L'idrossitirosolo, un polifenolo estratto dalle olive, attraverso una protezione dei mitocondri, le centrali energetiche della cellula, è in grado di aumentare la resistenza muscolare negli animali da esperimento

1. Free Radical Biology & Medicine 50 (2011) 1437–1446
Contents lists available at ScienceDirect
Free Radical Biology & Medicine
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Mitochondrial dynamic remodeling in strenuous exercise-induced muscle and
mitochondrial dysfunction: Regulatory effects of hydroxytyrosol
Zhihui Feng a,b,1, Liyuan Bai b,1, Jiong Yan b, Yuan Li b, Weili Shen a,b, Ying Wang c, Karin Wertz c,
Peter Weber c, Yong Zhang d, Yan Chen a, Jiankang Liu b,⁎
a
Institute for Nutritional Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, and Graduate School, Chinese Academy of Sciences, Beijing, China
b
Key Laboratory of Biomedical Information, Engineering, Ministry of Education, Institute of Mitochondrial Biology and Medicine, Xi'an Jiaotong University School of Life Science and
Technology, Xi'an 710049, China
c
DSM Nutritional Products, Inc., Basel, Switzerland
d
Tianjin Key Laboratory of Exercise Physiology and Sports Medicine, Department of Health and Exercise Science, Tianjin University of Sport, Tianjin, China
a r t i c l e i n f o a b s t r a c t
Article history: Physical exercise is considered to exert a positive effect on health, whereas strenuous or excessive exercise
Received 18 November 2010 (Exe) causes fatigue and damage to muscle and immune functions. The underlying molecular mechanisms are
Revised 19 February 2011 still unclear. We designed a protocol to mimic Exe and explore the ensuing cellular damage and involvement
Accepted 1 March 2011
of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damage
Available online 21 March 2011
to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA were
Keywords:
increased by Exe, accompanied by increased autophagy and mitochondrial fission in skeletal muscle. Exe
Mitochondrial biogenesis caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways and
Autophagy consequently induced p53, p21, and MnSOD expression in skeletal muscle. The involvement of oxidant-
Oxidative stress induced autophagy and mitochondrial dysfunction was confirmed in C2C12 myoblasts. Hydroxytyrosol (HT),
Apoptosis a natural olive polyphenol, efficiently enhanced endurance capacity and prevented Exe-induced renal and
JNK pathway immune system damage. Also, HT treatment inhibited both the Exe-induced increase in autophagy and
Erk1/2 pathway mitochondrial fission and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusion
Free radicals
and mitochondrial complex I and II activities in muscle of Exe rats. These results demonstrate that Exe-
induced fatigue and damage to muscle and immune functions may be mediated via the regulation of
mitochondrial dynamic remodeling, including the downregulation of mitochondrial biogenesis and
upregulation of autophagy. HT supplementation may regulate mitochondrial dynamic remodeling and
enhance antioxidant defenses and thus improve exercise capacity under Exe conditions.
© 2011 Elsevier Inc. All rights reserved.
An understanding of the molecular processes involved in physical disease that lowers muscle mass, thereby leading to muscle weakness
exercise is important for our health and for sports training programs. and affecting endurance capacity [9,10]. Loss of muscle mass occurs in
Many exercise models such as treadmill-paced exercise and swim- many conditions, ranging from denervation, inactivity, microgravity,
ming [1,2] have been used in numerous studies. Usually 3 to 5 and fasting to a multitude of systemic diseases such as cancer, AIDS,
consecutive days’ exercise is defined as short-term exercise. In diabetes, and cardiac and renal failure [11]. In all these conditions,
contrast, time periods for long-term exercise (LTE) range from enhanced protein breakdown and excessive protein degradation
weeks to months [3]. Moderate physical exercise has been correlated result in muscle atrophy. Excessive exercise such as marathon running
with a number of health benefits, including a reduced risk of is known to cause muscle dysfunction [12]. However, few studies link
developing many diseases, such as cardiovascular disease, cancer, excessive exercise directly to muscle atrophy. Atrogin-1 and MuRF1
and diabetes [4,5]. However, skeletal muscle, one of the most critical have been identified as markers of muscle atrophy [13,14]. Recently,
tissues involved in the development of exercise tolerance, shows autophagy has been found to be involved in the progression of muscle
different responses to various intensities of exercise [6–8]. atrophy. However, the role of autophagic pathways in skeletal muscle
Muscle mass and fiber function are major factors that affect muscle during endurance exercise still remains unclear. Studies show that
function during exercise. Muscle atrophy is defined as a muscle autophagy inhibition induces muscle atrophy and myopathy [15], and
excessive activation of autophagy aggravates muscle wasting by
⁎ Corresponding author.
degrading portions of cytoplasm, proteins, and organelles [16].
E-mail address: j.liu@mail.xjtu.edu.cn (J. Liu). Regardless of the controversial role of autophagy in muscle atrophy,
1
These authors contributed equally to this work. Forkhead box O3 (FoxO3), which belongs to the O subclass of the
0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2011.03.001

2. 1438 Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446
forkhead family of transcription factors, has been identified as the antibodies against GAPDH, LC3B, beclin-1, and Atg7 were from Cell
major factor that regulates autophagy in muscle [17]. Signaling Technology (Danvers, MA, USA); antibodies against complexes
Mitochondria are crucial organelles for the production of energy I, II, III, IV, and V were from Sigma; the Reverse Transcription System kit
by efficient aerobic energy metabolism and for the control of signaling was from Promega (Mannheim, Germany); SYBR green was from TaKaRa
cascades. Moreover, it is well established that regular exercise (Otsu, Shiga, Japan); PCR primers for MnSOD, transcription factor A
efficiently increases mitochondrial content in skeletal muscle by (Tfam), atrogin-1, MuRF1, and 18S rRNA were synthesized by Baiaoke
activation of PGC-1α [18] and, as a result, increases endurance and the Biotech (Beijing, China); HT was given as an olive extract powder
capacity for aerobic energy metabolism. Meanwhile, various studies formulation standardized to 15% HT (19% total polyphenols) from DSM
have demonstrated a connection between the increase in oxygen Nutritional Products Ltd. (Basel, Switzerland). TRIzol, JC-1, and other
consumption during exercise and the formation of reactive oxygen reagents were from Invitrogen (Carlsbad, CA, USA).
species (ROS). ROS and reactive nitrogen species are involved in
modulation of cell signaling pathways and control numerous redox- Animals
sensitive transcription factors. Low and physiological levels of ROS are
required for normal force production in skeletal muscle as well as for Sprague–Dawley (SD) male rats were purchased from a commer-
the adaptive response to exercise (training effect), but high levels of cial breeder (SLAC, Shanghai, China). The rats were housed in a
ROS promote contractile dysfunction, resulting in muscle weakness temperature- (22–28 °C) and humidity- (60%) controlled animal
and fatigue [19]. Accumulation of ROS leads to oxidative stress, room and maintained on a 12-h light/12-h dark cycle (light on from
thereby inducing activation of response genes such as p53, p21, and 8:00 AM to 8:00 PM) with free access to food and water throughout
SOD and activation of autophagy [20,21]. Recent studies also have the experiments. Eight-week-old male rats weighing 180–200 g were
shown that the mitochondrial network is remodeled in atrophying used and housed six rats per cage. Before the experiments were
muscles. Induction of mitochondrial fission and dysfunction activates begun, male rats were selected by their ability to perform 1 week of
the atrophy program, whereas inhibition of mitochondrial fission running exercise at low speed (10 m/min, 20 min/day), and those
prevents muscle loss during fasting [22]. Given that there is an inverse exhibiting high exercise activity were chosen for the experiments. The
relationship between the efficient energy-transducing activity of protocol used for rat selection was to exclude rats that were not
mitochondria and the formation of ROS in mitochondria, and that an willing to run even when subjected to external stimuli such as electric
excess of ROS induces mitochondrial fission as well as muscle shock. This protocol, including the selection procedure, is accepted by
autophagy, mitochondria are suggested as being a primary regulator all researchers in this kind of study. This procedure might produce
of autophagy in skeletal muscle. Mitochondria may not be the main adaptation to endurance training but the degrees of adaptation in the
source of ROS in active muscles, because mitochondria may make ROS control animals and the treated animals should be similar and will not
when they are not making ATP, e.g., when they are resting [23]. affect our observations.
Therefore, the contributions of NADPH oxidases and cytochrome P450
systems in muscles should not be ignored.
The Mediterranean diet is associated with a lower incidence of Endurance exercise procedure
atherosclerosis, cardiovascular disease, and certain types of cancer.
The apparent health benefits have been partially attributed to the Rats were randomly divided into four groups: sedentary, seden-
dietary consumption of virgin olive oil and recent interest has focused tary with HT supplementation (25 mg/kg/day), endurance exercise,
on biologically active phenolic compounds such as hydroxytyrosol and endurance exercise with HT supplementation (25 mg/kg/day).
(HT), tyrosol, and oleuropein [24]. The rationale for studying a single The diet was normal rat chow containing 20.5% protein and 4.6% fat.
component is that it is considered impossible to understand the No ingredient of the standard rat chow used in this study is known to
mechanism of a mixture and the best solution is to isolate the “active contain any measurable amount of HT. Normal diet was made fresh
component” so as to make it possible to study the molecular and stored at 4 °C to prevent oxidation; no specific antioxidants were
mechanism. Moreover, HT has been identified as the most active used. HT was administered by gavage 45 min before beginning the
compound in olive extracts in most assays. This study of HT in exercise exercise program for each animal. The HT dose was chosen based on
was prompted by two modes of action that are relevant in exercise: results of previous studies in mice (Raederstorff D, et al.; poster at
first, numerous studies showed that HT is a potent antioxidant Third Symposium of International Nutrition, Biologie de l'Oxygène et
[25–27]. Moreover, in our previous studies we found that HT could Médecine, 8–10 April 2009, Paris, France). Rats were run on a
induce phase II enzyme activation—i.e., induction of the endogenous motorized treadmill at a speed of 20 m/min and a grade of 5° for 1 h
antioxidant system (glutathione production)—in ARPE cells [28,29]. per day, 6 days per week. To prevent adaptation, the starting time of
This suggests that HT may be capable of reducing the negative effects exercise was chosen randomly each day, and rats were given
of pronounced ROS formation during exhaustive exercise. Second, we 5–10 min rest if they appeared exhausted during the latter weeks of
have discovered that HT is capable of stimulating mitochondrial exercise. After 8 weeks of exercise, endurance capacity was measured
biogenesis in ARPE and 3T3-L1 cells [28–30]. Mitochondrial mass is by running to exhaustion on the treadmill at a speed of 30 m/min and
linked to mitochondrial capacity for aerobic energy production, which a grade of 5°. Exhaustion was defined as the inability to continue
in muscles is an important determinant of endurance, because it is running and, consequently, failure to avoid sound and light irritation
much more efficient than anaerobic energy metabolism in terms of [31]. All procedures were carried out in accordance with the U.S.
moles ATP generated per mole glucose. In this study, we report the Public Health Services Guide for Care and Use of Laboratory Animals and
role of mitochondrial dynamic remodeling and the regulatory effects all efforts were made to minimize the number of animals used and
of HT after an 8-week excessive exercise (Exe) program in rats. their suffering.
Materials and methods Blood sample preparation
Chemicals At the end of 8 weeks, 1 h after HT gavage, tail vein blood was
collected for analysis. After 24 h recovery, endurance capacity was
Antibodies against PGC-1, Mfn1, Mfn2, Drp1, manganese–superoxide measured on the treadmill and tail vein blood was collected again
dismutase (MnSOD), p-Erk1/2, Erk1/2, p-JNK, JNK, p53, and p21 were immediately thereafter. Blood samples at both time points were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); analyzed for BUN (blood urea nitrogen), LYM (lymphocyte count),

3. Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446 1439
WBC (white blood cell count), and CREA (creatinine) with an treatments and concentrations. Plates were sealed and “read” on a
automatic biochemistry analyzer (Hitachi Ltd., Tokyo, Japan). fluorescence spectrometer (Fluoroskan Ascent; Thermo Fisher Scien-
tific) at 1-min intervals for 60 min at an excitation of 485 nm and
Isolation of skeletal muscle mitochondria emission of 615 nm. Results are expressed as the relative fluorescence
intensity.
Twenty-four hours after completing the endurance capacity test,
rats were sacrificed and the soleus muscle was removed from each leg. Western blot analyses
One portion was frozen in liquid N2 and used for extraction of total
RNA and protein. A second portion was trimmed of fat and connective Samples were lysed with Western and IP lysis buffer (Beyotime,
tissue, chopped finely with a pair of scissors, and used for isolation of Jiangsu, China). The lysates were homogenized and the homogenates
mitochondria as previously described [32]. Briefly, each aliquot was were centrifuged at 13,000g for 15 min at 4 °C. The supernatants were
rinsed in ice-cold medium A (120 mmol/L NaCl, 20 mmol/L Hepes, collected and protein concentrations were determined with a BCA
2 mmol/L MgCl2, 1 mmol/L EGTA, and 5 g/L bovine serum albumin, pH Protein Assay kit (Pierce, No. 23225). Equal aliquots (20 μg) of protein
7.4) to remove any residual blood. The disrupted muscle was samples were applied to 10% SDS–PAGE gels, transferred to pure
resuspended with medium A and homogenized with a hand-held nitrocellulose membranes (PerkinElmer Life Sciences, Boston, MA,
borosilicate glass homogenizer. The homogenate was centrifuged at USA), and blocked with 5% nonfat milk TBST (Tris-buffered Saline
600g for 10 min at 4 °C. The supernatant fluid was subsequently Tween-20) buffer. The membranes were incubated with anti-Mfn1,
recentrifuged at 17,000g for 10 min at 4 °C. The pellet containing the anti-Mfn2, anti-Drp1, anti-PGC-1, anti-MnSOD, anti-p-Erk1/2, anti-
mitochondria was resuspended in medium A and then centrifuged at Erk1/2, anti-p-JNK, anti-JNK, anti-p53, anti-p21 (1:1000; Santa Cruz
7000g for 10 min at 4 °C. The pellet obtained after the last Biotechnology); anti-Atg7, anti-LC3B (1:1000; Cell Signaling); or anti-
centrifugation was resuspended in medium B (300 mmol/L sucrose, complex I, II, III, IV, or V (1:10,000; Sigma) at 4 °C overnight. Then the
2 mmol/L Hepes, 0.1 mmol/L EGTA, pH 7.4) and recentrifuged (3500g, membranes were incubated with anti-rabbit or anti-mouse antibodies
10 min, 4 °C). The resulting pellet, which contained soleus muscle at room temperature for 1 h. Chemiluminescence detection was
mitochondria, was suspended in a small volume of medium B and performed using an ECL Western blotting detection kit (Pierce).
stored at − 70 °C. Individual rats show different induction levels. Ten rats in each group
were used for all analyses. We display a clear Western blot image to
Assays for mitochondrial complex activities show the trend and use statistical analysis to show the significance.
NADH–ubiquinone reductase (complex I) and succinate–CoQ
Real-time PCR
oxidoreductase (complex II) activities were measured spectrophoto-
metrically using conventional assays as described [33,34].
Total RNA was extracted from 30 mg of tissue using TRIzol
reagent (Invitrogen) according to the manufacturer's protocol. Two
C2C12 cell differentiation
micrograms of RNA was reverse transcribed into cDNA. Quantitative
PCR was performed using a real-time PCR system (Eppendorf,
Mouse C2C12 myoblasts were purchased from the ATCC (Manassas,
Hamburg, Germany). Reactions were performed with SYBR green
VA, USA) and maintained in Dulbecco's modified Eagle's medium
master mix (TaKaRa) using gene-specific primers. The primers were
supplemented with 10% fetal bovine serum (Invitrogen) at 60–70%
as follows: atrogin-1, 5′-CCATCAGGAGAAGTGGATCTATGTT-3′ (forward)
confluence. To initiate differentiation, cells were allowed to reach 100%
and 5′-GCTTCCCCCAAAGTGCAGTA-3′ (reverse); MuRF1, 5′-
confluence, and the medium was changed to Dulbecco's modified
GTGAAGTTGCCCCCTTACAA-3′ (forward) and 5′-TGGAGATGCAATTGCT-
Eagle's medium containing 2% horse serum (Invitrogen) and changed
CAGT-3′ (reverse); FoxO3a, 5′-TGCCGATGGGTTGGATTT-3′ (forward)
every 2 days. Full differentiation characterized by myotube fusion and
and 5′-CCAGTGAAGTTCCCCACGTT-3′ (reverse); 18S RNA, 5′-
spontaneous twitching was observed after 8 days.
CGAACGTCTGCCCTATCAACTT-3′ (forward) and 5′-CTTGGATGTGG-
TAGCCGTTTCT-3′ (reverse); Tfam, 5′-AATTGCAGCCATGTGGAGG-3′
JC-1 assay for mitochondrial membrane potential
(forward) and 5′-CCCTGGAAGCTTTCAGATACG-3′ (reverse); and
NRF1, 5′-TTACAGGGCGGTGAAATGAC-3′ (forward) and 5′-
Mitochondrial membrane potential was assessed in differentiated
GTTAAGGGCCATGGTGACAG-3′ (reverse).
myoblasts using the lipophilic cationic probe 5,5′,6,6′-tetrachloro-
mRNA contents were normalized to the mRNA of 18S RNA as a
1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). For
housekeeping gene and expressed as relative values using the 2− ΔΔCT
quantitative fluorescence measurements, cells were rinsed once
method.
after JC-1 staining and scanned with a microplate fluorimeter
(Fluoroskan Ascent; Thermo Fisher Scientific, Waltham, MA, USA) at
488 nm excitation and 535 and 590 nm emission, to measure green Total DNA isolation and real-time PCR
and red JC-1 fluorescence, respectively. Each well was scanned by
measuring the intensity of each of 25 squares (of 1 mm2 area) Total DNA was extracted using the QIAamp DNA Mini kit, and
arranged in a 5 × 5 rectangular array. quantitative PCR was performed using mitochondrial DNA and
genomic DNA-specific primers. The rat 18S rRNA gene served as the
Oxygen consumption endogenous reference gene. Melting curves were obtained to ensure
specific amplification. The standard curve method was used for
Cell respiration and oxygen consumption by intact cells were relative quantification. Final results are expressed as N-fold differ-
measured as an indication of mitochondrial respiration activity. We ences in mitochondrial D-loop expression relative to the 18S rRNA
used the BD Oxygen Biosensor System (BD Biosciences, San Diego, CA, gene.
USA). After treatment, C2C12 myotubes were washed in PBS buffer
plus 0.1% (w/v) bovine serum albumin. Cells from each condition Statistical analysis
were divided into aliquots in triplicate in a BD Oxygen Biosensor
System plate (BD Biosciences). The numbers of cells contained in All data are reported as means ± SEM. Statistical analysis was
equal volumes were not statistically significant in response to various performed using one-way ANOVA followed by least-significant difference

4. 1440 Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446
post hoc analysis. In all comparisons, the level of significance was set at and B). The Western images demonstrate that Atg7 and LC3B
pb 0.05. expression is decreased upon treatment with HT in most of the rats;
however, in one rat there were very high Atg7 and LC3B expression
Results levels for the sedentary + HT treatment. These high expression levels
resulted in trends toward higher average levels of Atg7 and LC3B and
Effects of Exe and HT supplementation on endurance capacity and greater SEMs than in control sedentary rats. Consequently, no
muscle atrophy statistically significant differences were found between the control
sedentary group and the sedentary + HT group for either of these
Exercise programs vary based on duration, frequency, speed, and factors. Furthermore, mRNA levels of the well-known autophagy
type of exercise. The capacity to perform endurance exercise has been upstream regulator FoxO3 were also increased by Exe (Fig. 2C). All of
reported as being directly affected by training frequency [35]. In these changes were efficiently eliminated by HT supplementation in
Hickson's study [35], the exercise program was designed with Exe rats (Figs. 2A–C).
gradually increased intensity and is therefore totally different from
our exercise protocol, which requires an excessive degree of exercise,
which has not been thoroughly studied. Therefore, there is a large Effects of Exe and HT supplementation on mitochondrial dynamic
disparity between this study and other published literature on remodeling
adaptations to endurance training. Exe was prone to reduce
endurance capacity, and HT supplementation was sufficient to Moderate exercise is known to induce mitochondrial biogenesis
improve it by 35% without having any effect on the endurance of through PGC-1α activation. However, the training protocol in our
sedentary rats (Fig. 1A). We also found that Exe significantly increased study was very different from protocols used in other reports with
mRNA expression levels of two well-known muscle atrophy markers, respect to duration, frequency, and speed; in our protocol, these
atrogin-1 and MuRF1 (Fig. 1B). HT supplementation significantly parameters were set to mimic excessive exercise. Similar to results for
inhibited the progression of muscle atrophy (Fig. 1B). Both Exe and HT endurance capacity, we found that Exe decreased PGC-1α and
supplementation had no obvious effect on body weight (data not complex I subunit expression, and HT supplementation inhibited
shown). these decreases (Figs. 3A and B). Complex II, III, IV, and V subunits
were not affected by Exe or HT supplementation. Mitochondrial DNA
Effects of Exe and HT supplementation on activation of autophagic copy numbers and Nrf1 mRNA levels were also not affected by LTE or
pathways HT supplementation (Fig. 3C). Interestingly, mRNA levels of the
mitochondrial Tfam were increased by Exe and inhibited by HT
Given the critical role of muscle atrophy regulation, autophagy supplementation (Fig. 3C). As we pointed out previously, whether
activation was determined by measuring levels of skeletal muscle exercise training causes an increase in endurance capacity depends on
proteins. Western blot results showed that the autophagy-related the design of the exercise training protocol. Our training protocol
proteins Atg7, beclin-1, and LC3 were highly induced by Exe (Figs. 2A subjects the animal to exhaustive/excessive exercise. Although this is
a type that has not been thoroughly studied, there are a few studies
reporting that excessive exercise does not increase endurance
capacity [36,37]. Therefore, the large disparities between the results
of this study and those of other published studies on mitochondrial
and physiological adaptations to endurance training can be under-
stood by taking note of the large differences in exercise intensity and
endurance utilized.
In addition to homeostatic regulation of mitochondrial biogenesis
by PGC-1α, mitochondrial homeostasis is also regulated by fusion and
fission processes that result in a continuous remodeling of the
mitochondrial network [38]. In our study, we found that Exe
significantly increased expression of the mitochondrial fission-related
protein Drp1 without affecting the mitochondrial fusion-related
proteins Mfn1 and Mfn2 (Figs. 4A and B). HT supplementation
inhibited the Exe-induced increase in Drp1 expression and also
significantly increased expression of Mfn1 and Mfn2 in Exe rats
(Figs. 4A and B). Meanwhile, mitochondrial complex I and II activities
were increased by HT supplementation in Exe rats (Fig. 4C). Higher
complex I and II activities would increase the mitochondrial electron
transport chain efficiency and therefore increase oxidative phosphor-
ylation and ATP production, while decreasing ROS generation.
Although the increase is small, the physiological significance is still
important.
Effects of Exe and HT supplementation on oxidative pathways
Fig. 1. Effects of HT supplementation and Exe on endurance capacity and muscle atrophy
markers. SD rats were either given saline or treated with HT (25 mg/kg/day) in both We examined proteins indicative of oxidative status and found
sedentary and exercise groups (Sed, sedentary; Exe, long-term endurance exercise; Sed + that Exe activated Erk1/2 and JNK (Fig. 5A). The oxidative response
HT, sedentary with 25 mg/kg HT treatment; and Exe + HT, Exe with 25 mg/kg HT proteins p53, p21, and MnSOD were also upregulated by Exe. HT
treatment). (A) After 8 weeks, rats were run to exhaustion on a treadmill, and run time was
recorded as endurance capacity. (B) Skeletal muscle mRNA was extracted and atrogin-1
supplementation, although having no effect on GSH and MDA (results
and MuRF1 were analyzed by real-time PCR. Values are means ± SEM from 10 rats; not shown), significantly inhibited the Exe-induced increases in Erk1/2,
^^p b 0.01 vs sedentary control; *p b 0.05, **p b 0.01 vs exercise control. JNK, p53, p21, and MnSOD (Figs. 5B and C).

5. Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446 1441
Fig. 2. Effects of HT supplementation and Exe on autophagy activation. SD rats were either given saline or treated with HT (25 mg/kg/day) in both sedentary and exercise groups
(Sed, sedentary; Exe, long-term endurance exercise; Sed + HT, sedentary with 25 mg/kg HT treatment; and Exe + HT, Exe with 25 mg/kg HT treatment). (A, B) After 8 weeks, rats
were sacrificed and the autophagy-related proteins Atg7, beclin-1, and LC3B were determined by Western blot (A, Western blot images; B, statistical results). (C) mRNA was
extracted from skeletal muscle and FoxO3 mRNA levels were analyzed by real-time RT-PCR. Values are means ± SEM from 10 rats; ^p b 0.05 vs sedentary control; *p b 0.05, **p b 0.01
vs exercise control.
Effects of Exe and HT supplementation on renal function and immune blot results showed that autophagy and mitochondrial fission were
system parameters activated after treatment for 6 h (Fig. 7A). After 24 h, mitochondrial
membrane potential and mitochondrial oxygen consumption de-
Blood samples were taken before and after the endurance capacity creased significantly at t-BHP concentrations of 50, 100, and 500 μM
(exhaustive exercise) test after 8 weeks of Exe. BUN levels and WBC (Figs. 7B and C). Moreover, after 48 h of treatment, even 1 μM t-BHP
numbers were significantly increased and LYM numbers significantly significantly decreased mitochondrial oxygen consumption capacity
decreased both pre- and post-exhaustive exercise. All of these (Fig. 7D).
changes were restored to normal levels by HT supplementation
(Figs. 6A–C). CREA levels were not affected in animals before Discussion
exhaustive exercise but significantly increased after it. HT supple-
mentation significantly inhibited this increase and also reduced CREA Exercise-induced adaptations in muscle are highly specific and
levels before exhaustive exercise (Fig. 6D). dependent upon the type of exercise, as well as its frequency, intensity,
and duration [38]. Usually exercise training is designed to reach
Effect of tert-butylhydroperoxide (t-BHP) on differentiated C2C12 myotubes maximum speed gradually over the whole exercise period, which
gives the rats time to adapt to increases in intensity. However, in our
Hydrogen peroxide is easily decomposed and hard to detect in protocol, rats were run immediately at maximum speed for 1 h, and
vivo. To our knowledge, so far no studies show specific hydrogen each day we changed the time when the exercise began, to prevent
peroxide production in muscle under any kind of exercise. However, a adaptation. We noticed that after nearly 4 weeks of training, the rats in
recent study shows that a concentration of hydrogen peroxide that is the control group were not able to finish 1 h of exercise, and we had to
low, yet sufficient to induce transient fragmentation, does not lower give them a 5-min break to keep going. We hypothesized that the
cell viability; this indicates that ROS generation may contribute to excessive exercise required by our training protocol would induce
exercise-induced changes in mitochondrial morphology in vivo [39]. dramatically higher ROS production, which in turn would trigger
In our study we use t-BHP instead of hydrogen peroxide for its activation of autophagy and mitochondrial fission—and not mitochon-
stability. Although t-BHP is not exactly the same as hydrogen drial biogenesis, which was not detected in the exercise group. Also, our
peroxide, both hydrogen peroxide and t-BHP induce oxidative stress study showed that Exe tended to decrease endurance capacity. Skeletal
and they are widely used as oxidants in all kinds of studies [40–43]. muscle function is one of the major determinants of exercise ability.
The C2C12 mouse cell line is more widely used than L6 myocytes in With this in mind, EDL, plantaris, white quadriceps, and soleus muscle
many reports, and new studies indicating that oxidative stress is are all important skeletal muscles in rat and all may contribute to
involved in exercise-induced mitochondrial morphology change and endurance capacity. However, soleus muscle is our primary interest
muscle wasting use the C2C12 cell model [39,44]. To mimic possible because first, soleus muscle has a large number of mitochondria under
oxidative stress induced by Exe in skeletal muscle, C2C12 myotubes basal conditions, and second, soleus mitochondria have been studied in
were challenged with the indicated concentrations of t-BHP. Western many models such as running, suspension, and immobilization [45–49],

6. 1442 Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446
Fig. 3. Effects of HT supplementation and Exe on mitochondria content. SD rats were either given saline or treated with HT (25 mg/kg/day) in both sedentary and exercise groups
(Sed, sedentary; Exe, long-term endurance exercise; Sed + HT, sedentary with 25 mg/kg HT treatment; and Exe + HT, Exe with 25 mg/kg HT treatment). (A, B) After 8 weeks, rats
were sacrificed and protein levels of muscle mitochondrial ETS subunits and PGC-1α were determined by Western blot (A, Western blot images; B statistical results of PGC-1α and
complex I subunit levels). (C) Mitochondrial DNA number (as D-loop DNA) or Nrf1 and Tfam RNA levels were measured by real-time PCR (DNA) or quantitative RT-PCR (RNA).
Values are means ± SEM from 10 rats; ^p b 0.05 vs sedentary control; *p b 0.05 vs exercise control.
and we designed our study to be compatible with these earlier results. well established that regular exercise activates PGC-1α, thereby
Although mitochondrial content has been well studied in those models, inducing nuclear respiratory factors (Nrf1 and 2), which in turn
neither alterations in mitochondrial dynamics nor activation of promote the expression of numerous nuclear genes encoding
autophagy has been well explored in soleus muscle; therefore, our mitochondrial proteins. Expression of one of these, mitochondrial
study mainly focused on soleus muscle adaption during Exe and as Tfam, leads directly to stimulation of mitochondrial DNA replication
influenced by HT supplementation. and transcription [18]. Furthermore, it has been found that PGC-1α
Autophagy is a catabolic process involving the degradation of a cell's activation during exercise is under ROS regulation during muscle
own components by lysosomal machinery and helps to maintain a contraction [21,54]. Oxidative stress impairs mitochondrial gene
balance between synthesis and degradation [50]. It was first described in transcription and protein expression, which impairment then stim-
the 1960s [51]. However, the role and regulation of the autophagic ulates the transcription factor and nuclear gene expression required
pathway in skeletal muscle are still insufficiently characterized. to activate biogenesis [55]. In our current study, instead of enhancing,
Autophagy clears damaged proteins and organelles so as to maintain Exe decreased PGC-1α and ETS (Electron Transport System) complex I
proper muscle function. Knockout of Atg7 (a crucial autophagy gene) in subunit expression. Mitochondrial DNA copy numbers were not
mice results in profound muscle atrophy and an age-dependent affected, but Tfam mRNA levels were increased. An increase in Tfam
decrease in muscle force [16]. Very recently, Mammucari et al. [52] mRNA levels indicates that mitochondrial biogenesis responded to
reported that overexpression of constitutively active FoxO3 activates exercise; however, the lack of change in mitochondrial subunits
autophagy, whereas knocking down the critical gene LC3 by RNAi indicates that the mitochondrial biogenesis response might be
partially prevents muscle loss. Measuring muscle fiber-type cross- blocked downstream of Tfam. The functional significance of increased
sectional area is important for determining whether muscle atrophy Tfam with no change in COX subunits needs to be studied further. We
results from a training protocol. However, atrogin-1 and MuRF1 have hypothesize that under Exe, accumulated ROS cause severe oxidative
been identified and widely used as muscle atrophy markers [13,14,53]. damage to proteins and at the same time stimulate mitochondrial
In our study, the muscle atrophy markers atrogin-1 and MuRF1, as well gene transcription. Consistent with this assumption, we found that
as the autophagy markers Atg7, beclin-1, LC3, and FoxO3, were highly Exe activates the oxidative response kinases Erk1/2 and JNK and the
induced by Exe. We conclude that exhaustive Exe activates autophagy, oxidative response proteins p53, p21, and MnSOD. Unlike other
which contributes to muscle atrophy and decreased endurance capacity. mitochondrial proteins, MnSOD is reported to be regulated by tumor
Mitochondria are highly dynamic organelles that are crucial for the suppressor p53, which therefore can affect mitochondrial ROS
production of energy and metabolic activity in skeletal muscle. It is production. Recent studies suggest that mitochondria can also

7. Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446 1443
Fig. 4. Effects of HT supplementation and Exe on mitochondrial dynamics. SD rats were either given saline or treated with HT (25 mg/kg/day) in both sedentary and exercise groups (Sed,
sedentary; Exe, long-term endurance exercise; Sed + HT, sedentary with 25 mg/kg HT treatment; and Exe + HT, Exe with 25 mg/kg HT treatment). (A, B) After 8 weeks, rats were sacrificed
and the muscle mitochondrial dynamics-related proteins Drp1, Mfn1, and Mfn2 were determined by Western blot (A, Western blot images; B, statistical results). (C) Mitochondria were
isolated and complex I and II activities were analyzed. Values are means± SEM from 10 rats; ^p b 0.05 vs sedentary control; *p b 0.05 vs exercise control.
Fig. 5. Effects of HT supplementation and Exe on oxidative status. SD rats were either given saline or treated with HT (25 mg/kg/day) in both sedentary and exercise groups (Sed, sedentary;
Exe, long-term endurance exercise; Sed + HT, sedentary with 25 mg/kg HT treatment; and Exe+ HT, Exe with 25 mg/kg HT treatment). After 8 weeks, the rats were sacrificed and
(A) proteins indicative of oxidative stress response pathway activation in muscle were determined by Western blot. (B, C) Protein levels of p53, p21, and MnSOD (B, Western blot images; C,
statistical results). Values are means ± SEM from 10 rats; ^p b 0.05, ^^p b 0.01 vs sedentary control; *p b 0.05, **p b 0.01 vs exercise control.

8. 1444 Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446
Fig. 6. Effects of HT supplementation and Exe on renal function and immune system parameters. SD rats were either given saline or treated with HT (25 mg/kg/day) in both
sedentary and exercise groups (Sed, sedentary; Exe, long-term endurance exercise; Sed + HT, sedentary with 25 mg/kg HT treatment; and Exe + HT, Exe with 25 mg/kg HT
treatment). After 8 weeks, blood was collected twice—1 day before and immediately after the endurance capacity test—to test (A) BUN levels, (B) WBC numbers, (C) LYM levels, and
(D) CREA levels. Values are means ± SEM from 10 rats; ^p b 0.05, ^^p b 0.01 vs sedentary control; *p b 0.05, **p b 0.01 vs exercise control.
regulate p53 activity and that assaults on the cell that affect reason(s) for the inconsistency in the results with respect to Tfam
mitochondrial ROS production and mitochondrial function can mRNA, PGC-1α (decreased), and mtDNA copy numbers (unchanged)
influence p53 activity [56,57]. The robust induction of MnSOD, but in Exe is unknown. It might be possible that the time of animal
not other proteins, may suggest a specific targeting to MnSOD. This termination (24 h after the exhaustive exercise test) evinces quick
specificity is interesting but needs more study in the future. The recovery of some parameters such as Tfam mRNA, but is too early to
Fig. 7. Effects of t-BHP on differentiated C2C12 myotubes. After 8 days of differentiation, C2C12 myotubes were treated with the indicated concentrations of t-BHP for 6, 24, or 48 h.
(A) After 6 h, proteins indicative of fission of mitochondria and autophagy activation were determined by Western blot. (B) After 24 h, mitochondrial membrane potential was
analyzed, and mitochondrial oxygen consumption capacity was determined after (C) 24 or (D) 48 h of treatment. Values are means ± SEM; *p b 0.05, **p b 0.01 vs control.

9. Z. Feng et al. / Free Radical Biology & Medicine 50 (2011) 1437–1446 1445
reveal changes in slower-responding ones such as PGC-1α protein. Acknowledgments
This issue warrants further study of the time-dependent changes in
both mRNA and protein expression of these factors. We thank Dr. Edward Sharman at the University of California at
Mitochondrial network dynamics are sensitive to exercise and Irvine for reading and editing the manuscript. This study was partially
central to cell function and survival, but have not been well studied. supported by DSM Nutritional Products Ltd.; the Open Project of the
Bo et al. reviewed their excellent work on mitochondrial network State Key Laboratory of Space Medicine Fundamentals and Applica-
remodeling response to acute and endurance exercise with a focus on tion, China Astronaut Research and Training Center, Beijing, China;
fusion and fission reactions [38]. Acute exercise, which increases ROS the National Natural Science Foundation of China, Key Program
generation and State 4 respiration, decreases mitochondrial fusion 30930105; and the 985 and 211 Projects of Xi'an Jiaotong University.
and increases mitochondrial fission [38]. Inhibition of mitochondrial Dr. Ying Wang, Dr. Karin Wertz, and Dr. Peter Weber are employees of
fission prevents muscle loss during fasting, and induction of DSM Nutritional Products, Ltd., a company produces and holds patents
mitochondrial fission and dysfunction activates an atrophy program for hydroxytyrosol.
[22]. Consistent with these studies, we found that Exe, although
having no apparent effect on mitochondrial fusion, activated the
mitochondrial fission machinery, leading to acceleration of mito- References
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