DNA AND CELL BIOLOGY ORIGINAL RESEARCH ARTICLES

Volume 33, Number 3, 2014

ª Mary Ann Liebert, Inc.
Pp. 128–135
DOI: 10.1089/dna.2013.2089

Simulated Microgravity Contributes to Autophagy Induction

by Regulating AMP-Activated Protein Kinase

Hyun-Wook Ryu,1,* Sang-Hun Choi,1,* Sim Namkoong,1 Ik-Soon Jang,2

Dong Hyun Seo,3 Inho Choi,1,4 Han-Sung Kim,3,4 and Junsoo Park1,4

Exposure to microgravity is supposed to affect almost all biological systems, and we speculated that micro-
gravity is potentially involved in autophagy regulation. A clinostat was used to simulate microgravity, and
HEK293 cells that stably express GFP-LC3 were used for sensitive monitoring of autophagy induction. The
clinorotation of GFP-LC3 cells resulted in autophagosome formation in the cytoplasm and a change in au-
tophagosomal marker expression. Autophagy induction was accompanied by phosphorylation of AMPK (Thr
172) and by the dephosphorylation of mammalian target of rapamycin. To elucidate the role of AMPK in
microgravity-induced autophagy, we suppressed AMPK expression by knockdown via siRNA, which inhibited
the induction of autophagy upon exposure to microgravity. In addition, the clinorotation of C2C12 myotube
cells resulted in the enlarged and distinctive LC3 spots in the cytoplasm and AMPK activation. These results
indicate that simulated microgravity possibly contributes to autophagy induction by regulating AMPK.

Introduction degradation system targets most soluble short-lived proteins,

autophagy targets long-lived proteins and organelles such as

B ased on the experience of human spaceflight, ex-

posure to microgravity is purported to affect almost all
biological systems. However, the effect of microgravity on
living organisms is not completely understood. Recently,
many efforts have been made to elucidate the effect of
microgravity on cellular processes, and accumulating data
show that microgravity alters the cytoskeleton, cellular
proliferation, and gene expression profiles (Ikemoto et al.,
2001; Arase et al., 2002; Grimm et al., 2002; Hirasaka et al.,
2005; Tamma et al., 2009; Kang et al., 2011).
Due to limited spaceflight access, alternative research
methods were developed. A clinostat (strictly a three-
dimensional [3D] clinostat, but hereafter referred to as clinostat
for simplicity) is considered a useful model system for simu-
lating microgravity in biological experiments (Hemdan et al.,
2009; Kang et al., 2011). A clinostat is equipped with two
independent rotating axes to disperse the gravity vector uni-
formly to a whole steric angle (Arase et al., 2002). Thus far,
simulated microgravity using the clinostat has provided insight
into biological processes and has been used to examine the
biological changes.
Autophagy (specifically, macroautophagy) is an intracel-
lular degradation and recycling pathway, conserved from
yeast to human life forms. While the ubiquitin-proteasomal
mitochondria (Levine and Klionsky, 2004). Autophagic
degradation is mediated by the formation of autophago-
somes, which are double-membrane vesicles that engulf the
cytoplasmic organelles and macromolecules. The autopha-
gosome is then fused with a lysosome to form an auto-
lysosome, in which the targeted protein is degraded via
lysosomal hydrolases at a low pH. The degradation products
such as amino acids can then be recycled (Levine and
Klionsky, 2004; Kroemer and Jaattela, 2005). Autophagy is
induced by various stimuli, including nutrient depletion,
accumulation of damaged organelles, and infection of cy-
toplasmic pathogens. A recent study identified the cellular
signaling pathways of autophagy and autophagy regulation
by nutrient depletion stimulus. Nutrient depletion promotes
autophagy by activating AMP-dependent protein kinase
(AMPK), a key energy sensor that responds to an increased
AMP/ATP ratio (Miranda et al., 2007; Mihaylova and
Shaw, 2011). AMPK is also activated by various cellular
stresses, such as hypoxia and heat shock (Corton et al.,
1994; Kudo et al., 1995). AMPK activation results to the
activation of FoxO3, which leads to an increase in the ex-
pression of Beclin, LC3-II, and Gabarap11 (Sanchez et al.,
2012). In addition, autophagy is negatively regulated by
mammalian target of rapamycin (mTOR), which responds to

1
Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea.
2
Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea.
3
Department of Biomedical Engineering, Yonsei University, Wonju, Republic of Korea.
4
Yonsei Institute of Space Bioscience, Yonsei University, Wonju, Republic of Korea.
*These authors contributed equally to this work.

128
MICROGRAVITY INDUCES AUTOPHAGY
the nutrient signal. Both AMPK activation and mTOR
suppression activates the autophagy-initiating kinase Ulk1
(ATG1 in yeast) (Kim et al., 2011; Sanchez et al., 2011).
Muscle atrophy is a decrease in muscle mass that is
typically caused by a variety of diseases or disuse (Sandri
et al., 2004). A decrease in muscle mass is caused by loss of
muscle protein. The proteasomes degrade myofibrillar pro-
teins via the ubiquitin-proteasome pathway, and autophagy
is responsible for long-lived protein and organelle degra-
dation (Solomon and Goldberg, 1996; Zhao et al., 2007).
Over the past decades, a considerable number of studies
have been conducted on the relationship between micro-
gravity and atrophy. In particular, ubiquitination in muscle
cells has been studied to elucidate the mechanism of protein
degradation in microgravity environments (Riley et al.,
1987; Nikawa et al., 2004).
Because autophagy is involved in diverse biological
processes, we speculated that microgravity is potentially
involved in autophagy regulation. To sensitively examine
autophagy induction due to microgravity, we generated
HEK293 cells with stable expression of GFP-LC3 (GFP-
LC3 cells), an autophagosomal marker, and used the
clinostat to evaluate the effect of microgravity on autop-
hagy induction. Incubation of GFP-LC3 cells in the
clinostat resulted in the alteration of autophagosomal
marker expression, which was accompanied by AMPK
activation and mTOR suppression. In addition, the
knockdown of AMPK with siRNA interfered with micro-
gravity-induced autophagy. These results suggest that mi-
crogravity positively regulates autophagy by regulating
AMPK-mTOR signaling pathways.
Materials and Methods
Cell culture and microscopy
HEK293 cells were grown in Leibovitz’s L-15 medium
(Welgene) supplemented with 10% fetal bovine serum
(Gibco). Leibovitz’s L-15 medium is formulated for carbon
dioxide (CO2)–free cell culture systems. The HEK293 cell
line that stably express GFP-LC3 was generated as de-
scribed previously (Yoon et al., 2011), and the GFP-LC3
plasmid was provided by T. Yoshimori (Kabeya et al.,
2000). C2C12 myoblast cells were grown in DMEM me-
dium (Welgene) with 10% fetal bovine serum. To induce the
differentiation, C2C12 myoblast cells were incubated in low
serum differentiation medium (DMEM and 2% horse serum)
as described previously (Ruest et al., 2002). Compound C
was purchased from Sigma. For microscopy, GFP-LC3 cells
were grown on 35-mm cover-glass-bottom dish (SPL Life
Science) fixed in 4% paraformaldehyde. Subsequently, the
slides were washed in PBS and mounted with mounting
media (Vector). Images were acquired with a Leica SP5
confocal microscope (Leica).
Three-dimensional clinorotation
The 3D clinostat is regarded as a useful tool to simulate
microgravity on the ground (Hemdan et al., 2009; Kang
et al., 2011). One day prior to clinorotation, GFP-LC3 cells
were plated into a T-25 flask or 35-mm cover-glass-bottom
dish. The dish was sealed with parafilm to avoid the leakage
of the medium, and the dish was placed on the 3D clinostat
129
with 3M tapes. After 24 h, the medium was replaced with
fresh medium and plated cells were subjected to clinorota-
tion for 24 or 72 h. The rotation cycles were controlled by an
external computer, and the sample stage was rotated with
two independent axes at a rate of 2 rpm. The entire system
was placed in an incubator set at 37C. Control cells were
placed in the incubator without clinorotation. For motion
control, cells were set in the laboratory platform rocker
(WiseMix RK-1D; WISD), and rotated at 5 rpm for 72 h.
siRNA transfection
The nucleotide sequence for AMPKa No. 1 was GCU
UGA UGC ACA CAU GAAU and the sequence for AMPKa
No. 2 was UGC CUA CCA UCU CAU AAUA. AMPK
siRNA and nonspecific (NS) siRNA were purchased from ST
Pharm. Transfections of siRNA were conducted using oligo-
fectamine reagent (Invitrogen) in accordance with the man-
ufacturer’s instructions. Twenty-four hours after transfection,
the cells were subjected to clinorotation.
Western blot
The cells were harvested and lysed in lysis buffer (150 mM
NaCl, 50 mM HEPES [pH 8.0], and 0.5% NP-40) containing
0.1 mM Na2VO3, 1 mM NaF, and a protease inhibitor cock-
tail (Roche). For protein immunoblots, polypeptides in the
whole-cell lysates were resolved via SDS-PAGE and trans-
ferred to nitrocellulose membrane filters. Immunodetection
was conducted with 1:1000–1:10,000 dilution of primary an-
tibody, using an enhanced chemiluminescence (ECL) system.
The images were acquired using the Chemidoc-it 410 imaging
system (UVP). The antibodies for LC3 and p62 were purchased
from Sigma, and antibodies for AMPK, phospho-AMPK
(T172), phospho-AMPK (S485), mTOR, and phospho-mTOR
(S2448) were purchased from Cell Signaling Technology. The
antibody for actin was purchased from Applied Biological
Materials (Richmond).
Results
Simulated microgravity-induced autophagy
in GFP-LC3 stable cells
Because microgravity conditions affect various cellular
processes, we attempted to examine whether microgravity is
involved in autophagy regulation. To simulate microgravity,
we used the clinostat as shown in Figure 1A. Initially, we
intended to validate whether our clinostat apparatus pro-
vided microgravity conditions by examining the expression
of MuRF-1 and ubiquitinated proteins, as previous studies
have shown that the expression of MuRF-1 and ubiquiti-
nated proteins changed after clinorotation (Hirasaka et al.,
2005; Hemdan et al., 2009). MuRF-1 is an E3 ubiquitin
ligase important in muscle atrophy (Bodine et al., 2001). As
reported before, the level of MuRF-1 in C2C12 myoblast
cells was significantly increased after 24 h of clinorotation
(Fig. 1B, C), and the level of ubiquitinated proteins in
HEK293 cells was decreased after 24 h of clinorotation (Fig.
1D). These results indicate that the current clinostat appa-
ratus adequately simulated microgravity.
For sensitive autophagy monitoring, we used the HEK293
cell lines that stably express GFP-LC3, an autophagosomal
marker. Various autophagy signals stimulate the formation
130 RYU ET AL.

FIG. 1. A clinostat was used to simulate microgravity. (A) Photograph of the three-dimensional clinostat. This apparatus
was used in the present study to simulate microgravity. (B) The increase in MuRF-1 expression upon exposure to simulated
microgravity. C2C12 myoblast cells were rotated for 24 h, and cell lysates were subjected to western blot analysis with anti-
MuRF-1 antibody. Experiments were repeated three times and the representative data were shown. (C) The density of
MuRF-1 bands was quantitated and plotted on the bar graph. *p < 0.001. (D) The decrease in ubiquitinated protein
expression upon exposure to simulated microgravity. HEK293 cells were rotated for 24 h, and cell lysates were subjected to
western blot analysis with anti-Ub antibody. Actin was used as an internal standard.

of cytoplasmic GFP-LC3 punctuates, which reflect autop-
hagy induction (Kabeya et al., 2000; Yoon et al., 2011).
GFP-LC3 cells were set on the clinostat to simulate mi-
crogravity and then rotated for 24 or 72 h. GFP-LC3 cells
exposed to microgravity for 72 h showed cytoplasmic GFP-
LC3 punctuates, which are reflective of the autophago-
somes, while cells exposed to other conditions did not (Fig.
2A, B). For motion control, we rotated GFP-LC3 cells using
the laboratory platform rocker at 5 rpm for 72 h; however,
we did not observe the cytoplasmic GFP-LC3 punctuates

FIG. 2. Simulated microgravity-induced autophagy. (A) Clinorotation for 72 h resulted in GFP-LC3 punctuates in the cytoplasm.
GFP-LC3 cells were set in the clinostat designed to simulate microgravity and rotated for 24 or 72 h, and control cells were kept under
the same environment without rotation. After clinorotation, cells were fixed and examined by confocal microscopy (White arrows
indicate autophagosome, scale bars =10 mM). (B) The number of GFP-LC3-dot–positive cells ( ‡ 5 dots per cell) was counted using a
fluorescent microscope (n = 300, *p < 0.005). (C) For motion control, GFP-LC3 cells were set in the laboratory platform rocker and
rotated at 5 rpm for 72 h, and control cells were kept under the same environment without rocking. (D) The number of GFP-LC3-dot–
positive cells was counted using a fluorescent microscope (n = 300).
MICROGRAVITY INDUCES AUTOPHAGY 131

(Fig. 2C, D). These results indicate that simulated micro-
gravity positively regulates autophagosome formation.
Expression of autophagy-related proteins under
simulated microgravity condition
To biochemically analyze autophagy induction under
simulated microgravity condition, we examined the ex-
pression patterns of LC3 and p62, which are widely used as
markers of autophagosomes. LC3 is initially produced in
an unprocessed form and then converted into LC3-I by C-
terminal domain cleavage. LC3-I is then modified into PE-
conjugated LC3-II, the autophagosomal marker (Klionsky
et al., 2008). GFP-LC3 cells placed in the clinostat for 24
or 72 h and untreated control cells were collected and the
cell lysates were subjected to western blot analysis with
anti-LC3 antibody. While other conditions did not show a
significant change in the density of GFP-LC3-II bands,
clinorotation for 72 h resulted in an elevation of GFP-LC3-
II level (Fig. 3A).
We then examined the level of p62, another autophagy
marker. Because p62 (also called SQSTM1) is degraded by
autophagy and inhibition of autophagy upregulates p62, p62
has been implicated as a potential autophagic marker
(Pankiv et al., 2007). Western blot analysis with anti-p62
antibody showed reduced levels of endogenous p62 after
72 h of clinorotation (Fig. 3A). Western blot results were
consistent with the microscopy data, and these results col-
lectively indicate that simulated microgravity positively
regulates autophagy induction. Inhibition of autophagic flux
using bafilomycin A1 resulted in the elevated level of en-
dogenous LC3-II, indicating that autophagosomes were not
formed due to inhibition of lysosomal degradation (Fig. 3B).
Since autophagy is induced by simulated microgravity
conditions, we attempted to determine the cellular signaling
pathway involved in autophagy induction. Recent studies
showed that AMPK and mTOR regulate autophagy directly
via phosphorylation of Ulk1 (yeast ATG1) (Kim et al.,
2011; Mihaylova and Shaw, 2011). We examined phos-
phorylation at two sites on AMPK (T172 and S485) and
mTOR. AMPK is activated by phosphorylation at threonine
172 (T172) within the a subunit activation loop and is
suppressed by phosphorylation at serine 485 (S485) (Woods
et al., 2003; Adams et al., 2004; Ning et al., 2011). Clin-
orotation for 24 and 72 h increased the level of phosphory-
lation of AMPK at T172 when compared with the control
(Fig. 3C). The phosphorylation of AMPK at S485 is reduced
by exposure to microgravity for 24 h; however, there was no

FIG. 3. Regulation of AMPK and mammalian target of rapamycin (mTOR) under simulated microgravity. (A) Expres-
sion of an autophagosome marker under simulated microgravity. The level of GFP-LC3-II was increased and the level of
p62 was decreased in cells subjected to simulated microgravity. After clinorotation, cells were collected for lysis and equal
amounts of cell lysates were subjected to western blot analysis with anti-LC3 antibody, anti-p62 antibody, and anti-
actin antibody. (B) GFP-LC3 cells were rotated for 72 h and autophagic flux was inhibited by bafilomycin A1 treatment
(100 nM, 4 h). (C) The phosphorylation of AMPKa and mTOR was changed by clinorotation. GFP-LC3 cells were set in the
clinostat and rotated for 24 or 72 h, and control cells were kept under the same conditions without rotation. After clino-
rotation, cells were collected for lysis and equal amounts of cell lysates were subjected to western blot analysis using the
corresponding antibodies. (D) For motion control, GFP-LC3 cells were set in the laboratory platform rocker and incubated
for 72 h, cells were collected for lysis, and equal amounts of cell lysates were subjected to western blot analysis.
132 RYU ET AL.

difference between the control and cells exposed to clinor-
otation at 72 h. Clinorotation decreased the level of phos-
phorylation at serine 2448 of mTOR in the same manner
(Fig. 3C). In addition, we examined the activation of AMPK
in the motion control; however, the level of phosphorylation
of AMPK at T172 was not significantly changed by rocking
for 72 h (Fig. 3D). These results suggest that simulated
microgravity activates AMPK and suppresses mTOR, and
these events are potentially involved in autophagy regula-
tion.
AMPK inhibition interferes
with microgravity-induced autophagy
Because AMPK is reported to respond to environmental
stresses, we hypothesized that AMPK activity may regulate
microgravity-induced autophagy. To examine the role of
AMPK in autophagy induction, we used AMPK siRNA to
suppress AMPK expression. GFP-LC3 cells were trans-
fected with control siRNA, AMPK siRNA No. 1, or AMPK
siRNA No. 2, and the transfected cells were set in the
clinostat to simulate microgravity and rotated for 72 h. We
examined the level of AMPK expression and AMPK ex-
pression was efficiently silenced by AMPK siRNA trans-
fection (Fig. 4A). While the control siRNA cells exhibited
autophagy induction by clinorotation, the AMPK-depleted
cells did not (Fig. 4B, C). AMPK-depleted cells did not
show autophagosome formation in the cytoplasm nor did
they exhibit changes in the expression of autophagosome
markers, GFP-LC3-II and p62 (Fig. 4B). These results in-
dicate that AMPK is involved in microgravity-induced au-
tophagy induction.
Simulated microgravity-induced autophagy
in C2C12 myotube cells
Next, we examined whether simulated microgravity in-
duced autophagy in C2C12 myotube cells. C2C12 myoblast
cells were incubated in the differentiation medium to form
the multinucleated myotube cells, and the cells were clin-
orotated for 72 h to simulate microgravity. Multinucleated
myotube cells exposed to microgravity showed the enlarged
and distinctive LC3 spots in the cytoplasm, suggesting that
the simulated microgravity induced autophagy in C2C12
myotube cells (Fig. 5A). To confirm the results, the C2C12
myotube cells exposed to microgravity were collected and
analyzed by western blots. The level of LC3-II was elevated,
suggesting that simulated microgravity induced autophagy

FIG. 4. Knockdown of
AMPK interferes with the
autophagosome formation
under microgravity. (A)
GFP-LC3 cells were trans-
fected with siRNA against
AMPK and autophagy mar-
ker (GFP-LC3-II and p62)
expression was examined by
western blot. The expression
of AMPK was suppressed by
two different siRNAs,
AMPK siRNA No. 1 and
AMPK siRNA No. 2. (B)
Autophagosome formation in
GFP-LC3 cells transfected
with AMPK siRNA. GFP-
LC3 cells were transfected
with control, AMPK siRNA
No. 1, or AMPK siRNA No.
2, fixed, and examined by
confocal microscopy (scale
bars = 10 mm). (C) The num-
ber of GFP-LC3-dot–positive
cells ( ‡ 5 dots per cell) was
counted using a fluorescent
microscope (n = 300,
*p < 0.01).
MICROGRAVITY INDUCES AUTOPHAGY
in C2C12 myotube cells (Fig. 5B). In addition, clinorotation
for 72 h increased the level of phosphorylation of AMPK at
T172 when compared with the control (Fig. 5B).
Discussion
Several studies have focused on the biological effect of
microgravity; however, little is known about the role of
microgravity in autophagy induction. We have demonstrated
that simulated microgravity induces autophagy in GFP-LC3
cells. In this study, a 3D-clinostat was used to simulate
microgravity to examine autophagy induction. The clinostat
is a useful apparatus to study gravitational biology (Arase
et al., 2002). Previously, a clinostat was used to examine the
inhibitory effect of simulated microgravity on cell differ-
entiation, as well as its stimulatory role in cellular apoptosis
(Sarkar et al., 2000; Grimm et al., 2002; Hirasaka et al.,
2005). The GFP-LC3 recombinant protein has been widely
used for monitoring autophagy microscopically due to its
sensitivity, and the detection of GFP-LC3 is especially
useful for in vivo studies (Kabeya et al., 2000; Galluzzi
et al., 2009). Because the transient expression of GFP-LC3
often results in artifacts due to overexpression, we used
GFP-LC3 stable cells (HEK293 cells that stably express
GFP-LC3) to reduce background and artifacts (Galluzzi
et al., 2009). This clinostat and GFP-LC3 stable cell system
seems to provide a reliable platform with which to examine
the effect of microgravity on autophagy induction.
AMPK is a key regulator of autophagy that responds to
the AMP/ATP ratio. Starvation conditions activate AMPK,
which induces autophagy for energy generation (Mihaylova
and Shaw, 2011). AMPK is activated by phosphorylation at
threonine 172 (T172) and is suppressed by phosphorylation
at serine 485 (S485) (Woods et al., 2003; Adams et al.,
2004; Ning et al., 2011). The inhibitory role of mTOR in
autophagy is also well established (Kim et al., 2011; Mi-
haylova and Shaw, 2011). Since AMPK also responds to
133
FIG. 5. Simulated micro-
gravity induced autophagy in
C2C12 myotube cells. (A)
C2C12 myoblast cells were
incubated in the differentia-
tion medium to form the
multinucleated myotubes,
and the differentiated cells
were rotated for 72 h. After
clinorotation, cells were fixed
and stained with anti-LC3
antibody. Scale bar = 10 mm.
White arrows indicate au-
tophagosome. (B) After ro-
tation for 72 h, C2C12
myotube cells were collected
for lysis and equal amounts
of cell lysates were subjected
to western blot analysis using
the corresponding antibodies.
cellular stresses, such as hypoxia and heat shock, we hy-
pothesized that AMPK plays an important role in autophagy
induction upon exposure to simulated microgravity. Because
simulated microgravity by clinorotation induces autophagy,
we examined AMPK in cells exposed to microgravity.
Clinorotation for both 24 and 72 h resulted in the phos-
phorylation of AMPK (T172) (Fig. 3A), suggesting that
AMPK is involved in microgravity-induced autophagy. To
prove the role of AMPK in microgravity-induced autop-
hagy, cells were treated with AMPK siRNA to block its
activity. AMPK-depleted cells did not exhibit autophagy
induction. These results collectively suggest that simulated
microgravity contributes to autophagy, possibly via AMPK
mediation. At this point, we question why clinorotation for
24 h did not induce autophagy, despite AMPK activation.
One possible explanation is that the activation of AMPK at
24 h does not reach an appropriate level to lead to autophagy
induction. As shown in Figure 3B, the level of AMPK
phosphorylation (T172) at 24 h was lower than the level of
AMPK phosphorylation of the control sample at 72 h.
Moreover, the different site of phosphorylation (S485), an
inhibitory phosphorylation site, was less phosphorylated at
72 h. Although the level of phosphorylation (S485) at 24 h
differed between the control cells and those exposed to
clinorotation, the level of phosphorylation (S485) at 72 h
was not significantly different. The phosphorylation of
AMPK (S485) is often mediated by growth factors such as
IGF-1; hence, the dephosphorylation of AMPK (S485) can
be mediated by growth factor deficiency (Ning et al., 2011).
These results suggest that prolonged culture can provide a
stressful condition to cells and also contributes to autophagy
induction. In other words, microgravity positively regulates
autophagy induction, and other environmental conditions
may be required to induce autophagy.
AMPK affects many cellular phenomena, such as cell
growth and metabolism (Mihaylova and Shaw, 2011).
Based on our study, it is clear that AMPK is positively
134
regulated by simulated microgravity. Clinorotation for 24 h
resulted in the activation of AMPK, although autophagy was
not observed under these conditions. For this reason, it will
be valuable to study the other effects of microgravity with
regard to AMPK activation. Additionally, autophagy in-
duction is just one phenomenon of microgravity-induced
AMPK activation. Further studies are required to evaluate
AMPK activation induced by microgravity exposure.
During atrophy, the ubiquitin/proteasomes degrade myo-
fibrillar proteins via a ubiquitin-proteasome pathway, and
autophagy is responsible for long-lived proteins and organ-
elles (Zhao et al., 2007). Our data suggest that microgravity
contributes to autophagy induction, which is potentially
linked to muscle atrophy. For this reason, it is possible that
microgravity affects muscle atrophy, at least in part by reg-
ulating autophagy. Moreover, it is possible that autophagy
inhibitors could serve as a potential solution to microgravity-
induced atrophy. Further studies are required to prove the
effect of microgravity-induced autophagy on muscle atrophy.
Acknowledgments
The authors would particularly like to thank T. Yoshimori
for providing the GFP-LC3 plasmid. This research was
supported by Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by
the Ministry of Education (MOE; 2013-053960) and by the
Leading Space Core Technology Development Program
through NRF funded by the Ministry of Science, ICT&Fu-
ture Planning (MSIP; 2013-042433). The development of
the clinostat was supported by the Leading Space Core
Technology Development Program through NRF funded by
MSIP (2011-0030888).
Disclosure Statement
No competing financial interests exist.
References
Adams, J., Chen, Z.P., Van Denderen, B.J., Morton, C.J., Par-
ker, M.W., Witters, L.A., Stapleton, D., and Kemp, B.E.
(2004). Intrasteric control of AMPK via the gamma1 subunit
AMP allosteric regulatory site. Protein Sci 13, 155–165.
Arase, Y., Nomura, J., Sugaya, S., Sugita, K., Kita, K., and
Suzuki, N. (2002). Effects of 3-D clino-rotation on gene ex-
pression in human fibroblast cells. Cell Biol Int 26, 225–233.
Bodine, S.C., Latres, E., Baumhueter, S., Lai, V.K., Nunez, L.,
Clarke, B.A., Poueymirou, W.T., Panaro, F.J., Na, E., Dhar-
marajan, K., et al. (2001). Identification of ubiquitin ligases
required for skeletal muscle atrophy. Science 294, 1704–
1708.
Corton, J.M., Gillespie, J.G., and Hardie, D.G. (1994). Role of
the AMP-activated protein kinase in the cellular stress re-
sponse. Curr Biol 4, 315–324.
Galluzzi, L., Aaronson, S.A., Abrams, J., Alnemri, E.S., An-
drews, D.W., Baehrecke, E.H., Bazan, N.G., Blagosklonny,
M.V., Blomgren, K., Borner, C., et al. (2009). Guidelines for
the use and interpretation of assays for monitoring cell death
in higher eukaryotes. Cell Death Differ 16, 1093–1107.
Grimm, D., Bauer, J., Kossmehl, P., Shakibaei, M., Schoberger,
J., Pickenhahn, H., Schulze-Tanzil, G., Vetter, R., Eilles, C.,
Paul, M., et al. (2002). Simulated microgravity alters differ-
RYU ET AL.
entiation and increases apoptosis in human follicular thyroid
carcinoma cells. FASEB J 16, 604–606.
Hemdan, D.I., Hirasaka, K., Nakao, R., Kohno, S., Kagawa, S.,
Abe, T., Harada-Sukeno, A., Okumura, Y., Nakaya, Y.,
Terao, J., et al. (2009). Polyphenols prevent clinorotation-
induced expression of atrogenes in mouse C2C12 skeletal
myotubes. J Med Invest 56, 26–32.
Hirasaka, K., Nikawa, T., Yuge, L., Ishihara, I., Higashibata, A.,
Ishioka, N., Okubo, A., Miyashita, T., Suzue, N., Ogawa, T.,
et al. (2005). Clinorotation prevents differentiation of rat
myoblastic L6 cells in association with reduced NF-kappa B
signaling. Biochim Biophys Acta 1743, 130–140.
Ikemoto, M., Nikawa, T., Takeda, S., Watanabe, C., Kitano, T.,
Baldwin, K.M., Izumi, R., Nonaka, I., Towatari, T., Teshima, S.,
et al. (2001). Space shuttle flight (STS-90) enhances degradation
of rat myosin heavy chain in association with activation of
ubiquitin-proteasome pathway. FASEB J 15, 1279–1281.
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako,
T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T.
(2000). LC3, a mammalian homologue of yeast Apg8p, is
localized in autophagosome membranes after processing.
EMBO J 19, 5720–5728.
Kang, C.Y., Zou, L., Yuan, M., Wang, Y., Li, T.Z., Zhang, Y.,
Wang, J.F., Li, Y., Deng, X.W., and Liu, C.T. (2011). Impact
of simulated microgravity on microvascular endothelial cell
apoptosis. Eur J Appl Physiol 111, 2131–2138.
Kim, J., Kundu, M., Viollet, B., and Guan, K.L. (2011). AMPK
and mTOR regulate autophagy through direct phosphoryla-
tion of Ulk1. Nat Cell Biol 13, 132–141.
Klionsky, D.J., Abeliovich, H., Agostinis, P., Agrawal, D.K.,
Aliev, G., Askew, D.S., Baba, M., Baehrecke, E.H., Bahr,
B.A., Ballabio, A., et al. (2008). Guidelines for the use and
interpretation of assays for monitoring autophagy in higher
eukaryotes. Autophagy 4, 151–175.
Kroemer, G., and Jaattela, M. (2005). Lysosomes and autop-
hagy in cell death control. Nat Rev Cancer 5, 886–897.
Kudo, N., Barr, A.J., Barr, R.L., Desai, S., and Lopaschuk, G.D.
(1995). High rates of fatty acid oxidation during reperfusion
of ischemic hearts are associated with a decrease in malonyl-
CoA levels due to an increase in 5¢-AMP-activated protein
kinase inhibition of acetyl-CoA carboxylase. J Biol Chem
270, 17513–17520.
Levine, B., and Klionsky, D.J. (2004). Development by self-
digestion: molecular mechanisms and biological functions of
autophagy. Dev Cell 6, 463–477.
Mihaylova, M.M., and Shaw, R.J. (2011). The AMPK signal-
ling pathway coordinates cell growth, autophagy and me-
tabolism. Nat Cell Biol 13, 1016–1023.
Miranda, N., Tovar, A.R., Palacios, B., and Torres, N. (2007).
[AMPK as a cellular energy sensor and its function in the
organism]. Rev Invest Clin 59, 458–469.
Nikawa, T., Ishidoh, K., Hirasaka, K., Ishihara, I., Ikemoto, M.,
Kano, M., Kominami, E., Nonaka, I., Ogawa, T., Adams,
G.R., et al. (2004). Skeletal muscle gene expression in space-
flown rats. FASEB J 18, 522–524.
Ning, J., Xi, G., and Clemmons, D.R. (2011). Suppression of
AMPK activation via S485 phosphorylation by IGF-I during
hyperglycemia is mediated by AKT activation in vascular
smooth muscle cells. Endocrinology 152, 3143–3154.
Pankiv, S., Clausen, T.H., Lamark, T., Brech, A., Bruun, J.A.,
Outzen, H., Overvatn, A., Bjorkoy, G., and Johansen, T.
(2007). p62/SQSTM1 binds directly to Atg8/LC3 to facilitate
degradation of ubiquitinated protein aggregates by autophagy.
J Biol Chem 282, 24131–24145.
MICROGRAVITY INDUCES AUTOPHAGY
Riley, D.A., Ellis, S., Slocum, G.R., Satyanarayana, T., Bain,
J.L., and Sedlak, F.R. (1987). Hypogravity-induced atrophy
of rat soleus and extensor digitorum longus muscles. Muscle
Nerve 10, 560–568.
Ruest, L.B., Marcotte, R., and Wang, E. (2002). Peptide elon-
gation factor eEF1A-2/S1 expression in cultured differentiated
myotubes and its protective effect against caspase-3-mediated
apoptosis. J Biol Chem 277, 5418–5425.
Sanchez, A.M., Candau, R.B., Csibi, A., Pagano, A.F., Raibon,
A., and Bernardi, H. (2012). The role of AMP-activated
protein kinase in the coordination of skeletal muscle turnover
and energy homeostasis. Am J Physiol Cell Physiol 303,
C475–C485.
Sanchez, A.M., Csibi, A., Raibon, A., Cornille, K., Gay, S.,
Bernardi, H., and Candau, R. (2011). AMPK promotes skel-
etal muscle autophagy through activation of Forkhead Fox-
O3a and interaction with Ulk1. J Cell Biochem 113,.695–710.
Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Pi-
card, A., Walsh, K., Schiaffino, S., Lecker, S.H., and Gold-
berg, A.L. (2004). Foxo transcription factors induce the
atrophy-related ubiquitin ligase atrogin-1 and cause skeletal
muscle atrophy. Cell 117, 399–412.
Sarkar, D., Nagaya, T., Koga, K., Kambe, F., Nomura, Y., and
Seo, H. (2000). Rotation in clinostat results in apoptosis of
osteoblastic ROS 17/2.8 cells. J Gravit Physiol 7, P71–P72.
Solomon, V., and Goldberg, A.L. (1996). Importance of the
ATP-ubiquitin-proteasome pathway in the degradation of
soluble and myofibrillar proteins in rabbit muscle extracts. J
Biol Chem 271, 26690–26697.
Tamma, R., Colaianni, G., Camerino, C., Di Benedetto, A.,
Greco, G., Strippoli, M., Vergari, R., Grano, A., Mancini, L.,
135
Mori, G., et al. (2009). Microgravity during spaceflight di-
rectly affects in vitro osteoclastogenesis and bone resorption.
FASEB J 23, 2549–2554.
Woods, A., Vertommen, D., Neumann, D., Turk, R., Bayliss, J.,
Schlattner, U., Wallimann, T., Carling, D., and Rider, M.H.
(2003). Identification of phosphorylation sites in AMP-
activated protein kinase (AMPK) for upstream AMPK
kinases and study of their roles by site-directed mutagenesis.
J Biol Chem 278, 28434–28442.
Yoon, J.H., Her, S., Kim, M., Jang, I.S., and Park, J. (2011). The
expression of damage-regulated autophagy modulator 2
(DRAM2) contributes to autophagy induction. Mol Biol Rep
39, 1087–1093.
Zhao, J., Brault, J.J., Schild, A., Cao, P., Sandri, M., Schiaffino,
S., Lecker, S.H., and Goldberg, A.L. (2007). FoxO3 coordi-
nately activates protein degradation by the autophagic/lyso-
somal and proteasomal pathways in atrophying muscle cells.
Cell Metab 6, 472–483.
Address correspondence to:
Junsoo Park, PhD
Division of Biological Science and Technology
Yonsei University
1 Yonseidae-gil
Wonju City 220-100
Republic of Korea
E-mail: junsoo@yonsei.ac.kr
Received for publication May 6, 2013; received in revised
form November 25, 2013; accepted November 26, 2013.