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
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 dOriginal ContributionMitochondrial dynamic remodeling in strenuous exercise-induced muscle andmitochondrial dysfunction: Regulatory effects of hydroxytyrosolZhihui 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, Chinab Key Laboratory of Biomedical Information, Engineering, Ministry of Education, Institute of Mitochondrial Biology and Medicine, Xian Jiaotong University School of Life Science andTechnology, Xian 710049, Chinac DSM Nutritional Products, Inc., Basel, Switzerlandd Tianjin Key Laboratory of Exercise Physiology and Sports Medicine, Department of Health and Exercise Science, Tianjin University of Sport, Tianjin, Chinaa r t i c l e i n f o a b s t r a c tArticle history: Physical exercise is considered to exert a positive effect on health, whereas strenuous or excessive exerciseReceived 18 November 2010 (Exe) causes fatigue and damage to muscle and immune functions. The underlying molecular mechanisms areRevised 19 February 2011 still unclear. We designed a protocol to mimic Exe and explore the ensuing cellular damage and involvementAccepted 1 March 2011 of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damageAvailable online 21 March 2011 to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA wereKeywords: increased by Exe, accompanied by increased autophagy and mitochondrial ﬁssion in skeletal muscle. ExeMitochondrial biogenesis caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways andAutophagy 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 andJNK pathway immune system damage. Also, HT treatment inhibited both the Exe-induced increase in autophagy andErk1/2 pathway mitochondrial ﬁssion and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusionFree 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 weaknessexercise is important for our health and for sports training programs. and affecting endurance capacity [9,10]. Loss of muscle mass occurs inMany 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 . In all these conditions,contrast, time periods for long-term exercise (LTE) range from enhanced protein breakdown and excessive protein degradationweeks to months . Moderate physical exercise has been correlated result in muscle atrophy. Excessive exercise such as marathon runningwith a number of health beneﬁts, including a reduced risk of is known to cause muscle dysfunction . However, few studies linkdeveloping many diseases, such as cardiovascular disease, cancer, excessive exercise directly to muscle atrophy. Atrogin-1 and MuRF1and 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 muscledifferent 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 thatfunction during exercise. Muscle atrophy is deﬁned as a muscle autophagy inhibition induces muscle atrophy and myopathy , and excessive activation of autophagy aggravates muscle wasting by ⁎ Corresponding author. degrading portions of cytoplasm, proteins, and organelles . E-mail address: firstname.lastname@example.org (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 the0891-5849/$ – 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) 1437–1446forkhead family of transcription factors, has been identiﬁed as the antibodies against GAPDH, LC3B, beclin-1, and Atg7 were from Cellmajor factor that regulates autophagy in muscle . 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 kitby efﬁcient aerobic energy metabolism and for the control of signaling was from Promega (Mannheim, Germany); SYBR green was from TaKaRacascades. Moreover, it is well established that regular exercise (Otsu, Shiga, Japan); PCR primers for MnSOD, transcription factor Aefﬁciently increases mitochondrial content in skeletal muscle by (Tfam), atrogin-1, MuRF1, and 18S rRNA were synthesized by Baiaokeactivation of PGC-1α  and, as a result, increases endurance and the Biotech (Beijing, China); HT was given as an olive extract powdercapacity for aerobic energy metabolism. Meanwhile, various studies formulation standardized to 15% HT (19% total polyphenols) from DSMhave demonstrated a connection between the increase in oxygen Nutritional Products Ltd. (Basel, Switzerland). TRIzol, JC-1, and otherconsumption during exercise and the formation of reactive oxygen reagents were from Invitrogen (Carlsbad, CA, USA).species (ROS). ROS and reactive nitrogen species are involved inmodulation of cell signaling pathways and control numerous redox- Animalssensitive transcription factors. Low and physiological levels of ROS arerequired 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 aROS promote contractile dysfunction, resulting in muscle weakness temperature- (22–28 °C) and humidity- (60%) controlled animaland fatigue . Accumulation of ROS leads to oxidative stress, room and maintained on a 12-h light/12-h dark cycle (light on fromthereby inducing activation of response genes such as p53, p21, and 8:00 AM to 8:00 PM) with free access to food and water throughoutSOD and activation of autophagy [20,21]. Recent studies also have the experiments. Eight-week-old male rats weighing 180–200 g wereshown that the mitochondrial network is remodeled in atrophying used and housed six rats per cage. Before the experiments weremuscles. Induction of mitochondrial ﬁssion and dysfunction activates begun, male rats were selected by their ability to perform 1 week ofthe atrophy program, whereas inhibition of mitochondrial ﬁssion running exercise at low speed (10 m/min, 20 min/day), and thoseprevents muscle loss during fasting . Given that there is an inverse exhibiting high exercise activity were chosen for the experiments. Therelationship between the efﬁcient energy-transducing activity of protocol used for rat selection was to exclude rats that were notmitochondria and the formation of ROS in mitochondria, and that an willing to run even when subjected to external stimuli such as electricexcess of ROS induces mitochondrial ﬁssion as well as muscle shock. This protocol, including the selection procedure, is accepted byautophagy, mitochondria are suggested as being a primary regulator all researchers in this kind of study. This procedure might produceof autophagy in skeletal muscle. Mitochondria may not be the main adaptation to endurance training but the degrees of adaptation in thesource of ROS in active muscles, because mitochondria may make ROS control animals and the treated animals should be similar and will notwhen they are not making ATP, e.g., when they are resting . affect our observations.Therefore, the contributions of NADPH oxidases and cytochrome P450systems in muscles should not be ignored. The Mediterranean diet is associated with a lower incidence of Endurance exercise procedureatherosclerosis, 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 . 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 tomechanism of a mixture and the best solution is to isolate the “active contain any measurable amount of HT. Normal diet was made freshcomponent” so as to make it possible to study the molecular and stored at 4 °C to prevent oxidation; no speciﬁc antioxidants weremechanism. Moreover, HT has been identiﬁed as the most active used. HT was administered by gavage 45 min before beginning thecompound in olive extracts in most assays. This study of HT in exercise exercise program for each animal. The HT dose was chosen based onwas prompted by two modes of action that are relevant in exercise: results of previous studies in mice (Raederstorff D, et al.; poster atﬁrst, numerous studies showed that HT is a potent antioxidant Third Symposium of International Nutrition, Biologie de lOxygène et[25–27]. Moreover, in our previous studies we found that HT could Médecine, 8–10 April 2009, Paris, France). Rats were run on ainduce 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 hantioxidant system (glutathione production)—in ARPE cells [28,29]. per day, 6 days per week. To prevent adaptation, the starting time ofThis suggests that HT may be capable of reducing the negative effects exercise was chosen randomly each day, and rats were givenof pronounced ROS formation during exhaustive exercise. Second, we 5–10 min rest if they appeared exhausted during the latter weeks ofhave discovered that HT is capable of stimulating mitochondrial exercise. After 8 weeks of exercise, endurance capacity was measuredbiogenesis 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 andlinked to mitochondrial capacity for aerobic energy production, which a grade of 5°. Exhaustion was deﬁned as the inability to continuein muscles is an important determinant of endurance, because it is running and, consequently, failure to avoid sound and light irritationmuch more efﬁcient than anaerobic energy metabolism in terms of . 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 androle of mitochondrial dynamic remodeling and the regulatory effects all efforts were made to minimize the number of animals used andof HT after an 8-week excessive exercise (Exe) program in rats. their suffering.Materials and methods Blood sample preparationChemicals 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 againdismutase (MnSOD), p-Erk1/2, Erk1/2, p-JNK, JNK, p53, and p21 were immediately thereafter. Blood samples at both time points werepurchased 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 1439WBC (white blood cell count), and CREA (creatinine) with an treatments and concentrations. Plates were sealed and “read” on aautomatic 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 andIsolation 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 analysesOne portion was frozen in liquid N2 and used for extraction of totalRNA 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 homogenatesmitochondria as previously described . Brieﬂy, each aliquot was were centrifuged at 13,000g for 15 min at 4 °C. The supernatants wererinsed in ice-cold medium A (120 mmol/L NaCl, 20 mmol/L Hepes, collected and protein concentrations were determined with a BCA2 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 protein7.4) to remove any residual blood. The disrupted muscle was samples were applied to 10% SDS–PAGE gels, transferred to pureresuspended 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 Saline600g 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 Cruz7000g 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 the2 mmol/L Hepes, 0.1 mmol/L EGTA, pH 7.4) and recentrifuged (3500g, membranes were incubated with anti-rabbit or anti-mouse antibodies10 min, 4 °C). The resulting pellet, which contained soleus muscle at room temperature for 1 h. Chemiluminescence detection wasmitochondria, 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 toAssays 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 PCRoxidoreductase (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 manufacturers protocol. TwoC2C12 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 greenVA, USA) and maintained in Dulbeccos modiﬁed Eagles medium master mix (TaKaRa) using gene-speciﬁc primers. The primers weresupplemented 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 Dulbeccos modiﬁed GTGAAGTTGCCCCCTTACAA-3′ (forward) and 5′-TGGAGATGCAATTGCT-Eagles 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 a1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). For housekeeping gene and expressed as relative values using the 2− ΔΔCTquantitative 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) at488 nm excitation and 535 and 590 nm emission, to measure green Total DNA isolation and real-time PCRand red JC-1 ﬂuorescence, respectively. Each well was scanned bymeasuring the intensity of each of 25 squares (of 1 mm2 area) Total DNA was extracted using the QIAamp DNA Mini kit, andarranged 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 theOxygen 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 rRNAused the BD Oxygen Biosensor System (BD Biosciences, San Diego, CA, gene.USA). After treatment, C2C12 myotubes were washed in PBS bufferplus 0.1% (w/v) bovine serum albumin. Cells from each condition Statistical analysiswere divided into aliquots in triplicate in a BD Oxygen BiosensorSystem plate (BD Biosciences). The numbers of cells contained in All data are reported as means ± SEM. Statistical analysis wasequal 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) 1437–1446post hoc analysis. In all comparisons, the level of signiﬁcance was set at and B). The Western images demonstrate that Atg7 and LC3Bpb 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 expressionResults levels for the sedentary + HT treatment. These high expression levels resulted in trends toward higher average levels of Atg7 and LC3B andEffects of Exe and HT supplementation on endurance capacity and greater SEMs than in control sedentary rats. Consequently, nomuscle 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 autophagytype of exercise. The capacity to perform endurance exercise has been upstream regulator FoxO3 were also increased by Exe (Fig. 2C). All ofreported as being directly affected by training frequency . In these changes were efﬁciently eliminated by HT supplementation inHicksons study , the exercise program was designed with Exe rats (Figs. 2A–C).gradually increased intensity and is therefore totally different fromour 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 dynamicdisparity between this study and other published literature on remodelingadaptations to endurance training. Exe was prone to reduceendurance capacity, and HT supplementation was sufﬁcient to Moderate exercise is known to induce mitochondrial biogenesisimprove it by 35% without having any effect on the endurance of through PGC-1α activation. However, the training protocol in oursedentary rats (Fig. 1A). We also found that Exe signiﬁcantly increased study was very different from protocols used in other reports withmRNA expression levels of two well-known muscle atrophy markers, respect to duration, frequency, and speed; in our protocol, theseatrogin-1 and MuRF1 (Fig. 1B). HT supplementation signiﬁcantly parameters were set to mimic excessive exercise. Similar to results forinhibited the progression of muscle atrophy (Fig. 1B). Both Exe and HT endurance capacity, we found that Exe decreased PGC-1α andsupplementation had no obvious effect on body weight (data not complex I subunit expression, and HT supplementation inhibitedshown). these decreases (Figs. 3A and B). Complex II, III, IV, and V subunits were not affected by Exe or HT supplementation. Mitochondrial DNAEffects of Exe and HT supplementation on activation of autophagic copy numbers and Nrf1 mRNA levels were also not affected by LTE orpathways 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, whetheractivation was determined by measuring levels of skeletal muscle exercise training causes an increase in endurance capacity depends onproteins. Western blot results showed that the autophagy-related the design of the exercise training protocol. Our training protocolproteins 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 . 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 pathwaysFig. 1. Effects of HT supplementation and Exe on endurance capacity and muscle atrophymarkers. SD rats were either given saline or treated with HT (25 mg/kg/day) in both We examined proteins indicative of oxidative status and foundsedentary and exercise groups (Sed, sedentary; Exe, long-term endurance exercise; Sed + that Exe activated Erk1/2 and JNK (Fig. 5A). The oxidative responseHT, 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. HTtreatment). (A) After 8 weeks, rats were run to exhaustion on a treadmill, and run time wasrecorded as endurance capacity. (B) Skeletal muscle mRNA was extracted and atrogin-1 supplementation, although having no effect on GSH and MDA (resultsand 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 1441Fig. 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, ratswere 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 wasextracted 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 0.01vs exercise control.Effects of Exe and HT supplementation on renal function and immune blot results showed that autophagy and mitochondrial ﬁssion weresystem 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-BHPnumbers were signiﬁcantly increased and LYM numbers signiﬁcantly signiﬁcantly decreased mitochondrial oxygen consumption capacitydecreased 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 Discussionexhaustive 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 andlevels before exhaustive exercise (Fig. 6D). dependent upon the type of exercise, as well as its frequency, intensity, and duration . Usually exercise training is designed to reachEffect 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, andvivo. To our knowledge, so far no studies show speciﬁc hydrogen each day we changed the time when the exercise began, to preventperoxide production in muscle under any kind of exercise. However, a adaptation. We noticed that after nearly 4 weeks of training, the rats inrecent 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 tolow, yet sufﬁcient to induce transient fragmentation, does not lower give them a 5-min break to keep going. We hypothesized that thecell viability; this indicates that ROS generation may contribute to excessive exercise required by our training protocol would induceexercise-induced changes in mitochondrial morphology in vivo . dramatically higher ROS production, which in turn would triggerIn 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, ourperoxide, both hydrogen peroxide and t-BHP induce oxidative stress study showed that Exe tended to decrease endurance capacity. Skeletaland 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 musclemany reports, and new studies indicating that oxidative stress is are all important skeletal muscles in rat and all may contribute toinvolved in exercise-induced mitochondrial morphology change and endurance capacity. However, soleus muscle is our primary interestmuscle wasting use the C2C12 cell model [39,44]. To mimic possible because ﬁrst, soleus muscle has a large number of mitochondria underoxidative stress induced by Exe in skeletal muscle, C2C12 myotubes basal conditions, and second, soleus mitochondria have been studied inwere 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) 1437–1446Fig. 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, ratswere 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 andcomplex 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, therebyAlthough mitochondrial content has been well studied in those models, inducing nuclear respiratory factors (Nrf1 and 2), which in turnneither alterations in mitochondrial dynamics nor activation of promote the expression of numerous nuclear genes encodingautophagy has been well explored in soleus muscle; therefore, our mitochondrial proteins. Expression of one of these, mitochondrialstudy mainly focused on soleus muscle adaption during Exe and as Tfam, leads directly to stimulation of mitochondrial DNA replicationinﬂuenced by HT supplementation. and transcription . Furthermore, it has been found that PGC-1α Autophagy is a catabolic process involving the degradation of a cells activation during exercise is under ROS regulation during muscleown components by lysosomal machinery and helps to maintain a contraction [21,54]. Oxidative stress impairs mitochondrial genebalance between synthesis and degradation . It was ﬁrst described in transcription and protein expression, which impairment then stim-the 1960s . However, the role and regulation of the autophagic ulates the transcription factor and nuclear gene expression requiredpathway in skeletal muscle are still insufﬁciently characterized. to activate biogenesis . 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 Iproper muscle function. Knockout of Atg7 (a crucial autophagy gene) in subunit expression. Mitochondrial DNA copy numbers were notmice results in profound muscle atrophy and an age-dependent affected, but Tfam mRNA levels were increased. An increase in Tfamdecrease in muscle force . Very recently, Mammucari et al.  mRNA levels indicates that mitochondrial biogenesis responded toreported that overexpression of constitutively active FoxO3 activates exercise; however, the lack of change in mitochondrial subunitsautophagy, whereas knocking down the critical gene LC3 by RNAi indicates that the mitochondrial biogenesis response might bepartially prevents muscle loss. Measuring muscle ﬁber-type cross- blocked downstream of Tfam. The functional signiﬁcance of increasedsectional area is important for determining whether muscle atrophy Tfam with no change in COX subunits needs to be studied further. Weresults from a training protocol. However, atrogin-1 and MuRF1 have hypothesize that under Exe, accumulated ROS cause severe oxidativebeen identiﬁed and widely used as muscle atrophy markers [13,14,53]. damage to proteins and at the same time stimulate mitochondrialIn our study, the muscle atrophy markers atrogin-1 and MuRF1, as well gene transcription. Consistent with this assumption, we found thatas the autophagy markers Atg7, beclin-1, LC3, and FoxO3, were highly Exe activates the oxidative response kinases Erk1/2 and JNK and theinduced by Exe. We conclude that exhaustive Exe activates autophagy, oxidative response proteins p53, p21, and MnSOD. Unlike otherwhich 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 ROSproduction 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 1443Fig. 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 sacriﬁcedand the muscle mitochondrial dynamics-related proteins Drp1, Mfn1, and Mfn2 were determined by Western blot (A, Western blot images; B, statistical results). (C) Mitochondria wereisolated 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) 1437–1446Fig. 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 bothsedentary 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 HTtreatment). 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 Tfammitochondrial 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 animalnot other proteins, may suggest a speciﬁc targeting to MnSOD. This termination (24 h after the exhaustive exercise test) evinces quickspeciﬁcity is interesting but needs more study in the future. The recovery of some parameters such as Tfam mRNA, but is too early toFig. 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 wasanalyzed, 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 1445reveal changes in slower-responding ones such as PGC-1α protein. AcknowledgmentsThis issue warrants further study of the time-dependent changes inboth 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 partiallycentral to cell function and survival, but have not been well studied. supported by DSM Nutritional Products Ltd.; the Open Project of theBo 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 . Acute exercise, which increases ROS the National Natural Science Foundation of China, Key Programgeneration and State 4 respiration, decreases mitochondrial fusion 30930105; and the 985 and 211 Projects of Xian Jiaotong University.and increases mitochondrial ﬁssion . Inhibition of mitochondrial Dr. Ying Wang, Dr. Karin Wertz, and Dr. Peter Weber are employees ofﬁssion prevents muscle loss during fasting, and induction of DSM Nutritional Products, Ltd., a company produces and holds patentsmitochondrial ﬁssion and dysfunction activates an atrophy program for hydroxytyrosol.. Consistent with these studies, we found that Exe, althoughhaving no apparent effect on mitochondrial fusion, activated themitochondrial ﬁssion machinery, leading to acceleration of mito- Referenceschondrial dysfunction by ROS generation. Exercise- and muscle  Al-Jarrah, M.; Pothakos, K.; Novikova, L.; Smirnova, I. V.; Kurz, M. J.; Stehno-Bittel,contraction-induced ROS production has been widely reported. L.; Lau, Y. 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C.; Latres, E.; Baumhueter, S.; Lai, V. K.; Nunez, L.; Clarke, B. A.;primary cause of mitochondrial ﬁssion and autophagy and muscle Poueymirou, W. T.; Panaro, F. J.; Na, E.; Dharmarajan, K.; Pan, Z. Q.; Valenzuela,atrophy. D. M.; DeChiara, T. M.; Stitt, T. N.; Yancopoulos, G. D.; Glass, D. J. Identiﬁcation of ubiquitin ligases required for skeletal muscle atrophy. Science 294:1704–1708; HT is a phytochemical with antioxidant properties. In our study HT 2001.was given as an olive extract powder formulation standardized to 15%  Gomes, M. D.; Lecker, S. H.; Jagoe, R. T.; Navon, A.; Goldberg, A. L. Atrogin-1, aHT (19% total polyphenols). HT plasma levels 45 min after dosing—i.e., muscle-speciﬁc F-box protein highly expressed during muscle atrophy. Proc. Natlbefore the start of exercise after 8 weeks of supplementation—were Acad. Sci. U. S. A. 98:14440–14445; 2001.  Masiero, E.; Sandri, M. Autophagy inhibition induces atrophy and myopathy in124.1 ± 30.8 μM in sedentary animals and 150 ± 18.7 μM in regularly adult skeletal muscles. Autophagy 6:307–309; 2010.exercised rats. The endogenous HT plasma level of control rats varies  Masiero, E.; Agatea, L.; Mammucari, C.; Blaauw, B.; Loro, E.; Komatsu, M.; Metzger,between 1 and 0.03 μM (data not shown). Though there was 4% D.; Reggiani, C.; Schiafﬁno, S.; Sandri, M. Autophagy is required to maintain muscle mass. Cell Metab. 10:507–515; 2009.unknown polyphenols in the diet, HT was still the most active and the  Zhao, J.; Brault, J. J.; Schild, A.; Cao, P.; Sandri, M.; Schiafﬁno, S.; Lecker, S. H.;major compound in the diet. Studies showed that HT efﬁciently Goldberg, A. L. FoxO3 coordinately activates protein degradation by thedecreased Exe-induced oxidative stress and protected against Exe- autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. 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