Acta Physiol 2014, 212, 75–85
Oestrogen-dependent satellite cell activation and
proliferation following a running exercise occurs via the
PI3K signalling pathway and not IGF-1
G. Mangan,1 E. Bombardier,2 A. S. Mitchell,2 J. Quadrilatero2 and P. M. Tiidus1
1 Departments of Kinesiology and Physical Education and Health Sciences, Wilfrid Laurier University, Waterloo, ON, Canada
2 Department of Kinesiology, University of Waterloo, Waterloo, ON, Canada
Received 12 February 2014,
revision requested 24 March
2014,
revision received 16 May 2014,
accepted 19 May 2014
Correspondence: P. M. Tiidus,
PhD, Health Sciences, Wilfrid
Laurier University, 75 University
Avenue West, Waterloo, ON,
Canada N2L3C5.
E-mail: [email protected]
Abstract
Aim: The purpose of this study was to determine whether 17b-estradiol
(E2) enhances the activation, proliferation and differentiation of muscle
satellite cells (SC) following eccentric exercise either via insulin-like
growth factor-1 (IGF-1) or through phosphatidylinositol 3-kinase (PI3K)
signalling.
Methods: This study used 64, 9-week-old, ovariectomized Sprague–Dawley rats that were divided into eight treatments groups based on oestrogen
status (0.25 mg oestrogen pellet or sham), exercise status (90 min run @
17 m min1, 13.5° or unexercised) and PI3K signalling inhibition
(0.7 mg wortmannin kg1 body weight or DMSO control).
Results: Significant increases in total SCs were found in both soleus and
white gastrocnemius muscles (immunofluorescent co-localization of Pax7+
nuclei) 72 h following eccentric exercise (P < 0.05). Oestrogen supplementation caused a further enhancement in total SCs in exercised rats
(P < 0.05). In animals where the PI3K pathway was inhibited, regardless
of oestrogen or exercise status, there was no significant enhancement of
SC number in both the soleus or white gastrocnemius muscles. Interestingly, oestrogen supplementation lowered muscle levels of IGF-1 with this
effect being most prominent in the soleus muscle. While IGF-1 was
increased following exercise (P < 0.05), oestrogen supplementation abrogated this increase back to sedentary levels.
Conclusion: These data suggest that the increase in SC population following exercise in oestrogen-supplemented females may be mediated via PI3K
pathway signalling and not IGF-1.
Keywords oestrogen, insulin-like growth factor-1, muscle, phosphatidylinositol-3 kinase, satellite cells.
Oestrogen treatment has been reported to enhance
muscle mass recovery following atrophy in rodents
(Brown et al. 2005, McClung et al. 2006) and to
maintain and enhance muscle mass in post-menopausal females (Ronkainen et al. 2009, Tiidus et al.
2013). There are a number of possible mechanisms by
which oestrogen may influence muscle mass including
enhancement of pro-anabolic signalling (Dieli-Conwright et al. 2009b), activating various other signalling pathways (Sitnick et al. 2006, Murray & Huss
2011), as well as reductions in post-exercise systemic
and muscle inflammatory responses and damage
(Dieli-Conwright et al. 2009a, Tiidus et al. 2013). In
addition, we have consistently demonstrated that
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
75
Oestrogen signalling via PI3K
· G Mangan et al.
oestrogen augments post-exercise muscle satellite cell
activation and proliferation in ovariectomized rodents
beyond that achieved by exercise alone (Enns &
Tiidus 2008, Enns et al. 2008).
It has also been demonstrated that oestrogen will
manifest this augmentation of muscle satellite cells and
muscle mass through binding of oestrogen receptors
alpha (ERa) and/or beta (ERb) (Enns et al. 2008,
Thomas et al. 2010, Velders et al. 2012). Augmented
activity following oestrogen supplementation in an
otherwise oestrogen-reduced state such as in ovariectomized rodents or potentially in post-menopausal
women leads to the increased involvement of satellite
cells in muscle adaptation (Thomas et al. 2010, Velders
et al. 2012, Tiidus et al. 2013, Velders & Diel 2013).
Communication between growth factors and signalling pathways increases the activity of certain transcription factors, leading to the activation of satellite
cells. Activation and subsequent proliferation and differentiation can cause the satellite cells to become
myoblasts that can then be fused with an existing myofibres (Hawke & Garry 2001, Yin et al. 2013). The
signalling pathways by which oestrogen communicates
with satellite cells are currently unknown. Links
between oestrogen and insulin-like growth factor-1
(IGF-1), as well as IGF-1 and the phosphatidylinositol3 kinase (PI3K)/Akt pathway, have been established.
Gao et al. (2004) described a link between IGF-1related induction of the PI3K/Akt signalling pathway
and satellite cell G1 cell cycle activation. Sitnick et al.
(2006) suggested an oestrogen-related relationship
between the PI3K/Akt pathway and augmentation of
post-unloading recovery of skeletal muscle mass. Kamanga-Sollo et al. (2008) have also suggested a role
for IGF-1 and oestrogen-related signalling in bovine
satellite cell proliferation. Oestrogen receptor interactions with the PI3K/Akt pathway have also been implicated in the regulation of cortical neurone regeneration
(Mannella & Brinton 2006).
Insulin-like growth factor-1 has been implicated as
a positive regulator of muscle growth and repair
(Barton et al. 2010). In addition, oestrogen receptor
agonists have been reported to augment muscle IGF-1
expression and muscle hypertrophy in rodent models,
suggesting a link between oestrogen signalling and
IGF-1-induced muscle satellite cell-related hypertrophy
(Velders et al. 2012). Nevertheless, resistance-exerciseinduced muscle hypertrophy can occur in animals
lacking IGF-1 receptors via other signalling pathways
(Spangenburg et al. 2008). While oestrogen replacement in post-menopausal females has been associated
with upregulation of IGF-1-signalling-related muscle
mass maintenance (Ahtiainen et al. 2012), oestrogen
has been shown to diminish muscle IGF-1 levels in
ovariectomized immature rodents (Tsai et al. 2007).
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Acta Physiol 2014, 212, 75–85
Hence, an association for oestrogen linked regulation
of muscle satellite cell function via PI3K/Akt signalling, while intriguing remains still unclear. The purpose of this study was to investigate the potential for
PI3K/Akt signalling and IGF-1 involvement in the
post-exercise oestrogen-related augmentation of muscle satellite cell proliferation in ovariectomized
rodents.
Methods
Animals
This study was approved by the Animal Care Committee at Wilfrid Laurier University and is in accordance with the Canada Council on Animal Care
standards for animal research. A total of 64 ovariectomized female Sprague–Dawley rats (Charles River
Laboratories, LaSalle, QC, Canada) were used in this
study. The ovaries were surgically removed at 8 weeks
of age at source and arrived at the laboratory 1 week
later. Animals were single-housed in a temperaturecontrolled environment with a standard 12 : 12 light/
dark cycle and allowed ad libitum access to food
(Tekland 22/5 Rodent Diet; Harlan-Tekland, Madison, WI, USA) and water. Upon arrival to the laboratory, the rats were divided into eight groups based on
three factors: oestrogen supplementation, PI3K inhibition and exercise level (Fig. 1). The animals were
acclimatized to the animal care facility for 1 week
prior to commencement of any study protocols.
Oestrogen supplementation protocol
The ovariectomized rats have basal levels of circulating oestrogen and were considered the control group.
The experimental group was similar to the control
group except for the addition of an oestrogen pellet
that was administered through surgery.
One week after arrival, each rat was subjected to
either an oestrogen pellet implantation or a sham
procedure (Enns & Tiidus 2008). Under aseptic conditions, the rats were anesthetized via inhalation of isoflurane. Excess hair on the incision site was removed
using electric trimmers. Prior to making the incision,
the analgesic Marcaine (8 g kg1 BW; 2.5 mg mL1;
Hospira Healthcare Corporation; Montreal, QC,
Canada) was injected into the skinfolds of the neck. A
small incision using a No. 10 blade was made in the
skin penetrating to the underlying fascia. For the experimental group, an oestrogen pellet (0.25 mg 17b-estradiol, 21 day release; Innovative Research of America,
Sarasota, FL, USA) was inserted. Each pellet released
on average 0.012 mg day1 of estradiol. The control
group underwent the same surgery albeit without
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
Acta Physiol 2014, 212, 75–85
G Mangan et al.
· Oestrogen signalling via PI3K
Ovariectomized female rats
(n = 64)
Sham procedure (S--)
(n = 32)
Control group (-C-)
(n = 16)
Saline
injecƟon
(--S)
(n = 8)
WTN
injecƟon
(--W)
(n = 8)
Estrogen supplementaƟon (E--)
(n = 32)
Exercise group (-E-)
(n = 16)
Saline
injecƟon
(--S)
(n = 8)
WTN
injecƟon
(--W)
(n = 8)
Control group (-C-)
(n = 16)
Saline
injecƟon
(--S)
(n = 8)
WTN
injecƟon
(--W)
(n = 8)
Exercise group (-E-)
(n = 16)
Saline
injecƟon
(--S)
(n = 8)
WTN
injecƟon
(--W)
(n = 8)
Figure 1 Schematic outline of protocol.
the insertion of the oestrogen pellet. After the incision
site was closed with a staple, the rat was taken off the
isoflurane and was placed into a separate cage to
recover.
After the oestrogen supplementation protocol (ESP),
the rats were returned to their cages for the next
seven days. Two five-minute treadmill acclimatization
sessions took place on the motorized treadmill
(Columbus Instruments, Columbus, OH, USA) on
days 3 and 4 following the ESP.
Exercise protocol
Seven days after the ESP, the rats in the exercise
group were placed on a motorized rodent treadmill
supplemented with an electric shock grid (Columbus
Instruments). The animals ran at approx. 17 m min1
at a grade of 13.5° for 5 min followed by 2 min of
rest. This was repeated until 90 min had been completed. This intermittent protocol was designed to be
non-fatiguing while still eliciting damage to muscles
that include the predominantly type I soleus muscle
and predominantly type IIb white gastrocnemius
(Komulainen et al. 1994). The damaging nature of the
protocol has been validated by the significant increases
in b-glucuronidase activity, following exercise in the
muscles used in this study (Salminen & Kihlstr€
om
1985, Enns & Tiidus 2008, Enns et al. 2008, Thomas
et al. 2010). The sedentary control rats were placed in
a stationary treadmill for the same total amount of
time as the exercise rats. After the exercise protocol,
the rats were returned to their cages (Enns et al.
2008, Enns & Tiidus 2008, Thomas et al. 2010).
PI3K inhibitor protocol
Wortmannin (WTN) is an highly specific inhibitor of
the PI3K signalling pathway as reflected by an IC50
between 2 and 4 nM, which is lower than other PI3K
inhibitors like LY294002 (Powis et al. 1994). Previous
studies have used WTN to investigate the PI3K signalling pathway. However, these studies either excised
the muscle following a damaging protocol and immediately placed it in a WTN bath or investigated the
short-term effects (~4–6 h) of a WTN injection following a damaging protocol (Kang et al. 2004, Zhang
et al. 2007, 2009, Tong et al. 2011, Ye et al. 2012).
As the number of activated satellite cell numbers
reaches a maximum 72 h post-exercise, it was necessary to determine a protocol that would inhibit PI3K
activity in vivo for this duration. We conducted a
preliminary experiment in which 1–3 days of daily
intraperitoneal injections of WTN (0.7 mg WTN
kg1 BW, 86 mg WTN mL1 DMSO, 0.7% DMSO
solution; Selleckchem, Houston, TX, USA) were given
to six female rats. Inhibition of the PI3K signalling
pathway was quantified via Western Blot of Akt phosphorylation (p-Akt). It was determined that 3 days of
daily WTN injections (which was the most appropriate
time course for our study) reduced the amount of
phosphorylated Akt by 71% with no overt negative
symptoms noted in the animals.
Four intraperitoneal injections of WTN were given
to the rats. The first injection occurred 15 min before
commencement of the treadmill exercise protocol. The
three remaining injections were given 20, 40 and 60 h
after the exercise protocol. This amount of WTM was
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
77
Oestrogen signalling via PI3K
· G Mangan et al.
greater than the cumulative amount received in a series
of injections reported in a similar study (de Lange et al.
2008). The control group followed the same schedule
albeit with intraperitoneal injections of saline. No
animals experienced overt negative symptoms related
to WTN exposure during the course of the study.
Killing and blood and tissue collection
Seventy-two hours after completion of the exercise
protocol, the animals were injected an overdose of
sodium pentobarbital (55 mg kg BW1). Blood was
collected from the right ventricle using a heparincoated syringe and deposited into an eppendorf tube.
The blood was centrifuged at 4500 relative centrifugal
force (centripetal acceleration relative to gravitational
acceleration) for 20 min at 4 °C to fractionate the
blood into erythrocytes and plasma. The plasma was
collected, placed in another eppendorf tube and frozen
at 80 °C until further analysis.
Soleus (SOL) and white gastrocnemius (WG) muscles were collected from both the right and left leg of
the animal. The WG is located superficially on both
the lateral and medial heads of the gastrocnemius
(Matsakas et al. 2006). SOL has a fibre-type composition that is 96.6% Type I while 88.1% of WG is comprised type IIB fibres (Bloemberg & Quadrilatero
2012). Muscle samples were placed in an ice-cold
PMSF buffer (250 mM sucrose, 5 mM HEPES, 10 mM
NaN3 and 0.2 mM phenylmethanesulfonyl fluoride,
pH 7.5). Muscle samples intended for immunohistochemical analysis were mounted and coated in optimal
cutting temperature medium (Tissue-Tek, Torrance,
CA, USA) and placed in liquid nitrogen cooled isopentane to freeze. Samples intended from enzyme and
protein analyses were flash frozen in liquid nitrogen
and stored at 80 °C.
Blood plasma analysis
Plasma oestrogen levels were determined in duplicate
using the Coat-a-Count radioimmunoassay kit (Inter
Medico, Markham, ON, USA). Within the Tiidus laboratory, the test–retest variability of this procedure is
4–7% (Enns & Tiidus 2008).
IGF-1 protein determination
To determine the amount of IGF-1 protein in SOL and
WG, a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was employed in combination
with the bicinchoninic acid (BCA) protein assay. Sample preparation required using approx. 15–20 mg of
muscle tissue diluted 1 : 25 (weight : volume) in
19 phosphate-buffered saline (PBS) that was then
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Acta Physiol 2014, 212, 75–85
manually homogenized and subsequently frozen in
liquid nitrogen and stored at 80 °C. A freeze/thaw
process allowed for the mechanical breakdown of cell
membranes. The samples were then centrifuged at
2650 g for 5 min allowing for extraction of the supernatant. IGF-1 concentrations within the supernatant of
the muscle homogenate were determined using an IGF1 ELISA kit (R&D Systems, Minneapolis, MN, USA)
following manufacturers’ instructions. For the ELISA,
50 lL of supernatant was diluted 1 : 4 (vol : vol) in
calibrator diluent (R&D Systems). For the BCA protein assay, 10 lL of supernatant was diluted 1 : 490
(vol : vol) in dH2O. The final value is represented as
the amount of IGF-1 protein with respect to total protein.
Muscle sectioning and immunofluorescence
Serial cross sections of the mounted SOL and WG
muscles (10 lm) were cut in a cryostat maintained at
20 °C. These muscle sections were transferred onto
Vectabond-coated (Vector Laboratories, Burlington,
ON, Canada) glass slides that were then dried at room
temperature and subsequently stored at 80 °C. Serial
sections were made to determine fibre type and satellite
cell activation/proliferation relationships within the
experiment (Bloemberg & Quadrilatero 2012).
Immunofluorescent analysis of paired box 7 (Pax7),
a transcription factor that is expressed in all satellite
cell whether they are in the quiescent or activated
state, and MyoD, a myogenic regulatory factor (MRF)
expressed in activated satellite cells and not quiescent
satellite cells (Hawke & Garry 2001, Yin et al. 2013),
was performed on both SOL and WG. Briefly, all
samples were fixed in 4% formaldehyde solution for
5 min and then submersed in the detergent, Triton
X-100, for 10 min to permeabilize the membranes.
These steps were each followed by three washes in a
19 PBS solution. The slides were then incubated for
1 h in a blocking solution consisting of 10% normal
goat serum in PBS to block non-specific sites. For
Pax7 immunostaining, slides were incubated with a
primary antibody Pax7 (Developmental Studies
Hybridoma Bank, University of Iowa, Iowa City, IA,
USA) for 1 h at a dilution of 1 : 40. For MyoD
immunostaining, slides were incubated with a primary
antibody MyoD (Dako Canada, Mississauga, ON,
Canada, M3512) at a dilution of 1 : 50. To detect
Pax7 and MyoD, a goat anti-mouse AlexaFluor 488
secondary was used on the respective muscle sections
(Abcam, Cambridge, MA, USA, ab150113) at a dilution of 1 : 500. Fifteen microlitre of the mounting
medium, Prolong Gold antifade reagent (Life Technologies, Burlington, ON, Canada, P36930), was added
to each slide and subsequently mounted with a #1
cover slip. Slides were visualized using an Axio
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
Acta Physiol 2014, 212, 75–85
Observer Z1 structured-illumination fluorescent
microscope equipped with an AxioCam HRm camera
and associated AXIOVISION software (Carl Zeiss)
(Bloemberg & Quadrilatero 2012).
Quiescent and activated satellite cells were identified and quantified from the immunostains in 5–8
muscle sections and at least 400 myofibres. From
these myofibres, the number of quiescent (Pax7+
nuclei) and activated (MyoD+ nuclei) cells per myofibre could be counted. This method of satellite cell
marker identification has been used in previous quantification of satellite cells in our laboratory and for
consistency was used in this study as well (Enns &
Tiidus 2008, Enns et al. 2008, Thomas et al. 2010).
This method of satellite cell quantification produces
results that are similar to other quantification methods. For example, Kurosaka et al. (2012) quantified
satellite cells either as a percentage of the sum of
myonuclei and satellite cells or satellite cells per fibre
with consistent trends seen between methods.
G Mangan et al.
· Oestrogen signalling via PI3K
the ELISA measured the concentration of total IGF-1 in
the muscle tissue. There was a significant main effect of
oestrogen supplementation in reducing the levels of
total IGF-1 in muscle tissue. More specifically, oestrogen significantly lowered total muscle IGF-1 levels in
SOL and WG tissues without WTN inhibition of the
PI3K pathway, while there is a trend for oestrogen to
lower total IGF-1 in muscle tissues subjected to WTN
inhibition of the PI3K pathway (Fig. 2). In the absence
of oestrogen, exercise produced an expected increase in
IGF-1 in both SOL and WG muscle as previously
reported (Bamman et al. 2001, Gregory et al. 2013).
However, oestrogen supplementation was able to significantly reduce IGF-1 in the SOL muscle of control
rats, as well as attenuate any increase of IGF-1 in the
SOL and WG of exercised rats.
Wortmannin also significantly decreased IGF-1 levels in SOL and WG of rats regardless of oestrogen
supplementation. Oestrogen supplementation also
decreased IGF-1 levels in WG of saline-administered
rats.
Statistical analyses
All data were presented as means SD. For all analyses, differences between groups were measured using a
three-way ANOVA with a level of significance of
P < 0.05. If significance was found, differences
between groups were evaluated using the Student–
Newman–Keuls test.
Results
Exogenous supplementation of oestrogen via subcutaneous pellet insertion significantly increased circulating
levels of oestrogen (pg mg1) in oestrogen-supplemented
rats (227.6 220.0) compared with non-supplemented rats (4.9 4.1). The different levels of oestrogen were also manifested in body weight differences.
Prior to the ESP, there were no significant differences in
body weight between rats that were randomly selected
for the sham procedure (265.03 18.62 g) and those
selected
for
supplementation
with
oestrogen
(262.84 12.90 g). The rats were then reweighed 1
week after the ESP. The weight of the rats that received
the sham procedure (295.75 22.89 g) was significantly higher than those who received oestrogen
(253.00 17.22 g). These changes in weight are typical of what are seen in these models (Enns & Tiidus
2008).
IGF-1 is significantly decreased following oestrogen
supplementation
The IGF-1 ELISA used polyclonal antibodies specific to
a region conserved between IGF-1 isoforms. As such,
Inhibition of the PI3K/Akt pathway leads to a decrease
in the number of total and activated satellite cells
following exercise
The use of immunofluorescence to identify Pax7+
nuclei allows investigators to determine the total number of satellite cells, regardless of their position in the
cell cycle. Immunofluorescence can also identify satellite cells that have exited the G0 phase of the cell
cycle (activated) by identification of MyoD+ nuclei
(Fig. 3). In SOL, exercise was able to significantly
increase the total number of satellite cells as well as
number of activated satellite cells in the absence of
WTN (Fig. 4). Oestrogen further augmented the
effects of exercise in total and activated satellite
cells in SOL. The PI3K inhibitor, WTN, abolished
both the exercise-induced and oestrogen-augmented
increases on the number of satellite cells to control
levels in SOL.
The effects of exercise and oestrogen supplementation on WG muscle were not as well defined. While
exercise tended to increase the number of total satellite cells, it significantly increased the number of activated satellite cells only in the absence of WTN
(Fig. 5). Oestrogen supplementation alone was only
able to significantly increase the total number of satellite cells and not the number of activated satellite cells
in the absence of WTN. Oestrogen supplementation
further amplified the effects of exercise in total but
not in activated satellite cells. As with the SOL data,
WTN did abolish any increases in Pax7+ and MyoD+
identified satellite cell nuclei that were caused by exercise and exogenous oestrogen.
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
79
Oestrogen signalling via PI3K
· G Mangan et al.
Sham-Saline
Estrogen-Saline
Sham-Wortmannin
[IGF-1]muscle
(pg mg–1 total protein)
(a)
250
Estrogen-Wortmannin
a,b
200
150
100
c
c
50
0
Sedentary
Exercise
Treatment groups
[IGF-1]muscle
(pg mg–1 total protein)
(b)
800
a,b
600
400
200
c
0
Sedentary
Exercise
Treatment groups
Figure 2 Effect of oestrogen supplementation, exercise and
wortmannin administration (PI3K/Akt inhibition) on insulinlike growth factor-1 (IGF-1) concentration in rat (a) soleus
and (b) white gastrocnemius 72 h following downhill running. n = 8 rats per group. Values are Means SD.
a
P < 0.05 elevated compared with sham implant–wortmannin (sedentary), bP < 0.05 elevated compared with oestrogen
implant–wortmannin (Sedentary), cP < 0.05 decreased compared with sham implant–saline (exercise and sedentary).
Discussion
This study has demonstrated that the PI3K/Akt pathway is centrally involved in the communication
between oestrogen and muscle satellite cells. An additional finding is that increases in total muscle IGF-1 are
not critical to estradiol’s ability to augment satellite cell
activity. These findings are the first to directly indicate
that the PI3K/Akt signalling pathway is integral to the
ability of oestrogen to influence satellite cell activity in
ovariectomized female rats following exercise.
This study built on and re-confirmed previous work
from our laboratory that demonstrated an oestrogeninduced augmentation in satellite cell activation and
proliferation following downhill running in ovariectomized female rats beyond that induced by exercise
80
Acta Physiol 2014, 212, 75–85
alone (Enns & Tiidus 2008, Enns et al. 2008). Previous studies have confirmed that this protocol produces
significant muscle damage as demonstrated through
increases in the activity of lysosomal enzyme b-glucuronidase, which is ameliorated with oestrogen
supplementation (Salminen & Kihlstr€
om 1985, Enns
& Tiidus 2008, Enns et al. 2008, Thomas et al.
2010). Additionally, we have previously demonstrated
that this protocol increases inflammatory-related
leucocyte infiltration in muscle, which is also ameliorated by oestrogen supplementation (Tiidus et al.
2001, Iqbal et al. 2008). Despite the evidence that
oestrogen seems to attenuate muscle damage and the
subsequent inflammatory response following exercise,
our studies have consistently shown a concomitant
increase in post-exercise activated satellite cells, which
is further augmented by oestrogen (Enns & Tiidus
2008, Enns et al. 2008, Thomas et al. 2010).
The expression of the Pax7 transcription factor is
vital for satellite cells (Sambasivan et al. 2011, von
Maltzahn et al. 2013). In accordance with previous
reports (Enns & Tiidus 2008, Enns et al. 2008, Thomas et al. 2010), the number of Pax7+ satellite cells
increased following exercise in ovariectomized rats
and was further increased in the presence of oestrogen. Following WTN-induced inhibition of the PI3K/
Akt signalling pathway, these increases were abrogated, suggesting that oestrogen interaction with satellite cells occurs primarily via this pathway. The effects
of inhibition of PI3K/Akt signalling were similar in
both primarily type I SOL and type IIB WG muscle,
although the results in the latter tended to be more
variable. Others have also linked PI3K/Akt signalling
to satellite cell function. Jia et al. (2012) used exogenous erythropoietin supplementation to demonstrate a
relationship between the increased levels of PI3K
activity and number of Pax7+ satellite cells per muscle
fibre. Interestingly, inhibition of the PI3K/Akt pathway by WTN resulted not only in the curtailment of
oestrogen augmentation of post-exercise satellite cell
activation and proliferation but the total negation of
exercise-induced satellite cell activation in the presence
or absence of oestrogen. This follows similar findings
of the absence of exercise-induced satellite cell
activation in the ovariectomized rodent model when
oestrogen receptor blockers were employed (Thomas
et al. 2010). This repeated finding of a total absence
of exercise-induced satellite cell activation when
aspects of oestrogen-associated signalling are interfered with is unexplained, curious and warrants further investigation. It should be noted that other
signalling pathways including the ERK/-MAP kinase
pathway may also be involved in oestrogen-related
communication with and regulation of myocyte activity (Murray & Huss 2011). How these other possible
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
Acta Physiol 2014, 212, 75–85
Figure 3 Representative images for
fibre-type composition and total satellite
cells. Fibre-type composition of rat (a)
soleus and (b) white gastrocnemius.
Type 1 – blue, Type IIA – green, Type
IIB – red. Immunofluorescent staining of
soleus muscle for (c) the membrane protein, dystrophin, (d) the total satellite
cell marker, Pax7, (e) the nuclear stain,
DAPI and (f) a merged picture.
G Mangan et al.
(a)
(b)
(c)
(d)
(e)
(f)
oestrogen-associated signalling pathways are related to
the results of this study is currently unknown.
Although it was initially hypothesized that IGF-1
would be the common link between oestrogen, PI3K/
Akt activity and satellite cell augmentation, our results
suggest otherwise. Levels of IGF-1 in muscle tissue
were increased following exercise but were significantly decreased following oestrogen supplementation
despite the latter resulting in further augmentation of
muscle satellite cells. As our ELISA measured total
muscle IGF-1 and did not distinguish isoforms, we
cannot ascertain the origins of the muscle IGF-1, only
that it was attenuated by oestrogen. Tsai et al. (2007)
used ovariectomized female rats supplemented with
estradiol and also reported decreased levels of IGF-1
in muscle from these animals. In our study, Pax7+
satellite cells increased in the muscle tissue of exercised and oestrogen-supplemented rats despite the
reduction of IGF-1 in muscle tissue. IGF-1 and its
effect on muscle hypertrophy and growth has been a
contentious issue in recent years. A recent point–counterpoint series in the Journal of Applied Physiology
produced no <15 commentaries on either side of the
initial debate (Flueck & Goldspink 2010, Stewart &
Pell 2010). It has been shown that IGF-1 has positive
effects on myogenesis during growth (Liu et al. 1993)
· Oestrogen signalling via PI3K
and that IGF-1 can alter satellite cell proliferation in
cell culture (Chakravarthy et al. 2000). However,
IGF-1’s role in adult muscle tissue following exercise
is less clear. Criswell et al. (1998) demonstrated that
overexpression of IGF-1 in skeletal muscle could not
counteract the atrophy induced by muscle unloading.
However, Taaffe et al. (1996) showed that resistance
exercise in elderly men produced improved muscle
strength associated with increased IGF-1 expression.
Alternatively, it has been shown that a reduction in
the IGF-1 receptor did not affect the ability of the
muscle to exhibit hypertrophy following overload
(Spangenburg et al. 2008). Additionally, it is important to consider the impact of IGF-1 isoform on myogenesis. It has been suggested that IGF-1Ea and the
mouse IGF-1Eb/human IGF-1Ec, which is also called
MGF, have distinct roles in muscle growth. Studies
have suggested that IGF-1Ea has roles in proliferation
and differentiation (Engert et al. 1996), while MGF
has a role in proliferation alone (Philippou et al.
2009). However, a recent study by Fornaro et al.
(2014) has disputed this role for MGF using C2C12
and primary cell lines. Futhermore, MGF levels may
have already peaked before the time of our muscle
excision at 72 h following exercise (Philippou et al.
2009) thus possibly reducing its influence in this
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
81
# of Pax7+ nuclei/myofibre
(a)
0.4
· G Mangan et al.
(a)
Sham-Saline
Estrogen-Saline
Sham-Wortmannin
Estrogen-Wortmannin
0.3
Acta Physiol 2014, 212, 75–85
a,b,c
a
d
0.2
d
0.1
# of Pax7+ Nuclei/myofibre
Oestrogen signalling via PI3K
0.0
0.4
0.3
0.2
d
d
0.1
Exercise
Control
Treatment groups
Exercise
Treatment groups
(b)
(b)
0.4
a,b,c
0.3
a
0.2
d
d
0.1
0.0
Sedentary
Exercise
Treatment group
Figure 4 Effect of oestrogen supplementation, exercise and
wortmannin administration (PI3K/Akt inhibition) on (a) total
satellite cell markers (Pax7+ nuclei) and (b) activated satellite
cell markers (MyoD+ nuclei) in rat soleus 72 h following
downhill running. n = 8 rats per group. Values are
Means SD. aP < 0.05 elevated compared with sham
implant–saline (sedentary), bP < 0.05 elevated compared with
oestrogen implant–saline (sedentary), cP < 0.05 elevated compared with sham implant–saline (exercise), dP < 0.05
decreased compared with oestrogen implant–saline (exercise).
study. As such, the single collection point of the current study and a lack of data on specific IGF-1 isoforms may be considered a limitation. Nevertheless,
any possible effects that muscle IGF-1 and its isoforms
may have had on exercise-related muscle hypertrophy
seem to be negligible in the context of oestrogen supplementation. This is characterized by the oestrogendependent reduction in total IGF-1 to a basal level not
only in sedentary animals but also in those that were
exercised. As such, the current study further suggests
that the effects of oestrogen on satellite cell activation
are independent of muscle IGF-1 following exercise.
The enhancement of the satellite cell pool following
oestrogen supplementation and exercise is a significant
finding even in the absence of a direct measure of
hypertrophy or hyperplasia. While endurance exercise
# of MyoD+ nuclei/myofiber
# of MyoD+ nuclei/myofiber
a,b,c
0.0
Control
82
Sham-Saline
Estrogen-Saline
Sham-Wortmannin
Estrogen-Wortmannin
0.4
0.3
0.2
*
d,e
d,e
0.1
0.0
Sedentary
Exercise
Treatment group
Figure 5 Effect of oestrogen supplementation, exercise and
wortmannin administration (PI3K/Akt inhibition) on (a) total
satellite cell markers (Pax7+ nuclei) and (b) activated satellite
cell markers (MyoD+ nuclei) in rat white gastrocnemius 72 h
following downhill running. n = 8 rats per group. Values are
Means SD. aP < 0.05 elevated compared with sham
implant–saline (sedentary), bP < 0.05 elevated compared with
oestrogen implant–saline (sedentary), cP < 0.05 elevated compared with sham implant–saline (exercise), dP < 0.05
decreased compared with oestrogen implant–saline (exercise),
e
P < 0.05 decreased compared with sham implant–saline
(exercise).
increases the satellite cell pool, it does not necessarily
lead to increased muscle mass or mean fibre area
(Kurosaka et al. 2012). It is also true that any
increases in muscle mass are proceeded by an
increased satellite cell population (Bruusgaard et al.
2010). This is especially vital in an ageing population
as the satellite cell pool experiences an age-dependent
decline. The combined effect of a less anabolic local
environment and the reduced number of satellite cells
causes older adults experience age-associated muscle
atrophy, called sarcopenia (Karakelides & Nair 2005,
Shefer et al. 2006), which is more pronounced in
females (Day et al. 2010). Oestrogen supplementation
could be beneficial to post-menopausal women by
© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12317
Acta Physiol 2014, 212, 75–85
increasing their satellite cell pool and thus creating an
environment that is more conducive to muscle growth.
In summary, exercise was able to increase activated
and total number of satellite cells in both SOL and
WG. Oestrogen supplementation further increased
activated and total numbers of satellite cells in SOL
and to a lesser extent in WG following exercise in
ovariectomized rodents. Variance in results between
SOL and WG muscles may suggest that a fibre-typedependent response is present but the explicit mechanism has not yet been elucidated.
Wortmannin-induced PI3K inhibition resulted in the
negation of satellite cell activation and proliferation
following exercise and oestrogen supplementation,
suggesting that the PI3K pathway may be an important signalling connection between oestrogen receptora and muscle satellite cells. In addition, the complete
ablation of exercise-induced muscle satellite cell activation and proliferation due to the inhibition of this
pathway supported previous findings of a relationship
between oestrogen and its communication with muscle
satellite cells (Thomas et al. 2010) as a potentially
necessary requirement for satellite cell activation and
proliferation in our ovariectomized rodent model.
It was also confirmed that oestrogen supplementation in ovariectomized immature rats creates a distinct
model where IGF-1 levels in the muscle tissue are
decreased while post-exercise satellite cell numbers are
still augmented. This finding is opposite to the effects
of oestrogen supplementation on IGF-1 levels in
humans (Ahtiainen et al. 2012, Velders & Diel 2013).
However, this rodent model does provide evidence that
IGF-1 may not be a critical signalling factor in satellite
cell activation and proliferation in all situations.
Conflict of interest
The authors report no conflict of interests.
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