ORIGINAL
E n d o c r i n e
ARTICLE
R e s e a r c h
Impaired Muscle AMPK Activation in the Metabolic
Syndrome May Attenuate Improved Insulin Action
after Exercise Training
Andrew S. Layne, Sami Nasrallah, Mark A. South, Mary E. A. Howell,
Melanie P. McCurry, Michael W. Ramsey, Michael H. Stone,
and Charles A. Stuart
The Center of Excellence for Sport Science and Coach Education (A.S.L., M.A.S., M.W.R., M.H.S.),
Department of Kinesiology, Leisure, and Sports Science, Clemmer College of Education, and Department
of Internal Medicine (S.N., M.E.A.H., M.P.M., C.A.S.), Quillen College of Medicine, East Tennessee State
University, Johnson City, Tennessee 37614
Context: Strength training induces muscle remodeling and may improve insulin responsiveness.
Objective: This study will quantify the impact of resistance training on insulin sensitivity in subjects
with the metabolic syndrome and correlate this with activation of intramuscular pathways mediating mitochondrial biogenesis and muscle fiber hypertrophy.
Design: Ten subjects with the metabolic syndrome (MS) and nine sedentary controls underwent 8
wk of supervised resistance exercise training with pre- and posttraining anthropometric and muscle biochemical assessments.
Setting: Resistance exercise training took place in a sports laboratory on a college campus.
Main Outcome Measures: Pre- and posttraining insulin responsiveness was quantified using a
euglycemic clamp. Changes in expression of muscle 5-AMP-activated protein kinase (AMPK) and
mammalian target of rapamycin (mTOR) pathways were quantified using immunoblots.
Results: Strength and stamina increased in both groups. Insulin sensitivity increased in controls
(steady-state glucose infusion rate ⫽ 7.0 ⫾ 2.0 mg/kg 䡠 min pretraining training vs. 8.7 ⫾ 3.1
mg/kg 䡠 min posttraining; P ⬍ 0.01) but did not improve in MS subjects (3.3 ⫾ 1.3 pre vs. 3.1 ⫾ 1.0
post). Muscle glucose transporter 4 increased 67% in controls and 36% in the MS subjects. Control
subjects increased muscle phospho-AMPK (43%), peroxisome proliferator-activated receptor ␥
coactivator 1␣ (57%), and ATP synthase (60%), more than MS subjects (8, 28, and 21%, respectively).
In contrast, muscle phospho-mTOR increased most in the MS group (57 vs. 32%).
Conclusion: Failure of resistance training to improve insulin responsiveness in MS subjects was
coincident with diminished phosphorylation of muscle AMPK, but increased phosphorylation of
mTOR, suggesting activation of the mTOR pathway could be involved in inhibition of exercise
training-related increases in AMPK and its activation and downstream events. (J Clin Endocrinol
Metab 96: 1815–1826, 2011)
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2010-2532 Received October 27, 2010. Accepted March 11, 2011.
First Published Online April 20, 2011
Abbreviations: AMPK, 5-AMP-activated protein kinase; GIR, glucose infusion rate; GLUT4,
glucose transporter 4; HDL, high-density lipoprotein; IPF, isometric PF; LBM, lean body
mass; LDL, low-density lipoprotein; MS, metabolic syndrome; mTOR, mammalian target of
rapamycin; PF, peak force; PGC-1␣, peroxisome proliferator-activated receptor ␥ coactivator 1␣; RFD, rate of force development; S6K1, 70-kDa S6 protein kinase; VO2max,
maximal oxygen consumption.
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
jcem.endojournals.org
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Layne et al.
Muscle AMPK Activation in Metabolic Syndrome
he prevalences of obesity, the metabolic syndrome
(MS), and diabetes have increased globally in the past
two decades. Low physical activity levels (1) and low cardiovascular fitness (2) are associated with increased risk of
the MS. Exercise is considered one of the cornerstones of
diabetes prevention and treatment. Both endurance and
strength training ameliorate insulin resistance (3–5) and
improve blood sugar control (3, 6). However, the skeletal
muscle adaptations and the signaling pathways through
which these adaptations occur appear to be specific to the
type of exercise performed (7).
Endurance training increases the oxidative capacity of
both type 1 (slow twitch, red) and type 2 (fast twitch,
white) muscle fibers primarily by increasing oxidative enzyme content through up-regulation of mitochondrial biogenesis (8). These adaptations enhance the efficiency of
energy production from fatty acids and glucose. 5-AMPactivated protein kinase (AMPK) is a key energy sensor in
most cells and is activated during exercise by an increase
in the AMP:AMP ratio (9). Activation of peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣), a
downstream target of AMPK, turns on expression of mitochondrial genes, both in mitochondria and the nucleus
(10). Activation of the AMPK signaling pathway is involved in the exercise-related acute translocation of glucose transporter 4 (GLUT4) to the muscle plasma membrane (11) and may be involved in increased GLUT4
expression seen with endurance training (12).
Resistance training results in skeletal muscle hypertrophy, particularly in type 2 fibers, with a concomitant increase in muscular strength (13). Increased protein synthesis occurs through a cell signaling pathway involving
mammalian target of rapamycin (mTOR). mTOR integrates intracellular and extracellular signals from growth
factors, substrate availability and cellular energy levels to
regulate metabolic responses within the cell (14). Upstream of mTOR, growth factors, including insulin and
IGF-I, activate a cascade involving phosphatidylinositol
3-kinase, phosphoinositide-dependent kinase 1, and Akt.
Akt activation results in phosphorylation of mTOR.
mTORC1 in turn phosphorylates 70-kDa S6 protein kinase (S6K1) and 4E binding proteins resulting in increased
protein synthesis (14).
The aim of the current study was to evaluate the impact
of a resistance training program on insulin responsiveness
in subjects with the MS and to quantify the changes in the
intramyocyte pathways that mediate changes in insulin
action through mitochondrial biogenesis and/or muscle
fiber hypertrophy in these subjects.
T
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
Subjects and Methods
Subject selection
Nineteen sedentary subjects were recruited to undergo 8 wk
of supervised resistance exercise. None of the subjects had performed regular exercise for at least 1 yr. The research protocol
and the consent documents were approved by the East Tennessee
State University Institutional Review Board. The exercise program was performed at the East Tennessee State University Exercise and Sports Sciences Laboratory supervised by students and
faculty from the Department of Kinesiology. Subjects were recruited into two groups: high risk for type 2 diabetes [body mass
index (BMI) ⱖ30 kg/m2; waist circumference ⱖ40 in. for males
or ⱖ35 in. for females; family history of type 2 diabetes] and low
risk for type 2 diabetes (BMI ⬍30 kg/m2; no family history of
type 2 diabetes). The 10 subjects at high risk for diabetes qualified for the designation MS, as set forward by the American
Diabetes Association and the World Health Organization (15).
All 10 had BMI higher than 30 kg/m2 and waist circumference
over 40 in. and exhibited insulin resistance in insulin infusion
studies. Subjects were instructed to maintain weight during the
study period. Muscle biopsies, insulin infusions, blood pressure,
blood lipids, body composition, and strength and endurance
measurements were performed before and after the full 8-wk
training program.
Exercise protocol
Training consisted of large muscle mass free weight exercises
(Supplemental Table 1, published on The Endocrine Society’s
Journals Online web site at http://jcem.endojournals.org). A
light familiarization and baseline measurement period was followed by 4 wk of high-volume, low-intensity training (phase I).
The intensity of the exercise (based on an estimate of relative
repetition maximum) increased approximately 10% each week
for the first 3 wk, followed by a 10% drop in intensity from wk
3 to wk 4 to allow for recovery. During phase II (wk 5– 8), volume
decreased and intensity increased to allow for greater strength
gains. Similar to phase I, intensity increased approximately 10%
week to week and was decreased by 10% during wk 8 to allow
for recovery. All sets (warm-up and target sets) were recorded
and calculated as total volume load (sets ⫻ repetitions ⫻ weight).
Subject assessments
Several anthropometric descriptors were measured at baseline and after 8 wk of resistance training. Body composition was
measured by air displacement plethysmography (BodPod, Concord, CA). Waist circumference was measured just above the
iliac crest to the nearest 0.1 cm.
Strength testing
Strength was assessed isometrically using a custom-built lifting rack. The lifting rack was set so that the subjects pulled from
the mid-thigh pull position used in training. Peak force (PF) was
measured on a force plate (Rice Lake Weighing Systems, Rice
Lake, WI) with a sampling rate of 1000 Hz. Data were collected
and analyzed for PF and rate of force development (RFD) using
custom Labview version 8.6 software (National Instruments,
Upper Saddle River, NJ).
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
Endurance testing
Endurance was measured using a Monark Ergomedic 874E
cycle ergometer (Monark Exercise AB, Vansbro, Sweden). Expired air was analyzed using a TrueOne 2400 Metabolic Measurement System (ParvoMedics, Sandy, UT). Heart rate, maximal oxygen consumption (VO2max), respiratory exchange
ratio, and time to exhaustion were recorded.
Muscle biopsies
Percutaneous needle biopsies of vastus lateralis were performed after an overnight fast and 2 h of quiet recumbency as
previously described, using a Bergstrom-Stille 5-mm muscle biopsy needle with suction (16). The second biopsy was performed
24 – 48 h after the last training session. The sample was divided
in half, with one piece frozen immediately in liquid nitrogen for
later analysis and the second piece mounted on cork and quickly
frozen in a slurry of isopentane cooled in liquid nitrogen. The
cork-mounted piece was stored at ⫺80 C and later sectioned on
a cryostat (Leica, Wetzlar, Germany) for evaluation of fiber type
composition.
Euglycemic-hyperinsulinemic clamp
After a 2-h baseline period, a single infusion of regular insulin
was performed at 15 mU/m2 䡠 min for 3 h to achieve a physiological increment in insulin concentration of about 50 ␮U/ml and
a stable glucose infusion rate (GIR) to quantify insulin sensitivity
as previously described (17).
Quantification of muscle fiber type composition
and fiber size
Fiber composition was determined using methods described
by Behan et al. (18). Muscle sections were stained for light microscopy in a two-step method using commercial monoclonal
antibodies to fast and slow isoforms of myosin heavy chain. After
acetone fixation and incubation with 20% normal rabbit serum,
the slow myosin antibody (Sigma clone NOQ7.5.4D; SigmaAldrich, St. Louis, MO) was applied, followed by a peroxidaseconjugated rabbit antimouse IgG antibody. The fast myosin antibody (Sigma clone MY-32 alkaline phosphatase conjugate)
was then applied (18). Slides were alcohol dehydrated, cleared
with xylene, and preserved in synthetic medium. This technique
allows discrimination of type 1, type 2a, and type 2b. All sections
were coded and then quantified independently by three observers
who were unaware of which subject or treatment the image
represented.
Preparation of muscle homogenates
A small piece of muscle was removed from the ⫺80 C freezer
and slowly thawed on ice. Muscle homogenate was prepared by
placing 25–50 mg muscle in 500 ␮l 0.25 M sucrose, 20 mM
HEPES (pH 7.4) containing protease inhibitors (Halt Protease
Inhibitor Cocktail Kit; Pierce, Rockford, IL) and homogenized
with two 30-sec bursts of a hand-held homogenizer (Pellet Pestle
Motor; Kontes, Vineland, NJ).
Mitochondrial markers
A mixture of antibodies to five different mitochondrial components (no. MS604) were purchased from MitoSciences (Eugene, OR). These antibodies were directed against complexes I,
II, III, and IV and to ATP synthase subunit ␣. The principal
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component used for these analyses was ATP synthase. For these
studies, 4 –20% gradient gels (Thermo Scientific, Rockford, IL)
were loaded with 7.5 ␮g protein from muscle homogenates. Blots
were blocked with 0.25% nonfat dry milk.
Key regulatory factors (AMPK, PGC-1␣, mTOR, and
S6K1)
Comparison of expression of these proteins were also quantified in immunoblots as previously described (19). Antibodies
for AMPK, phospho-AMPK␣1, mTOR, phospho-mTOR, and
phospho-p70 S6 kinase (2532, 2531, 2972, 2971, and 9205)
were purchased from Cell Signaling Technology (Danvers, MA).
The antibody for PGC-1␣ (AB3242) was purchased from Millipore (Billerica, MA). For AMPK and phospho-AMPK, samples
containing 5 ␮g protein from muscle homogenates were applied
to 10% polyacrylamide gels. Immunoblots were blocked with
5% nonfat dry milk. Gradient gels (3– 8%) (Invitrogen, Carlsbad, CA) were used for mTOR and phospho-mTOR immunoblots. These samples were 10 ␮g per lane, and blocking was
0.25% nonfat dry milk. PGC-1␣ immunoblots were from 10%
polyacrylamide gels and were blocked with 0.5% nonfat milk.
Immunoblots for phospho-S6K1 also used 10% polyacrylamide
gels and 0.5% nonfat milk for blocking.
Expression of principal muscle hexose transporters
(GLUT4 and GLUT5)
The techniques for quantifying these glucose transporters
were described previously (16, 17). Affinity-purified rabbit antibodies against human GLUT5 (GT52-A) were purchased from
Alpha Diagnostics (San Antonio, TX). GLUT4 antibodies
(AB1049, goat antihuman) were purchased from Chemicon (Temecula, CA).
Statistics
All data are displayed as mean ⫾ SD, except as explicitly indicated. ANOVA was used for data assessment, and for each
study group, the paired t test was used for comparing mean levels
before and after training. Comparing mean data between the
two groups was performed using the independent t test. Effect
size correlations were calculated using Cohen’s d (20). Rela-
TABLE 1. Subject characteristics at baseline
Controls
9
5
36.4 ⫾ 12.2
69.2 ⫾ 16.2
24.3 ⫾ 3.6
113 ⫾ 10
MS subjects
10
5
45.0 ⫾ 8.6
99.5 ⫾ 13.8a
33.7 ⫾ 3.0a
131 ⫾ 20
Number (n)
Female gender (n)
Age (yr)
Body mass (kg)
BMI (kg/m2)
Resting systolic blood pressure
(mm Hg)
Resting diastolic blood pressure
77 ⫾ 10
82 ⫾ 9
(mm Hg)
Fasting blood sugar (mM)
4.9 ⫾ 0.7
5.7 ⫾ 0.7
Fasting serum insulin (pmol/liter)
53 ⫾ 25
103 ⫾ 67a
VO2max (ml/kg 䡠 min)
31.0 ⫾ 4.5
23.3 ⫾ 3.1a
IPF (N/kg2/3)
134 ⫾ 37
135 ⫾ 40
RFD (N/sec)
3180 ⫾ 1320 2940 ⫾ 2170
Unless indicated otherwise, data are mean ⫾ SD.
a
Significant difference from controls, P ⬍ 0.01.
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Muscle AMPK Activation in Metabolic Syndrome
TABLE 2. The impact of 8 wk resistance training on
anthropometrics
Controls (n ⴝ 9)
Body Mass (kg)
Pre
Post
d
BMI (kg/m2)
Pre
Post
d
Waist circumference (cm)
Pre
Post
d
Fat mass (kg)
Pre
Post
d
LBM (kg)
Pre
Post
d
% Body fat
Pre
Post
d
MS (n ⴝ 10)
69.2 ⫾ 6.2
69.2 ⫾ 16.1
0.004
99.5 ⫾ 13.8a
99.3 ⫾ 4.3a
⫺0.020
24.3 ⫾ 3.6
24.5 ⫾ 3.3
0.057
33.7 ⫾ 3.0a
34.0 ⫾ 3.3a
0.099
93 ⫾ 11
90 ⫾ 2
⫺0.319
116 ⫾ 6a
114 ⫾ 8a
⫺0.375
19.8 ⫾ 9.3
19.5 ⫾ 9.1
⫺0.024
41.4 ⫾ 8.1a
41.3 ⫾ 8.4a
⫺0.006
48.4 ⫾ 9.9
49.7 ⫾ 9.9c
0.141
56.8 ⫾ 9.5a
58.0 ⫾ 10.1a,b
0.127
28.3 ⫾ 9.4
27.5 ⫾ 8.4
⫺0.095
42.2 ⫾ 0.4a
41.7 ⫾ 5.3a,c
⫺0.102
Data are mean ⫾ SD. d, Cohen’s d effect size.
a
P ⬍ 0.01, compared with controls, independent t test.
b
P ⬍ 0.05, compared with baseline, paired t test.
c
P ⬍ 0.01, compared with baseline, paired t test.
tionships between select variables were assessed using a Pearson correlation coefficient. Statistical procedures were performed using SigmaStat version 3.11 from Systat Software
(San Jose, CA).
Results
Subject characteristics
The baseline characteristics of the MS subjects were
very different from the sedentary control subjects in key
variables as listed in Table 1. Body mass and BMI were 30
and 35% higher in MS subjects. Mean fasting insulin concentration was 94% higher in MS subjects. VO2max at
baseline was 25% lower in the MS group. Age, blood
pressure, and fasting glucose concentration tended to be
higher in MS subjects, but these differences were not statistically significant. Additional baseline data are included
in subsequent tables. The fasting glucose, insulin, lipids,
and blood pressures were determined on the day of the
muscle biopsy and euglycemic clamp study before and after the resistance training protocol.
Anthropometrics, functional capabilities, and
volume load
Eight weeks of resistance training had a positive impact
on body composition (Table 2). Overall, training had little
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
effect on body mass or fat mass; however, the percent
change in body mass was strongly correlated with the percent gain in lean body mass (LBM) in both groups (r ⫽
0.532). Although the increase in LBM was relatively small
(d ⫽ 0.127 and 0.141), the change was statistically significant in both groups. The decrease in waist circumference was statistically significant (P ⫽ 0.022); however, the
effect size was moderate in MS and controls (⫺0.375 and
⫺0.319, respectively). Training tended to decrease body
fat percentage in both groups, but the change was statistically significant only in MS (P ⫽ 0.010).
Overall, VO2max increased 10% and time to exhaustion increased approximately 35% in both groups. The
increase in VO2max was statistically significant in both
groups, and both groups achieved relatively large effect
sizes (d ⫽ 0.819 in MS subjects and 0.535 in controls). The
increase in time to exhaustion was also statistically significant (P ⬍ 0.001). MS subjects improved in isometric PF
(IPF) by 13% and RFD by 28% after training. Controls
tended to increase in IPF (7%) and RFD (15%), but the
increases were not statistically significant.
Volume load, calculated as sets ⫻ repetitions ⫻
weight, was tracked for each subject. Average weekly
volume load is listed in Table 3. Total volume load was
16% higher in MS subjects than in controls (93,800 ⫾
32,200 and 80,700 kg ⫾ 27,000 kg, respectively); however, this difference was not statistically significant. IPF
measured before training and total volume load were
strongly correlated (r ⫽ 0.681), indicating that initially
stronger subjects were able to handle heavier loads during training. Allometric scaling (divide by weight in kilograms raised to the 2/3 power) adjusts for body size to
allow males and females to be similar when combined.
Because the MS subjects were much heavier, their unscaled volume load was 16% higher, but when allometrically scaled, the volume load was 9% lower than
that of controls.
There was variability in initial strength among subjects
in both groups (SD of allometrically scaled baseline peak
power was 28%). To consider whether there was an impact of the differences in workload among subjects, we
quantified for each subject the volume load of the initial
week, the peak volume load (usually in wk 7), and the
total volume load from 8 wk of training sessions. Those
data (raw data and allometrically scaled) were compared with changes in strength, LBM, insulin responsiveness, VO2max, and changes in muscle expression of
GLUT4, ATP synthase, phospho-AMPK, and phosphomTOR using the Pearson product moment correlation.
We found no statistically significant correlation of the minor subject differences in workload to the differences in
response in any of these outcome variables.
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
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TABLE 3. Average weekly volume load
Week
1
2
3
4
5
6
7
8
Controls (n ⴝ 9)
5860 ⫾ 2,570
10,100 ⫾ 3,870
10,300 ⫾ 4,550
11,200 ⫾ 3,910
11,000 ⫾ 3,750
12,200 ⫾ 3,810
13,600 ⫾ 3,870
6,460 ⫾ 1,910
MS (n ⴝ 10)
7,010 ⫾ 5,160
11,200 ⫾ 4,980
12,600 ⫾ 5,050
12,800 ⫾ 4,460
13,200 ⫾ 3,740
11,500 ⫾ 3,870
15,000 ⫾ 4,500
7,460 ⫾ 2,090
Volume load is an estimate of total work performed and is a product
of the sets, repetitions, and load lifted.
12
10
**
8
6
4
*
*
2
0
ne
eli
d
ine
tra
s
ba
d
ine
tra
Skeletal muscle fiber composition
Percutaneous muscle biopsies of the vastus lateralis
were performed at baseline and after training, and muscle
metabolic
syndrome
14
ne
eli
Euglycemic-hyperinsulinemic clamp
Insulin sensitivity was assessed using the euglycemic-hyperinsulinemic clamp technique described in the Subjects and
Methods. Fasting insulin was higher in MS subjects both at
baseline and after training (103 ⫾ 67 vs. 97 ⫾ 58 pmol/liter).
Fasting insulin decreased 22% (53 ⫾ 25 vs. 42 ⫾ 27 pmol/
liter) in controls after training (P ⬍ 0.05). Fasting blood
glucose was higher in MS subjects than controls before
and after training.
As shown in Fig. 1, MS subjects showed marked insulin
resistance at baseline (47% of control baseline, P ⬍ 0.01
by independent t test), and training had little effect on GIR
(P ⫽ 0.37, paired t test). In contrast, controls demonstrated normal insulin sensitivity before training (GIR ⫽
7.0 ⫾ 2.0 mg/kg 䡠 min), and GIR increased 25% after resistance training (P ⫽ 0.03, paired t test). The increment
in insulin concentration achieved by the insulin infusion
was similar in both groups before and after training. The
insulin concentration increment averaged 52 and 52
␮U/ml for MS subjects and controls at baseline and 52 and
49 ␮U/ml after training, respectively.
sedentary
controls
s
ba
Blood lipids, glucose, and insulin
Circulating levels of triglycerides and high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were measured at baseline and after training. Triglyceride levels were 114 ⫾ 52 mg/dl in controls and 198 ⫾
173 mg/dl in MS subjects at baseline. Controls had HDL
and LDL cholesterol concentrations of 48 ⫾ 11 and 87 ⫾
16 mg/dl at baseline, whereas MS subjects had lower HDL
cholesterol (40 ⫾ 9 mg/dl) and higher LDL cholesterol
(112 ⫾ 41 mg/dl). Triglycerides and total cholesterol
tended to be lower in both groups after resistance training, but the differences were not statistically significant
and the effect sizes were small (d ⫽ ⫺0.158 and ⫺0.148,
respectively).
gluc
cose infus
sion rate (mg/kg.mi
(
n)
Average weekly volume load (kg)
16
1819
FIG. 1. Changes in insulin responsiveness after 8 wk of resistance
exercise training. The data shown here are the individual data and the
means and SE of the steady-state GIR in the last 30 min of a 3-h insulin
infusion at 40 mU/m2 䡠 min. The ANOVA table indicated an important
interaction effect between the group factor and training. Because the
interaction factor is significant, interpretation of the main effects can
be replaced by examination of simple effects of training in each group
(which is the paired t test applied to before and after values in controls
and MS). The control group shows a significant change in mean level
(P ⫽ 0.03), and the MS group does not show a statistically significant
change in mean level (P ⫽ 0.37). *, Significant difference from control
means; **, significant difference from mean baseline. The figure
shows the control group does have a training effect, whereas the MS
group does not. The difference in response to training between the
two groups, either absolute or percentage, is significant (P ⫽ 0.02,
independent-sample t test).
fiber composition was determined using monoclonal antibodies for fast and slow myosin (18). As shown in Fig. 2,
MS subjects had a lower percentage of type 1 muscle fibers
than controls at baseline (36.3 ⫾ 10.2 vs. 50.0 ⫾ 17.7%,
P ⫽ 0.03). Percentage of type 1 fibers was unchanged after
training. Training tended to cause a shift in fiber composition from type 2b to type 2a in some MS subjects; however, the effect was small (d ⫽ 0.119) and not statistically
significant.
Hexose transport proteins
Expression of hexose transport proteins GLUT4 and
GLUT5 were also quantified. As shown in Fig. 3A, MS
subjects had slightly higher GLUT4 expression than controls before training (2.80 ⫾ 1.41 fmol/10 ␮g membrane
protein, and 2.38 ⫾ 1.11 fmol/10 ␮g membrane protein,
respectively). GLUT4 expression increased significantly
after training in both groups (P ⱕ 0.05). The percent increase in muscle GLUT4 content was greater in controls
than MS subjects (67%, d ⫽ 1.634, vs. 36%, d ⫽ 0.843,
respectively). GLUT5 expression increased significantly in
MS subjects (P ⱕ 0.05; d ⫽ 1.0512) but was unchanged in
controls (Fig. 3B).
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Layne et al.
fiber type
t
portion (%)
A
Muscle AMPK Activation in Metabolic Syndrome
control subjects (n
(n=9)
9)
Metabolic Syndrome (n=10)
60
50
40
p=0.39
p=0.03
30
p=0.10
20
10
0
type 1
type 2a
type 2b
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
Peroxisome proliferator-activated receptor ␥
coactivator 1␣
PGC-1␣ muscle expression tended to increase in both
groups after resistance training (Fig. 3E); however, the
increase was only statistically significant in controls
(P ⫽ 0.029, d ⫽1.181). The increase in PGC-1␣ expression to resistance training was muted in MS subjects
compared with controls (28 and 57%, respectively),
and the effect size was moderate in MS subjects (d ⫽
0.542).
muscle fiber type
fiber type porttion (%)
B
control subjects
60
baseline
trained
50
40
30
20
10
0
type
yp 1
type
yp 2a
type
yp 2b
muscle fiber type
fiber type portion (%)
C
60
50
Metabolic Syndrome
baseline
trained
40
30
20
10
0
type 1
type 2a
type 2b
muscle fiber type
FIG. 2. The effect of resistance training on muscle fiber type
composition. Percutaneous needle biopsies of vastus lateralis were
obtained before and after 8 wk of supervised resistance training. A,
Comparison of the baseline fiber composition in the two groups. MS
subjects had 28% less (P ⫽ 0.03) type 1 fiber content than the control
subjects. B and C, Pre- and posttraining fiber composition for the
control subjects and the MS subjects, respectively. Some individuals
showed modest changes in fiber composition, but in groups, there was
no significant change after training.
5-AMP-activated protein kinase
Total and activated AMPK expression were quantified
by immunoblot analysis as shown in Fig. 3, C and D. Total
AMPK expression increased significantly in both groups,
with very strong effect sizes (d ⫽ 1.338 in MS subjects, and
d ⫽ 1.77 in controls). Phospho-AMPK expression increased 50% in controls (P ⬍ 0.001; d ⫽ 1.924). PhosphoAMPK expression increased 13% in MS subjects; however, the increase was not statistically significant, and the
effect size was relatively small (d ⫽ 0.368).
ATP synthase
ATP synthase expression, a marker of mitochondrial
enzyme activity, increased significantly in both groups in
response to training as shown in Fig. 3F. MS subjects
tended to have higher ATP synthase expression at baseline
than controls (P ⫽ 0.112), but training increased ATP
synthase expression by 63% in controls compared with
25% in MS subjects.
Mammalian target of rapamycin
Total and activated mTOR, a molecular mediator of
protein synthesis, increased significantly in MS subjects in
response to training (Fig. 3, G and H). Although the increase in total mTOR expression was not statistically significant in controls, total mTOR expression increased
32%, and the effect size was strong (d ⫽ 0.772). Total
mTOR expression increased 57% in MS subjects in response to resistance training. Phospho-mTOR expression
had a similar response to resistance training. PhosphomTOR increased 55% in MS subjects (d ⫽ 1.49; P ⬍ 0.01)
and 39% in controls. Again, the effect size was large in
controls (d ⫽ 0.754), but the increase was not statistically
significant.
Discussion
In the present study, 8 wk of supervised resistance training
improved several physical and metabolic parameters in
healthy, previously sedentary subjects as well as previously
sedentary subjects with the MS. The sedentary control subjects improved insulin responsiveness and decreased fasting
serum insulin concentrations after resistance training. Despite improvements in strength, endurance, and body composition, MS subjects’ insulin resistance was unchanged by 8
wk of resistance training.
The two groups that were studied for this report were
very different from each other based on the entry criteria.
The MS subjects were obese and had a family history of
type 2 diabetes, whereas the sedentary controls were
nonobese and had no close family members with diabetes.
The baseline data were dramatically different in several of
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
p=0.04
4
3
2
1
0.4
0.2
50
0
p<0.01
150
MS
p<0.01
100
50
0
total mTOR
A
B
A
200
controls
150
p=0.13
p<0 01
p<0.01
150
B
MS
p<0.01
100
50
0
p=0.39
100
50
0
t
controls
G
MS
s
po
e
pr
0
100
controls
t
50
200
ATP synthase
B
A
B
p<0 01
p<0.01
200
s
po
e
pr
100
p<0.01
p
150
0.0
A
MS
1821
phospho AMPK
B
A
B
A
t
p=0.236
150
controls
200
st
po
e
pr
PGC-1α
eline)
(% of base
0.6
t
MS
p=0.029
p=0.04
0.8
F
B
MS
1.0
s
po
e
pr
PGC-1α
B
A
controls
200
p=0.95
t
t
A
1.2
s
po
e
pr
s
po
e
pr
E
1.4
controls
D
total AMPK
B
A
B
A
B
s
po
e
pr
0
A
phospho-AMP
PK
(% of control baseline)
p=0.01
B
H
phospho-mTOR
A
B
A
200
controls
150
p=0.12
B
MS
p=0.03
100
50
0
t
st
po
e
pr
s
po
e
pr
t
st
po
e
pr
s
po
e
pr
st
po
e
pr
st
po
e
pr
t
t
s
po
e
pr
s
po
e
pr
p-mTOR
R content
(% of contrrol baseline)
MS
C
GLUT5
A
n
GLUT5 expression
(fmol/5µg protein)
controls
5
B
B
total AMPK
(% of control baseline)
A
ATP synth
hase
(% of control b
baseline)
B
st
po
e
pr
GLUT4 expression
n
(fmol/5µg protein)
A
total mTOR
(% of contrrol baseline)
GLUT4
A
jcem.endojournals.org
FIG. 3. Training-related changes in GLUT4, GLUT5, AMPK, phospho-AMPK, PGC-1␣, ATP synthase, total mTOR, and phospho-mTOR. Muscle
glucose transporter expression was increased after resistance training. Panels A and B, Examples of typical immunoblots for GLUT4 and GLUT5 as
well as bar graphs with the results of image analysis of blots from all of the subjects. In each of the immunoblots displayed, a control subject is on
the left and a metabolic subject is shown on the right. The designation A represents the baseline muscle biopsy homogenate, and B indicates the
posttraining biopsy sample. Panel C, GLUT4 protein content increased in both groups after training. These data represent the mean expression
determined in at least three separate quantitative immunoblots (using chimeric protein standards) of muscle homogenates for each individual (nine
controls and 10 MS subjects). Panel B displays the data from GLUT5 immunoblots. GLUT5 expression did not change in the control subjects but
increased slightly but significantly in the MS subjects. The P values shown in each panel were calculated as paired t tests. Panels C and D display
results of similar analyses of expression of total AMPK and activated phospho-AMPK. Without quantitative protein standards, these data are
expressed relative to the control baseline signal intensity. Panel E, Pre- and posttraining data for PGC-1␣ expression. Panel F, Training-related
changes in ATP synthase expression as a direct indicator of mitochondrial enzyme increases. As is displayed, each of these measurements tended
to increase after resistance training, but the smaller increases in phospho-AMPK and PGC-1␣ were not statistically significant for the MS subjects.
Panels G and H, Typical immunoblots and summary bar graphs showing the changes that occurred in total mTOR and activated phospho-mTOR
expression in response to 8 wk of resistance exercise training. The total mTOR and phospho-mTOR measurements increased significantly in the MS
subjects, but the smaller increases in the control group did not achieve statistical significance. In each panel, MS indicates the MS group, and pre
and post indicate pretraining and posttraining data.
the measured parameters. The MS subjects had higher
BMI, higher LBM, higher fat mass and percent body fat,
and greater waist circumference. The MS subjects had
lower VO2max, nearly double the fasting serum insulin,
and less than half the insulin response to a euglycemic
insulin infusion.
The lack of improvement in insulin resistance in the
present study may have contributions from age, body fat,
and baseline strength. Previous studies have suggested that
the quantitative adaptations to resistance exercise are reduced with age (21). In the present study, however, older
subjects (both controls and MS) showed comparable improvements to younger subjects in strength and endurance. Many studies that suggest resistance training may be
effective at improving insulin resistance also demonstrate
a decrease in fat mass as a result of training (3, 6). Our
study took care to maintain body weight throughout so
that the effects observed were primarily due to exercise
training and were not confounded by weight loss. In the
present study, fat mass did not change in either group.
Homogeneity was not present within either group. In
fact, there was overlap in many variables, including GIR,
despite no overlap in BMI (dictated by entry criteria). The
high variability in the subjects at baseline may have obscured smaller changes that might have been seen if the
groups were larger and more homogeneous. Stone and
co-workers (22) showed that strength at baseline correlated with response to resistance training in college athletes. Our MS subjects started training slightly stronger
(not significant), which likely was related to their higher
LBM and the higher proportion of type 2b fibers in their
vastus lateralis. When corrected for body mass, though,
1822
Layne et al.
Muscle AMPK Activation in Metabolic Syndrome
they were slightly lower in PF development. The percent
increase in strength was the same in both groups. Training
workload increased each week by about the same increment in all subjects in both groups. No difference was seen
in the overall workload, and there was no evidence that the
higher responses in insulin responsiveness were the most
trained. There was no statistically significant correlation
of difference in volume load and GIR response to training.
The lack of improvement in insulin responsiveness may
have been related to a low level of baseline fitness from a
previous extremely sedentary lifestyle in some subjects.
Some of the MS subjects may not have been strong enough
at the beginning of the training program to achieve workloads high enough during training to cause significant adaptations in only 8 wk. Higher-volume work increases
general fitness and is associated with greater improvements in body composition compared with low-volume
training. Some of the subjects may have needed to achieve
a higher threshold of strength before they could lift with
enough intensity to force adaptation to the higher-volume
phase of training. Longer duration and/or higher intensity
may be necessary to cause sufficient muscle remodeling in
MS subjects, as suggested by Sriwijitkamol et al. (23).
However, strength quantified by RFD and by allometrically scaled IPF was essentially the same at baseline in both
groups (Table 2). There were seven of the nine control
subjects and four of the MS subjects who increased their
insulin responsiveness after strength training. Baseline
VO2max or RFD data did not identify those who would
respond. Surprisingly, the positive responders among the
MS subjects tended to have lower VO2max and RFD at
baseline, suggesting these parameters of fitness did not
correlate with improvement in insulin resistance after
strength training.
Lovell and co-workers performed 16 wk of resistance
training of older men (24). They found improved strength
and improved cardiovascular fitness manifested as increase VO2max, suggesting that in these previously sedentary subjects, the impact of resistance training was
mixed (24). There was increased strength and increased
endurance. The evidence in our subjects also suggests a
mixed benefit. If we had started with recreational athletes
(more fit subjects), the impact of resistance training might
have been more restricted to increased strength and fiber
hypertrophy.
Normal human skeletal muscle contains a mixture of
type 1, 2a, and 2b muscle fibers. Each fiber type is suited
for different types of physical activity. Type 1 (slowtwitch, red) fibers generally contain more mitochondria
and are well suited for oxidative energy production and
sustained activity. Although type 1 fibers can provide energy for long periods of time, their relatively slow con-
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
traction speed and low force production make them best
suited for long-term, low-intensity activity (25). Conversely, type 2b (fast-twitch, white) fibers are best suited
for energy production via phosphagens and fast glycolysis.
Type 2b fibers have the fastest speed of contraction and
highest force production capabilities, making them well
suited for short-term, high-intensity activity such as
sprints or heavy lifting. Type 2a fibers are an intermediate
fiber type with properties of both type 1 and type 2b fibers.
Training can cause a shift from type 2b muscle fibers toward type 2a fibers, indicative of the altered energy need
of the trained muscles (25). However, even long-term
training has not resulted in alteration of the baseline proportion of type 1 and type 2 fibers in humans (26). In view
of the divergent functional capabilities of each skeletal
muscle fiber type, it is not surprising that many of the
adaptations to training are fiber type dependent (27) and
may occur through the largely separate cell signaling pathways that predominate in each of the two principal fiber
types (7). Our subjects with the MS had a higher proportion of type 2 fibers, which might have made them more
adaptable to strength training, but this study did not find
a training advantage in this group.
Endurance training is associated with improved efficiency of substrate uptake and use by skeletal muscle.
These improvements are brought about by increases in
mitochondrial biogenesis, oxidative enzymes, and fatty
acid oxidation (28). In addition, endurance training appears to increase glucose uptake into the cell by increasing
the expression of GLUT4 in skeletal muscle (29). Many of
these adaptations are mediated by AMPK and its downstream targets (30). Resistance training results in increased
protein synthesis (31) and may increase GLUT4 expression in skeletal muscle (19, 32). The mTOR signaling pathway appears to be crucial in mediating the fiber hypertrophy response to resistance exercise; however, mTOR’s
role in improving insulin sensitivity after resistance training has remained elusive.
A bout of exercise will acutely increase AMPK activation, but this increase is transient (33, 34). Endurance exercise training of healthy young men has been shown to
increase skeletal muscle total AMPK and activated AMPK
(35, 36), but after endurance training, the augmentation
induced by an acute exercise bout is less than in untrained
subjects (35). An increase in basal activated AMPK does
not always occur after exercise training and may be attenuated based on the type of training and the pretraining
fitness level of the subjects. Clark and co-workers (37)
employed 3 wk of intensified training of well-trained athletes and found despite a shift to increased fat oxidation,
that both basal and acute exercise-stimulated AMPK activation did not change. We previously found no change in
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
phospho-AMPK in six very sedentary subjects who underwent 6 wk of stationary cycle training (19). The muscle
adaptation responses in the subjects of this earlier study
resemble those of the MS group in the current study in that
mTOR activation predominated. The current study design
was quite different, however, because in this protocol, the
duration was longer and the whole-body workload was
much higher.
Another way of expressing the impact of the exercise
intervention on phosphorylation of AMPK is to adjust the
percent increase in phospho-AMPK by the percent increase in the total AMPK, assuming that this expression
would better reflect the activity change of the upstream
AMPK-kinase (LKB1). Applying this expression to our
data by subtracting the percent increase in total AMPK
from the increase in phospho-AMPK would result in essentially no increase in adjusted phospho-AMPK in controls (⫹2%), but the adjusted change in MS subjects
would become negative (⫺22%). Similar adjustment to
the phospho-mTOR data to reflect change in the upstream
Akt activity would result in a much smaller increase in the
MS subject muscle (⫹7%) and almost no change in the
control subjects’ adjusted phospho-mTOR (⫺2%). However, it is clear that the activation of AMPK and/or mTOR
will amplify changes in the activity of their upstream kinases and, in this case, is also reflected in downstream
effects on PGC-1␣ and ATP synthase.
Transgenic and knockout mouse studies involving key
elements of the AMPK and mTOR pathways have provided some insight into potential mechanisms for the results we observed in these subjects. Chronic activation
of AMPK increases muscle mitochondrial content in
5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranosidetreated mice (38). A gain-of-function mutation in the ␥3
regulatory subunit of AMPK expressed exclusively in
mouse type 2 muscle fibers resulted in increased expression of PGC-1␣ and mitochondria in type 2 fibers without
changing the fiber-specific type of myosin heavy chain
expression (39). Overexpression of PGC-1␣ in a transgenic mouse results in a more than 2-fold increase in mitochondria but paradoxically caused muscle insulin resistance, perhaps because of increased intramyocyte lipid
(40), whereas a muscle-specific knockout of PGC-1␣ results in a shift in muscle fiber type from the mitochondriarich types 1 and 2a to the glycolytic types 2x and 2b (41).
These mice exhibited increased inflammatory cytokines
and impaired glucose tolerance but normal peripheral insulin sensitivity. Expression of activated Akt1 (a kinase
upstream of mTOR) in muscle resulted in mTOR activation and improved metabolic parameters. In this model,
Akt1 activation decreased fat pad mass and normalized
fasting blood glucose levels, insulin levels, and glucose
jcem.endojournals.org
1823
uptake with a concomitant increase in type 2b muscle fiber
size (42). Recently, Selman and co-workers (43) demonstrated increased activation of AMPK in S6K1-null mice
and suggested that the beneficial effects of knocking out
S6K1, including lower body fat and increased life span,
were due to higher AMPK activity in the absence of S6K1
tonic inhibition of LKB1 (AMPK-kinase).
The low-grade chronic inflammation seen in obesity
may also impair the activation of AMPK (44). Steinberg
and coworkers showed that TNF␣ suppresses AMPK activity by up-regulation of a phosphatase (protein phosphatase 2C). They further demonstrated that the TNF␣related suppression of AMPK activation was completely
abolished in mice lacking both types of TNF receptors
showing that the TNF␣ effect was specific (44). We did not
measure TNF␣ of other inflammatory cytokines in our
subjects.
In studies of muscle from lean, obese, and type 2 diabetic subjects, Sriwijitkamol and co-workers (23) found
that an acute exercise bout increased phosphorylation of
AS160 (a protein thought to be involved in GLUT4 translocation), presumably mediated by AMPK activation.
They found that even though obese and diabetic subjects
tended to have higher baseline phospho-AMPK in muscle,
their increase in the ratio of phospho-AMPK to total
AMPK was much less in response to the acute exercise
challenges. Biopsies were obtained at times 0, 10, and 40
min of either 50% VO2max or 70% VO2max on a cycle
ergometer. The higher-intensity exercise caused increases
in phosphorylation of AS160 that paralleled the increased
phospho-AMPK in the three groups. The maximum increase in phospho-AMPK was about 40% of that of the
lean controls in the obese group and about 30% in the
diabetic subjects (23). Their obese subject data on muscle AMPK activation by acute exercise is qualitatively
comparable to our 8 wk strength training where total
AMPK increased significantly, but the increment in
baseline phospho-AMPK was much less. This is consistent with a defect in the activation of AMPK with exercise in obesity/MS (45).
Bandyopadhyay and co-workers (46) measured basal
activity and insulin suppression of the activity of muscle
AMPK in lean, obese, and type 2 diabetic subjects. They
reported basal activity of AMPK and the ratio of phosphoAMPK were decreased in the obese and the diabetic subjects. Insulin suppressed AMPK activity and increased malonyl-coenzyme A levels in lean controls but had no effect
in the obese or diabetic groups (46). Unlike their obese
subjects, our baseline (untrained) MS AMPK and phospho-AMPK data do not show a difference from controls.
Their euglycemic clamp data, however, suggest that dysregulation of AMPK activation is a component of the in-
1824
Layne et al.
Muscle AMPK Activation in Metabolic Syndrome
sulin resistance of obesity and diabetes (46). Further evidence of dysregulation of AMPK activation in obesity was
shown by Bruce and co-workers (47). They found that
adiponectin stimulation of AMPK␣1 and AMPK␣2 was
diminished and/or delayed in isolated muscle strips from
rectus abdominus from obese females who underwent
elective hysterectomies (47).
Reports from the HERITAGE Family Study have suggested that the benefits of aerobic exercise training are
better correlated with weight loss than to increases in aerobic fitness measurements (48, 49). In this study, 105 of
621 participants were classified as MS. All volunteers were
subjected to 20 wk of supervised cycle ergometer training
after which the mean VO2max increased by 15 and 18%
(male and female) and fat mass decreased by 3– 4%. Of
105 MS subjects, 30% improved sufficiently that they no
longer met the criteria for the designation of MS (49).
The STRRIDE Study (Studies of a Targeted Risk Reduction Intervention through Defined Exercise) determined the effectiveness of three different exercise training
programs in reversing the parameters defining the MS
(50). Sixteen to 18 subjects with at least three criteria for
the MS were subjected to no intervention, low amount of
moderate activity, low amount of vigorous activity, or
high amount of vigorous activity for 8 months. The group
with low amount of vigorous activity was not different
from the no-exercise controls, but the other two groups
significantly decreased the number of criteria for the MS
(50). Analysis of the actual time spent in organized exercise for each subject suggested that the critical issue was
time spent exercising, rather than intensity, because the
group with a low amount of vigorous activity averaged
less time than the other two groups.
In these studies, sedentary control subjects improved
insulin responsiveness, whereas MS subjects did not, despite both groups participating side by side in 8 wk of
supervised resistance training. The mechanisms for the
lack of equivalent improvement in insulin action in the MS
subjects may be related to the intrinsic differences in their
untrained muscle. Control subjects had more type 1 fibers
in their vastus lateralis, whereas MS subjects had more
type 2 fibers, at a level similar to that reported in type 2
diabetes (51, 52).
Increases in GLUT4 and ATP synthase occurred coincident with increased insulin responsiveness in controls;
half or less of the increases in GLUT4 and ATP synthase
were seen in MS subjects, and GIR increased modestly in
only four of 10. Phosphorylation of mTOR was more in
the MS group, suggesting mTOR activation is not sufficient to improve insulin responsiveness. We conclude that
increases in AMPK activation mitochondrial expression
and GLUT4 are either directly involved in improved in-
J Clin Endocrinol Metab, June 2011, 96(6):1815–1826
sulin action or are increased in parallel to a key insulin
pathway.
Acknowledgments
We express our appreciation to research nurse Mary Ward, who
coordinated the clinical portion of this project, and to Ashley
Kavanaugh, Lauren Huskey, Anna Swisher, Chris Plourd, Matt
Shifflet, Henry Nowell, Travis Livingston, Brian Hobbs, Trey
Maughan, Jacob Wheeler, Jason Eble, Keith Painter, and Guy
Hornsby, the students in Kinesiology who acted as training
coaches to our subjects.
Address all correspondence and requests for reprints to: Charles
A. Stuart, M.D., East Tennessee State University, P.O. Box 70622,
Johnson City, Tennessee 37614. E-mail: [email protected].
Funding for these studies came from the National Institutes of
Health, National Institute of Diabetes and Digestive and Kidney
Diseases, DK080488 (to C.A.S.).
Disclosure Summary: A.S.L., S.N., M.A.S., M.E.A.H.,
M.P.M., M.W.R., M.H.S., and C.A.S. have nothing to declare.
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