Int J Clin Exp Med 20169(3)5677-5687wwwijcemcom ISSN1940-5901IJCEM0019751

Original ArticleThe role of the PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy induced by COPD

Yanli Li1 Yan Li1 Fengfeng Han1 Haiyang Yu1 Tianyun Yang1 Wenbin Guan2 Xuejun Guo1

Departments of 1Respiratory Medicine 2Pathology Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine China Equal contributors and co-first authors

Received November 14 2015 Accepted February 2 2016 Epub March 15 2016 Published March 30 2016

Abstract Objective To confirm the role of PI3KAKTmTOR protein synthesis pathway in skeletal muscle atrophy induced by chronic obstructive pulmonary disease (COPD) and to explore the possible targets in the signaling path-way inhibiting muscle atrophy Methods Rats exposed to chronic cigarette smoke (CS) were selected for our study divided into two exposure groups sham-exposed and 12 weeks CS Total RNA and protein were extracted from exten-sor digitorum longus muscle (EDLM) for Real-time PCR and Western blot analysis to assess the MHC expression The proteins expression associated with PI3KAKTmTOR signaling pathway (PI3K Akt mTOR GSK-3β p70S6K1 and 4EBP1) were assessed by Western blot analysis 2 FBS induces L6 cells to differentiate IGF-1 (PI3K agonist) and 10 CSE exposed L6 myoblasts for 24 hours then detect the expression of MHC and the proteins associated with PI3KAKTmTOR signaling pathway Results Chronic CS exposure decreased the MHC expression The phosphory-lation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after Chronic CS exposure and no significant changes were observed in the protein levels of PI3K CSE treatment also decreased MHC expres-sion PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment Not only the phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 decreased significantly after CSE treatment but also PI3K Moreover IGF-1 increased the phosphorylation levels of the protein synthesis pathway in comparison with simple CSE treatment Conclusion Chronic CS exposure induces muscle at-rophy and activates PI3KAKTmTOR signaling pathway Using IGF-1 activates PI3KAKTmTOR signaling pathway inhibits L6 myoblasts atrophy induced by CSE

Keywords PI3KAKTmTOR signaling pathway skeletal muscle atrophy L6 myoblasts COPD

Introduction

Chronic obstructive pulmonary disease (COPD) a common preventable and treatable disease is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways [1] Cigarette smoking is the most common risk factor The Global Burden of Disease Study projected that COPD will become the third leading cause of death worldwide by 2020 a newer projection estimated COPD will be the fourth leading cause of death in 2030 [2] COPD is associated with significant eco-nomic burden Not only do COPD causes pulmo-nary destruction but also systemic effects among which muscle atrophy is one of the most

important Muscle atrophy results in skeletal muscle dysfunction and respiratory muscle weakness directly affecting the progression and prognosis of the disease [3] So the re- search of mechanisms of muscle atrophy in COPD is vital to prevent muscle atrophy and improve the quality of life in patients with COPD Multi-factors and multi-molecular mechanisms participate in muscle atrophy causing imbal-ance between the rates of muscle protein syn-thesis and degradation eventually resulting in muscle atrophy [4] Most domestic and foreign studies [5-9] focused on the mechanism of pro-tein degradation pathways in COPD muscle atrophy including the ubiquitin-proteasome system (UPS) lysosomal pathway peroxisome proliferator-activated receptor (PPAR) and Cal-

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5678 Int J Clin Exp Med 20169(3)5677-5687

cium-dependent pathway of which the UPS is particularly important However the role of pro-tein synthesis pathway in the mechanism of muscle atrophy in COPD is unclear so we focused on phosphoinositide 3-kinase (PI3K)AKTmechanistic target of rapamycin (mTOR) protein synthesis pathway to observe how it acted in COPD muscle atrophy

PI3KAKTmTOR signaling pathway is an impor-tant intracellular transduction pathways PI3K can be activated by extracellular signal (cyto-kines growth factors hormones etc) then as a second messenger binding of Akt PH area acti-vates Akt [10] Activated Akt promotes the phosphorylation of mTOR glycogen synthase kinase-3β (GSK-3β) and other downstream substrate playing a wide range of biological effects such as anti-apoptosis promoting cell survival and other functions [11] phospho-mTOR initiates the phosphorylated translation of eukaryotic initiation factor 4E binding protein 1 (4EBP1) and 70 kD ribosomal protein S6 kinase (p70S6k1) MTOR specifically via phos-phorylation of p70S6K1 and 4EBP1 plays a central role in regulating the rate of protein syn-thesis in skeletal muscle [12-16]

Considering the preponderant role of cigarette smoke (CS) as a causative factor and lacking skeletal muscle cells in cellular atrophy models induced by cigarette smoke extract (CSE) which has been used to research the precise molecular mechanisms of the disorders trig-gered and regulated by CS at the cellular level rats exposed to chronic CS and L6 myotubes treated with various concentrations of CSE were chosen for our study resemble the previ-ous study [17] Our research show that in vivo chronic CS and in vitro CSE incubation induced muscle atrophy which were associated with the activity of PI3KAKTmTOR signaling path-way an agent believed to be critical in the regu-lation of muscle protein metabolism Muscle atrophy induced by CSE was associated with activation of PI3KAKTmTOR signaling path-way as specific activator inhibited muscle atrophy

Materials and methods

Animal cigarette smoke exposure and histo-logical analysis

Adult male Sprague-Dawley (S-D) rats weighing about 150 g were purchased from the Shanghai

Laboratory Animal Center and fed in the animal experimental center of Xinhua Hospital Affi- liated to Shanghai Jiaotong University School of Medicine The local institutional animal care committees approved the animal facilities and protocols All rats were divided into two groups (a) control group and (b) CS exposure group All rats were fed adaptively for one week then the control group was exposed to normal air while CS exposure group were exposed to CS gener-ated from 24 DaQianMen cigarettes per day (ShangHai China) CS exposure group were exposed for 12 weeks (Two times of CS expo-sure a day each time three cycles a cycle of four cigarettes) All rats were measured weight every 4 weeks Rats were given anesthesia by intra-peritoneal injection of 1 sodium pento- barbital

For histological analysis firstly the lungs and extensor digitorum longus muscle (EDLM) were immersed in 4 paraformaldehyde for 24 h Secondly they were imbedded in paraffin Finally they were stained with hematoxylin and eosin (HampE)

Cell culture and ultrastructural analysis under transmission electron microscope (TEM)

L6 myotubes were purchased from cell bank of Chinese Academy of Science To preserve the differential characteristics of the L6 myotubes L6 myotubes were split a maximum of seven times L6 myotubes were cultured in Dulbeccorsquos modified Eaglersquos medium (DMEM) consisting of 10 fetal bovine serum (FBS) and incubated at 37degC in a humidified 5 CO2 95 air atmo-sphere in 100-mm 6-well or 96-well plates Cells were covered bottom 70 and induced differentiate into multinucleated myotubes by using the 2 FBS for 6 days Observe charac-teristics of the differentiated L6 myotubes by an inverted Olympus CK40-F200 microscope

Differentiated L6 myotubes were fixed with 3 glutaraldehyde for 30 minutes on ice We col-lected the fixed cells using cell scrapers and centrifuged the fixed cells The cell pellets were fixed in 2 glutaraldehyde for 2 hours and in 1 osmium tetroxide 2 hours dehydrated in an ascending ethanol series and embedded in Epon 618 The ultrathin sections were observed through a PHILIPS CM120 electron microscope (magnifications 3500 to 15000)

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5679 Int J Clin Exp Med 20169(3)5677-5687

CSE preparation and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay

CSE was prepared according to the method of Liu Qian [17] Briefly a DaQianMen cigarette without filters was connected to a channel of three-limb tubes a 50 ml syringe was connect-ed to another channel and a 50 ml centrifugal tube with 10 ml DMEM was connected to the last channel by a catheter The smoke was released into the DMEM by pulling the syringe slowly until the unburned butt was less than 1 cm long This solution represented ldquo100rdquo strength Smoked medium was adjusted PH to 74 with NaOH and sterilized through a 022 μm

pore filter Smoked medium was used for the experiment within 30 min and diluted by DMEM to the required strength

Cells were grown in 96-well plates (2times103 cellswell) Cells were exposed to control medi-um or medium containing various concentra-tions of CSE (5 10 20 30) Cell survival was tested by the MTT assay after 24 hours After removing culture medium we stained cells with 20 ul 5 mgml sterile MTT dye (Sigma USA) at 37degC for 4 hours then added 200 ul dimethylsulfoxide (DMSO) to each well for 10 minutes The spectrometric absorbance was measured at 490 nm by automatic multiwell spectrophotometer

Figure 1 Chronic cigarette smoke (CS) exposure induces muscle atrophy A Weight were measured every 4 weeks and presented as the mean plusmn SD B Lungs were stained with hematozylin and eosin (HampE) (times100 magnification) for histological examination Twelves of CS exposure produced emphysema alveolar rupture bullae formation inflam-matory cell infiltration C Skeletal cells of extensor digitorum longus muscle (EDLM) were stained with HampE (times100 magnification) for histological examination The number of cells was calculated per high-power (HP) lens D MHC mRNA expression level was assessed by qPCR and protein expression level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5680 Int J Clin Exp Med 20169(3)5677-5687

Immunocytochemistry

L6 myotubes were seeded in 6-well plates containing sterile cover glasses incubated overnight and treated with 2 FBS for 6 days The cells were washed with phosphate-buff-ered saline (PBS) fixed with 4 paraformalde-hyde 30 minutes permeabilized with 03 TritonX-100 for 30 minutes and blocked with peroxidase blocking solution (DAKO) and bovine serum albumin (BSA) for 30 minutes respec-tively Then cells were incubated with anti-smooth muscle actin (SMA) antibody (1200) (sigma USA) at 4degC overnight After washing in PBS the cells were incubated with a goat anti-rabbit IgGHRP antibody for 1 h incubated with DAB peroxidase substrate counterstained with hematoxylin Images were acquired by an inverted Olympus CK40-F200 microscope

Western blot

The rat extensor digitorum longus tissues and L6 myotubes were lysed in lysis buffer (RIPA 1 mM PMSF and phosphatase inhibitors (Roche Germany)) on ice for 30 minutes and centri-fuged at 12000 rpm 4degC for 30 minutes The protein concentration was tested using BCA protein assay Protein extracts (60 ug) were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes After blocking for 2 hours at room temperature in 5 non-fat milk the membranes were incubated with primary anti-bodies GAPDH (Beyotime China) MHC (Santa Cruz USA) PI3K (CST USA) phospho-Akt (CST USA) phospho-mTOR (CST USA) phospho-GSK-3β (CST USA) phospho-p70S6K1 (CST USA) phospho-4EBP1 (CST USA) Akt (CST USA) mTOR (CST USA) GSK-3β (CST USA) p70S6K1 (CST USA) 4EBP1 (CST USA) over-night at 4degC Then the membranes were incu-bated with the appropriate secondary antibody conjugated to HRP for 1 h at room temperature and detected by enzyme-linked chemilumines-cence (ECL) The protein expression levels were determined by ChemiDoc XRS + Systems and analyzed by Image Lab 20 Software GAPDH was used as internal control

Comparative analysis of quantitative real-time polymerase chain reaction PCR (qPCR)

Total RNA was extracted from the EDLM tissues and L6 myotubes using Trizol reagent and 1 ug

of total RNA was reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara Japan) and RNase Free dH2O in a 20-ml final volume qPCR was then performed using an ABI Prism 7500 Sequence Detection System and SYBR Premix Ex Taq (Takara Japan) Sequences of the primers were for GAPDH forward 5rsquo-AAG GTC GGA GTC AAG GGA TTT-3rsquo and reverse 5rsquo-AGA TGA TGA CCC TTT TGG CTC-3rsquo MHC for-ward 5rsquo-GCG GAA AGA AAG GTG GCA AGA-3rsquo and reverse 5rsquo-TGG GAA TGA GGC ATC GGA CAA-3rsquo Strand cDNA was used as template in 20 ul reactions including 10 ul of 2timesSYBRRP remix Ex TaqTM and 500 nM of each primer Cycling conditions were performed as follows Step 1 30 s at 95degC step 2 5 s at 95degC and step 3 34 s at 60degC with 40 repeats of step 2 to step 3 Relative expression of mRNA was calculated using the 2-ACt method with GAPDH as an internal reference gene

Statistical analysis

All data are presented as the mean plusmn SD from three independent experiments SPSS statis-tics v190 software was used for statistical analysis Statistical analyses were performed using Studentrsquos-t test or one-way ANOVA fol-lowed by Dunnettrsquos tests P lt 005 was consid-ered statistically significant

Results

Chronic CS exposure causes muscle atrophy

The mean bodyweights of the rats in the 8 weeks and 12 weeks of CS exposure were lower significantly than those in the control groups (Figure 1A P lt 001) Twelve weeks of CS exposure to rats led to pulmonary lesions that morphologically resembled human chro- nic bronchial inflammation and emphysema (Figure 1B) Compared with the control group skeletal cell numbers per high-power (HP) lens increased after 12 weeks of CS exposure (Fig- ure 1C P lt 001) suggesting muscle wasting Chronic CS exposure decreased the mRNA and MHC protein level of MHC in the 12-week groups (Figure 1D P lt 001) Taken together these data show muscle atrophy

Chronic CS exposure activates PI3KAKTmTOR signaling pathway

Western blot analysis was used to assess the protein levels of PI3K and phosphorylation lev-

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5681 Int J Clin Exp Med 20169(3)5677-5687

Figure 2 Chronic CS exposure activates PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5682 Int J Clin Exp Med 20169(3)5677-5687

els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 As shown in Figure 2 Chronic CS expo-sure activates PI3KAKTmTOR signaling path-way The phosphorylation levels of Akt (Figure 2B P lt 001) mTOR (Figure 2C P lt 005) GSK-3β (Figure 2D P lt 001) p70S6K1 (Figure 2E P lt 005) and 4EBP1 (Figure 2F P lt 005) were increased significantly after Chronic CS expo-

sure and no significant changes were observed in the protein levels of PI3K (Figure 2A)

2 FBS induces L6 cells to differentiate into multinucleated myotubes

After cultured with 2 FBS L6 myotubes exhib-ited an elongated cell shape with branched

Figure 3 2 FBS induces L6 cells to differentiate A The morphology of L6 myotubes were evaluated by inverted microscope (times100 magnification) B The structures of L6 myotubes were presented by magnification of electron micrographs (times3500-12000 magnification) C SMA expression was examined by immunocytochemistry (times100 magnification)

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5683 Int J Clin Exp Med 20169(3)5677-5687

and multi-nucleated and even displayed fusion (Figure 3A) Moreover a large number of fila-ments were longitudinally arranged in L6 myo-tubes (Figure 3B) The SMA-stained myoblasts were increased obviously in differential L6 myo-tubes (Figure 3C) Taken together Figure 3 shows that L6 myotubes differentiate into mul-tinucleated myotubes successfully

MTT and L6 myotubes atrophy

Exposed to various concentrations of CSE L6 myotubes survival was tested by MTT assay 24 hours later CSE produced a decrease in cell survival at the concentration of 20 (P lt 005) and 30 (P lt 001) Conversely treated with 5 10 and 20 CSE the survival rates of cells had no change (Figure 4A) 10 CSE was selected for subsequent experiments CSE treatment decreased the mRNA and protein level of MHC 24 hours later (Figure 4B P lt 001) suggesting L6 myotubes atrophy However CSE and PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment (Figure 4B P lt 001)

CSE suppresses PI3KAKTmTOR signaling pathway

As shown in Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway After treated

with CSE 24 hours later not only the phos- phorylation levels of Akt (Figure 5B P lt 005) mTOR (Figure 5C P lt 001) GSK-3β (Figure 5D P lt 001) p70S6K1 (Figure 5E P lt 001) and 4EBP1 (Figure 5F P lt 001) decreased signifi-cantly but also PI3K Moreover IGF-1 increased the the phosphorylation levels in comparison with simple CSE treatment (Figure 5)

Discussion

COPD results in significant systemic effects among which muscle dysfunctionwasting is one of the most important [17] Many studies have demonstrated the importance of the mechanism of protein degradation pathways such as the UPS lysosomal pathway PPAR and Calcium-dependent pathway However little is known about the role of protein synthesis path-way especially PI3KAKTmTOR signaling path-way in this process

We obtained the evidence that PI3KAKTmTOR signaling pathway was activated in animal mod-els of COPD induced by CS Both 8 and 12 weeks-CS exposure caused significant weight loss and 12 weeks-CS exposure produced emphysema and decreased in muscle size Similar to our research chronic exposure to the smoke of 20 cigarettes per day for 12 weeks

Figure 4 MTT and L6 myotubes atrophy A Cell survival was tested by MTT assay after 24 hours B MHC mRNA ex-pression level was assessed by qPCR and protein expres-sion level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 com-pared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5679 Int J Clin Exp Med 20169(3)5677-5687

CSE preparation and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay

CSE was prepared according to the method of Liu Qian [17] Briefly a DaQianMen cigarette without filters was connected to a channel of three-limb tubes a 50 ml syringe was connect-ed to another channel and a 50 ml centrifugal tube with 10 ml DMEM was connected to the last channel by a catheter The smoke was released into the DMEM by pulling the syringe slowly until the unburned butt was less than 1 cm long This solution represented ldquo100rdquo strength Smoked medium was adjusted PH to 74 with NaOH and sterilized through a 022 μm

pore filter Smoked medium was used for the experiment within 30 min and diluted by DMEM to the required strength

Cells were grown in 96-well plates (2times103 cellswell) Cells were exposed to control medi-um or medium containing various concentra-tions of CSE (5 10 20 30) Cell survival was tested by the MTT assay after 24 hours After removing culture medium we stained cells with 20 ul 5 mgml sterile MTT dye (Sigma USA) at 37degC for 4 hours then added 200 ul dimethylsulfoxide (DMSO) to each well for 10 minutes The spectrometric absorbance was measured at 490 nm by automatic multiwell spectrophotometer

Figure 1 Chronic cigarette smoke (CS) exposure induces muscle atrophy A Weight were measured every 4 weeks and presented as the mean plusmn SD B Lungs were stained with hematozylin and eosin (HampE) (times100 magnification) for histological examination Twelves of CS exposure produced emphysema alveolar rupture bullae formation inflam-matory cell infiltration C Skeletal cells of extensor digitorum longus muscle (EDLM) were stained with HampE (times100 magnification) for histological examination The number of cells was calculated per high-power (HP) lens D MHC mRNA expression level was assessed by qPCR and protein expression level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5680 Int J Clin Exp Med 20169(3)5677-5687

Immunocytochemistry

L6 myotubes were seeded in 6-well plates containing sterile cover glasses incubated overnight and treated with 2 FBS for 6 days The cells were washed with phosphate-buff-ered saline (PBS) fixed with 4 paraformalde-hyde 30 minutes permeabilized with 03 TritonX-100 for 30 minutes and blocked with peroxidase blocking solution (DAKO) and bovine serum albumin (BSA) for 30 minutes respec-tively Then cells were incubated with anti-smooth muscle actin (SMA) antibody (1200) (sigma USA) at 4degC overnight After washing in PBS the cells were incubated with a goat anti-rabbit IgGHRP antibody for 1 h incubated with DAB peroxidase substrate counterstained with hematoxylin Images were acquired by an inverted Olympus CK40-F200 microscope

Western blot

The rat extensor digitorum longus tissues and L6 myotubes were lysed in lysis buffer (RIPA 1 mM PMSF and phosphatase inhibitors (Roche Germany)) on ice for 30 minutes and centri-fuged at 12000 rpm 4degC for 30 minutes The protein concentration was tested using BCA protein assay Protein extracts (60 ug) were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes After blocking for 2 hours at room temperature in 5 non-fat milk the membranes were incubated with primary anti-bodies GAPDH (Beyotime China) MHC (Santa Cruz USA) PI3K (CST USA) phospho-Akt (CST USA) phospho-mTOR (CST USA) phospho-GSK-3β (CST USA) phospho-p70S6K1 (CST USA) phospho-4EBP1 (CST USA) Akt (CST USA) mTOR (CST USA) GSK-3β (CST USA) p70S6K1 (CST USA) 4EBP1 (CST USA) over-night at 4degC Then the membranes were incu-bated with the appropriate secondary antibody conjugated to HRP for 1 h at room temperature and detected by enzyme-linked chemilumines-cence (ECL) The protein expression levels were determined by ChemiDoc XRS + Systems and analyzed by Image Lab 20 Software GAPDH was used as internal control

Comparative analysis of quantitative real-time polymerase chain reaction PCR (qPCR)

Total RNA was extracted from the EDLM tissues and L6 myotubes using Trizol reagent and 1 ug

of total RNA was reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara Japan) and RNase Free dH2O in a 20-ml final volume qPCR was then performed using an ABI Prism 7500 Sequence Detection System and SYBR Premix Ex Taq (Takara Japan) Sequences of the primers were for GAPDH forward 5rsquo-AAG GTC GGA GTC AAG GGA TTT-3rsquo and reverse 5rsquo-AGA TGA TGA CCC TTT TGG CTC-3rsquo MHC for-ward 5rsquo-GCG GAA AGA AAG GTG GCA AGA-3rsquo and reverse 5rsquo-TGG GAA TGA GGC ATC GGA CAA-3rsquo Strand cDNA was used as template in 20 ul reactions including 10 ul of 2timesSYBRRP remix Ex TaqTM and 500 nM of each primer Cycling conditions were performed as follows Step 1 30 s at 95degC step 2 5 s at 95degC and step 3 34 s at 60degC with 40 repeats of step 2 to step 3 Relative expression of mRNA was calculated using the 2-ACt method with GAPDH as an internal reference gene

Statistical analysis

All data are presented as the mean plusmn SD from three independent experiments SPSS statis-tics v190 software was used for statistical analysis Statistical analyses were performed using Studentrsquos-t test or one-way ANOVA fol-lowed by Dunnettrsquos tests P lt 005 was consid-ered statistically significant

Results

Chronic CS exposure causes muscle atrophy

The mean bodyweights of the rats in the 8 weeks and 12 weeks of CS exposure were lower significantly than those in the control groups (Figure 1A P lt 001) Twelve weeks of CS exposure to rats led to pulmonary lesions that morphologically resembled human chro- nic bronchial inflammation and emphysema (Figure 1B) Compared with the control group skeletal cell numbers per high-power (HP) lens increased after 12 weeks of CS exposure (Fig- ure 1C P lt 001) suggesting muscle wasting Chronic CS exposure decreased the mRNA and MHC protein level of MHC in the 12-week groups (Figure 1D P lt 001) Taken together these data show muscle atrophy

Chronic CS exposure activates PI3KAKTmTOR signaling pathway

Western blot analysis was used to assess the protein levels of PI3K and phosphorylation lev-

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5681 Int J Clin Exp Med 20169(3)5677-5687

Figure 2 Chronic CS exposure activates PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5682 Int J Clin Exp Med 20169(3)5677-5687

els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 As shown in Figure 2 Chronic CS expo-sure activates PI3KAKTmTOR signaling path-way The phosphorylation levels of Akt (Figure 2B P lt 001) mTOR (Figure 2C P lt 005) GSK-3β (Figure 2D P lt 001) p70S6K1 (Figure 2E P lt 005) and 4EBP1 (Figure 2F P lt 005) were increased significantly after Chronic CS expo-

sure and no significant changes were observed in the protein levels of PI3K (Figure 2A)

2 FBS induces L6 cells to differentiate into multinucleated myotubes

After cultured with 2 FBS L6 myotubes exhib-ited an elongated cell shape with branched

Figure 3 2 FBS induces L6 cells to differentiate A The morphology of L6 myotubes were evaluated by inverted microscope (times100 magnification) B The structures of L6 myotubes were presented by magnification of electron micrographs (times3500-12000 magnification) C SMA expression was examined by immunocytochemistry (times100 magnification)

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5683 Int J Clin Exp Med 20169(3)5677-5687

and multi-nucleated and even displayed fusion (Figure 3A) Moreover a large number of fila-ments were longitudinally arranged in L6 myo-tubes (Figure 3B) The SMA-stained myoblasts were increased obviously in differential L6 myo-tubes (Figure 3C) Taken together Figure 3 shows that L6 myotubes differentiate into mul-tinucleated myotubes successfully

MTT and L6 myotubes atrophy

Exposed to various concentrations of CSE L6 myotubes survival was tested by MTT assay 24 hours later CSE produced a decrease in cell survival at the concentration of 20 (P lt 005) and 30 (P lt 001) Conversely treated with 5 10 and 20 CSE the survival rates of cells had no change (Figure 4A) 10 CSE was selected for subsequent experiments CSE treatment decreased the mRNA and protein level of MHC 24 hours later (Figure 4B P lt 001) suggesting L6 myotubes atrophy However CSE and PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment (Figure 4B P lt 001)

CSE suppresses PI3KAKTmTOR signaling pathway

As shown in Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway After treated

with CSE 24 hours later not only the phos- phorylation levels of Akt (Figure 5B P lt 005) mTOR (Figure 5C P lt 001) GSK-3β (Figure 5D P lt 001) p70S6K1 (Figure 5E P lt 001) and 4EBP1 (Figure 5F P lt 001) decreased signifi-cantly but also PI3K Moreover IGF-1 increased the the phosphorylation levels in comparison with simple CSE treatment (Figure 5)

Discussion

COPD results in significant systemic effects among which muscle dysfunctionwasting is one of the most important [17] Many studies have demonstrated the importance of the mechanism of protein degradation pathways such as the UPS lysosomal pathway PPAR and Calcium-dependent pathway However little is known about the role of protein synthesis path-way especially PI3KAKTmTOR signaling path-way in this process

We obtained the evidence that PI3KAKTmTOR signaling pathway was activated in animal mod-els of COPD induced by CS Both 8 and 12 weeks-CS exposure caused significant weight loss and 12 weeks-CS exposure produced emphysema and decreased in muscle size Similar to our research chronic exposure to the smoke of 20 cigarettes per day for 12 weeks

Figure 4 MTT and L6 myotubes atrophy A Cell survival was tested by MTT assay after 24 hours B MHC mRNA ex-pression level was assessed by qPCR and protein expres-sion level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 com-pared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5680 Int J Clin Exp Med 20169(3)5677-5687

Immunocytochemistry

L6 myotubes were seeded in 6-well plates containing sterile cover glasses incubated overnight and treated with 2 FBS for 6 days The cells were washed with phosphate-buff-ered saline (PBS) fixed with 4 paraformalde-hyde 30 minutes permeabilized with 03 TritonX-100 for 30 minutes and blocked with peroxidase blocking solution (DAKO) and bovine serum albumin (BSA) for 30 minutes respec-tively Then cells were incubated with anti-smooth muscle actin (SMA) antibody (1200) (sigma USA) at 4degC overnight After washing in PBS the cells were incubated with a goat anti-rabbit IgGHRP antibody for 1 h incubated with DAB peroxidase substrate counterstained with hematoxylin Images were acquired by an inverted Olympus CK40-F200 microscope

Western blot

The rat extensor digitorum longus tissues and L6 myotubes were lysed in lysis buffer (RIPA 1 mM PMSF and phosphatase inhibitors (Roche Germany)) on ice for 30 minutes and centri-fuged at 12000 rpm 4degC for 30 minutes The protein concentration was tested using BCA protein assay Protein extracts (60 ug) were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes After blocking for 2 hours at room temperature in 5 non-fat milk the membranes were incubated with primary anti-bodies GAPDH (Beyotime China) MHC (Santa Cruz USA) PI3K (CST USA) phospho-Akt (CST USA) phospho-mTOR (CST USA) phospho-GSK-3β (CST USA) phospho-p70S6K1 (CST USA) phospho-4EBP1 (CST USA) Akt (CST USA) mTOR (CST USA) GSK-3β (CST USA) p70S6K1 (CST USA) 4EBP1 (CST USA) over-night at 4degC Then the membranes were incu-bated with the appropriate secondary antibody conjugated to HRP for 1 h at room temperature and detected by enzyme-linked chemilumines-cence (ECL) The protein expression levels were determined by ChemiDoc XRS + Systems and analyzed by Image Lab 20 Software GAPDH was used as internal control

Comparative analysis of quantitative real-time polymerase chain reaction PCR (qPCR)

Total RNA was extracted from the EDLM tissues and L6 myotubes using Trizol reagent and 1 ug

of total RNA was reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara Japan) and RNase Free dH2O in a 20-ml final volume qPCR was then performed using an ABI Prism 7500 Sequence Detection System and SYBR Premix Ex Taq (Takara Japan) Sequences of the primers were for GAPDH forward 5rsquo-AAG GTC GGA GTC AAG GGA TTT-3rsquo and reverse 5rsquo-AGA TGA TGA CCC TTT TGG CTC-3rsquo MHC for-ward 5rsquo-GCG GAA AGA AAG GTG GCA AGA-3rsquo and reverse 5rsquo-TGG GAA TGA GGC ATC GGA CAA-3rsquo Strand cDNA was used as template in 20 ul reactions including 10 ul of 2timesSYBRRP remix Ex TaqTM and 500 nM of each primer Cycling conditions were performed as follows Step 1 30 s at 95degC step 2 5 s at 95degC and step 3 34 s at 60degC with 40 repeats of step 2 to step 3 Relative expression of mRNA was calculated using the 2-ACt method with GAPDH as an internal reference gene

Statistical analysis

All data are presented as the mean plusmn SD from three independent experiments SPSS statis-tics v190 software was used for statistical analysis Statistical analyses were performed using Studentrsquos-t test or one-way ANOVA fol-lowed by Dunnettrsquos tests P lt 005 was consid-ered statistically significant

Results

Chronic CS exposure causes muscle atrophy

The mean bodyweights of the rats in the 8 weeks and 12 weeks of CS exposure were lower significantly than those in the control groups (Figure 1A P lt 001) Twelve weeks of CS exposure to rats led to pulmonary lesions that morphologically resembled human chro- nic bronchial inflammation and emphysema (Figure 1B) Compared with the control group skeletal cell numbers per high-power (HP) lens increased after 12 weeks of CS exposure (Fig- ure 1C P lt 001) suggesting muscle wasting Chronic CS exposure decreased the mRNA and MHC protein level of MHC in the 12-week groups (Figure 1D P lt 001) Taken together these data show muscle atrophy

Chronic CS exposure activates PI3KAKTmTOR signaling pathway

Western blot analysis was used to assess the protein levels of PI3K and phosphorylation lev-

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5681 Int J Clin Exp Med 20169(3)5677-5687

Figure 2 Chronic CS exposure activates PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5682 Int J Clin Exp Med 20169(3)5677-5687

els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 As shown in Figure 2 Chronic CS expo-sure activates PI3KAKTmTOR signaling path-way The phosphorylation levels of Akt (Figure 2B P lt 001) mTOR (Figure 2C P lt 005) GSK-3β (Figure 2D P lt 001) p70S6K1 (Figure 2E P lt 005) and 4EBP1 (Figure 2F P lt 005) were increased significantly after Chronic CS expo-

sure and no significant changes were observed in the protein levels of PI3K (Figure 2A)

2 FBS induces L6 cells to differentiate into multinucleated myotubes

After cultured with 2 FBS L6 myotubes exhib-ited an elongated cell shape with branched

Figure 3 2 FBS induces L6 cells to differentiate A The morphology of L6 myotubes were evaluated by inverted microscope (times100 magnification) B The structures of L6 myotubes were presented by magnification of electron micrographs (times3500-12000 magnification) C SMA expression was examined by immunocytochemistry (times100 magnification)

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5683 Int J Clin Exp Med 20169(3)5677-5687

and multi-nucleated and even displayed fusion (Figure 3A) Moreover a large number of fila-ments were longitudinally arranged in L6 myo-tubes (Figure 3B) The SMA-stained myoblasts were increased obviously in differential L6 myo-tubes (Figure 3C) Taken together Figure 3 shows that L6 myotubes differentiate into mul-tinucleated myotubes successfully

MTT and L6 myotubes atrophy

Exposed to various concentrations of CSE L6 myotubes survival was tested by MTT assay 24 hours later CSE produced a decrease in cell survival at the concentration of 20 (P lt 005) and 30 (P lt 001) Conversely treated with 5 10 and 20 CSE the survival rates of cells had no change (Figure 4A) 10 CSE was selected for subsequent experiments CSE treatment decreased the mRNA and protein level of MHC 24 hours later (Figure 4B P lt 001) suggesting L6 myotubes atrophy However CSE and PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment (Figure 4B P lt 001)

CSE suppresses PI3KAKTmTOR signaling pathway

As shown in Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway After treated

with CSE 24 hours later not only the phos- phorylation levels of Akt (Figure 5B P lt 005) mTOR (Figure 5C P lt 001) GSK-3β (Figure 5D P lt 001) p70S6K1 (Figure 5E P lt 001) and 4EBP1 (Figure 5F P lt 001) decreased signifi-cantly but also PI3K Moreover IGF-1 increased the the phosphorylation levels in comparison with simple CSE treatment (Figure 5)

Discussion

COPD results in significant systemic effects among which muscle dysfunctionwasting is one of the most important [17] Many studies have demonstrated the importance of the mechanism of protein degradation pathways such as the UPS lysosomal pathway PPAR and Calcium-dependent pathway However little is known about the role of protein synthesis path-way especially PI3KAKTmTOR signaling path-way in this process

We obtained the evidence that PI3KAKTmTOR signaling pathway was activated in animal mod-els of COPD induced by CS Both 8 and 12 weeks-CS exposure caused significant weight loss and 12 weeks-CS exposure produced emphysema and decreased in muscle size Similar to our research chronic exposure to the smoke of 20 cigarettes per day for 12 weeks

Figure 4 MTT and L6 myotubes atrophy A Cell survival was tested by MTT assay after 24 hours B MHC mRNA ex-pression level was assessed by qPCR and protein expres-sion level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 com-pared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5681 Int J Clin Exp Med 20169(3)5677-5687

Figure 2 Chronic CS exposure activates PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5682 Int J Clin Exp Med 20169(3)5677-5687

els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 As shown in Figure 2 Chronic CS expo-sure activates PI3KAKTmTOR signaling path-way The phosphorylation levels of Akt (Figure 2B P lt 001) mTOR (Figure 2C P lt 005) GSK-3β (Figure 2D P lt 001) p70S6K1 (Figure 2E P lt 005) and 4EBP1 (Figure 2F P lt 005) were increased significantly after Chronic CS expo-

sure and no significant changes were observed in the protein levels of PI3K (Figure 2A)

2 FBS induces L6 cells to differentiate into multinucleated myotubes

After cultured with 2 FBS L6 myotubes exhib-ited an elongated cell shape with branched

Figure 3 2 FBS induces L6 cells to differentiate A The morphology of L6 myotubes were evaluated by inverted microscope (times100 magnification) B The structures of L6 myotubes were presented by magnification of electron micrographs (times3500-12000 magnification) C SMA expression was examined by immunocytochemistry (times100 magnification)

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5683 Int J Clin Exp Med 20169(3)5677-5687

and multi-nucleated and even displayed fusion (Figure 3A) Moreover a large number of fila-ments were longitudinally arranged in L6 myo-tubes (Figure 3B) The SMA-stained myoblasts were increased obviously in differential L6 myo-tubes (Figure 3C) Taken together Figure 3 shows that L6 myotubes differentiate into mul-tinucleated myotubes successfully

MTT and L6 myotubes atrophy

Exposed to various concentrations of CSE L6 myotubes survival was tested by MTT assay 24 hours later CSE produced a decrease in cell survival at the concentration of 20 (P lt 005) and 30 (P lt 001) Conversely treated with 5 10 and 20 CSE the survival rates of cells had no change (Figure 4A) 10 CSE was selected for subsequent experiments CSE treatment decreased the mRNA and protein level of MHC 24 hours later (Figure 4B P lt 001) suggesting L6 myotubes atrophy However CSE and PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment (Figure 4B P lt 001)

CSE suppresses PI3KAKTmTOR signaling pathway

As shown in Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway After treated

with CSE 24 hours later not only the phos- phorylation levels of Akt (Figure 5B P lt 005) mTOR (Figure 5C P lt 001) GSK-3β (Figure 5D P lt 001) p70S6K1 (Figure 5E P lt 001) and 4EBP1 (Figure 5F P lt 001) decreased signifi-cantly but also PI3K Moreover IGF-1 increased the the phosphorylation levels in comparison with simple CSE treatment (Figure 5)

Discussion

COPD results in significant systemic effects among which muscle dysfunctionwasting is one of the most important [17] Many studies have demonstrated the importance of the mechanism of protein degradation pathways such as the UPS lysosomal pathway PPAR and Calcium-dependent pathway However little is known about the role of protein synthesis path-way especially PI3KAKTmTOR signaling path-way in this process

We obtained the evidence that PI3KAKTmTOR signaling pathway was activated in animal mod-els of COPD induced by CS Both 8 and 12 weeks-CS exposure caused significant weight loss and 12 weeks-CS exposure produced emphysema and decreased in muscle size Similar to our research chronic exposure to the smoke of 20 cigarettes per day for 12 weeks

Figure 4 MTT and L6 myotubes atrophy A Cell survival was tested by MTT assay after 24 hours B MHC mRNA ex-pression level was assessed by qPCR and protein expres-sion level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 com-pared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5682 Int J Clin Exp Med 20169(3)5677-5687

els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 As shown in Figure 2 Chronic CS expo-sure activates PI3KAKTmTOR signaling path-way The phosphorylation levels of Akt (Figure 2B P lt 001) mTOR (Figure 2C P lt 005) GSK-3β (Figure 2D P lt 001) p70S6K1 (Figure 2E P lt 005) and 4EBP1 (Figure 2F P lt 005) were increased significantly after Chronic CS expo-

sure and no significant changes were observed in the protein levels of PI3K (Figure 2A)

2 FBS induces L6 cells to differentiate into multinucleated myotubes

After cultured with 2 FBS L6 myotubes exhib-ited an elongated cell shape with branched

Figure 3 2 FBS induces L6 cells to differentiate A The morphology of L6 myotubes were evaluated by inverted microscope (times100 magnification) B The structures of L6 myotubes were presented by magnification of electron micrographs (times3500-12000 magnification) C SMA expression was examined by immunocytochemistry (times100 magnification)

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5683 Int J Clin Exp Med 20169(3)5677-5687

and multi-nucleated and even displayed fusion (Figure 3A) Moreover a large number of fila-ments were longitudinally arranged in L6 myo-tubes (Figure 3B) The SMA-stained myoblasts were increased obviously in differential L6 myo-tubes (Figure 3C) Taken together Figure 3 shows that L6 myotubes differentiate into mul-tinucleated myotubes successfully

MTT and L6 myotubes atrophy

Exposed to various concentrations of CSE L6 myotubes survival was tested by MTT assay 24 hours later CSE produced a decrease in cell survival at the concentration of 20 (P lt 005) and 30 (P lt 001) Conversely treated with 5 10 and 20 CSE the survival rates of cells had no change (Figure 4A) 10 CSE was selected for subsequent experiments CSE treatment decreased the mRNA and protein level of MHC 24 hours later (Figure 4B P lt 001) suggesting L6 myotubes atrophy However CSE and PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment (Figure 4B P lt 001)

CSE suppresses PI3KAKTmTOR signaling pathway

As shown in Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway After treated

with CSE 24 hours later not only the phos- phorylation levels of Akt (Figure 5B P lt 005) mTOR (Figure 5C P lt 001) GSK-3β (Figure 5D P lt 001) p70S6K1 (Figure 5E P lt 001) and 4EBP1 (Figure 5F P lt 001) decreased signifi-cantly but also PI3K Moreover IGF-1 increased the the phosphorylation levels in comparison with simple CSE treatment (Figure 5)

Discussion

COPD results in significant systemic effects among which muscle dysfunctionwasting is one of the most important [17] Many studies have demonstrated the importance of the mechanism of protein degradation pathways such as the UPS lysosomal pathway PPAR and Calcium-dependent pathway However little is known about the role of protein synthesis path-way especially PI3KAKTmTOR signaling path-way in this process

We obtained the evidence that PI3KAKTmTOR signaling pathway was activated in animal mod-els of COPD induced by CS Both 8 and 12 weeks-CS exposure caused significant weight loss and 12 weeks-CS exposure produced emphysema and decreased in muscle size Similar to our research chronic exposure to the smoke of 20 cigarettes per day for 12 weeks

Figure 4 MTT and L6 myotubes atrophy A Cell survival was tested by MTT assay after 24 hours B MHC mRNA ex-pression level was assessed by qPCR and protein expres-sion level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 com-pared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5683 Int J Clin Exp Med 20169(3)5677-5687

and multi-nucleated and even displayed fusion (Figure 3A) Moreover a large number of fila-ments were longitudinally arranged in L6 myo-tubes (Figure 3B) The SMA-stained myoblasts were increased obviously in differential L6 myo-tubes (Figure 3C) Taken together Figure 3 shows that L6 myotubes differentiate into mul-tinucleated myotubes successfully

MTT and L6 myotubes atrophy

Exposed to various concentrations of CSE L6 myotubes survival was tested by MTT assay 24 hours later CSE produced a decrease in cell survival at the concentration of 20 (P lt 005) and 30 (P lt 001) Conversely treated with 5 10 and 20 CSE the survival rates of cells had no change (Figure 4A) 10 CSE was selected for subsequent experiments CSE treatment decreased the mRNA and protein level of MHC 24 hours later (Figure 4B P lt 001) suggesting L6 myotubes atrophy However CSE and PI3K agonist insulin-like growth factor (IGF-1) treatment increased the MHC expression in comparison with simple CSE treatment (Figure 4B P lt 001)

CSE suppresses PI3KAKTmTOR signaling pathway

As shown in Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway After treated

with CSE 24 hours later not only the phos- phorylation levels of Akt (Figure 5B P lt 005) mTOR (Figure 5C P lt 001) GSK-3β (Figure 5D P lt 001) p70S6K1 (Figure 5E P lt 001) and 4EBP1 (Figure 5F P lt 001) decreased signifi-cantly but also PI3K Moreover IGF-1 increased the the phosphorylation levels in comparison with simple CSE treatment (Figure 5)

Discussion

COPD results in significant systemic effects among which muscle dysfunctionwasting is one of the most important [17] Many studies have demonstrated the importance of the mechanism of protein degradation pathways such as the UPS lysosomal pathway PPAR and Calcium-dependent pathway However little is known about the role of protein synthesis path-way especially PI3KAKTmTOR signaling path-way in this process

We obtained the evidence that PI3KAKTmTOR signaling pathway was activated in animal mod-els of COPD induced by CS Both 8 and 12 weeks-CS exposure caused significant weight loss and 12 weeks-CS exposure produced emphysema and decreased in muscle size Similar to our research chronic exposure to the smoke of 20 cigarettes per day for 12 weeks

Figure 4 MTT and L6 myotubes atrophy A Cell survival was tested by MTT assay after 24 hours B MHC mRNA ex-pression level was assessed by qPCR and protein expres-sion level was assessed by western blot analysis GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 com-pared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5684 Int J Clin Exp Med 20169(3)5677-5687

Figure 5 CSE suppresses PI3KAKTmTOR signaling pathway A Western blotting for PI3K protein B Western blotting for phospho-Akt and Akt protein C Western blotting for phospho-mTOR and mTOR protein D Western blotting for phospho-GSK-3β and GSK-3β protein E Western blotting for phospho-p70S6K1 and p70S6K1 protein F Western blotting for phospho-4EBP1 and 4EBP1 protein GAPDH served as the internal standard P lt 005 compared with control group P lt 001 compared with control group P lt 005 compared with CSE group P lt 001 compared with CSE group

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5685 Int J Clin Exp Med 20169(3)5677-5687

induced parenchymal destruction enlarge-ment of airways and weight loss in rats [17] In our study chronic CS exposure decreased the mRNA and protein level of MHC in the EDLM suggesting that chronic CS exposure induce muscle wasting Factors considered potential-ly relevant to skeletal muscle atrophy induced by chronic CS exposure include hypoxia hyper-capnia systemic or local inflammation anabol-iccatabolic hormone imbalance nutritional depletion and oxidative stress [18-20] PI3KAKTmTOR signaling pathway is involved in reg-ulation of cell growth and apoptosis [11] All along Akt is considered to play an important role in rodent skeletal muscle atrophy and hypertrophy Leacuteger B et al [21] found that AKT and its downstream signals mTOR and GSK-3β act in the regulation of human skeletal muscle atrophy and hypertrophy for the first time Knockouting PI3K gene caused the weight of mouse skeletal muscle loss and muscle fiber atrophy [22] PI3KAKTmTOR signaling path-way regulates the skeletal muscle protein syn-thesis The phosphorylation levels of Akt 4EBP1 p70S6K1 and total Akt were increased in atrophic denervated muscle Increased pro-tein degradation rather than decreased pro-tein synthesis is likely to be responsible for the loss of muscle mass in denervated atrophic muscles [23] Our study showed that the phos-phorylation levels of Akt mTOR GSK-3β p70S6K1 4EBP1 total mTOR and total GSK-3βwere increased significantly in chronic CS exposure rats indicating that PI3KAKTmTOR signaling pathway was activated by chronic CS exposure which increased protein synthesis Although protein synthesis increased skeletal muscle still atrophied suggesting that skeletal muscle protein degradation rather than pro-tein synthesis seems to be responsible for the loss of muscle mass in chronic CS exposure rats Activated PI3KAKTmTOR signaling path-way may be the bodyrsquos self-protection against skeletal muscle atrophy and is of great signifi-cance to skeletal muscle atrophy in chronic CS exposure rats

CS induces muscle wasting which is obtained from the studies in animals Moreover CSE has direct effects within cells To investigate the mechanism of the disorders caused by CS at the cellular level we checked the effect of CSE on L6 myotubes We differentiated L6 myo-tubes with 2 FBS for six days prior to experi-

mentations The differential L6 myotubes ex- hibite an elongated cell shape with branched and multi-nucleated and even display fusion Moreover a large number of filaments are lon-gitudinally arranged in L6 myotubes The SMA-stained myotubes increased obviously in differ-ential L6 myotubes SMA is a marker of skeletal muscle cell differentiation [24] All the above results indicated that L6 myotubes differenti-ated successfully The mRNA and protein level of MHC was significantly lower after 10 CSE treatments suggesting muscle wasting The phosphorylation levels of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were decreased signifi-cantly after 10 CSE treatments suggesting that CSE could inhibit the phosphorylation of PI3KAKTmTOR protein synthesis pathway and thus make skeletal muscle atrophy which is an attractive possibility for future investigation However we noted that the result of L6 myo-tubes experiment was inconsistent with the animal experiment The possible reasons were as follows To begin with the different prepara-tions of CS and CSE which was prepared only contained the hydrophilic components of ciga-rette smoke were used respectively in animal experiment and L6 myotubes experiment Secondly the concentration of CSE used may be too high and the treatment time may be too short leading that L6 myotubes had no time to compensate and PI3KAKTmTOR sig-naling pathway wasnrsquot activated furthermore it was possible that in early CS exposure pro-tein synthesis was indeed reduced and then some kind of compensatory mechanisms acted in the body activating the PI3KAKTmTOR sig-naling pathway Finally in vitro CSE exposure bypasses the airways and precludes the filter-ing action of the lungs [17] while in vivo ciga-rette smoking might stimulate the body produc-ing a variety of inflammatory cytokines hor-mones cytokines which activate the immune and endocrine system and then activate the PI3KAKTmTOR signaling pathway through some unknown mechanism

To further verify the role of PI3KAKTmTOR signaling pathway in skeletal muscle atrophy we used PI3K agonist IGF-1 treating CSE expo-sure-L6 myotubes for 24 hours and then detected the protein of PI3KAKTmTOR signal-ing pathway IGF-1 a survival factor to the smooth muscle cells fibroblasts and macro-phagesis involved in protein synthesis cell

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5686 Int J Clin Exp Med 20169(3)5677-5687

migration and mitosis [25 26] IGF-1R acti-vated by a variety of signaling pathways induc-es the growth differentiation and migration of smooth muscle cells IGF-1 is a commonly acti-vator of PI3K pathway [27 28] Compared with simple CSE exposure the phosphorylation lev-els of Akt mTOR GSK-3β p70S6K1 and 4EBP1 were increased significantly after IGF-1 and CSE treatment Moreover the mRNA and pro-tein level of MHC increased significantly when we used IGF-1 indicating the degree of muscle atrophy reduced Our study showed that acti-vating PI3KAKTmTOR signaling pathway inhib-ited skeletal muscle atrophy induced by CSE

In summary PI3KAKTmTOR signaling path-way is activated in chronic CS exposure rats Activating PI3KAKTmTOR signaling pathway inhibited skeletal muscle atrophy induced by CSE Selecting the activators of PI3KAKTmTOR signaling pathway is crucial for the devel-opment of new and individualized therapeutic strategies to prevent skeletal muscle atrophy in COPD patients Since studies on PI3KAKTmTOR signaling pathway targets are still fewer we need further study on these targets

Acknowledgements

This study was supported by Foundation of Shanghai Science and Technology Commission (No 14ZR1426800)

Disclosure of conflict of interest

None

Address correspondence to Fengfeng Han De- partment of Respiratory Medicine Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine 1665 Kong Jiang Road Shanghai 200092 China Tel 86-13816386372 Fax 86-21-65153984 E-mail fengfh86sinacom

References

[1] Vestbo J Hurd SS Agustiacute AG Jones PW Vogelmeier C Anzueto A Barnes PJ Fabbri LM Martinez FJ Nishimura M Stockley RA Sin DD Rodriguez-Roisin R Global strategy for the diagnosis management and prevention of chronic obstructive pulmonary disease GOLD executive summary Am J Respir Crit Care Med 2013 187 347-65

[2] Mathers CD Loncar D Projections of global mortality and burden of disease from 2002 to 2030 PLoS Med 2006 3 e442

[3] Kim HC Mofarrahi M Hussain SN Skeletal muscle dysfunction in patients with chronic ob-structive pulmonary disease Int J Chron Obstruct Pulmon Dis 2008 3 637-658

[4] Sandri M Signaling in muscle atrophy and hy-pertrophy Physiology (Bethesda) 2008 23 60-170

[5] Plant PJ Brooks D Faughnan M Bayley T Bain J Singer L Correa J Pearce D Binnie M Batt J Cellular markers muscle atrophy in chronic ob-structive pulmonary disease Am J Respir Cell Mol Biol 2010 42 461-471

[6] Lewis A Riddoch-Contreras J Natanek SA Donaldson A Man WD Moxham J Hopkinson NS Polkey MI Kemp PR Downregulation of the serum response factormiR-1 axis in the quadriceps of patients with COPD Thorax 2012 67 26-34

[7] Vogiatzis I Simoes DC Stratakos G Kourepini E Terzis G Manta P Athanasopoulos D Roussos C Wagner PD Zakynthinos S Effect of pulmonary rehabilitation on muscle remod-elling in cachectic patients with COPD Eur Respir J 2010 36 301-310

[8] Wuumlst RC Degens H Factors contributing to muscle wasting and dysfunction in COPD pa-tients Int J Chron Obstruct Pulmon Dis 2007 2 289-300

[9] Verhees KJ Pansters NA Schols AM Langen RC Regulation of skeletal muscle plasticity by glycogen synthase kinase-3β a potential tar-get for the treatment of muscle wasting Curr Pharm Des 2013 19 3276-3298

[10] Toker A Cantley LC Signalling through the lipid products of phosphoinositide-3-OH kinase Nature 1997 387 673-676

[11] Nojima H Tokunaga C Eguchi S Oshiro N Hidayat S Yoshino K Hara K Tanaka N Avruch J Yonezawa K The mammalian target of ra-pamycin (mTOR) partner raptor binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif J Biol Chem 2003 278 15461-15464

[12] Winter JN Jefferson LS Kimball SR ERK and Akt signaling pathways function through paral-lel mechanisms to promote mTORC1 signaling Am J Physiol Cell Physiol 2011 300 C1172-80

[13] Weigl LG Lost in translation regulation of skel-etal muscle protein synthesis Curr Opin Pharmacol 2012 12 377-82

[14] Schakman O Kalista S Barbeacute C Loumaye A Thissen JP Glucocorticoid-induced skeletal muscle atrophy Int J Biochem Cell Biol 2013 45 2163-72

[15] Shimizu N Yoshikawa N Ito N Maruyama T Suzuki Y Takeda S Nakae J Tagata Y Nishitani S Takehana K Sano M Fukuda K Suematsu M Morimoto C Tanaka H Crosstalk between

PI3KAKTmTOR protein synthesis pathway in the skeletal muscle atrophy

5687 Int J Clin Exp Med 20169(3)5677-5687

glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle Cell Metab 2011 13 170-82

[16] Salto R Viacutelchez JD Cabrera E Guinovart JJ Giroacuten MD Activation of ERK by sodium tung-state induces protein synthesis and prevents protein degradation in rat L6 myotubes FEBS Lett 2014 588 2246-54

[17] Liu Q Xu WG Luo Y Han FF Yao XH Yang TY Zhang Y Pi WF Guo XJ Cigarette smoke-in-duced skeletal muscle atrophy is associated with up-regulation of USP-19 via p38 and ERK MAPKs J Cell Biochem 2011 112 2307-16

[18] Man WD Kemp P Moxham J Polkey MI Skeletal muscle dysfunction in COPD Clinical and laboratory observations Clin Sci (Lond) 2009 117 251-64

[19] Kolsum U Roy K Starkey C Borrill Z Truman N Vestbo J Singh D The repeat ability of inter-leukin-6 tumornecrosis factor-alpha and C-reactive protein in COPD patients over one year Int J Chron Obstruct Pulmon Dis 2009 4 149-156

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