Acute resistance exercise‐induced IGF1 expression and subsequent GLUT4 translocation

Abstract

Acute aerobic exercise (AE) is a major physiological stimulus for skeletal muscle glucose uptake through activation of 5′ AMP-activated protein kinase (AMPK). However, the regulation of glucose uptake by acute resistance exercise (RE) remains unclear. To investigate the intracellular regulation of glucose uptake after acute RE versus acute AE, male Sprague–Dawley rats were divided into three groups: RE, AE, or nonexercise control. After fasting for 12 h overnight, the right gastrocnemius muscle in the RE group was exercised at maximum isometric contraction via percutaneous electrical stimulation (3 × 10 sec, 5 sets). The AE group ran on a treadmill (25 m/min, 60 min). Muscle samples were taken 0, 1, and 3 h after completion of the exercises. AMPK, Ca2+/calmodulin-dependent protein kinase II, and TBC1D1 phosphorylation were increased immediately after both forms of exercise and returned to baseline levels by 3 h. Muscle IGF1 expression was increased by RE but not AE, and maintained until 3 h after RE. Additionally, Akt and AS160 phosphorylation were sustained for 3 h after RE, whereas they returned to baseline levels by 3 h after AE. Similarly, GLUT4 translocation remained elevated 3 h after RE, although it returned to the baseline level by 3 h after AE. Overall, this study showed that AMPK/TBC1D1 and IGF1/Akt/AS160 signaling were enhanced by acute RE, and that GLUT4 translocation after acute RE was more prolonged than after acute AE. These results suggest that acute RE-induced increases in intramuscular IGF1 expression might be a distinct regulator of GLUT4 translocation.

Full text

ORIGINAL RESEARCH
Acute resistance exercise-induced IGF1 expression and
subsequent GLUT4 translocation
Kohei Kido
1
, Satoru Ato
1
, Takumi Yokokawa
2
, Yuhei Makanae
3
, Koji Sato
4
& Satoshi Fujita
1
1 Faculty of Sport and Health Science, Ritsumeikan University, Kusatsu, Japan
2 Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Japan
3 Department of Physical Education, National Defense Academy, Yokosuka, Japan
4 Graduate School of Human Development and Environment, Kobe University, Kobe, Japan
Keywords
Aerobic exercise, glucose transporter type 4,
insulin-like growth factor 1, resistance
exercise.
Correspondence
Satoshi Fujita, Faculty of Sport and Health
Science, Ritsumeikan University, 1-1-1
Nojihigashi, Kusatsu 525-8577, Japan.
Tel: +81-77-561-3760;
Fax: +81-77-561-3761;
E-mail: safujita@fc.ritsumei.ac.jp
Funding Information
This work was supported by JSPS KAKENHI
Grant nos 25282200 and 25560379 to S.
Fujita. This work was also supported by the
Japanese Council for Science, Technology
and Innovation, SIP (Project ID 14533567),
“Technologies for creating next-generation
agriculture, forestry and fisheries” (funding
agency: Bio-oriented Technology Research
Advancement Institution, NARO).
Received: 18 July 2016; Accepted: 21 July
2016
doi: 10.14814/phy2.12907
Physiol Rep, 4 (16), 2016, e12907,
doi: 10.14814/phy2.12907
Acute aerobic exercise (AE) is a major physiological stimulus for skeletal mus-
cle glucose uptake through activation of 50AMP-activated protein kinase
(AMPK). However, the regulation of glucose uptake by acute resistance exer-
cise (RE) remains unclear. To investigate the intracellular regulation of glucose
uptake after acute RE versus acute AE, male Sprague–Dawley rats were
divided into three groups: RE, AE, or nonexercise control. After fasting for
12 h overnight, the right gastrocnemius muscle in the RE group was exercised
at maximum isometric contraction via percutaneous electrical stimulation
(3 910 sec, 5 sets). The AE group ran on a treadmill (25 m/min, 60 min).
Muscle samples were taken 0, 1, and 3 h after completion of the exercises.
AMPK, Ca
2+
/calmodulin-dependent protein kinase II, and TBC1D1 phospho-
rylation were increased immediately after both forms of exercise and returned
to baseline levels by 3 h. Muscle IGF1 expression was increased by RE but not
AE, and maintained until 3 h after RE. Additionally, Akt and AS160 phospho-
rylation were sustained for 3 h after RE, whereas they returned to baseline
levels by 3 h after AE. Similarly, GLUT4 translocation remained elevated 3 h
after RE, although it returned to the baseline level by 3 h after AE. Overall,
this study showed that AMPK/TBC1D1 and IGF1/Akt/AS160 signaling were
enhanced by acute RE, and that GLUT4 translocation after acute RE was more
prolonged than after acute AE. These results suggest that acute RE-induced
increases in intramuscular IGF1 expression might be a distinct regulator of
GLUT4 translocation.
Introduction
Aerobic exercise (AE) is a major physiological stimulus that
induces skeletal muscle glucose uptake and improves insu-
lin sensitivity. Acute and chronic AE affect glucose metabo-
lism differently. Specifically, acute AE induces skeletal
muscle glucose uptake incrementally and transiently
(Goodyear et al. 1990). A previous study suggests that the
blood glucose level is decreased approximately 30% for 4 h
by acute AE, as compared with nonexercised controls (Bac-
chi et al. 2012). Daily blood glucose control is critical for
diabetes patients to avoid diabetic complications (Ameri-
can Diabetes Association 2015b). Chronic AE improves
insulin sensitivity, fasting blood glucose, and HbA1c levels
continuously (Umpierre et al. 2011; Mann et al. 2014). The
improvement of these parameters can lead to a complete
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the American Physiological Society and The Physiological Society.
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2016 | Vol. 4 | Iss. 16 | e12907
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Physiological Reports ISSN 2051-817X

reversal of type II diabetes. Thus, both acute and chronic
AE are significant for improving glucose metabolism. In a
recent study, chronic resistance exercise (RE), which is a
skeletal muscle hypertrophy model, also improved hyper-
glycemia and HbA1c levels in diabetic patients (Umpierre
et al. 2011). Therefore, recent exercise guidelines from the
American Diabetes Association recommend RE for control-
ling blood glucose (American Diabetes Association 2015a).
However, acute RE-induced augmentation of skeletal mus-
cle glucose uptake and the regulation of this process are still
poorly understood.
AMP-activated protein kinase (AMPK), which is acti-
vated by increases in the cellular AMP/ATP ratio, is a key
intracellular signaling protein for glucose uptake (Musi
et al. 2003; Jessen and Goodyear 2005; O’Neill 2013). The
activation of AMPK is mediated by acute AE that induces
glucose transporter type 4 (GLUT4) translocation to the
cell membrane, resulting in glucose uptake (Musi et al.
2003; Jessen and Goodyear 2005; O’Neill et al. 2011;
O’Neill 2013), but no study confirmed the signaling
response after RE. Ca
2+
/calmodulin-dependent protein
kinase II (CaMKII) is another important signaling protein
for glucose uptake following acute AE. Previous studies
demonstrated that CaMKII activates AMPK as an
upstream event in response to AE or muscle contraction
(Raney and Turcotte 2008; Morales-Alamo et al. 2013),
but other studies indicated that CaMKII may also induce
glucose uptake independent of AMPK signaling pathways
(Raney and Turcotte 2008; Witczak et al. 2010). Although
CaMKII may have important roles in skeletal muscle glu-
cose uptake following AE, the regulation of CaMKII sig-
naling remains unclear. Some previous studies showed
that both AMPK and CaMKII were phosphorylated fol-
lowing acute RE, which might contribute to glucose
uptake (Witczak et al. 2010; Ogasawara et al. 2014).
However, no study showed the roles of AMPK and CaM-
KII in acute RE-induced glucose uptake.
Phosphorylation of TBC1D1 and AS160 occurs down-
stream of AMPK and CaMKII (Funai and Cartee 2008;
Vendelbo et al. 2014). Through the phosphorylation of
TBC1D1 and AS160, AMPK and CaMKII enhance GLUT4
translocation (Chavez et al. 2008; Witczak et al. 2010).
However, the relationship between AMPK/CaMKII phos-
phorylation and TBC1D1/AS160 phosphorylation remains
unknown. Moreover, acute RE-induced TBC1D1/AS160
responses are not fully clarified.
Insulin-like growth factor 1 (IGF1) is increased in
skeletal muscle by acute RE (Ogasawara et al. 2013a,b). In
contrast, there is no evidence of acute AE inducing the
expression of skeletal muscle IGF1. In a cell culture study,
IGF1 phosphorylated Akt which then phosphorylated
AS160 at Thr642 resulting in enhanced GLUT4 transloca-
tion and glucose uptake (Ciaraldi et al. 2002; Roach et al.
2007; Baus et al. 2008; Taylor et al. 2008; Morissette et al.
2009; Peck et al. 2009). Accordingly, IGF1 may play cru-
cial roles as a stimulator of both AMPK- and CaMKII-
independent glucose uptake through the activation of Akt
and AS160 signaling pathways. In general, it is poorly
understood how different modes of exercise, that is, acute
RE and AE, differentially regulate IGF1/Akt/AS160 signal-
ing and subsequent GLUT4 translocation.
The purpose of this study was to identify specific cellu-
lar signal responses to acute RE, including IGF1 signaling,
as compared with those to acute AE in respect with glu-
cose metabolism. We additionally investigated the role of
IGF1 on TBC1D1/AS160 phosphorylation and glucose
uptake by using an in vitro model.
Materials and Methods
In vitro experiments
Mouse C2C12 myoblasts (American Type Culture Collec-
tion, Manassas, VA) were cultured as described previously
(Yokokawa et al. 2015). Briefly, cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM; 4.5 g glucose/L,
Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine
serum and 1% penicillin-streptomycin (P/S). To initiate
myogenic differentiation, the culture medium was replaced
by DMEM containing 2% horse serum and 1% P/S. After
4 days of differentiation, myotubes were serum-starved
overnight and then incubated for 30 min in serum-free
medium containing 5 lmol/L 5-aminoimidazole-4-carbox-
amide ribonucleoside (AICAR; Wako, Osaka, Japan),
1lmol/L insulin (Novolin; Novo Nordisk, Bagsvaerd, Den-
mark), or 200 ng/mL IGF1 (PeproTech, Rocky Hill, NJ).
In vivo experiments
The study protocol was approved by the Ethics Commit-
tee for Animal Experiments at Ritsumeikan University,
and was conducted in accordance with the Declaration of
Helsinki. Forty-two male Sprague–Dawley rats, aged
10 weeks (320–360 g), were obtained from CLEA Japan
(Tokyo, Japan). The rats were divided into three groups:
nonexercise control, RE, or AE. All rats were housed for
1 week in an environment maintained at 22–24°C with a
12:12-h light–dark cycle, and were allowed food (CE2;
CLEA Japan) and water ad libitum.
The time course of changes in signaling protein levels
was evaluated following RE and AE initiated after a 12-h
overnight fast, as detailed below. Rats were sacrificed 0, 1,
or 3 h after completion of the exercise routine. Control
rats were sacrificed at the basal state. Dissected gastrocne-
mius muscles were frozen rapidly in liquid nitrogen and
stored at 80°C until use.
2016 | Vol. 4 | Iss. 16 | e12907
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ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Glucose Uptake Regulation by Resistance Exercise K. Kido et al.

RE protocol
Under isoflurane anesthesia, hair was shaved off the
right lower leg of each rat; the area was then cleaned
with alcohol wipes. The rats were kept in a prone posi-
tion with their right foot on the footplate and the
ankle joint angle positioned at 90°. The triceps surae
muscle was stimulated percutaneously with electrodes
(Vitrode V, Ag/AgCl; Nihon Kohden, Tokyo, Japan)
that were cut to 10 95 mm and connected to an elec-
tric stimulator and an isolator (SS-104J; Nihon Koh-
den) (Nakazato et al. 2010). The right gastrocnemius
muscle was exercised isometrically by stimulation with
ten 3-sec contractions per set for 5 sets. There was a
7-sec interval between contractions and 3-min rest
intervals between sets. Voltage (~30 V) and stimulation
frequency (100 Hz) were adjusted to produce maximal
isometric tension. This exercise protocol is used widely
as a RE model for animals (Ogasawara et al. 2013a,b,
2014; Tsutaki et al. 2013; Kido et al. 2015) and
induces significant muscle hypertrophy (Ogasawara et al.
2013a,b).
AE protocol
Rats in the AE group were habituated to the treadmill by
running for 30 min at 15 m/min, 45 min at 20 m/min,
and 60 min at 25 m/min over a week. Three to five days
after the last running habituation, rats were placed on a
flat treadmill and made to run for 60 min at 25 m/min
(Langfort et al. 1996).
Analyses
In vitro glucose uptake assay
Glucose uptake was determined by measuring the glucose
concentration of the medium as described previously
(Yokokawa et al. 2015). In brief, culture media were col-
lected and the glucose concentrations assayed spectropho-
tometrically using a Glucose II test kit (Wako).
Measurements of serum IGF1, insulin, and
glucose, and muscle IGF1 concentrations
Insulin-like growth factor 1 levels in the serum and skele-
tal muscle were determined using the mouse/rat IGF1
Quantikine ELISA kit (R&D Systems, Minneapolis, MN).
Serum insulin levels were detected using a rat insulin
ELISA kit (Shibayagi, Gunma, Japan), according to the
manufacturer’s instructions. Serum glucose concentrations
were measured by the YSI 2300 STAT Plus analyzer (Yel-
low Springs Instrument, Yellow Springs, OH).
Western blotting analyses
Western blotting analyses were performed as reported
previously (Goodman et al. 2011). Briefly, stimulated
C2C12 myotubes were washed once with cold phosphate-
buffered saline (PBS) and lysed in radioimmunoprecipita-
tion assay buffer containing 10 mmol/L Tris HCl (pH
7.4), 1% NP-40, 1% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS), 150 mmol/L NaCl, and 5 mmol/L
EDTA. The extracts were centrifuged at 13,700 gfor
20 min at 4°C. Protein concentrations of the supernatants
were determined using a protein assay kit (Nacalai Tes-
que). The lysates were mixed with 69sample buffer con-
taining 350 mmol/L TrisHCl (pH 6.8), 10% SDS, 30%
glycerol, 9.3% dithiothreitol, and 0.03% bromophenol
blue, then boiled at 95°C for 5 min.
Gastrocnemius muscles were homogenized in buffer
containing 100 mmol/L TrisHCl (pH 7.8), 1% NP-40,
0.1% SDS, 0.1% sodium deoxycholate, 1 mmol/L EDTA,
150 mmol/L NaCl, and protease and phosphatase inhibi-
tor cocktail (Thermo Fisher Scientific, Waltham, MA).
Homogenates were centrifuged at 13,700 gfor 20 min at
4°C. The supernatant was removed, and the protein con-
centration determined using the Protein Assay Rapid kit
(Wako).
Samples were diluted in 39sample buffer (1.0%
b-mercaptoethanol, 4.0% SDS, 0.16 mol/L TrisHCl (pH
6.8), 43% glycerol, and 0.2% bromophenol blue), and
boiled at 95°C for 5 min. Using 8–12% SDS-polyacryla-
mide gels, 5 lg (for cell lysates) or 20 lg of protein (for
muscle lysates) was separated by electrophoresis and
transferred to polyvinylidene difluoride membranes. After
the transfer, membranes were washed in Tris-buffered
saline containing 0.1% Tween 20 (TBST). Membranes
were then blocked with 5% powdered milk in TBST for
1 h at room temperature. After blocking, the membranes
were washed and incubated overnight at 4°C with pri-
mary antibodies against p-Akt (Thr308), p-Akt (Ser473),
total Akt, p-AMPK (Thr172), total AMPK, p-AS160
(Thr642), total AS160, p-CaMKII (Thr286), a-tubulin
(Cell Signaling Technology, Danvers, MA), total CaMKII
(Santa Cruz Biotechnology, Santa Cruz, CA), or
p-TBC1D1 (Ser237) (Merck Millipore, Damstadt, Ger-
many). The membranes were then washed again in TBST
and incubated for 1 h at room temperature with the
appropriate secondary antibodies. Chemiluminescent
reagents (Luminata Forte Western HRP Substrate; Merck
Millipore) were used to facilitate the detection of protein
bands. Images were scanned using a chemiluminescence
detector (ImageQuant LAS 4000; GE Healthcare, Buck-
inghamshire, UK). Band intensities were quantified using
ImageJ 1.46 software (National Institutes of Health,
Bethesda, MD).
ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2016 | Vol. 4 | Iss. 16 | e12907
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K. Kido et al.Glucose Uptake Regulation by Resistance Exercise

To assess the plasma membrane localization of GLUT4,
plasma membranes were extracted as modified methods of
previous study (Sato et al. 2009). Briefly, gastrocnemius
muscles were homogenized into buffer A (20 mmol/L
Tris [pH 7.4], 1 mmol/L EDTA, 0.25 mmol/L EGTA,
0.25 mol/L sucrose, 1 mmol/L DTT, 50 mmol/L
NaF, 25 mmol/L sodium pyrophosphate, and 40 mmol/L
b-glycerophosphate). The homogenates were centrifuged
at 400 gfor 15 min at 4°C. The supernatant was
centrifuged again at 249,138 g(Hitachi CS100GXII, Ibar-
aki, Japan) for 1 h at 4°C. The fractions were resus-
pended in buffer A and then homogenized to add equal
volume buffer B containing 20 mmol/L Tris (pH 7.4),
1 mmol/L EDTA, 0.25 mmol/L EGTA, 2% Triton X-100,
50 mmol/L NaF, 25 lmol/L sodium pyrophosphate, and
40 mmol/L b-glycerophosphate. The homogenates were
centrifuged at 274,052 gfor 1 h at 4°C (Hitachi
CS100GXII) and the supernatant was used as plasma
membrane fraction. Prepared plasma membrane fraction
was used for the measurement of plasma membrane
GLUT4 level, which was determined by Western blotting
analysis using antibodies against GLUT4 (Merck Milli-
pore) (Sato et al. 2009).
Statistical analysis
All results are expressed as means SE. A one-way
ANOVA with a least significant difference post hoc test
was used to evaluate changes among multiple groups, and
the unpaired Student t-test was used for two-group com-
parisons (Yang et al. 2012).
Results
In vitro experiments
AICAR stimulation
AICAR, an AMPK activator, significantly increased the
phosphorylation of AMPK (Thr172), TBC1D1 (Ser237),
and AS160 (Thr642). Additionally, AICAR induced signif-
icant decreases in media glucose concentrations indicating
an increase in glucose uptake (Fig. 1).
Insulin and IGF1 stimulation
Akt phosphorylation at Thr308 and Ser473, and AS160
phosphorylation at Thr642, were increased significantly
by stimulation with insulin and IGF1, whereas TBC1D1
phosphorylation at Ser237 was not changed. Additionally,
insulin and IGF1 induced significant decreases in glucose
concentrations in the media indicating an increase in glu-
cose uptake (Fig. 2).
In vivo experiments
Blood parameters
Table 1 shows the changes in serum insulin, IGF1, and
glucose levels. Serum insulin was not changed by RE and
AE. Serum IGF1 was decreased significantly at 3 h after
AE (P<0.05) but not changed by RE. In the RE group,
serum glucose was increased significantly immediately
after exercise (P<0.05), and returned to the baseline
level by 3 h after exercise. In the AE group, serum glucose
was increased significantly immediately after exercise
(P<0.05). Glucose dropped to below the baseline level at
1h(P<0.05) and returned to the baseline level by 3 h
after AE.
Skeletal muscle parameters
AMPK was phosphorylated significantly immediately after
both RE and AE (P<0.05). AMPK phosphorylation
remained high at 1 h after RE (P<0.05). This was in
contrast to AE-induced AMPK phosphorylation that
returned to baseline by 1 h after exercise (Fig. 3).
CaMKII phosphorylation was increased significantly
immediately after RE and returned to the baseline level
by 1 h after exercise. In the AE group, CaMKII was phos-
phorylated significantly immediately after exercise and
returned to the baseline level at 3 h after AE (Fig. 4).
TBC1D1 was phosphorylated significantly at Ser237
immediately after RE. The phosphorylation level returned
to baseline by 3 h after RE. In the AE group, TBC1D1
phosphorylation tended to increase immediately after
exercise and reached significance at 1 h. Phosphorylation
levels returned to baseline by 3 h after exercise (Fig. 5).
Resistance exercise induced a significant (63%) increase
in IGF1 expression in skeletal muscle at 1 h after exercise.
This increase was maintained until 3 h after exercise.
However, AE-induced IGF1 expression was decreased sig-
nificantly at every time point (P<0.05) (Fig. 6).
Both RE and AE induced significant phosphorylation
of Akt at Thr308 immediately after exercise (P<0.05). p-
Akt (Thr308) levels returned to baseline by 1 h after exer-
cise. Akt was also phosphorylated at Ser473 immediately
after RE and AE (P<0.05). Although AE-induced p-Akt
(Ser473) returned to the baseline level at 1 h after exer-
cise, this phosphorylation was maintained for 3 h after
RE (P<0.05) (Fig. 7).
Phosphorylation of AS160 at Thr642 increased signifi-
cantly at 1 h after RE (P<0.05). This increase was sus-
tained for 3 h (P<0.05). In contrast, AE-induced
phosphorylation of AS160 occurred immediately after
exercise (P<0.05) and returned to the baseline level by
3 h (Fig. 8).
2016 | Vol. 4 | Iss. 16 | e12907
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ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Glucose Uptake Regulation by Resistance Exercise K. Kido et al.

The expression of GLUT4 on the plasma membrane
was increased significantly 1 h after RE (P<0.05). This
increase was maintained until 3 h after RE (P<0.05). AE
induced a significant increase in plasma membrane
GLUT4 immediately and 1 h after exercise (P<0.05).
Plasma membrane GLUT4 expression returned to the
baseline level by 3 h after AE (Fig. 9).
Discussion
In this study, we investigated intracellular signaling and
GLUT4 translocation in response to an acute bout of RE
or AE in rat skeletal muscle. Skeletal muscle IGF1 expres-
sion was increased after RE but not AE, and subsequent
GLUT4 translocation was sustained longer after RE as
compared with AE. Additionally, we used an in vitro
study to show that activation of AMPK/TBC1D1 and
IGF1/Akt/AS160 signaling enhanced glucose uptake inde-
pendently. These data provided crucial new information
to explain the regulation of glucose uptake by acute RE
through specific signals, and showed differences between
acute RE and AE at the molecular level.
The results of this study indicated that RE- and AE-
induced AMPK phosphorylation were similar. In previous
studies, AMPK was also found to be phosphorylated
immediately after exercise by both RE and AE, and no
significant difference was observed between the responses
to RE and AE (Rasmussen et al. 1998; McConell et al.
2008; Vissing et al. 2013; Ogasawara et al. 2014; Ahti-
ainen et al. 2015). The activation of AMPK was found
previously to depend upon skeletal muscle contraction
tension (Ihlemann et al. 1999). Additionally, the
16
17
18
19
20
21
22
CON AICAR
Glucose concentraon (mmol/L)
0
0.5
1
1.5
2
2.5
CON AICAR
Phospho/total AMPK (AU)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
CON AICAR
Phospho-TBC1D1/α-tubulin
(AU)
0
0.5
1
1.5
2
2.5
3
3.5
CON AICAR
Phospho/total AS160 (AU)
*
*
**
Phospho
Total
Phospho
Phospho
Total
α-tubulin
AB
CD
Figure 1. Effects of AICAR on mouse C2C12 myotubes. Phosphorylation of AMPK at Thr172 (A), TBC1D1 at Ser237 (B), and AS160 at Thr642
(C) following stimulation with AICAR. Media glucose concentration indicating glucose uptake (D) relative to control (CON) following stimulation
with AICAR. Data are presented as means SE (n=5). *P<0.05 versus CON.
ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2016 | Vol. 4 | Iss. 16 | e12907
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K. Kido et al.Glucose Uptake Regulation by Resistance Exercise

phosphorylation of AMPK depended upon the intensity
of AE (i.e., %VO
2
max) (Chen et al. 2003; Sriwijitkamol
et al. 2007). According to these previous studies, the
magnitude of AMPK phosphorylation in response to RE
and AE depends on exercise intensity. Therefore, further
studies are needed to identify the magnitude of AMPK
0
2
4
6
8
10
12
AB
C
E
D
Thr308
Ser473
Total
0
2
1
3
4
5
6
Phospho/total Akt at Thr308
(AU)
0
0.2
0.4
0.6
0.8
1
1.2
Phospho-TBC1D1/α-Tubulin
(AU)
16
17
18
19
20
21
22
Glucose concentraon (mmol/L)
Phospho/total Akt at Ser473
(AU)
CON INSULIN IGF1
CON INSULIN IGF1 CON INSULIN IGF1
CON INSULIN IGF1
CON INSULIN IGF1
Phospho
Total
0
0.5
1
1.5
2
2.5
Phospho/total AS160 (AU)
Phospho
α-tubulin
Figure 2. Effects of insulin and IGF1 on mouse C2C12 myotubes. Phosphorylation of Akt at Thr308 (A) and Ser473 (B), AS160 at Thr642 (C),
and TBC1D1 at Ser237 (D) following stimulation by insulin or IGF1. Media glucose concentration indicating glucose uptake (E) relative to
control (CON) following stimulation by insulin or IGF1. Data are expressed as means SE (n=6). *P<0.05 versus CON.
2016 | Vol. 4 | Iss. 16 | e12907
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ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Glucose Uptake Regulation by Resistance Exercise K. Kido et al.

phosphorylation following RE and AE with various inten-
sities.
This is the first study comparing the time course of
changes in CaMKII phosphorylation between RE and AE.
The results showed that CaMKII was phosphorylated
immediately after both acute RE and AE. This was consis-
tent with a previous study showing that the phosphoryla-
tion of CaMKII was increased significantly immediately
after acute AE (Rose et al. 2006). Together, these results
demonstrate that both acute RE and AE induce CaMKII
phosphorylation immediately after exercise.
The phosphorylation of CaMKII apparently depended
on Ca
2+
released from the sarcoplasmic reticulum. Ca
2+
release is increased by higher skeletal muscle contraction
tension (Chin and Allen 1997). Accordingly, RE, which
induces higher contraction tension than AE, might
phosphorylate CaMKII to a greater extent than AE. How-
ever, the phosphorylation of CaMKII after acute RE
returned to the baseline level faster than after acute AE.
Thus, contraction tension might not be the most signifi-
cant regulatory factor for CaMKII phosphorylation after
AE.
As a downstream signal of AMPK and CaMKII, the
phosphorylation of TBC1D1 (Ser237) was measured after
acute RE and AE and found to match the time course of
changes in AMPK phosphorylation. To better support
the functional relationship between these changes,
TBC1D1 phosphorylation was measured in response to
AICAR-induced AMPK phosphorylation or insulin/IGF1
stimulation in vitro. The results showed that AMPK
phosphorylation induced TBC1D1 phosphorylation. In
contrast, insulin/IGF1-induced Akt signal activation did
Table 1. Blood parameters.
CON RE AE
0H 1H 3H 0H 1H 3H
Insulin (pmol/l) 11.3 3.0 9.7 1.4 7.7 3.2 12.7 2.1 10.3 1.8 10.9 1.0 9.12 0.9
IGF1 (ng/mL) 1281.4 51.6 1203.9 115.6 1262.3 60.1 1200.0 54.1 1491.9 57.1 1178.1 115.3 937.9 72.7*
Glucose (mmol/L) 3.7 0.3 7.2 0.8*9.9 1.0*4.0 1.7 7.4 0.3*2.6 0.1*3.0 0.2
Data are presented as means SE (n=6).
CON, control; RE, resistance exercise; AE, aerobic exercise.
*
P<0.05 versus CON.
0
0.5
1
1.5
2
2.5
3
3.5
CON RE0H RE1H RE3H
Phospho/total AMPK (AU)
0
0.5
1
1.5
2
2.5
3
3.5
CON AE0H AE1H AE3H
Phospho/total AMPK (AU)
*
*
*
Phospho
Total
AB
Figure 3. Effects of RE and AE on AMPK. Phosphorylation of AMPK at Thr172 relative to total AMPK protein content following RE (A) or AE
(B). Data are expressed as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise.
ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2016 | Vol. 4 | Iss. 16 | e12907
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K. Kido et al.Glucose Uptake Regulation by Resistance Exercise

not affect TBC1D1 phosphorylation at Ser237. A previous
animal study showed that p-TBC1D1 (Ser237) increased
immediately after acute AE, and was diminished in
AMPK knockout mice (Fentz et al. 2015). Based on these
results, acute RE-induced TBC1D1 phosphorylation at
Ser237 may be also caused by AMPK phosphorylation.
0
0.5
1
1.5
2
2.5
3
3.5
4
CON AE0H AE1H AE3H
Phospho/total CaMKII (AU)
0
0.5
1
1.5
2
2.5
3
3.5
4
CON RE0H RE1H RE3H
Phospho/total CaMKII (AU)
Phospho
Total
***
AB
Figure 4. Effects of RE and AE on CaMKII. Phosphorylation of CaMKII at Thr286 relative to total CaMKII protein content following RE (A) or
AE (B). Data are expressed as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise.
0
0.5
1
1.5
2
2.5
3
CON RE0H RE1H RE3H
Phospho-TBC1D1/α-tubulin (AU)
Phospho-TBC1D1/α-tubulin (AU)
0
0.5
1
1.5
2
2.5
3
CON AE0H AE1H AE3H
*
*
*
P = 0.07
Phospho
α-tubulin
AB
Figure 5. Effects of RE and AE on TBC1D1. Phosphorylation of TBC1D1 at Ser237 relative to total TBC1D1 protein content following RE (A) or
AE (B). Data are expressed as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise.
2016 | Vol. 4 | Iss. 16 | e12907
Page 8
ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Glucose Uptake Regulation by Resistance Exercise K. Kido et al.

Acute RE-induced IGF1 expression was assessed previ-
ously as a regulator of skeletal muscle hypertrophy
(Ogasawara et al. 2013b). As a glucose uptake regulator,
IGF1 was measured after acute RE and AE. The results
showed that skeletal muscle IGF1 was increased 1 h after
acute RE and maintained until 3 h. In contrast, acute
0
50
100
150
200
250
CON RE0H RE1H RE3H
Skeletal muscle IGF1
expression (ng/protein)
0
50
100
150
200
250
CON AE0H AE1H AE3H
Skeletal muscle IGF1
expression (ng/protein)
*
**
*
*
AB
Figure 6. Effects of RE and AE on IGF1. The expression of skeletal muscle IGF1 relative to CON following RE (A) or AE (B). Data are expressed
as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise.
0
1
2
3
4
5
6
7
CON AE0HAE1HAE3H
0
1
2
3
4
5
6
7
CON RE0H RE1H RE3H
Phospho/total Akt at
Ser473 (AU)
0
1
2
3
4
5
6
7
CON RE0H RE1H RE3H
Phospho/total Akt at
Thr308 (AU)
Phospho/total Akt at
Ser473 (AU)
Phospho/total Akt at
Thr308 (AU)
0
1
2
3
4
5
6
7
CON AE0HAE1HAE3H
*
*
*
*
*
*
Thr308
Ser473
Tot a l
AB
CD
Figure 7. Effects of RE and AE on Akt. Phosphorylation of Akt at Thr308 and Ser473 relative to total Akt protein content following RE (A and
C) or AE (B and D). Data are expressed as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic
exercise.
ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
2016 | Vol. 4 | Iss. 16 | e12907
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K. Kido et al.Glucose Uptake Regulation by Resistance Exercise

AE did not augment intramuscular IGF1. Following aug-
mented IGF1 expression, the increase in Akt phosphory-
lation seen in the RE was more prolonged than that in
the AE group. As a downstream signal of Akt, AS160
phosphorylation was also maintained longer than AE.
Moreover, using an in vitro model, we also determined
that IGF1 stimulated Akt and AS160 phosphorylation.
According to these results, Akt and AS160 phosphoryla-
tion after RE may be modulated by RE-induced IGF1
expression, which may have contributed to prolonged
increase in Akt and AS160 phosphorylation. Taken
together, the IGF1 signaling, including prolonged Akt
and AS160 phosphorylation, may be a specific signal
response to acute RE.
The early-phase phosphorylation of Akt by both RE
and AE may occur independently of IGF1 expression
because there was no change in IGF1 expression immedi-
ately after exercise. Moreover, according to a previous
study, the integrin/Akt signaling response occurred imme-
diately after exercise (Klossner et al. 2009). Thus, Akt
phosphorylation immediately after acute RE and AE may
be caused by an integrin/Akt signal.
0
0.5
1
1.5
2
2.5
3
CON RE0H RE1H RE3H
Phospho/total AS160 (AU)
0
0.5
1
1.5
2
2.5
3
CON AE0H AE1H AE3H
Phospho/total AS160 (AU)
****
Phospho
Total
AB
P = 0.05
Figure 8. Effects of RE and AE on AS160. Phosphorylation of AS160 at Thr642 relative to total AS160 protein content following RE (A) or AE
(B). Data are expressed as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
CON RE0H RE1H RE3H
GLUT4 in plasma membrane (AU)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
CON AE0H AE1H AE3H
GLUT4 in plasma membrane (AU)
***
*
AB
Figure 9. Effects of RE and AE on GLUT4. Total GLUT4 expression in plasma membrane following RE (A) or AE (B) relative to CON. Data are
expressed as means SE (n=6). *P<0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise.
2016 | Vol. 4 | Iss. 16 | e12907
Page 10
ª2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
the American Physiological Society and The Physiological Society.
Glucose Uptake Regulation by Resistance Exercise K. Kido et al.

This is the first study which directly compared AS160
phosphorylation in response to RE and AE. Previous
studies showed that AS160 phosphorylation in rat skeletal
muscle after swimming increased immediately after exer-
cise, and it maintained until 3 h after exercise (Arias et al.
2007; Funai et al. 2009; Castorena et al. 2014). This is
contrary to this study which demonstrated that AS160
phosphorylation returned to baseline level by 3 h after
exercise. However, these previous studies used different
mode of exercise (i.e., swimming vs. running) and ana-
lyzed epitrochlearis muscle. Acute swimming exercise-
induced PGC1aexpression, which is downstream of
AMPK, was different with acute running exercise on sev-
eral skeletal muscles including epitrochlearis and gastroc-
nemius muscle (Terada and Tabata 2004). Additionally,
epitrochlearis and gastrocnemius muscle have different
muscle fiber composition (Castorena et al. 2011). These
methodological differences between previous and this
studies may have caused the differences in AS160 phos-
phorylation level and time course of response.
This is also the first report that compared the time
course of changes in GLUT4 translocation and upstream
signal responses after acute RE and AE. Interestingly,
enhanced GLUT4 translocation after acute RE was found
at later time points, but was maintained longer than after
acute AE. Previously, acute AE was reported to augment
GLUT4 translocation immediately after exercise, and
GLUT4 translocation returned to the baseline level by 2 h
after exercise (Goodyear et al. 1990). Additional impor-
tant results were that the RE-induced increase in AS160
phosphorylation was more prolonged than TBC1D1 phos-
phorylation, and that acute RE-induced GLUT4 transloca-
tion was also prolonged until the same time point.
Furthermore, the induction of AS160/TBC1D1 phospho-
rylation and GLUT4 translocation was not as prolonged
with AE as with RE. These findings may underscore the
significance of AS160 signal activation on RE-induced
GLUT4 translocation. In the future study, we need to
directly assess the glucose uptake to confirm our findings
on GLUT4 translocation.
Overall, our data showed that AMPK/TBC1D1 and
IGF1/Akt/AS160 signal activation enhanced glucose
uptake independently, and that acute RE activated these
signals. Moreover, acute RE increased the expression of
skeletal muscle IGF1 as a RE-specific GLUT4 transloca-
tion regulator. Further studies might clarify the contribu-
tion of IGF1 signals to acute RE-induced GLUT4
translocation by using an IGF1 knockout model.
Conflicts of Interest
The authors declare no conflicts of interest, financial or
otherwise.
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