research article high fat diet-induced skeletal muscle...

Research ArticleHigh Fat Diet-Induced Skeletal Muscle Wasting Is Decreased byMesenchymal Stem Cells Administration Implications onOxidative Stress Ubiquitin Proteasome Pathway Activationand Myonuclear Apoptosis

Johanna Abrigo12 Juan Carlos Rivera12 Javier Aravena12 Daniel Cabrera34

Felipe Simon25 Fernando Ezquer6 Marcelo Ezquer6 and Claudio Cabello-Verrugio12

1Laboratorio de Biologia y Fisiopatologia Molecular Departamento de Ciencias BiologicasFacultad de Ciencias Biologicas y Facultad de Medicina Universidad Andres Bello 8370146 Santiago Chile2Millennium Institute on Immunology and Immunotherapy 8370146 Santiago Chile3Departamento de Ciencias Quimicas y Biologicas Facultad de Salud Universidad Bernardo O Higgins 8370993 Santiago Chile4Departamento de Gastroenterologıa Facultad de Medicina Pontificia Universidad Catolica de Chile 8330024 Santiago Chile5Laboratorio de Fisiologia Integrativa Departamento de Ciencias Biologicas Facultad de Ciencias Biologicas y Facultad de MedicinaUniversidad Andres Bello 8370146 Santiago Chile6Centro de Medicina Regenerativa Facultad de Medicina Clinica Alemana Universidad del Desarrollo 7710162 Santiago Chile

Correspondence should be addressed to Claudio Cabello-Verrugio claudiocabellounabcl

Received 17 March 2016 Accepted 14 June 2016

Academic Editor Saeid Golbidi

Copyright copy 2016 Johanna Abrigo et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Obesity can lead to skeletal muscle atrophy a pathological condition characterized by the loss of strength and muscle mass Afeature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation asevidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1 Additionally other mechanismsare related to muscle wasting including oxidative stress myonuclear apoptosis and autophagy Stem cells are an emerging therapyin the treatment of chronic diseases such as high fat diet-induced obesity Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals The presentstudy is the first to analyze the effects of systemicMSC administration on high fat diet-induced skeletal muscle atrophy in the tibialisanterior of mice Treatment with MSCs reduced losses of muscle strength and mass decreases of fiber diameter and myosin heavychain protein levels and fiber type transitions Underlying these antiatrophic effects MSC administration also decreased ubiquitinproteasome pathway activation oxidative stress and myonuclear apoptosis These results are the first to indicate that systemicallyadministered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes

1 Introduction

Skeletal muscle is the most abundant tissue in the humanbody and has a wide variety of physiological functionsThereforemuscle loss results not only in physical dysfunctionbut also in metabolic impairment [1 2] Related to thisrecent studies have shown that obesity is associated withskeletal muscle loss and dysfunction and the development

of muscle atrophy [3] Indeed mice fed with a high fat diet(HFD) develop the typical features of muscle wasting suchas weakness the loss of muscle mass and decreased fiberdiameter [4]

Skeletal muscle is composed of a variety of fast and slowfiber types and subtypes (ie slow I fast IIb IId and IIaor mixtures) [5] Moreover muscle fibers are versatile andcapable of changing phenotypic properties in response to

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 9047821 13 pageshttpdxdoiorg10115520169047821

2 Oxidative Medicine and Cellular Longevity

muscle wasting showing modifications in the expression ofMHC isoforms Therefore when fast muscles atrophy a fast-to-slow transition occurs (eg IIbrarrIIdrarrIIararrI) [5 6]

Ubiquitin proteasome pathway (UPP) overactivationoxidative stress and myonuclear apoptosis are some of themechanisms involved in muscle atrophy induced by obesityand other causes [4 7] Overactivation of the UPP in muscleatrophy is characterized by an increased expression of twomuscle-specific ubiquitin E3-ligase F-box proteins muscleatrophy F-box (MAFbx)atrogin-1 and muscle ring-fingerprotein 1 (MuRF-1) These proteins increase the ubiquitina-tion of targets such as the myosin heavy chain (MHC) whichare further degraded by the proteasome [8ndash11]

Oxidative stress is produced by increased reactive oxygenspecies (ROS) andor decreased antioxidant mechanisms[12] Oxidative stress has been associated with several modelsof muscle atrophy including that induced by obesity [313 14] Another mechanism involved in muscle atrophy ismyonuclear apoptosis which is related to muscle wastinginduced by cachexia and obesity [4] In atrophic muscleseveral parameters of myonuclear apoptosis increase such asthe BaxBcl2 ratio caspase-3 levels and activity and apoptoticnuclei [15]

Stem cell-based interventions act throughmultiplemech-anisms and provide a clear advantage when treating diseaseswith a complex pathophysiology such as HFD-inducedobesity This type of intervention is arguably better in ther-apeutic terms than single-agent drug-based treatments [1617] Multipotent mesenchymal stromal cells also referredto as mesenchymal stem cells (MSCs) are a heterogeneousadult stem cell population These are a potentially ideal toolfor stem cell-based interventions since they can be isolatedfrombonemarrow and othermesenchymal tissues includingadipose tissue dental pulp the placenta and the umbilicalcord and subsequently rapidly expanded ex vivo [18 19] Asimmunomodulatory cells MSCs can limit inflammation indamaged tissue [20] produce a broad range of trophic factorsthat protect parenchymal cells from apoptotic death andpromote the proliferation and differentiation of endogenousprecursors [21]

The present study is the first to provide evidence thatsystemic MSC administration can prevent the loss of mus-cle strength and mass the decrease in fiber diameter andMHC protein levels and the transition of fiber type in amurine model of HFD-induced muscle wasting Preventionof the several parameters of muscle atrophy among themUPP overactivation increased ROS levels and activation ofmyonuclear apoptosis was also found in this model whichwould explain the observed antiatrophic effects of MSCadministration

2 Materials and Methods

21 Animals Male 12-week-old C57BL10 mice were housedat a constant temperature (22 plusmn 2∘C) with 60 relativehumidity and with a 12 12 light dark cycle Mice had adlibitum access to food and autoclaved water Mice were fedwith either a standard diet (control group 10 cal fat 20 cal

proteins and 70 cal carbohydrates Champion SA Chile)or a high fat diet (HFD group 60 cal fat 20 cal proteinsand 20 cal carbohydrates D12492 Research Diets IncUSA) After 30 weeks of HFD feeding mice were separatedinto two groups matched by average body weight Thenfor an additional eight weeks one group received a vehicletreatment (HFD) while the other was treated with MSCs(HFD + MSCs) At the end of the 38-week experimentalperiod the mice were euthanized under anesthesia and thetibialis anterior (TA) muscles were dissected removed andrapidly frozen and stored at minus80∘C until processing Allprotocols were conducted in strict accordance with and withthe formal approval of the Animal Ethics Committees ofUniversidad Andres Bello and Universidad del Desarrollo

22 Isolation and Administration of MSCs Six-to-eight-week-old C57BL10 male mice were sacrificed by cervicaldislocation Bone marrow cells were obtained by flushing thefemurs and tibias with sterile PBS After centrifugation thecells were resuspended in an 120572-Minimum Essential Medium(Gibco USA) supplemented with 10 selected fetal bovineserum (Hyclone USA) and 80 120583gmL gentamicin (Labora-torio Sanderson Chile) and plated at a density of 1 times 106nucleated cells per square centimeter Nonadherent cells wereremoved after 72 h by changing the medium When the focireached confluence adherent cells were detached with 025trypsin and 265mM EDTA centrifuged and subculturedat 7000 cells per square centimeter After two subculturesadherent cells were phenotypified and characterized accord-ing to the adipogenic and osteogenic differentiation potentialas previously described [6] Then 05 times 106MSCs wereresuspended in 02mL of 5 mouse plasma (vehicle) andadministered via the tail-vein to anesthetizedmice Untreatedmice were given 02mL of vehicle

23 Weightlifting Strength Test At the end of the treatmentthe muscle strength of the mice was measured through aweightlifting test as previously described [22] Briefly theapparatus consisted in a series of increasingly long chain linksattached to a ball of tangled fine wire The number of linksranged from two to seven with total weights between 155 and541 g Before performing the test and prior to treatments themice were trained once per day for twoweeks To perform thetest themouse grasped the different weights with its forepawsand a score was assigned The final score was calculated asthe summation of the product between the link weight andthe time the weight was held The average of three measuresfrom each mouse was normalized against body weight [23]

24 Contractile Properties The TA muscles removed fromsacrificed mice were placed in a dish containing oxygenatedKrebs-Ringer solution (in mmolL NaCl 118 NaHCO

3 25

D-glucose 20 KCl 47 CaCl2 32 KH

2PO4 12 MgSO

4 12)

The TA muscles were firmly tied with surgical silk at thebottom end of the tendon and at a portion of the kneebone The muscle was transferred to a custom-built Plexiglasbath filled with oxygenated Krebs-Ringer solution that wasthermostaticallymaintained at room temperature for optimal

Oxidative Medicine and Cellular Longevity 3

oxygen diffusion The muscles were vertically aligned andtied directly between a fixed hook and a force transducer(MLT 1030D AD Instruments USA) Two platinum plateelectrodes were positioned in the organ bath so as to flankthe length of the muscles Muscles were field-stimulated bysupramaximal square wave pulses (S48 Stimulator GrassUSA) which were amplified to increase and sustain currentintensity at a level sufficient for producing a maximumisometric tetanic contraction Optimum muscle length (Lo)and stimulation voltage were determined from the microma-nipulation of muscle length to produce maximum isometrictwitch force All stimulation parameters and contractileresponses were controlled and measured using Power Lab435 (AD Instruments USA) All obtained data were com-puted with the LabChart analysis software (AD InstrumentsUSA)Maximum isometric tetanic force (Po)was determinedfrom the plateau of the frequency-force relationship aftersuccessive stimulations between 10 and 150Hz for 850mswith 2min rest periods between the stimuli After testing themuscles were removed from the bath and the tendons andany adherent nonmuscle tissues were trimmed blotted onceon filter paper and weighed Muscle mass and Lo were usedto calculate specific net force or the normalized force of eachtotal muscle fiber cross-sectional area (mNmm2) [24ndash27]

25 Blood Triglycerides Cholesterol Glucose and InsulinDetermination After 4 h of fasting mice were sacrificedand blood samples were collected Serum triglycerides andcholesterol levels were determined in the ARCHITECTc8000 Clinical Chemistry Analyzer (Abbott USA) Bloodglucose levels were measured with the Accu-Chek Perfor-mance glucometer system (Roche Diagnostic Germany)Plasma insulin levels were assayed using the UltrasensitiveMouse-Insulin ELISA Kit (Mercodia Sweden)

26 RNA Isolation Reverse Transcription and QuantitativeReal-Time PCR Total RNAwas isolated from the TAmusclesusing TRIzol (Invitrogen USA) The total RNA (1 120583g) wasreverse transcribed to cDNA using random hexamers andSuperscript II reverse transcriptase (Invitrogen USA) Taq-Man quantitative real-time PCR reactions were performedin triplicate using an Eco Real-Time PCR System (IlluminaUSA) with predesigned primer sets for mouse atrogin-1MuRF-1 and the housekeeping gene 18S (TaqManAssays-on-Demand Applied Biosystems USA) The mRNA expressionwas quantified using the comparative ΔCt method (2minusΔΔCt)with 18S as the reference gene The mRNA levels wereexpressed relative to the mean expression in the vehicle-treated mice [23 28]

27 Western Blot Analysis The muscles were homogenizedin Tris-EDTA buffer with a cocktail of protease inhibitorsand 1mM phenylmethanesulfonyl fluoride Proteins weresubjected to SDS-PAGE transferred onto polyvinylidenedifluoride membranes (Millipore USA) and probed withmouse anti-MHC (1 1000) (MF-20 Developmental Studies

Hybridoma Bank University of Iowa USA) mouse anti-tubulin (1 5000)mouse anti-GAPDH(1 5000) rabbit anti-Bax (1 500) rabbit anti-Bcl2 (1 500) rabbit anti-caspase-3(1 500) rabbit anti-Ubproteins (1 500) (SantaCruzBiotech-nology USA) rabbit anti-LC3B (1 1500) (Cell SignalingUSA) and rabbit anti-p62 (1 1500) (Abcam USA) Allimmunoreactions were visualized by enhanced chemilumi-nescence (Thermo Scientific USA) Images were acquiredusing Fotodyne FOTOAnalyst Luminary Workstation Sys-tems (Fotodyne Inc USA)

28 Muscle Histology and Fiber Diameter Determination andQuantification Fresh-frozen TA muscles were sectionedand cryosections (8 120583m) were stained with hematoxylin andeosin (HampE) or Alexa-Fluor 594 tagged wheat germ agglu-tinin (WGA) (Life Technologies USA) according to stan-dard procedures Wheat germ agglutinin-stained fibers wereassessed through blind analysis using the Image J software(NIH USA) with fiber sizes determined for seven randomlycaptured images from each experimental condition Fiberswere handled manually and the minimal Feret diameter ofeach fiber was computed by the software [28 29]

29 Immunohistochemical Analysis For immunohistochem-istry fresh-frozen TA muscle cryosections (8120583m) were fixedin acetone and incubated overnight in 1 bovine serumalbumin in PBS with anti-MHC IIa (clone SC-71-s 1 20) andanti-MHC IIb (clone BF-F3-s 1 20) (Developmental StudiesHybridoma Bank University of Iowa USA) Then cryosec-tions were blocked for 15min in 3 methanol-H

2O2and

incubated for 30min with the Envision Dual Link System-HRP (Dako USA) Enzyme activity was detected with a 3101584031015840-diaminobenzidine tetrahydrochloride liquid system (DakoUSA) Nuclei were stained with hematoxylin [26 30]

210 ROS Detection For ROS detection fresh-frozen TAmuscle cryosections (8120583m) were incubated for 30min at37∘C with 10 120583M CM-H

2DCF-DA dye (Molecular Probes

Eugene Oregon USA) in Hankrsquos buffered salt solutionThenthe muscles were fixed in 4 paraformaldehyde in PBS for10min at room temperature The nuclei were stained withHoechst 33258 (1 5000) in PBS After rinsing the muscleswere mounted with a fluorescent mounting medium (DakoUSA) under a glass slide and viewed and photographed usingthe Motic BA310 epifluorescence microscope [31]

211 Terminal Deoxynucleotidyl Transferase Mediated dUTPNick End Labelling (TUNEL) Technique Breaks in the DNAstrand as a result of endonuclease activity were detected bythe TUNEL labelling technique using the commercial Dead-EndColorimetric TUNELKit (Promega USA)The label wasdeveloped with a DAB 331015840-diaminobenzidine horseradishperoxidase substrate that produces a dark-brown reactionproduct A blinded field quantification of TUNEL-positivenuclei was then performed on five randomly captured imagesof the TA taken from four mice and for each experimentalcondition [15]


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Figure 1 Mesenchymal stem cells (MSCs) administration inhibits the decreased muscle strength induced by a high fat diet (HFD) in miceC57BL10J male mice were fed with a standard chow (control) or HFD for 38 weeks At week 30 a subgroup of HFD mice received MSCinjected through the tail-vein At week 38 all mice were subjected to the following (a) A weightlifting test to determine limbmuscle strengthvalues represent the percentage of muscle strength reached by the mice with each weight and correspond to the mean plusmn SD (119899 = 8 lowast119875 lt 005versus control 119875 lt 005 versus HFD two-way ANOVA) (b) A weightlifting test the values represent the scores normalized by body weightThe values correspond to the mean plusmn SD (119899 = 8 lowast119875 lt 005 versus control 119875 lt 005 versus HFD two-way ANOVA) (c) Maximal isometricstrengths (mNmm2) against stimulation frequencies (Hz) in the tibialis anterior (TA) muscles values represent the mean plusmn SD (119899 = 8lowast

119875 lt 005 versus HFD two-way ANOVA)

212 Caspase-3 Activity The activity of caspase-3 was deter-mined in the TA protein extract using the commercialCaspase-3 Colorimetric Assay Kit (BioVision Inc USA) A405 nm absorbance value for each experimental conditionwas expressed as a fold of induction relative to control muscle[15]

213 Statistics For statistical analysis two-way analysis of var-iance (ANOVA) was used with a post hocmultiple-comparison

Bonferroni test (Prisma) Differences were considered statis-tically significant at 119875 lt 005

3 Results

31 Systemic Administration of Mesenchymal Stem CellsReverts the Decreased Muscle Strength Induced by a High FatDiet After receiving aHFD for 38weeks livemice evidenceddecreased muscle strength as evaluated by a weightlifting


Control HFD HFD + MSC

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Figure 2 Systemic mesenchymal stem cells (MSCs) administration inhibits fiber type transitions in the tibialis anterior (TA) muscle ofmice fed with a high fat diet (HFD) C57BL10J male mice were fed with a standard chow (control) or HFD for 38 weeks At week 30a subgroup of HFD mice received MSC injected through the tail-vein At week 38 all mice were sacrificed and the TA was analyzed todetermine fiber type through the immunohistochemical detection of myosin heavy chain isoforms (IIa and IIb) Images obtained at 10x (a)and 40x (b) magnification show fiber types IIa (upper panel) and IIb (lower panel) Quantitative analysis of the fiber type is shown in (c)Graph representing the percentage of specific fiber types relative to the total fibers counted per field Values represent the mean plusmn SD (119899 = 8lowast

119875 lt 005 versus control 119875 lt 005 versus HFD two-way ANOVA)



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Figure 3 Mesenchymal stem cells (MSCs) administration prevents the decreased fiber diameter of the tibialis anterior (TA) muscle in highfat diet- (HFD-) induced skeletal muscle atrophy TA muscles were obtained from C57BL10J male mice fed with standard chow (control) aHFD or a HFD-MSC treated Muscle cross sections were stained with wheat germ agglutinin to delimit muscle fiber sarcolemma (a) Imageswere obtained at 10x and 40x magnification Bars correspond to 150120583m (b) Minimal Feret diameters were determined in TA cross sectionsfrom (b) Fiber diameters were grouped and ranged from 0 to 70120583m Values are expressed as a percentage of the total quantified fibers Valuescorrespond to the mean plusmn SD (119899 = 8 lowast119875 lt 005 versus control two-way ANOVA)

assay (Figure 1(a)) However HFD + MSC mice showeda partial recovery of muscle strength after MSC treatment(Figures 1(a) and 1(b))

Muscle strength was also measured through electro-physiological assays of tetanic force in isolated TA musclesWhile the muscle force of HFD mice decreased in all of theassessed frequency ranges (Figure 1(c)) muscle strength was

recovered albeit not completely following MSC treatmentThis recovery reached approximately 50 for frequenciesfrom 40 to 150Hz and was without differences in lowerfrequencies

These results suggest that MSC administration can pre-vent the decreased muscle strength induced by a HFD inmice


HFD + MSCControl(kDa)

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Figure 4 Systemic mesenchymal stem cells (MSCs) administration prevents the decreased myosin heavy chain (MHC) levels in the tibialisanterior (TA) muscle of mice fed with a high fat diet (HFD) C57BL10J male mice were fed with a standard chow (control) or HFD for 38weeks At week 30 a subgroup of HFD mice received MSC injected through the tail-vein After eight weeks all mice were sacrificed and theTA was excised and homogenized to evaluate the following (a) MHC protein levels throughWestern blot analysis (GAPDH levels were usedas the loading control molecular weight markers are shown in kDa) and (b) quantitative analysis of the experiments from (a) The levels ofMHC normalized to GAPDH are expressed relative to control mice (lowast119875 lt 005 versus control 119875 lt 005 versus HFD)

32 Mesenchymal Stem Cells Prevent Fiber Type TransitionDecreased Fiber Diameter and theMyosin Heavy Chain LevelsInduced by a High Fat Diet Themost abundant fiber type inthe TA is IIb When the TA is atrophied IIb fibers transitiontowards IIa fibers [5 6] This was observed in the presentstudy where a HFD produced a shift to IIa fibers in affectedmice increasing the quantity of IIa fibers from 43 to 245and decreasing the quantity of IIb fibers from 928 to 694(Figures 2(a) and 2(b)) This transition was prevented in theHFD+MSC group which showed a similar proportion of IIbfibers as the control group (Figures 2(a) and 2(b))The graphsin Figure 2(c) show the quantitative analysis of Figures 2(a)and 2(b)

The architecture of theTAmuscleswas conservedwithoutevident changes and without foci of necrotic or damagedfibers (Supplemental Figure S1) (see Supplementary Materialavailable online at httpdxdoiorg10115520169047821)However when fiber size was evaluated (Figure 3(a)) theHFD mice showed a notable displacement towards smallersized fibers a situation that was reversed to the normal valuesin the HFD + MSC group (Figures 3(a) and 3(b))

A characteristic feature of muscle atrophy is a decreasein the levels of the myofibrillar proteins such as MHC In linewith this theHFDdecreasedMHCprotein levels in the TA toonly 40 of levels in control TA In contrast in HFD +MSCmice the administration of MSC partially prevented loweredMHC levels which reached 82 of the levels presented incontrol TA (Figures 4(a) and 4(b))

33 Mesenchymal Stem Cell Administration Reverts UbiquitinProteasome Pathway Overactivation the Increases in Reactive

Oxygen Species and the Myonuclear Apoptosis Induced by aHigh Fat Diet Increased UPP activity was demonstrated byhigh levels of ubiquitinated proteins in the TA of HFD mice(Figures 5(a) and 5(b)) In contrast HFD + MSC mice wereable to decrease protein ubiquitination reachingUPP activitylevels similar to the control group To corroborate this resultatrogin-1 andMuRF-1 expression which increase under mostmuscle atrophy conditions were also evaluated A HFDinduced increased atrogin-1 and MuRF-1 gene expression(Figures 5(c) and 5(d)) However MSC treatment in HFD +MSC mice completely prevented increased atrogin-1 expres-sion and partially prevented increased MuRF-1 expressionin the TA (Figures 5(c) and 5(d)) When autophagy wasevaluated the levels and processing of LC3B and p62 didnot show differences between the controls HFD and HFD+ MSC mice (Supplemental Figures S2A B and C)

The levels of ROS were also evaluated in the TA of HFDmice For this a DCF probe was used to detect (Figure 6(a))and quantify (Figure 6(b)) ROS HFD increased ROS levels722-fold relative to the control while MSC administrationpartially prevented this increase (383-fold relative to thecontrol)

Another mechanism recently described in relation toHFD-induced muscle wasting is myonuclear apoptosis [4]Proapoptotic Bax levels and antiapoptotic Bcl2 levels weremeasured (Figure 7(a)) The BaxBcl2 ratio increased inthe TA of HFD mice as compared to the control group(Figure 7(b)) However HFD + MSC mice presented adecreased BaxBcl2 ratio in the TA as compared to the HFDgroup Additionally HFD-induced caspase-3 protein levels


HFD + MSCControl

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Figure 5 Mesenchymal stem cells (MSCs) administration inhibits increases in ubiquitinated proteins and atrogin-1 and MuRF-1 geneexpression levels induced by a high fat diet (HFD) in mice C57BL10J male mice were fed with a standard chow (control) or HFD for 38weeks At week 30 a subgroup of HFDmice receivedMSC injected through the tail-vein At week 38 all mice were sacrificed and the TA wasexcised and homogenized to evaluate the following (a) total ubiquitinated (Ub) protein levels through Western blot analysis (tubulin levelswere used as the loading control molecular weight markers are shown in kDa) and (b) quantitative analysis of the experiments from (a) Thelevels of Ub normalized to tubulin are expressed relative to control mice (lowast119875 lt 005 versus control 119875 lt 005 versus HFD) Detection ofatrogin-1 (c) and MuRF-1 (d) mRNA levels through RT-qPCR using 18S as the reference gene Expressions are shown as the fold of inductionrelative to the TA from control mice and the values correspond to the mean plusmn SD (lowast119875 lt 005 versus control 119875 lt 005 versus HFD)

(Figure 7(c)) were decreased by MSC administration (Fig-ure 7(d)) Similarly MSC injection decreased HFD-inducedcaspase-3 activity in the TA (Figure 7(e)) Finally apoptoticnuclei were evaluated by TUNEL analysis (Figure 7(f))The results revealed that a HFD increased the number ofapoptotic nuclei in the TA (1654 relative to the control)while MSC injection fully prevented this increase in HFD +MSC mice (Figure 7(g)) Together these results indicate thatMSC administration decreases myonuclear apoptosis in theTA of HFD mice

4 Discussion

It is well reported that HFD impairs muscle function andspecifically produces muscle atrophy [32ndash34] HFD inducesskeletal muscle atrophy through the induction of severalmechanisms such as UPP overactivation increased oxidativestress and the generation of myonuclear apoptosis [4 7]In this context the present study corroborated that HFDcan produce phenotypical changes in skeletal muscle manyof which also occur in muscle atrophy caused by other



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Figure 6 Systemic mesenchymal stem cells (MSCs) administration prevents increased reactive oxygen species (ROS) levels in the tibialisanterior (TA) of mice fed with a high fat diet (HFD) C57BL10J male mice were fed with a standard chow (control) or HFD for 38 weeks Atweek 30 a subgroup of HFDmice receivedMSC injected through the tail-vein At week 38 all mice were sacrificed (a) Cryosections obtainedfrom the TAwere incubated with a DCF probe for ROS detection through fluorescencemicroscopy Nuclei were labelled via Hoechst staining(b) Quantification of ROS levels from experiments showed in (a)The values are expressed as the fold of induction of the DCF probe intensityValues correspond to the mean plusmn SD (119899 = 8 lowast119875 lt 005 versus control 119875 lt 005 versus HFD two-way ANOVA)

stimuli such as by immobilization sepsis or cachexia Thesechanges include decreased muscle strength and fiber sizefiber type transitions (eg IIbrarrIIa) the downregulation ofMHC levels the upregulation of atrogenes such as atrogin-1and MuRF-1 and increased oxidative stress and myonuclearapoptosis Interestingly all of these features of HFD-inducedmuscle wasting were prevented when MSC was systemicallyadministrated to mice

One feature of obesity is an increase of some circulatingfactors that can affect muscle function and structure pro-ducing muscle atrophy These factors include angiotensin IIand TNF-120572 [7 35 36] Thus the next step in our modelof study is to know if the MSC administration alters thecirculating and tissue levels of angiotensin II and TNF-120572The mechanisms through which angiotensin II and TNF-120572 induce muscle wasting include increased oxidative stressUPP overactivation and myonuclear apoptosis [7 37ndash39]These studies are in line with the present results whichshow that MSC treatment prevented oxidative stress UPPoveractivation and myonuclear apoptosis Building on thisresult it is important that future studies elucidate the possibleparticipation of the angiotensin II and TNF-120572 inducedpathways in the improvements observed when MSC is givento obese mice

Despite the fact that one of the effects observed by MSCtreatment is the decrease of myonuclear apoptosis the celltype responsible for or sensible to this effect is not studiedWe cannot rule out the possibility that apoptotic signalingmay have occurred by a combination of muscle (fiber orsatellite cells) and nonmuscle cells (eg endothelial cellsand resident fibroblast) that reside inside muscles in vivoMoreover we cannot be sure that apoptotic myonuclei are

intramuscular or associated with satellite cells Howeverconsidering that the main and more abundant cell type inskeletal muscle is the muscle fibers and the evidences thatdescribe the fibers as main source of apoptotic myonuclei inmodels of muscle atrophy by unloading [40] then we canspeculate that at least this cell type significantly contributesto the changes in apoptotic signaling induced by high fatdiet (HFD) and therefore it is sensitive to MSC treatmentThis suggestion is supported by other studies that showthe muscle fiber as the main cell source of the myonuclearapoptosis induced by HFD [4 41] Despite these evidenceswe cannot discard that part of the atrophic effect observedunder HFD can be attributed to another cell type specificallyto satellite cells whose dysfunction has been reported inatrophied muscles [42 43] opening another cell target tothe effect of MSC administration Thus further studies suchas specific immunolocalization of apoptotic marker must beperformed to detect and clarify the cell types responsible forthe myonuclear apoptosis in the skeletal muscle under HFDevaluating in addition the effect of MSC treatment

The use of stem cell therapy to improve muscle dis-orders has been previously evaluated in models of muscleregeneration or dystrophy mainly using the graft strategy[44 45] However the efficiency of these procedures has beenpoor In turn the present results indicate that systemic MSCadministration improves skeletal muscle atrophy associatedwith a HFD Importantly the effects of MSC administrationwere not related to obesity reversion since HFD miceremained obese hypercholesterolemic hyperglycemic andhyperinsulinemic (Supplemental Table S1)

Also worth noting the MSCs incorporated into themuscle tissue of HFDmice were undetected in posttreatment


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lowast


(g)

Figure 7 Mesenchymal stem cells (MSCs) administration inhibits the increased myonuclear apoptosis induced by a high fat diet (HFD) inmice C57BL10J male mice were fed with a standard chow (control) or HFD for 38 weeks At week 30 a subgroup of HFDmice receivedMSCinjected through the tail-vein At week 38 all mice were sacrificed and the tibialis anterior (TA) was excised and homogenized to evaluate(a) Bax and Bcl2 protein levels through Western blot analysis (b) BaxBcl2 ratio analysis (the values are expressed as fold of inductionrelative to the control and correspond to the mean plusmn SD (119899 = 8 lowast119875 lt 005 versus control 119875 lt 005 versus HFD two-way ANOVA)) (c)cleaved caspase-3 levels detected by Western blot analysis (d) quantification of caspase-3 levels from experiments shown in (c) The valuesare expressed as fold of induction relative to the control and correspond to the mean plusmn SD (119899 = 8 lowast119875 lt 005 versus control 119875 lt 005 versusHFD two-way ANOVA) For (a) and (c) the levels of tubulin are shown as the loading control The molecular weights are shown in kDa(e) Caspase-3 activity was measured and expressed as fold of induction relative to the control with values corresponding to the mean plusmn SD(119899 = 8 lowast119875 lt 005 versus control 119875 lt 005 versus HFD two-way ANOVA) (f) Cryosections of TA were used to perform the TUNEL assayThe bar corresponds to 50 120583m (g) Blinded quantification of TUNEL-positive nuclei per field in five randomly captured images The valuesare expressed as the fold of induction relative to the control and correspond to the mean plusmn SD (119899 = 8 lowast119875 lt 005 versus control 119875 lt 005versus HFD two-way ANOVA)


analyses (data not shown) Diverse studies have shown thatlt1 of systemically administered MSCs remain present oneweek after administration in any organ including the lungsheart kidneys liver spleen and gut [16 46 47] Howeverthe clinical benefits associated with MSC administration canbe observed for a much longer period Considering this itis possible to speculate that the impaired muscle functionand features of muscle atrophy were prevented by someindirect event dependent on the MSCs For example MSCssecrete a broad range of bioactive growth factors such asVEGF bFGF IGFHGF and EGF [21]ThereforeMSCs couldprovide trophic support for injured tissue by modifying themicroenvironment thus inducing local precursor prolifera-tion and differentiation to improve damaged tissue irrigationand prevent parenchymal cell apoptosis [17 21] Consideringthis the impaired muscle function and features of muscleatrophy could be prevented by some indirect or paracrineevent dependent on MSCs as has been previously found inrelation to the secretion of trophic factors [21 48] If this is themechanism by which the effects of MSCs occur it is possiblethat MSCs increase the secretion of trophic factors such asEGF and bFGF [21] In skeletal muscle EGF and bFGF alsostimulate growth and prevent muscle atrophy [49 50]

Through MSC administration the muscle function af-fected by sepsis-induced skeletalmuscle wastingwas partiallyrecovered mainly through improving the function of satellitecells the primary cell population responsible for repairingskeletal muscle [51] Considering differences in MSC admin-istration between Gao et al [46] and the present studyit is possible that in the present model of HFD-inducedmuscle wasting MSC treatment could induce the secretionof soluble factors that finally improve muscle strength Thispossible mechanism could be mediated by an effect onsatellite cells or another cell population that participates inmuscle repair One such population is a muscle-residentpopulation of nonsatellite progenitor cells that are termedbipotent fibroadipogenic progenitors and are located in themuscle interstitium and neighbor muscle-associated bloodvessels These cells could contribute to preventing muscleatrophy by secreting paracrine factors such as IL-6 IGF-1 andWnt1 [52 53]

Alternatively MSC administration produces an anti-inflammatory response in several models and pathologiesSystemic inflammation can increase during obesity resultingin increased proinflammatory cytokines such as IL-1120573 andTNF-120572 [54] which induce muscle wasting [55] Thereforeit is possible that the presently obtained results are dueto decreased TNF-120572 plasma levels which in turn coulddecrease the atrophic effect on skeletal muscle

5 Conclusions

The present study is the first to demonstrate that systemicallyadministeredMSCs could prevent themuscle wasting associ-ated with HFD-induced obesity by preventing the decreasedMHC levels increased UPP activity increased myonuclearapoptosis and oxidative stress

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Johanna Abrigo and Juan Carlos Rivera contributed equallyto the present work

Acknowledgments

This study was supported by research grants from theAssociation-Francaise Contre Les Myopathies AFM 16670(Claudio Cabello-Verrugio) FONDECYT 1161646 (ClaudioCabello-Verrugio) 1150589 (Marcelo Ezquer) 1161288 (FelipeSimon) and 3140396 (Daniel Cabrera) the MillenniumInstitute on Immunology and Immunotherapy P09-016-F(Claudio Cabello-Verrugio and Felipe Simon) and UNABDI-741-15N (Claudio Cabello-Verrugio and Felipe Simon)Johanna Abrigo and Juan Carlos Rivera thank CONICYT fora PhD Scholarship 21161353 and 21141242

References

[1] R T Jagoe S H Lecker M Gomes and A L GoldbergldquoPatterns of gene expression in atrophying skeletal musclesresponse to food deprivationrdquo The FASEB Journal vol 16 no13 pp 1697ndash1712 2002

[2] J L Bailey ldquoInsulin resistance and muscle metabolism inchronic kidney diseaserdquo ISRN Endocrinology vol 2013 ArticleID 329606 14 pages 2013

[3] B A Bhatt J J Dube N Dedousis J A Reider and R MOrsquoDoherty ldquoDiet-induced obesity and acute hyperlipidemiareduce I120581B120572 levels in rat skeletal muscle in a fiber-type depen-dentmannerrdquoAmerican Journal of PhysiologymdashRegulatory Inte-grative and Comparative Physiology vol 290 no 1 pp R233ndashR240 2006

[4] B Sishi B Loos B Ellis W Smith E F Du Toit and A-MEngelbrecht ldquoDiet-induced obesity alters signalling pathwaysand induces atrophy and apoptosis in skeletal muscle in aprediabetic rat modelrdquo Experimental Physiology vol 96 no 2pp 179ndash193 2011

[5] D Pette and R S Staron ldquoMyosin isoforms muscle fiber typesand transitionsrdquoMicroscopy Research and Technique vol 50 no6 pp 500ndash509 2000

[6] K Higashino T Matsuura K Suganuma K Yukata TNishisho and N Yasui ldquoEarly changes in muscle atrophy andmuscle fiber type conversion after spinal cord transection andperipheral nerve transection in ratsrdquo Journal of NeuroEngineer-ing and Rehabilitation vol 10 no 1 article 46 2013

[7] C Cabello-Verrugio M G Morales J C Rivera D Cabreraand F Simon ldquoRenin-angiotensin system an old player withnovel functions in skeletal musclerdquoMedicinal Research Reviewsvol 35 no 3 pp 437ndash463 2015

[8] S C Bodine E Latres S Baumhueter et al ldquoIdentification ofubiquitin ligases required for skeletal muscle atrophyrdquo Sciencevol 294 no 5547 pp 1704ndash1708 2001

[9] B A Clarke D DrujanM SWillis et al ldquoThe E3 ligaseMuRF1degrades myosin heavy chain protein in dexamethasone-treated skeletal musclerdquo Cell Metabolism vol 6 no 5 pp 376ndash385 2007


[10] M J Eddins J G Marblestone K G Suresh Kumar et alldquoTargeting the ubiquitin E3 ligase MuRF1 to inhibit muscleatrophyrdquo Cell Biochemistry and Biophysics vol 60 no 1-2 pp113ndash118 2011

[11] V C Foletta L JWhite A E Larsen B Leger andA P RussellldquoThe role and regulation of MAFbxatrogin-1 and MuRF1 inskeletal muscle atrophyrdquo Pflugers Archiv European Journal ofPhysiology vol 461 no 3 pp 325ndash335 2011

[12] P G Arthur M D Grounds and T Shavlakadze ldquoOxidativestress as a therapeutic target duringmusclewasting consideringthe complex interactionsrdquoCurrent Opinion in Clinical Nutritionand Metabolic Care vol 11 no 4 pp 408ndash416 2008

[13] D E Kelley J He E V Menshikova and V B Ritov ldquoDys-function of mitochondria in human skeletal muscle in type 2diabetesrdquo Diabetes vol 51 no 10 pp 2944ndash2950 2002

[14] C S Stump E J Henriksen Y Wei and J R SowersldquoThe metabolic syndrome role of skeletal muscle metabolismrdquoAnnals of Medicine vol 38 no 6 pp 389ndash402 2006

[15] CMeneses M GMorales J Abrigo F Simon E Brandan andC Cabello-Verrugio ldquoThe angiotensin-(1ndash7)Mas axis reducesmyonuclear apoptosis during recovery from angiotensin II-induced skeletal muscle atrophy in micerdquo Pflugers Archiv vol467 pp 1975ndash1984 2015

[16] S Schrepfer T Deuse H ReichenspurnerM P Fischbein R CRobbins andM P Pelletier ldquoStem cell transplantation the lungbarrierrdquo Transplantation Proceedings vol 39 no 2 pp 573ndash5762007

[17] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferenti-ation andmodes of tissue repairmdashcurrent viewsrdquo STEMCELLSvol 25 no 11 pp 2896ndash2902 2007

[18] R J Deans andA BMoseley ldquoMesenchymal stem cells biologyand potential clinical usesrdquo Experimental Hematology vol 28no 8 pp 875ndash884 2000

[19] M JHoogduijnMGH Betjes andCC Baan ldquoMesenchymalstromal cells for organ transplantation different sources andunique characteristicsrdquo Current Opinion in Organ Transplan-tation vol 19 no 1 pp 41ndash46 2014

[20] I Rasmusson ldquoImmune modulation by mesenchymal stemcellsrdquo Experimental Cell Research vol 312 no 12 pp 2169ndash21792006

[21] A I Caplan and J E Dennis ldquoMesenchymal stem cells astrophic mediatorsrdquo Journal of Cellular Biochemistry vol 98 no5 pp 1076ndash1084 2006

[22] R M Deacon ldquoMeasuring the strength of micerdquo Journal ofVisualized Experiments no 76 article e2610 2013

[23] M G Morales H Olguın G Di Capua E Brandan FSimon and C Cabello-Verrugio ldquoEndotoxin-induced skeletalmuscle wasting is prevented by angiotensin-(1ndash7) through a p38MAPK-dependent mechanismrdquo Clinical Science vol 129 no 6pp 461ndash476 2016

[24] C Cabello-Verrugio M G Morales D Cabrera C P Vio andE Brandan ldquoAngiotensin II receptor type 1 blockade decreasesCTGFCCN2-mediated damage and fibrosis in normal anddystrophic skeletal musclesrdquo Journal of Cellular and MolecularMedicine vol 16 no 4 pp 752ndash764 2012

[25] M G Morales D Cabrera C Cespedes et al ldquoInhibition ofthe angiotensin-converting enzyme decreases skeletal musclefibrosis in dystrophic mice by a diminution in the expressionand activity of connective tissue growth factor (CTGFCCN-2)rdquo Cell and Tissue Research vol 353 no 1 pp 173ndash187 2013

[26] M G Morales C Cabello-Verrugio C Santander D CabreraR Goldschmeding and E Brandan ldquoCTGFCCN-2 over-expression can directly induce features of skeletal muscledystrophyrdquo The Journal of Pathology vol 225 no 4 pp 490ndash501 2011

[27] P Gregorevic D R Plant K S Leeding L A Bach and G SLynch ldquoImproved contractile function of the mdx dystrophicmouse diaphragm muscle after insulin-like growth factor-IadministrationrdquoTheAmerican Journal of Pathology vol 161 no6 pp 2263ndash2272 2002

[28] F Cisternas M G Morales C Meneses et al ldquoAngiotensin-(1ndash7) decreases skeletal muscle atrophy induced by angiotensinII through a Mas receptor-dependent mechanismrdquo ClinicalScience vol 128 no 5 pp 307ndash319 2015

[29] CMeneses M GMorales J Abrigo F Simon E Brandan andC Cabello-Verrugio ldquoThe angiotensin-(1ndash7)Mas axis reducesmyonuclear apoptosis during recovery from angiotensin II-induced skeletal muscle atrophy in micerdquo Pflugers ArchivmdashEuropean Journal of Physiology vol 467 no 9 pp 1975ndash19842015

[30] M G Morales J Abrigo C Meneses F Cisternas F Simonand C Cabello-Verrugio ldquoExpression of the Mas receptor isupregulated in skeletal muscle wastingrdquoHistochemistry and CellBiology vol 143 no 2 pp 131ndash141 2014

[31] J Abrigo J C Rivera F Simon D Cabrera and C Cabello-Verrugio ldquoTransforming growth factor type beta (TGF-120573)requires reactive oxygen species to induce skeletal muscleatrophyrdquo Cellular Signalling vol 28 no 5 pp 366ndash376 2016

[32] N H Le C-S Kim T Park et al ldquoQuercetin protects againstobesity-induced skeletal muscle inflammation and atrophyrdquoMediators of Inflammation vol 2014 Article ID 834294 10pages 2014

[33] S-R Lee A V Khamoui E Jo et al ldquoEffects of chronic high-fat feeding on skeletalmusclemass and function inmiddle-agedmicerdquo Aging Clinical and Experimental Research vol 27 no 4pp 403ndash411 2015

[34] Y Shpilberg J L Beaudry A DrsquoSouza J E Campbell APeckett and M C Riddell ldquoA rodent model of rapid-onsetdiabetes induced by glucocorticoids and high-fat feedingrdquoDisease Models andMechanisms vol 5 no 5 pp 671ndash680 2012

[35] I Peluso and M Palmery ldquoThe relationship between bodyweight and inflammation lesson from anti-TNF-120572 antibodytherapyrdquo Human Immunology vol 77 no 1 pp 47ndash53 2016

[36] H Khodabandehloo S Gorgani-Firuzjaee G Panahi andR Meshkani ldquoMolecular and cellular mechanisms linkinginflammation to insulin resistance and 120573-cell dysfunctionrdquoTranslational Research vol 167 no 1 pp 228ndash256 2016

[37] P Plomgaard A R Nielsen C P Fischer et al ldquoAssociationsbetween insulin resistance and TNF-120572 in plasma skeletalmuscle and adipose tissue in humans with and without type 2diabetesrdquo Diabetologia vol 50 no 12 pp 2562ndash2571 2007

[38] Y-P Li Y Chen J John et al ldquoTNF-120572 acts via p38 MAPK tostimulate expression of the ubiquitin ligase atrogin1MAFbx inskeletalmusclerdquo FASEB Journal vol 19 no 3 pp 362ndash370 2005

[39] N Carbo S Busquets M van Royen B Alvarez F J Lopez-Soriano and J M Argiles ldquoTNF-120572 is involved in activatingDNA fragmentation in skeletal musclerdquo British Journal ofCancer vol 86 no 6 pp 1012ndash1016 2002

[40] Y Hao J R Jackson Y Wang N Edens S L Pereira andS E Alway ldquo120573-hydroxy-120573-methylbutyrate reduces myonu-clear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged ratsrdquo American Journal of


PhysiologymdashRegulatory Integrative and Comparative Physiologyvol 301 no 3 pp R701ndashR715 2011

[41] S J Lessard D A Rivas K So et al ldquoThe AMPK-relatedkinase SNARK regulates muscle mass and myocyte survivalrdquoThe Journal of Clinical Investigation vol 126 no 2 pp 560ndash5702016

[42] X Fu M Zhu S Zhang M Foretz B Viollet and M DuldquoObesity impairs skeletal muscle regeneration through inhi-bition of AMPKrdquo Diabetes vol 65 no 1 pp 188ndash200 2016

[43] M L Messi T Li Z M Wang A P Marsh B Nicklasand O Delbono ldquoResistance training enhances skeletal muscleinnervation without modifying the number of satellite cells ortheir myofiber association in obese older adultsrdquo Journals ofGerontology Series A Biological Sciences and Medical Sciences2015

[44] F S Tedesco A Dellavalle J Diaz-Manera G Messina andG Cossu ldquoRepairing skeletal muscle regenerative potential ofskeletal muscle stem cellsrdquoThe Journal of Clinical Investigationvol 120 no 1 pp 11ndash19 2010

[45] A Bareja and A N Billin ldquoSatellite cell therapymdashfrom mice tomenrdquo Skeletal Muscle vol 3 article 2 2013

[46] J Gao J E Dennis R F Muzic M Lundberg and A I CaplanldquoThe dynamic in vivo distribution of bone marrow-derivedmesenchymal stem cells after infusionrdquo Cells Tissues Organsvol 169 no 1 pp 12ndash20 2001

[47] R H Lee A A Pulin M J Seo et al ldquoIntravenous hMSCsimprove myocardial infarction in mice because cells embolizedin lung are activated to secrete the anti-inflammatory proteinTSG-6rdquo Cell Stem Cell vol 5 no 1 pp 54ndash63 2009

[48] S Jose M L Hughbanks B Y K Binder G C Ingavle andJ K Leach ldquoEnhanced trophic factor secretion by mesenchy-mal stemstromal cells with Glycine-Histidine-Lysine (GHK)-modified alginate hydrogelsrdquo Acta Biomaterialia vol 10 no 5pp 1955ndash1964 2014

[49] S Chen J Murphy R Toth D G Campbell N A Morriceand C Mackintosh ldquoComplementary regulation of TBC1D1and AS160 by growth factors insulin and AMPK activatorsrdquoBiochemical Journal vol 409 no 2 pp 449ndash459 2008

[50] S Yamada N Buffinger J DiMario and R C StrohmanldquoFibroblast growth factor is stored in fiber extracellular matrixandplays a role in regulatingmuscle hypertrophyrdquoMedicine andScience in Sports and Exercise vol 21 no 5 pp S173ndashS180 1989

[51] P Rocheteau L Chatre D Briand et al ldquoSepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunc-tion amenable by mesenchymal stem cell therapyrdquo NatureCommunications vol 6 article 10145 2015

[52] A W B Joe L Yi A Natarajan et al ldquoMuscle injury activatesresident fibroadipogenic progenitors that facilitate myogene-sisrdquo Nature Cell Biology vol 12 no 2 pp 153ndash163 2010

[53] M D Boppart M D Lisio K Zou and H D HuntsmanldquoDefining a role for non-satellite stem cells in the regulation ofmuscle repair following exerciserdquo Frontiers in Physiology vol 4article 310 2013

[54] M Debnath S Agrawal A Agrawal and G P DubeyldquoMetaflammatory responses during obesity pathomechanismand treatmentrdquoObesity Research amp Clinical Practice vol 10 no2 pp 103ndash113 2016

[55] S Cohen J A Nathan and A L Goldberg ldquoMuscle wastingin disease molecular mechanisms and promising therapiesrdquoNature Reviews Drug Discovery vol 14 no 1 pp 58ndash74 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


muscle wasting showing modifications in the expression ofMHC isoforms Therefore when fast muscles atrophy a fast-to-slow transition occurs (eg IIbrarrIIdrarrIIararrI) [5 6]

Ubiquitin proteasome pathway (UPP) overactivationoxidative stress and myonuclear apoptosis are some of themechanisms involved in muscle atrophy induced by obesityand other causes [4 7] Overactivation of the UPP in muscleatrophy is characterized by an increased expression of twomuscle-specific ubiquitin E3-ligase F-box proteins muscleatrophy F-box (MAFbx)atrogin-1 and muscle ring-fingerprotein 1 (MuRF-1) These proteins increase the ubiquitina-tion of targets such as the myosin heavy chain (MHC) whichare further degraded by the proteasome [8ndash11]

Oxidative stress is produced by increased reactive oxygenspecies (ROS) andor decreased antioxidant mechanisms[12] Oxidative stress has been associated with several modelsof muscle atrophy including that induced by obesity [313 14] Another mechanism involved in muscle atrophy ismyonuclear apoptosis which is related to muscle wastinginduced by cachexia and obesity [4] In atrophic muscleseveral parameters of myonuclear apoptosis increase such asthe BaxBcl2 ratio caspase-3 levels and activity and apoptoticnuclei [15]

Stem cell-based interventions act throughmultiplemech-anisms and provide a clear advantage when treating diseaseswith a complex pathophysiology such as HFD-inducedobesity This type of intervention is arguably better in ther-apeutic terms than single-agent drug-based treatments [1617] Multipotent mesenchymal stromal cells also referredto as mesenchymal stem cells (MSCs) are a heterogeneousadult stem cell population These are a potentially ideal toolfor stem cell-based interventions since they can be isolatedfrombonemarrow and othermesenchymal tissues includingadipose tissue dental pulp the placenta and the umbilicalcord and subsequently rapidly expanded ex vivo [18 19] Asimmunomodulatory cells MSCs can limit inflammation indamaged tissue [20] produce a broad range of trophic factorsthat protect parenchymal cells from apoptotic death andpromote the proliferation and differentiation of endogenousprecursors [21]

The present study is the first to provide evidence thatsystemic MSC administration can prevent the loss of mus-cle strength and mass the decrease in fiber diameter andMHC protein levels and the transition of fiber type in amurine model of HFD-induced muscle wasting Preventionof the several parameters of muscle atrophy among themUPP overactivation increased ROS levels and activation ofmyonuclear apoptosis was also found in this model whichwould explain the observed antiatrophic effects of MSCadministration

2 Materials and Methods

21 Animals Male 12-week-old C57BL10 mice were housedat a constant temperature (22 plusmn 2∘C) with 60 relativehumidity and with a 12 12 light dark cycle Mice had adlibitum access to food and autoclaved water Mice were fedwith either a standard diet (control group 10 cal fat 20 cal

proteins and 70 cal carbohydrates Champion SA Chile)or a high fat diet (HFD group 60 cal fat 20 cal proteinsand 20 cal carbohydrates D12492 Research Diets IncUSA) After 30 weeks of HFD feeding mice were separatedinto two groups matched by average body weight Thenfor an additional eight weeks one group received a vehicletreatment (HFD) while the other was treated with MSCs(HFD + MSCs) At the end of the 38-week experimentalperiod the mice were euthanized under anesthesia and thetibialis anterior (TA) muscles were dissected removed andrapidly frozen and stored at minus80∘C until processing Allprotocols were conducted in strict accordance with and withthe formal approval of the Animal Ethics Committees ofUniversidad Andres Bello and Universidad del Desarrollo

22 Isolation and Administration of MSCs Six-to-eight-week-old C57BL10 male mice were sacrificed by cervicaldislocation Bone marrow cells were obtained by flushing thefemurs and tibias with sterile PBS After centrifugation thecells were resuspended in an 120572-Minimum Essential Medium(Gibco USA) supplemented with 10 selected fetal bovineserum (Hyclone USA) and 80 120583gmL gentamicin (Labora-torio Sanderson Chile) and plated at a density of 1 times 106nucleated cells per square centimeter Nonadherent cells wereremoved after 72 h by changing the medium When the focireached confluence adherent cells were detached with 025trypsin and 265mM EDTA centrifuged and subculturedat 7000 cells per square centimeter After two subculturesadherent cells were phenotypified and characterized accord-ing to the adipogenic and osteogenic differentiation potentialas previously described [6] Then 05 times 106MSCs wereresuspended in 02mL of 5 mouse plasma (vehicle) andadministered via the tail-vein to anesthetizedmice Untreatedmice were given 02mL of vehicle

23 Weightlifting Strength Test At the end of the treatmentthe muscle strength of the mice was measured through aweightlifting test as previously described [22] Briefly theapparatus consisted in a series of increasingly long chain linksattached to a ball of tangled fine wire The number of linksranged from two to seven with total weights between 155 and541 g Before performing the test and prior to treatments themice were trained once per day for twoweeks To perform thetest themouse grasped the different weights with its forepawsand a score was assigned The final score was calculated asthe summation of the product between the link weight andthe time the weight was held The average of three measuresfrom each mouse was normalized against body weight [23]

24 Contractile Properties The TA muscles removed fromsacrificed mice were placed in a dish containing oxygenatedKrebs-Ringer solution (in mmolL NaCl 118 NaHCO

3 25

D-glucose 20 KCl 47 CaCl2 32 KH

2PO4 12 MgSO

4 12)

The TA muscles were firmly tied with surgical silk at thebottom end of the tendon and at a portion of the kneebone The muscle was transferred to a custom-built Plexiglasbath filled with oxygenated Krebs-Ringer solution that wasthermostaticallymaintained at room temperature for optimal









2O2and







0

50

100

ControlHFDHFD + MSC

Mus

cle st

reng

th (

)


lowast

lowast

lowast

(a)

0

50

100

150


Perc

enta

ge o

f mus

cle st

reng

th(s

core

bod

y w

eigh

t)

lowast

(b)

10 20 30 40 60 80 90 100 1500

10

20

30

40

ControlHFDHFD + MSC

Frequency (Hz)

lowast

lowastlowast

lowastlowast lowast

Spec

ific i

som

etric

stre

ngth

(mN

mm

2 )

(c)






3 Results




IIaII

b

(a)

IIaII

b


(b)

0

50

75

100

Fibe

r typ

e IIa

()

25

0

50

75

100

Fibe

r typ

e IIb

()

25

lowast

lowast



(c)





10x

40x

(a)

0

5

10

15

20

25

ControlHFDHFD + MSC

Perc

enta

ge o

f fibe

rs lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lt20

20

ndash25

25

ndash30

30

ndash35

35

ndash40

40

ndash45

45

ndash50

50

ndash55

55

ndash60

60

ndash65

65

ndash70

gt70


(b)








190

GAPDH

MHC

32

HFD

(a)

00

05

10

15


lowast

MH

CG

APD

H (f

old

of in

duct

ion)

(b)










HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2O2and







0

50

100

ControlHFDHFD + MSC

Mus

cle st

reng

th (

)


lowast

lowast

lowast

(a)

0

50

100

150


Perc

enta

ge o

f mus

cle st

reng

th(s

core

bod

y w

eigh

t)

lowast

(b)

10 20 30 40 60 80 90 100 1500

10

20

30

40

ControlHFDHFD + MSC

Frequency (Hz)

lowast

lowastlowast

lowastlowast lowast

Spec

ific i

som

etric

stre

ngth

(mN

mm

2 )

(c)






3 Results




IIaII

b

(a)

IIaII

b


(b)

0

50

75

100

Fibe

r typ

e IIa

()

25

0

50

75

100

Fibe

r typ

e IIb

()

25

lowast

lowast



(c)





10x

40x

(a)

0

5

10

15

20

25

ControlHFDHFD + MSC

Perc

enta

ge o

f fibe

rs lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lt20

20

ndash25

25

ndash30

30

ndash35

35

ndash40

40

ndash45

45

ndash50

50

ndash55

55

ndash60

60

ndash65

65

ndash70

gt70


(b)








190

GAPDH

MHC

32

HFD

(a)

00

05

10

15


lowast

MH

CG

APD

H (f

old

of in

duct

ion)

(b)










HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

ControlHFDHFD + MSC

Mus

cle st

reng

th (

)


lowast

lowast

lowast

(a)

0

50

100

150


Perc

enta

ge o

f mus

cle st

reng

th(s

core

bod

y w

eigh

t)

lowast

(b)

10 20 30 40 60 80 90 100 1500

10

20

30

40

ControlHFDHFD + MSC

Frequency (Hz)

lowast

lowastlowast

lowastlowast lowast

Spec

ific i

som

etric

stre

ngth

(mN

mm

2 )

(c)






3 Results




IIaII

b

(a)

IIaII

b


(b)

0

50

75

100

Fibe

r typ

e IIa

()

25

0

50

75

100

Fibe

r typ

e IIb

()

25

lowast

lowast



(c)





10x

40x

(a)

0

5

10

15

20

25

ControlHFDHFD + MSC

Perc

enta

ge o

f fibe

rs lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lt20

20

ndash25

25

ndash30

30

ndash35

35

ndash40

40

ndash45

45

ndash50

50

ndash55

55

ndash60

60

ndash65

65

ndash70

gt70


(b)








190

GAPDH

MHC

32

HFD

(a)

00

05

10

15


lowast

MH

CG

APD

H (f

old

of in

duct

ion)

(b)










HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















IIaII

b

(a)

IIaII

b


(b)

0

50

75

100

Fibe

r typ

e IIa

()

25

0

50

75

100

Fibe

r typ

e IIb

()

25

lowast

lowast



(c)





10x

40x

(a)

0

5

10

15

20

25

ControlHFDHFD + MSC

Perc

enta

ge o

f fibe

rs lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lt20

20

ndash25

25

ndash30

30

ndash35

35

ndash40

40

ndash45

45

ndash50

50

ndash55

55

ndash60

60

ndash65

65

ndash70

gt70


(b)








190

GAPDH

MHC

32

HFD

(a)

00

05

10

15


lowast

MH

CG

APD

H (f

old

of in

duct

ion)

(b)










HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10x

40x

(a)

0

5

10

15

20

25

ControlHFDHFD + MSC

Perc

enta

ge o

f fibe

rs lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lt20

20

ndash25

25

ndash30

30

ndash35

35

ndash40

40

ndash45

45

ndash50

50

ndash55

55

ndash60

60

ndash65

65

ndash70

gt70


(b)








190

GAPDH

MHC

32

HFD

(a)

00

05

10

15


lowast

MH

CG

APD

H (f

old

of in

duct

ion)

(b)










HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















190

GAPDH

MHC

32

HFD

(a)

00

05

10

15


lowast

MH

CG

APD

H (f

old

of in

duct

ion)

(b)










HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HFD + MSCControl

Tubulin

(kDa)

55 -

Ub proteins

HFD

75 -58 -

46 -

100 -

130 -

(a)HFD + MSC

00

10

20

30

Control HFD

lowast

Ub

prot

eins

tubu

lin (f

old

of in

duct

ion)

(b)

00

10

20

30


Atro

gin-

118

S (fo

ld o

f ind

uctio

n)

lowast

(c)

00

10

20

30


MuR

F-1

18S

(fold

of i

nduc

tion)

lowast

lowast

(d)



4 Discussion




10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10x

40x

(a)

0

2

4

6

8

DCF

fluo

resc

ence

(fol

d of

indu

ctio

n)

lowast

lowast

Control HFD +MSC

HFD

(b)









Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Tubulin

Bax

Tubulin

Bcl-2

(kDa)25 -

55 -

(kDa)

25 -

55 -



(a)

00

05

10

15

20

25

Control HFD

Bax

Bcl2

ratio

(fol

d of

indu

ctio

n)

lowast

HFD +MSC

(b)

Caspase-3

(kDa)

17 -

55 - Tubulin


(c)

00

05

10

15

20

Cas

pase

-3tu

bulin

(fol

d of

indu

ctio

n)

lowast


(d)

0

50

100

150

200

Cas

pase

-3 ac

tivity

( re

lativ

e to

cont

rol)

lowast


(e)


(f)

Apop

totic

nuc

lei p

er fi

eld

(fold

relat

ive t

o co

ntro

l)

0

1

2

3

4

lowast


(g)






5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusions


Competing Interests




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article high fat diet-induced skeletal muscle...

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