose production was determined by dividing the tritiated glucose infusion rate (dpm/min) by the steady-state plasma tritiated glucose radioactivity (dpm/ml). During the euglycemic insulin clamp, the rate of total body glucose appearance was calculated using Steele's equation (46) and a distribution volume of 0.65. Hepatic glucose production was calculated by subtracting the exogenous glucose infusion rate from the tracer-derived rate of total body glucose production. The rate of insulin-mediated total body glucose disposal was determined by adding the rate of residual hepatic glucose production to the rate of exogenous glucose infusion. Rates of glucose and lipid oxidation were calculated from respiratory gas exchange measurements (44). Insulin-mediated nonoxidative glucose disposal, which primarily represents glycogen synthesis, was calculated by subtracting the rate of glucose oxidation from the rate of whole body glucose disposal.
Statistical analyses included ANOVA with repeated measures over time followed by Fisher's t-test. Pearson correlation coefficients were used for correlation analyses. ![]() |
RESULTS |
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The basal plasma insulin concentration was 54 ± 14 pmol/l
(9 ± 2 mU/l) and increased to 121 ± 9 pmol/l (20 ± 1 mU/l) after 24 h of continuous low-dose insulin infusion
(n = 20, P < 0.01). No further
increase was observed after 48 (136 ± 8 pmol/l or 23 ± 1 mU/l) and 72 h (141 ± 23 pmol/l or 24 ± 3 mU/l). The
basal plasma glucose was 5.1 ± 0.01 mmol/l (92 ± 1 mg/dl)
and did not change after 24 (5.2 ± 0.1 mmol/l or 93 ± 2 mg/dl) and 72 h (5.2 ± 0.1 mmol/l or 93 ± 2 mg/dl).
Before start of the continuous low-dose insulin infusion, total body
insulin-mediated glucose uptake during the euglycemic insulin clamp was
10.6 ± 1.0 mg · kg
FFM1 · min
1 in the 24-h group and
9.69 ± 0.71 mg · kg
FFM
1 · min
1 in the 72-h group and
decreased to 9.0 ± 1.2 (P < 0.05) and 7.50 ± 0.69 (P < 0.001) mg · kg
FFM
1 · min
1, respectively, after 24 and 72 h of low-dose insulin infusion (Fig.
1). Plasma insulin levels during the
insulin clamp studies averaged 419 ± 25 pmol/l (70 ± 4 mU/l
before) and 408 ± 36 pmol/l (68 ± 6 mU/l after) in the 24-h
group [P = nonsignificant (NS)] and 424 ± 44 pmol/l (70 ± 7 mU/l before) and 444 ± 48 pmol/l (74 ± 8 mU/l after) in the 72-h group (P = NS). During the
baseline insulin clamp, glucose uptake was negatively correlated with
the BMI (r =
0.69, P < 0.01) and the
basal plasma insulin concentration (r =
0.84,
P < 0.01).
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During the insulin clamp studies performed before, after 24 h, and after 72 h of low-dose continuous insulin infusion, the mean plasma glucose concentrations were 5.11 ± 0.08 (92 ± 1 mg/dl), 5.00 ± 0.12 (90 ± 2 mg/dl), and 5.05 ± 0.04 mmol/l (91 ± 1 mg/dl), respectively. The fasting plasma glucose concentrations during the insulin clamps performed before, after 24 h, and after 72 h of low-dose insulin infusion were 5.19 ± 0.4 (93 ± 7 mg/dl), 5.05 ± 0.16 mmol/l (91 ± 3 mg/dl), and 5.17 ± 0.12 mmol/l (93 ± 2 mg/dl), respectively. In all insulin clamp studies, the coefficient of variation in plasma glucose concentration was <5%.
Before start of the low-dose insulin infusion, basal hepatic glucose
production (which equals total body glucose disposal under
postabsorptive conditions) was 3.03 ± 0.37 and 2.85 ± 0.22 mg · kg FFM1 · min
1 in the
24- and 72-h groups, respectively, and did not change after 24 (3.25 ± 0.25 mg · kg
FFM
1 · min
1) or 72 h
(3.31 ± 0.15 mg · kg
FFM
1 · min
1; P = NS) of experimental hyperinsulinemia. It should be noted that the
repeat measurement of basal hepatic glucose production was performed
2 h after the chronic low-dose insulin infusion was stopped. The
ability of insulin to suppress hepatic glucose production during the
insulin clamp was not impaired by long-term insulin infusion (before
vs. after in 24-h group: 0.78 ± 0.37 vs. 0.51 ± 0.28 mg · kg FFM
1 · min
1,
P = NS; 72-h group: 0.35 ± 0.14 vs. 0.31 ± 0.18 mg · kg FFM
1 · min
1,
P = NS). During the euglycemic insulin clamp studies
performed before low-dose insulin infusion, total body glucose disposal was 10.6 ± 1.1 (24-h group) and 9.69 ± 0.71 mg · kg
FFM
1 · min
1 (72-h group; Fig. 1).
During the insulin clamp studies performed after 24 and 72 h of
low-dose insulin infusion, total body glucose disposal was reduced to
9.06 ± 1.1 (P < 0.05) and 7.50 ± 0.69 mg · kg FFM
1 · min
1
(P < 0.01), respectively (Fig. 1).
Basal glucose oxidation increased from 2.38 ± 0.30 to 4.30 ± 0.36 mg · kg FFM1 · min
1
(P < 0.01) after 24 h of continuous low-dose
insulin infusion and from 2.70 ± 0.11 to 4.54 ± 0.34 mg · kg FFM
1 · min
1
(P < 0.01) after 72 h of low-dose insulin
infusion. Insulin-mediated glucose oxidation increased from 2.38 ± 0.30 to 4.74 ± 0.24 mg · kg
FFM
1 · min
1 (P < 0.05) during the baseline insulin clamp and to 5.28 ± 0.17 during
the insulin clamp performed after 24 h of insulin infusion (Fig.
1). Insulin-mediated glucose oxidation increased from 2.70 ± 0.11 to 4.66 ± 0.35 (P < 0.01) during the baseline
insulin clamp and to 5.40 ± 0.51 mg · kg
FFM
1 · min
1 during the insulin
clamp performed after 72 h of insulin infusion (Fig. 1). However,
the increment in glucose oxidation above baseline during the insulin
clamp was lower after 24 h (
= 0.98 ± 0.30 mg · kg FFM
1 · min
1) and
after 72 h (0.81 ± 0.39 mg · kg
FFM
1 · min
1) of sustained low-dose
insulin infusion compared with the increment during the baseline study
(
= 2.36 ± 0.21, P < 0.01, and 1.96 ± 0.29 mg · kg
FFM
1 · min
1, P < 0.05, during the 24- and 72-h baseline studies, respectively). Insulin-stimulated nonoxidative glucose disposal during the baseline insulin clamp was 5.82 ± 1.16 and 5.22 ± 0.76 mg · kg FFM
1 · min
1 in the
24- and 72-h groups, respectively (Fig. 1). During the repeat insulin
clamp study after 24 and 72 h of constant low-dose insulin
infusion, insulin-stimulated nonoxidative glucose disposal declined to
3.77 ± 1.16 (P < 0.05) and to 2.18 ± 0.44 (P < 0.01) mg · kg
FFM
1 · min
1, respectively (Fig. 1).
In both groups, the decrement in nonoxidative glucose metabolism was
strongly correlated with the decrement in total body insulin-mediated
glucose disposal (24-h group: r = 0.90, P < 0.01; 72-h group: r = 0.91, P < 0.001).
Basal lipid oxidation (24-h group: 0.96 ± 0.14; 72-h group:
0.82 ± 0.06 mg · kg
FFM1 · min
1) decreased
significantly after 24 h (0.22 ± 0.16 mg · kg
FFM
1 · min
1, P < 0.01) and 72 h (
0.23 ± 0.13 mg · kg
FFM
1 · min
1, P < 0.01) of continuous low-dose insulin infusion. In both groups, the
decrement in basal lipid oxidation was strongly correlated with the
increment in basal glucose oxidation (24-h group: r = 0.96, P < 0.01; 72-h group: r = 0.83, P < 0.01). Insulin-mediated suppression of lipid
oxidation during the insulin clamp study performed after 72 h of
constant insulin infusion was significantly greater than during the
baseline study (0.08 ± 0.15 vs.
0.59 ± 0.13 mg · kg FFM
1 · min
1,
P < 0.01). The decline in lipid oxidation during the
insulin clamp study performed after 24 h of constant insulin
infusion also was significantly greater than during the baseline study (
0.09 ± 0.15 vs.
0.27 ± 0.11 mg · kg
FFM
1 · min
1, P = NS).
To examine the molecular and biochemical events by which physiological
hyperinsulinemia inhibits insulin-stimulated glucose disposal, HKII and
HKI mRNA content/enzyme activity, GLUT-4 protein, and GS activity were
assayed on muscle biopsy samples obtained before and after the
euglycemic insulin clamp studies performed before and after 24 and
72 h of experimental hyperinsulinemia. Before the low-dose insulin
infusion was started, HKII mRNA increased from 0.177 ± 0.025 to
0.310 ± 0.059 units (relative to the 28S internal control signal;
P < 0.01) during the euglycemic insulin clamp (Fig.
2). Similarly, during the insulin clamp
performed after 24 h of experimental hyperinsulinemia, HKII mRNA
increased from 0.189 ± 0.056 to 0.279 ± 0.061 units
(P < 0.05) and after 72 h of hyperinsulinemia
from 0.192 ± 0.440 to 0.344 ± 0.079 (P < 0.05 vs. baseline; P = NS vs. 24-h value; Fig. 2).
During the baseline euglycemic insulin clamp, HKII activity in the
soluble (cytosolic) fraction increased from 2.12 ± 0.37 to
3.18 ± 0.35 pmol · min1 · µg
protein
1 (P < 0.01; Fig. 2). During the
insulin clamp studies carried out after 24 and 72 h of
experimental hyperinsulinemia, cytosolic HKII activity rose from
2.33 ± 0.38 to 3.21 ± 0.69 (P < 0.05) and
from 2.11 ± 0.52 to 3.16 ± 0.58 pmol · min
1 · µg protein
1
(P < 0.05; Fig. 2). HKI mRNA content and activity
(Table 1) remained unchanged during the low-dose insulin infusion and insulin clamp studies.
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In the baseline euglycemic insulin clamp study, GSFV
increased from 0.080 ± 0.01 to 0.210 ± 0.02 (P < 0.001; Fig. 2), and the increment in GS activity
was correlated with both the increment in nonoxidative glucose disposal
(r = 0.54, P < 0.01) and the increment
in total body glucose uptake (r = 0.52, P < 0.01). After 24 h of experimental
hyperinsulinemia, the increment (change from baseline) in
GSFV (from 0.088 ± 0.013 to 0.152 ± 0.013, P < 0.001) during the insulin clamp was significantly
impaired compared with the baseline insulin clamp study
(P < 0.05). After 72 h of experimental hyperinsulinemia, the increment in GSFV (from 0.069 ± 0.008 to 0.157 ± 0.023, P < 0.001)
during the insulin clamp remained impaired (Fig.
3). The decrease in insulin-mediated
stimulation of GSFV after 72 h of experimental
hyperinsulinemia was slightly, but not significantly, greater than the
decrease observed after 24 h of experimental hyperinsulinemia. The
decrease in GS activity at 72 h was not correlated
(r = 0.074, P = NS) with the decrease in nonoxidative glucose metabolism or the decrease in total body glucose uptake during the insulin clamp.
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To determine whether decreased GLUT-4 expression, induced by
hyperinsulinemia, could account for the decrease in insulin-stimulated whole body glucose uptake and nonoxidative glucose disposal, GLUT-4 protein content was compared in muscle biopsies before and after experimental hyperinsulinemia in five subjects from whom sufficient biopsy material remained after HK and GS assays. Three subjects had
participated in the 24-h and two in the 72-h insulin infusion studies.
Baseline GLUT-4 protein was 0.94 ± 0.31 units and remained essentially unchanged, 0.91 ± 0.22 units, in the five subjects after 24-72 h of experimental hyperinsulinemia. We have previously shown that, during a 4-h 40 mU · m2 · min
1 euglycemic
insulin clamp, hyperinsulinemia had no effect on GLUT-4 protein content
(26).
In six subjects who participated in the 72-h insulin infusion, muscle
glycogen concentration, measured before the start of each euglycemic
insulin clamp, did not change (116 ± 28 vs. 126 ± 29 mmol/kg wet wt, P = NS). Nonetheless, total body
glucose disposal (9.29 ± 0.98 to 7.36 ± 0.63 mg · kg FFM1 · min
1) and
nonoxidative glucose disposal (4.69 ± 0.68 to 2.62 ± 0.34 mg · kg FFM
1 · min
1)
consistently decreased in all of the six subjects (both
P < 0.05).
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DISCUSSION |
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We previously have shown that sustained physiological euglycemic hyperinsulinemia for 48-72 h reduced insulin-mediated total body glucose uptake and nonoxidative glucose metabolism in healthy subjects (11). In the present study, we demonstrate that 24 and 72 h of sustained physiological hyperinsulinemia specifically inhibits the ability of insulin to increase nonoxidative glucose disposal [which primarily represents glycogen formation (44)] by 34 (P < 0.05) and 58% (P < 0.01), respectively, in association with an impaired ability of insulin to stimulate glycogen synthase activity. There was no significant difference between the magnitude of decrease in nonoxidative glucose disposal at 24 and 72 h, indicating that the insulin-induced insulin resistance is maximally established within 24 h of exposure to hyperinsulinemia. The constant, low-dose insulin infusion rate was chosen to achieve plasma insulin concentrations (15-30 µU/ml) similar to those in obese nondiabetic and both lean and obese type 2 diabetic subjects (8, 36). These results provide the first documentation in humans that physiological hyperinsulinemia inhibits insulin-stimulated muscle glycogen synthase in association with a decrease in nonoxidative glucose disposal (glycogen synthesis).
Glycogen synthase plays a pivotal role in the regulation of muscle glycogen synthesis. In the present study, a preserved ability of insulin to stimulate glycolysis in the presence of a reduced ability to augment nonoxidative glucose disposal (glycogen synthesis) suggests a postglucose transport/postphosphorylation defect in insulin action that selectively involves the glycogen synthetic pathway. Consistent with this hypothesis, glycogen synthase fractional velocity was reduced during both the 24- and 72-h insulin infusion studies. However, unlike the initial insulin clamp study (r = 0.54, P < 0.01), the normal correlation between muscle glycogen synthase activity and in vivo nonoxidative glucose disposal was not observed during the insulin clamp performed after 24-72 h of low-dose insulin infusion (r = 0.074, P = NS). Several possible explanations could explain the lack of correlation. 1) Basal lipid oxidation (and presumably basal FFA concentration, not measured) declined after 24-72 h of sustained physiological hyperinsulinemia. Decreased FFA oxidation/FFA concentrations are known to augment nonoxidative glucose disposal (glycogen synthesis) (42). Thus opposing effects (decreased plasma FFA concentration/oxidation to augment glucose disposal) and chronic physiological hyperinsulinemia (to inhibit nonoxidative glucose disposal) may have obscured the expected correlation between glycogen synthase activity and nonoxidative glucose disposal. 2) Considerable evidence supports the existence of multiple intracellular pathways of insulin receptor signal transduction (1, 28, 30, 38, 39), as well as subcellular compartmentalization of these insulin-signaling pathways (17). Thus one could postulate that hyperinsulinemia altered pathways of insulin signaling that specifically led to impaired glucose transport, while having less of an effect on pathways involved in the regulation of glycogen synthase. Alternatively, hyperinsulinemia conceivably could have interfered with the processes leading to the fusion of glucose transport vesicles with the plasma membrane (47, 49). 3) Chronic stimulation of the insulin receptor by sustained physiological hyperinsulinemia causes a compensatory increase in serine phosphorylation, leading to downregulation of insulin receptor tyrosine phosphorylation (5, 7, 20). The activation of serine kinases also could inhibit glycogen synthesis (nonoxidative glucose disposal) by mechanisms that do not involve glycogen synthase, thereby disrupting the normal relationship between glycogen synthase activity and in vivo glycogen synthesis. 4) Stimulation of glycolytic flux by 24-72 h of hyperinsulinemia could lead to a decline in intracellular UDGP and G-6-P concentrations, causing a decrease in in vivo muscle glycogen synthesis (41).
Several studies have suggested that increased intracellular muscle glycogen concentration can inhibit glycogen synthase activity through modulation of glycogen synthase phosphatase (16, 48). However, an equal number of studies have failed to support a physiological role for changes in muscle glycogen concentration in the regulation of glycogen synthase activity (25, 29). In the present study, muscle glycogen concentration did not change after 72 h of low-dose insulin infusion, and there was no correlation between the change in glycogen synthase activity and the change in nonoxidative glucose disposal. Therefore, it seems unlikely that the insulin-induced decreases in glycogen synthase activity and nonoxidative glucose disposal can be explained by an increase in muscle glycogen content. Our failure to observe an increase in muscle glycogen concentration is consistent with previous observations in humans that elevations in the plasma insulin concentration of <30 µU/ml do not stimulate nonoxidative glucose disposal (14). Similarly, in vitro studies with human muscle cells failed to demonstrate any effect of low insulin concentrations for 4 days on muscle glycogen content (18). Although it seems unlikely that the level of sustained hyperinsulinemia employed in the present study had any stimulatory effect on muscle glycogen formation, it should be noted that the muscle glycogen assay employed in the present study cannot detect increments in muscle glycogen concentration <8%. Therefore, we cannot exclude the possibility that sustained physiological hyperinsulinemia caused a small increase in muscle glycogen concentration, which subsequently caused an inhibition of glycogen synthase activity.
Experiments in transgenic mice (37) indicate that the early steps of intracellular metabolism, i.e., glucose transport and glucose phosphorylation, play a crucial role in the regulation of glycogen formation. Similarly, in humans, Shulman et al. (43) have suggested that activation of glycogen synthase is primarily regulated by substrate availability, i.e., the rates of glucose transport and glucose phosphorylation. Therefore, we quantified HKII and HKI activity and mRNA levels in the basal state and in response to insulin before and after 72 h of sustained physiological hyperinsulinemia. Both HKII activity and mRNA levels increased normally (26) during the 4-h insulin clamp performed after 3 days of low-dose insulin infusion. HKI activity and mRNA levels were not altered by 72 h of physiological hyperinsulinemia. Similarly, hyperinsulinemia did not alter the levels of GLUT-4 protein in muscle. However, these studies do not exclude defects in GLUT-4 translocation or intrinsic activity of the glucose transporter. The possibility that sustained physiological hyperinsulinemia inhibits the insulin receptor signal transduction system was not examined in the present study but remains an attractive hypothesis.
As expected, the increase in basal glucose oxidation was accompanied by a proportional decrease in lipid oxidation, as expected by the Randle cycle (35). Basal glucose oxidation increased threefold in response to the sustained low-dose insulin infusion, consistent with previously published results (11). During the insulin clamp performed after 24-72 h of experimental hyperinsulinemia, glucose oxidation was increased compared with the baseline clamp study. However, the increment in glucose oxidation above baseline during the insulin clamp was markedly reduced, most probably reflecting the achievement of near-maximal stimulation of the glycolytic/oxidative pathway by the continuous low-dose insulin infusion.
Unlike in muscle, sustained physiological hyperinsulinemia for 24-72 h did not induce hepatic resistance to insulin. Basal hepatic glucose production did not change after 24-72 h of experimental hyperinsulinemia, and the ability of insulin to suppress hepatic glucose production was not impaired. Whether more prolonged exposure of the liver would result in the development of hepatic insulin resistance remains to be determined.
In summary, 24-72 h of sustained physiological hyperinsulinemia, in the range of fasting insulin concentrations observed in insulin-resistant conditions such as obesity and type 2 diabetes mellitus, impaired the ability of insulin to increase glycogen synthase activity and to augment insulin-mediated nonoxidative glucose metabolism. These experimentally induced abnormalities in glycogen synthase activity and nonoxidative glucose disposal closely mimic disturbances seen in conditions characterized by hyperinsulinemia and insulin resistance. including obesity, impaired glucose tolerance, and type 2 diabetes mellitus (8). When viewed in the context of the present results, the "compensatory" hyperinsulinemia observed in all of the preceding conditions may represent a self-perpetuating or aggravating cause of the insulin resistance.
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ACKNOWLEDGEMENTS |
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We thank the nurses in the General Clinical Research Center (GCRC) for the diligent care of our volunteers, and especially Patricia Wolff and Norma Diaz for their expert assistance in carrying out the insulin clamp/muscle biopsy studies. We gratefully acknowledge the technical assistance of Andrea Barrentine, Kathy Camp, Jean Finlayson, Gilbert Gomez, Cindy Munoz, and Sheila Taylor. Lorrie Albarado and Jerry Lynn Beesley provided skilled secretarial support in the preparation of the manuscript.
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FOOTNOTES |
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This work was supported by GCRC Grant RR-01346, National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK-24092 (R. A. DeFronzo) and R01 DK-47936 (L. Mandarino), by a Veterans Affairs (VA) Merit Award (R. A. DeFronzo), and by the VA Medical Research fund.
Address for reprint requests and other correspondence: R. A. DeFronzo, Diabetes Division, Dept. of Medicine, Univ. of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7886.
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. Section 1734 solely to indicate this fact.
Received 17 March 2000; accepted in final form 18 January 2001.
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