Research Article The Effects of Omega-3 Fatty Acid …downloads.hindawi.com/journals/bmri/2014/961438.pdf · 2019-07-31 · Research Article The Effects of Omega-3 Fatty Acid Supplementation

Research ArticleThe Effects of Omega-3 Fatty Acid Supplementation onDexamethasone-Induced Muscle Atrophy

Alan Fappi Tiago S Godoy Jessica R Maximino Vanessa R RizzatoJuliana de C Neves Gerson Chadi and Edmar Zanoteli

Department of Neurology University of Sao Paulo No 455 Dr Arnaldo Avenue 01246-903 Sao Paulo SP Brazil

Correspondence should be addressed to Edmar Zanoteli zanoteliterracombr

Received 18 March 2014 Accepted 28 April 2014 Published 25 May 2014

Academic Editor Emanuele Marzetti

Copyright copy 2014 Alan Fappi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Corticosteroids cause muscle atrophy by acting on proteasomal and lysosomal systems and by affecting pathways related tomuscular trophysm such as the IGF-1PI-3kAktmTOR Omega-3 fatty acid (n-3) has been used beneficially to attenuate muscleatrophy linked to sepsis and cachexia however its effect on dexamethasone-induced muscle atrophy has not been evaluatedObjectivesWe evaluated whether n-3 supplementation couldmitigate the development of dexamethasone-inducedmuscle atrophyMethods Two groups of Wistar rats were orally supplemented with n-3 or vehicle solution for 40 days In the last 10 daysdexamethasone or saline solution was administrated establishing four groups control dexamethasone n-3 and dexamethasone +n-3 The cross-sectional areas of muscle fibers gene expression (MyoD Myogenin MuRF-1 and Atrogin-1) and protein expression(Akt GSK3120573 FOXO3a and mTOR) were assessed Results Dexamethasone induced a significant loss in body and muscle weightatrophy in type 2B fibers and decreased expression of P-Akt P-GSK3120573 and P-FOXO3a N-3 supplementation did not attenuatethe negative effects of dexamethasone on skeletal muscle instead it caused atrophy in type 1 2A reduced the expression ofMyogenin and increased the expression of Atrogin-1 Conclusion Food supplements containing n-3 are usually healthful but theymay potentiate some of the side effects of glucocorticoids

1 Introduction

Muscle atrophy is the loss of muscle mass resulting from areduction in muscle fiber area or density due to a decreasein protein synthesis and an increase in muscle proteinbreakdown these processes activate two major systems theproteasomal (ubiquitin-proteasome system or UPS) and theautophagic-lysosomal system [1ndash3] Many conditions areassociated with skeletal muscle atrophy such as prolongedfasting inactivity or pathological conditions such as sepsiscachexia AIDS cancer and glucocorticoid treatment amongothers [1 2]

Glucocorticoids are one of the most widely prescribedtherapeutic compounds used in the treatment of inflam-matory autoimmune lymphoproliferative and neuromus-cular disorders [4 5] Despite its benefits in inflammatoryresponses treatment with glucocorticoids harms the skeletalmuscle [6]

Many studies have shown that glucocorticoids not onlycause muscle atrophy through the IGF-1PI-3KAktmTORand myostatinSmad23 pathways [7 8] but also throughthe regulation of muscle transcription factors atrogenes andcathepsins [8 9] Some of these mechanisms involve thereduction inmTOR activity induced by an increase of REDD1(regulated in development and DNA damage responses 1)[10] and an increase in the GSK3120573 protein Increased levelsof GSK3120573 are associated with the proteasomal system leadingto increased protein degradation and decreased proteinsynthesis [11] Glucocorticoids reduce the activity of Aktwhich among other functions acts as a negative regulator ofthe forkhead transcription factor (FOXO) FOXOs activationleads to a rapid protein degradation and stimulates thetranscription of several components of the lysosomal andubiquitin-proteasome systems [12ndash16] Activated FOXO1 andFOXO3a lead to the upregulation of Atrogin-1 MuRF-1REDD1 and 4EBP1 which lead to muscle atrophy [17]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 961438 13 pageshttpdxdoiorg1011552014961438

2 BioMed Research International

Polyunsaturated fatty acids (PUFAs) are considered ben-eficial food supplements to human health particularly effec-tive in the cardiovascular and central nervous systems [18ndash20] Two particular PUFAs families the Omega-3 (n-3) andOmega-6 (n-6) fatty acids are the most consumed worldwideand are linked to effects widely discussed in the scien-tific community The n-3 as supplement is commercializedworldwide with free access of the population in capsulesranging from 500ndash2000mg of fish oil extract per capsuleTheWorld Health Organization (WHO) considers 025 to 2 g ofEPADHA per day as part of a healthy diet [21] and the USFood and Drug Administration (FDA) recommends a maxi-mum intake of 3 g per day of n-3 (EPADHA) [22] equivalentto 50mgkgday in a 60 kg person The American HeartAssociation (AHA) recommends the intake of 2 to 4 g ofEPADHA per day to patients with increased levels of serumtriglycerides provided as capsules under a physicianrsquos care[23] The n-3 PUFAs such as EPA (eicosapentaenoic acid)DHA (docosahexaenoic acid) andALA (120572-linolenic acid) areincorporated into the cell membrane after absorption andcan modulate various cellular functions such as signalingand gene expression [24] and decrease the activity of certainUPS components as seen in cancer cachexia [25] Howeverto our knowledge the effects of n-3 PUFA on glucocorticoid-induced muscle atrophy have not been tested

This study evaluated the effects of n-3 PUFA (capsuleformulation) on the development of dexamethasone-inducedmuscle atrophy through histological analysis expression ofgenes involved in protein synthesis and degradation andassessment of expression of proteins involved in the IGF-1PI-3kAktmTOR pathway

2 Methods

21 Animal Treatment We used in this study 24 maleWistarrats aged between 10 to 12 weeks with body weights from 330to 370 grams All experiments were performed according tothe National Institutes of Health (NIH) guidelines on carehandling and use of laboratory animals and approved bythe local ethics committee Animals were housed in fourcages (6 animals each) and placed under controlled light (12-hour lightdark cycle) and temperature (25∘C) conditionsAnimals were fed ad libitum with a standard commercialdiet (Nuvilab CR1) with an approximated composition of220 crude protein 45 ether extract 80 fibrous matter14 calcium and 08 phosphorus and amino acids (DL-Methionine 300mg lysine 100mg)

In the first part of the study 24 animals were divided intotwo initial groups 12 animals were supplemented daily for 40days via gavage (vg) with n-3 capsules (Proepa Ache labo-ratories) diluted in vehicle solution (tween-20 and ultrapurewater) and the other 12 animals received the same volumeof vehicle solution for the same period of time According tothe manufacturer each n-3 capsule contained energetic value(94 Kcal = 395 KJ) total fats (10 g) monounsaturated fats(03 g) polyunsaturated fats (04 g) eicosapentaenoic acid(180mg) and docosahexaenoic acid (120mg) The amount

of EPA and DHA administrated was adjusted in vehiclesolution to the dosage of 100mgkgday per animal Afterday 30 six animals from each group received 5mgkgdayof subcutaneous (sc) dexamethasone (Decadron Ache) for10 days to induce muscle atrophy along with n-3 supple-mentationThe six remaining animals in each group receivedsaline solution as a control The experimental protocolsutilized in these four groups are summarized as DX +n-3 group [n-3 PUFA vg (40 days) + dexamethasone sc (last10 days)] n-3 group [n-3 PUFAvg (40 days) + saline solutionsc (last 10 days)] DX group [vehicle solution vg (40 days)+ dexamethasone sc (last 10 days)] and CT group [vehiclesolution vg (40 days) + saline solution sc (last 10 days)]Theanimals were weighed on days 1 10 20 30 32 34 36 and40

Animals were euthanized after 40 days by intraperitonealinjection with sodium pentobarbital (30mgkg) Bilateralgastrocnemius (GA) and tibialis anterior (TA) muscles werecollected and weighed Right-side samples were mounted inTissue-Tek immersed in isopentane and cooled in liquidnitrogen for histological analysis The remaining muscleswere frozen in liquid nitrogen and stored atminus80∘C for subseq-uent gas chromatography and extraction of protein andRNA

22 Analysis of PUFAs and Fat Content by Gas Chromatogra-phy GAmuscle samples were analyzed by gas chromatogra-phy to evaluate PUFAs and fat content Fat extractionwas car-ried out by the Bligh and Dyer methodology [26] accordingto Iverson et al (2001) [27] Initially 125mg of samples washomogenized (Ultra-turrax IKA T10 Basic) with a mixturecontaining chloroform (125 120583L) and methanol (200120583L) Thesolution was homogenized again with chloroform (125 120583L)and saline (125 120583L of 088 NaCl) The final emulsion wascentrifuged at 13000 g for 5minutes at 25∘CThe upper phasewas removed and the lower phase evaporated under nitrogenstream Fat samples were derivatized using the techniqueof direct esterification described by Shirai et al (2005) [28]and their compositionwas determined by gas chromatograph(Agilent 7890 AGC System Agilent Technologies Inc SantaClara USA) A fused silica capillary column (JampW DB-23Agilent 122-236 60m times 250mm inner diameter) was usedfor injection High-purity helium was used as the carrier gasat a flow rate of 1mLminute and a split injection of 50 1The programming of column temperature was starting at80∘C a gradient step at a heating rate of 5∘Cminute to reach175∘C a gradient step at a heating rate of 3∘Cminute to reach230∘C and maintenance of 230∘C for 5 minutes The injectorand detector (FID) temperatures were 250∘C and 280∘Crespectively The fatty acids were identified by comparingtheir retention times to those of four purified standardmixtures of fatty-acid methyl esters (Sigma Chemical Co4-7801 47085-U 49453-U and 47885-U) The results wereexpressed as percentages of total fatty acids Muscle tissuecontents of total PUFAs and fat were determined DX groupmuscles were not evaluated because this group did not receiven-3 supplementation

BioMed Research International 3

23 Analysis of Cross-Sectional Areas in the Skeletal MuscleCross-sectional areas frommuscle fibers (types 1 2A and 2B)were calculated and analyzed TA muscles were sectioned ina Leica CM3000 cryostat (8 120583m) and sections were treatedusing the metachromatic dye-ATPase method (mATPase)according to Ogilvie and Feeback (1990) [29] Slides withdried frozen sections were placed in a preincubation solutioncomposed of distilled water (475mL) dehydrated potassiumacetate (245 g) and calcium chloride (130 g) at pH 45 for 2minutes slides were subsequently rinsed in Tris buffer Slideswere then incubated for 25 minutes in an incubation solutioncomposed of distilled water (30mL) potassium chloride(185mg) ATP disodium salt (76mg) dehydrated calciumchloride (018M = 5mL) and Sigma 221 buffer (15M 2-amino-2-methyll-propanol) (335mL) at pH 94 Slides wererinsed by dipping in three changes of 1 calcium chloridesolution stained with 1 aqueous toluidine blue for 10seconds and immediately rinsed in distilled water Images ofstained muscle sections were acquired on an Olympus AX70light microscope (Olympus Melville NY) Cross-sectionalareas from types 1 2A and 2B fibers from five slides peranimal (300 to 400 fibersanimal) were measured using theImage J software (NIH)

24 Immunoblot Expression of mTOR Akt GSK3120573 andFOXO3a Proteins The mTOR Akt GSK3120573 and FOXO3aprotein levels were assessed by quantitative Western blotanalysis in GA muscle tissue samples collected as describedTissues were homogenized in a Potter homogenizer in lysingbuffer containing 1 protease and phosphatase inhibitorcocktail (Sigma-Aldrich P5726 and A-1153 resp) 1mMEDTA (Sigma) and 1 NP40 (Sigma) diluted in phosphate-buffered saline (PBS pH 74) and stored at minus80∘C until useTotal protein concentrations were quantified by Bradfordmethod (1976) [30] Samples with up to 50 to 100 120583g oftotal protein were submitted to Western blot The primaryantibodies used were Akt (1 2000 Cell Signaling 4691) P-Akt (1 2000 Cell Signaling 4060 Ser473) mTOR (1 500Cell Signaling 2972) P-mTOR (1 500 Cell Signaling 2971Ser2448) GSK3120573 (1 2000 Cell Signaling 9315) P-GSK3120573(1 2000 Cell Signaling 9322 Ser9) FoXO3a (1 500 CellSignaling 2497) P-FoXO3a (1 500 Cell Signaling 9466Ser253) and 120572-tubulin (1 30000 Hybridoma bank) in 5BSATBS-T The secondary conjugated antibodies were IgG-ECL anti-rabbit (1 10000 KPL) or anti-mouse (1 6000Santa Cruz Biotechnology) Finally membranes were incu-bated with Western Lightning Chemiluminescence ReagentPlus (PerkinElmer Life Science USA) for 1min and exposedto an X-ray film (Hyperfilm ECL Amersham BiosciencesUSA) for visualization of the protein bands The filmswere scanned (HP Scanjet G4000 series) and protein levelswere quantified by densitometry using a computer-assistedimage analyzer through the Image J software (version 143uNational Institute of Health USA) The density values werenormalized by the 120572-tubulin density values

25 Expression of MyoD Myogenin REDD1 REDD2 MuRF-1 and Atrogin-1 Measured by Quantitative PCR Total RNA

was extracted fromGAmuscle samples with TRIZOL reagent(Invitrogen) according to the manufacturerrsquos instructionsRNA quantity and integrity were assessed by spectrophotom-etry (Nanodrop Thermo Scientific USA) and microfluidics-based electrophoresis (Agilent 2100 Bioanalyzer AgilentTechnologies USA) respectively RNA samples with 260280OD ratios of approximately 20 and RINgt 70 were used in thequantitative PCR (qPCR) reactions cDNA was synthesizedfrom 1 120583g of total RNA using TaqMan Reverse TranscriptionReagentsN808-0234 (AppliedBiosystemsLife Technologies)according to the manufacturerrsquos instructions

The qPCR reactions were carried out in duplicate with50 ng cDNA SYBR Green PCR master mix (Fermentas)and 800 to 900 nM of each primer in a final volume reac-tion of 25 120583L in the Step One Plus device (Applied Biosys-tems USA) Real-time PCR was performed to investigatethe expression of the following genes MyoD [31] (for-ward 51015840-TGTAACAACCATACCCCACTCTC-31015840 reverse51015840-AGATTTTGTTGCACTACACAGCA-31015840)Myogenin [32](forward 51015840-CACATCTGTTCGACTCTCTTCT-31015840 reverse51015840-ACCTTGGTCAGATGACAGCTTTA-31015840) REDD-1 [33](forward 51015840-CACCGGCTTCAGAGTCATCA-31015840 reverse 51015840-CGGGTCTCCACCACA GAAAT-31015840) REDD-2 [33] (for-ward 51015840-CTTCAGCGTCTGGTGAAATCC-31015840 reverse 51015840-ATGCTGGCCGTGTTCTTACTG-31015840) MuRF-1 [34] (for-ward 51015840-TCGACATCTACAAGCAGGAA-31015840 reverse 51015840-CTGTCCTTGGAAGATGCTTT-31015840) and Atrogin-1 [34](forward 51015840-TGAAGACCGGCTACTGTGGAAGAGAC-31015840reverse 51015840-TTGGGGTGAAAGTGAGACGGAG CAG-31015840)The results were normalized individually by the expression ofGAPDH [35] (forward 51015840-ACGCCAGTAGACTCCACGAC-31015840 reverse 51015840-ATGACTCTACCCACGGCAAG-31015840) and fur-ther compared by logarithmic normalization (2minusΔΔCT ABIPRISM 7700 Sequence Detection System protocol AppliedBiosystems)

26 Statistical Analysis Differences between means wereanalyzed by unpaired Studentrsquos t-test whereas differencesamong means were analyzed by One- or Two-way ANOVAfollowed by Bonferroni post hoc testing All analyses wereperformed using the Graph Pad Prism 50 software forWindows (Graph Pad Software USA)The data are presentedas mean plusmn SEM with the significance level set at 119875 lt 005

3 Results

31 n-3 PUFA Does Not Alleviate Body and Muscle WeightLoss or Muscle Atrophy Induced by Dexamethasone All ani-mals presented appropriate weight gain according to naturalgrowth during the first 30 days of the study with no statisticaldifference in weight gain between the four experimentalgroups during this period A significant difference in bodyweight became evident on the second day after dexametha-sone administration (groups DX and DX + n-3) (Figure 1)One animal from the DX group died on the fifth day ofdexamethasone administration The mean body weight inthe CT group was 35633 plusmn 47 g at the beginning of thestudy and 42766 plusmn 118 g at the end of the study indicating


450

400

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1 10 20 30 32 34 36 40

(days)

(g)

Body weight

CTDX

n-3DX + n-3

versus CTlowastlowastlowast

Figure 1 Changes in body weight (grams) in animals treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last 10days) The dotted line indicates the start of dexamethasone administration CT = control group (119899 = 6) DX = dexamethasone group (119899 = 5)n-3 = n-3 PUFA group (119899 = 6) and DX + n-3 = dexamethasone + n-3 PUFA group (119899 = 6) Values represent means plusmn SEM analyzed bytwo-way ANOVA repeated measure followed by Bonferroni post hoc test lowastlowastlowast119875 lt 0001

lowastlowastlowastlowastlowastlowast15

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05

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(g)

CT DX n-3 DX + n-3

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(a)

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(g)

30

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00CT DX n-3 DX + n-3

Gastrocnemius muscle weight

(b)

Figure 2Muscle weights in the DX andDX+ n-3 groups were significantly decreased compared to the CT group CT = control group (119899 = 6)DX = dexamethasone group (119899 = 5) n-3 = n-3 PUFA group (119899 = 6) and DX + n-3 = dexamethasone + n-3 PUFA group (119899 = 6) Valuesrepresent means plusmn SEM analyzed by one-way ANOVA followed by Bonferroni post hoc test lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

a gain of 2001The weight gain in the n-3 group was 1461(from 3455 plusmn 88 g to 3960 plusmn 164 g) which has no statisticaldifference to CT group Animals from the DX and DX + n-3groups showed both body weight loss compared to the initialto the final day of study corresponding 854 in theDXgroup(initial weight 3395 plusmn 68 g and final weight 3105 plusmn 56 g) and988 in the DX + n-3 group (initial weight 33400 plusmn 552 gand final weight 3010 plusmn 98 g) Whereas the body weight losspre- and postdexamethasone administration was of 2175(DX group) and 1991 (DX + n-3 group) both significantlydecreased compared to CT group (119875 lt 0001) There wereno statistical differences in body weight comparing DX to theDX + n-3 group and comparing CT to the n-3 group

The TA and GA muscle samples were weighed immedi-ately after euthanasia the weights of muscle samples from theDX and DX + n-3 groups was significantly smaller than thosefrom the CT group (119875 lt 001 and119875 lt 0001 resp) (Figure 2)There was no difference in muscle weight comparing CT tothe n-3 groupThese findings indicate that the administrationof n-3 did not effectively mitigate the weight loss either inbody or muscle tissues induced by dexamethasone

In the muscle atrophy analysis we observed that theadministration of dexamethasone induced a significant TAmuscle atrophy of type 2B muscle fibers (fast-twitch) reduc-ing their cross-sectional area by 2503 in the DX group andby 3271 in the DX + n-3 group compared to the CT group


lowastlowastlowast

lowastlowastlowastlowastlowast

lowast

lowast lowast

Cross section areas2000

1500

1000

500

0

1 2A 2BFiber type

Are

a (120583

m2)

CTDX

n-3DX + n-3

(a)

lowast

lowast

(b)

Figure 3 Means of cross-sectional areas of tibialis anterior muscle fibers (type 1 2A and 2B) in rats treated with n-3 PUFA or vehicle for 40days and dexamethasone or vehicle (last 10 days) (a) Fiber type size distribution (b) representative mATPase dye histological images of TAmuscle from the control group (left) and from the DX + n-3 group (right) Small arrow = type 1 fiber large arrow = type 2A fiber asterisk =type 2B fiber Scale bar (100 120583m) CT = control group (119899 = 6) DX = dexamethasone group (119899 = 5) n-3 = n-3 PUFA group (119899 = 6) and DX +n-3 = dexamethasone + n-3 PUFA group (119899 = 6) Values represent means plusmn SEM analyzed by One-way ANOVA (lowast) followed by Bonferronipost hoc test or unpaired Studentrsquos t-test () lowast119875 lt 005 lowastlowast or 119875 lt 001 and lowastlowastlowast or 119875 lt 0001

(119875 lt 001 and 119875 lt 0001 resp) In addition the DX + n-3group showed a significant muscle atrophy of type 1 and 2Amuscle fibers compared to the CT (2466 and 2053 resp)(119875 lt 005) and to the DX group (1962 and 1795 in fibers1 and 2A resp) (119875 lt 0001 and 119875 lt 001 resp) (Figures 3(a)and 3(b)) These findings confirmed that the administrationof dexamethasone induces atrophy preferentially in type 2Bmuscle fibers In addition the concomitant administrationof n-3 PUFA induced atrophy in muscle fiber types that areusually more resistant to atrophy induced by dexamethasonesuch as type 1 and 2A fibers

32 n-3 PUFA Induces an Increase in PUFA and ALA and aDecrease in Fat and AA in Skeletal Muscles PUFAs contentincreased by 917 and fat content decreased by 1263 in then-3 group showing a significant elevation compared to theCT group (119875 lt 005) Similar increase in PUFAs and decreasein fat content was observed in the DX + n-3 group comparedto the CT group (+600 and minus821 resp) (119875 lt 005)(Figure 4(a))

Weobserved a significant decrease in the arachidonic acid(AA) content in the n-3 and DX + n-3 groups compared

to the CT group (119875 lt 0001) (Figure 4(b)) and the 120572-linolenic acid (ALA) content was significantly increased inthe n-3 group compared to the CT group (119875 lt 001) witha mild increase in the DX + n-3 group compared to the CTgroup (119875 lt 005) (Figure 4(c)) These findings indicated thatthe oral administration of n-3 PUFA was effective for theincorporation of PUFAs in the skeletal muscle

33 The Concomitant Administration of Dexamethasone andn-3 PUFA Induces Suppression of Muscle TranscriptionalFactors The effects of dexamethasone and n-3 PUFA admin-istration on muscle regeneration were evaluated through theexpression of muscle transcriptional factors The expressionof MyoD was increased significantly in the n-3 group onlywhen compared to the DX + n-3 group (119875 lt 005)(Figure 5(a)) suggesting that dexamethasone abrogated theexpression of MyoD through interaction with n-3 Con-versely Myogenin expression was significantly decreased inthe DX + n-3 group in comparison to the CT group (119875 lt005) (Figure 5(b)) In DX group there were no statisticaldifferences in the expressions of MyoD or Myogenin asobserved in DX + n-3 group These results suggest that


lowast

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(g100

g)

+917

minus1263 minus821

+600

Fat

PUFA and fat (skeletal muscle)

PUFA Fat PUFA Fat PUFA

(a)

lowastlowastlowastlowastlowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)

Arachidonic acid (AA) (C204 n-6)20

15

10

5

0

(b)

lowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)

120572-Linolenic acid (ALA) (C183 n-3)15

10

05

00

(c)

Figure 4 Gas chromatography of GA muscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle(last 10 days) (a) Percentages of total fat and PUFAs per experimental group Percentages above the bar indicate decrease or increase inPUFA and fat content compared to the same type of fatty acid in the CT group ((b) and (c)) percentages of arachidonic and 120572-linolenic acidsrespectively CT = control group (119899 = 6) n-3 = n-3 PUFA group (119899 = 6) andDX + n-3 = dexamethasone + n-3 PUFA group (119899 = 6) Statisticalanalysis is in comparison with the CT group Values represent means plusmn SEM analyzed by one-way ANOVA (lowast) followed by Bonferroni posthoc test or Studentrsquos t-test () lowast or 119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001

the administration of n-3 PUFA together with dexametha-sone may affect the muscular regeneration process

34 Dexamethasone Associated with n-3 PUFA Induces HigherAtrogin-1 Expression in Muscle Fibers Than DexamethasoneAlone We evaluated the expression of the Atrogin-1 andMuRF-1 atrogenes after dexamethasone and n-3 administra-tion Both genes showed increased expression in the DX +n-3 group compared to the CT group (119875 lt 001 and 119875 lt 005resp) as well as in the DX group inMuRF-1 expression (119875 lt001) The n-3 group did not show any increase in atrogenesexpression (Figures 5(c) and 5(d))The expression ofMuRF-1in the DX + n-3 group was 274-fold higher than DX group(not statistically different) and in Atrogin-1 expression was346-fold higher (119875 lt 005) The expression of REDD2

genes was increased significantly only in the DX groupcompared to the CT group (119875 lt 005) without any increasein DX + n-3 group (Figure 5(e)) No significant differencein REDD1 expression was observed between the groups(data not shown) These data suggest that dexamethasoneadministration may affect REDD2 and atrogenes expressionin skeletal muscles and that the concomitant administrationof n-3 PUFA can increase this effect in Atrogin-1 expression

35 Dexamethasone Induces a Decrease in Akt GSK3-120573 andFOXO3a Phosphorylation We evaluated the phosphorylated(P) total (t) and phosphorylatedtotal ratio forms of AktGSK3120573 FOXO3a and mTOR proteins in GAmuscle samplesto assess protein expression in the IGF-1PI-3KAktmTORpathway (Figures 6 and 7)


lowastt

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Figure 5 Quantitative PCR results of gene expression in GA muscle samples from rats treated with n-3 PUFA or vehicle for 40 days anddexamethasone or vehicle (last 10 days) Relative fold change values for (a)MyoD (b)Myogenin (c) Atrogin-1 (d)MuRF-1 and (e) REDD2Results are represented as means plusmn SEM CT = control group (119899 = 6) DX = dexamethasone group (119899 = 5) n-3 = n-3 PUFA group (119899 = 6)and DX + n-3 = dexamethasone + n-3 PUFA group (119899 = 6) Values represent means plusmn SEM analyzed by one-way ANOVA (lowast) followed byBonferroni post hoc test or Studentrsquos t-test () lowast or 119875 lt 005 and 119875 lt 001


tAkt20

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lue

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(a)

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(kD

a)

(g)

Figure 6 Protein expression inGAmuscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last10 days) Relative optical density of total Akt (tAkt) and phosphorylated Akt (P-Akt) ((a) and (c)) total GSK3120573 (tGSK3120573) and phosphorylatedGSK3120573 (P-GSK3120573) ((b) and (d)) and tAktP-Akt and tGSK3120573P-GSK3120573 ratios ((e) and (f) resp) Bands representing tAkt P-Akt tGSK3and P-GSK3120573 are illustrated in (g) 120572-Tubulin (55 kDa) was used as control The numbers represent the mean plusmn SEM CT = control group(119899 = 6) DX = dexamethasone group (119899 = 5) n-3 = n-3 PUFA group (119899 = 6) and DX + n-3 = dexamethasone + n-3 PUFA group (119899 = 6)Values represent means plusmn SEM analyzed by Studentrsquos t-test 119875 lt 005


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CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)

Figure 7 Protein expression in GAmuscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last10 days) Relative optical density of total FOXO3a (tFOXO3a) and phosphorylated FOXO3a (P-FOXO3a) ((a) and (c)) total mTOR (t-mTOR)and phosphorylated (P-mTOR) ((b) and (d)) and P-FOXO3atFOXO3a and P-mTORtmTOR ratios ((e) and (f) resp) Bands representingtFOXO3a P-FOXO3a t-mTOR andP-mTORare illustrated in (g)120572-Tubulin (55 kDa)was used as controlThenumbers represent themean plusmnSEM CT = control group (119899 = 6) DX = dexamethasone group (119899 = 5) n-3 = n-3 PUFA group (119899 = 6) and DX + n-3 = dexamethasone +n-3 PUFA group (119899 = 6) Values represent means plusmn SEM analyzed by Studentrsquos t-test 119875 lt 005


In the DX group the t-AKT P-Akt and P-GSK3120573 expres-sion as well as P-AkttAkt and P-GSK3120573tGSK3120573 ratios weresignificantly decreased compared to the CT group (119875 lt 005)(Figures 6(a) 6(c) 6(d) 6(e) and 6(f))

The P-FOXO3a and P-FOXO3atFOXO3a ratio weresignificantly decreased (119875 lt 005) compared to the CTgroup (Figures 7(c) and 7(e)) No significant differencewas observed in mTOR expression between the four groups(Figures 7(b) 7(d) and 7(f))

DX + n-3 group showed intermediate values in theexpression of P-Akt and P-FOXO3a between CT and DXgroups however with no statistical difference comparingboth groups The expression of all forms of Akt GSK3120573mTOR and FOXO3a was not affected in the groups supple-mented with n-3 These findings suggest that the phospho-rylation of Akt GSK3120573 and FOXO3a is inhibited by dexam-ethasone administration and that the n-3 supplementationapparently contributes to attenuate these protein changes

4 Discussion

Some studies have shown that EPA and n-3 PUFA supple-mentation can alleviate muscle atrophy related to cancerfasting and septicemia [36ndash38] Therefore we sought toverify whether n-3 would influence the development ofmuscle atrophy induced by dexamethasone We observedthat dexamethasone administration induced significant lossof body and muscle weight and caused important muscleatrophy in type 2B fibers in rats and that the concomitantsupplementation of n-3 did not attenuate these alterations

Other studies have shown that n-3 is absorbed effectivelyby skeletal muscles when administrated orally [39ndash41] Inour study the gas chromatography analysis showed that n-3 supplementation for 40 days was effective to increase thelevels of PUFAs and fatty acids in the skeletal muscle Thuswe believe that the lack of an effective n-3 attenuation ofthe dexamethasone-induced muscle atrophy was not relatedto deficient intestinal absorption of n-3 The dose of n-3 used in our study similar to that recommended by theUS FDA WHO and AHA for humans (approximated ratioof 50mgkgday) [21ndash23] could not have been effectivehowever much higher dose of n-3 (EPA) up to 25 gkgdayhas been tried by others [42] Thus further studies testingdifferent concentrations of n-3 and dexamethasone are neces-sary to confirm the effects of n-3 in the skeletal muscle underdexamethasone treatment found in the present study

The 2B muscle fiber atrophy observed in this study hasalready been described as resulting from dexamethasoneadministration [43] Some studies postulate that the high sen-sitivity of 2Bfibers is due to the lower content of PGC1120572 in thistype of fiber [44] Interestingly in our study the concomitantadministration of n-3 induced significant muscle atrophy oftypes 1 and 2Amuscle fibers which are usually more resistantto dexamethasone

According to another study AA (arachidonic acid) lev-els stimulate an increase of protein synthesis with releaseof PGF2120572 [45] Sohal et al 1992 [46] showed that EPAadministration by changing fatty acid composition ofmuscle

phospholipids causes a decrease in PGF2120572 synthesis andthis change might suppress the rate of protein synthesis onthe muscles AA is the immediate precursor of PGF [47]and EPADHA administration is associated with a decreasein levels of AA [48] In our study we observed a reductionof 42 in AA levels in the animals treated with n-3 anddexamethasoneThis findingmight indicate a possible doubleeffect on the muscles of these animals with a reduction ofprotein synthesis (by n-3 supplementation which reducesAAlevels) and increase of protein degradation (by dexametha-sone administration which inhibits IGF-1 pathway-relatedproteins)

Concerning cell signaling mechanisms Le Foll et al(2007) [40] showed that the administration of a diet richin n-3 to rats eliminates insulinrsquos ability to stimulate PI-3Kactivity and slightly reduces the level of insulin-induced Aktphosphorylation inmuscle tissueThedecrease inAkt activityby PI-3k results in a higher activation of transcription factorssuch as FOXOs which might lead to a higher transcriptionof E3 ligases involved in the muscle atrophy process [14]According to the literature the atrophy induced by glucocor-ticoids occurs through genetic and molecular mechanismsinvolved in protein degradation including reduction in AktGSK3120573 P70S6K and mTOR phosphorylation and increasein the transcription of atrogenes [49 50] In our studythe levels of Akt GSK3120573 and FOXO3a phosphorylationwere decreased in the DX group in addition DX + n-3 group showed higher activation of atrogenes such asAtrogin-1 and MuRF-1 This last finding indicates that thesupplementation of n-3 might aggravate the side effects ofdexamethasone by affecting upstream pathways of atrogenesactivation Considering that many studies have reported thatthe administration of n-3 alters the lipid composition incell membranes [46 51] we might speculate that the n-3 supplementation influences dexamethasone-induced mus-cle atrophy affecting lipid composition in the sarcolemmaand leading to decreased protein synthesis associated withthe effects of hypercortisolism on skeletal muscle Thesealterations could have modified the activity of receptors incell membranes thus altering the activation of intracellularpathways and downstream regulatory proteins such as thoserelated to the IGF-1PI3-KAktmTOR pathway which arecompartmentalized and activated through their translocationto the plasma membrane [52] Nevertheless these effects aretissue- and cell-specific because activation or inactivation ofAkt by the administration of fatty acids seems to be cell-typedependent [40]

Wang et al (2006) [10] showed that dexamethasoneincreases REDD1 expression in skeletal muscle which sub-sequently activates the tuberin-hamartin complex and sup-presses mTOR activity Moreover the same study observeda decrease in REDD2 mRNA expression ControversiallyFrost et al (2009) [53] showed that IGF-1 a stimulatorof muscle synthesis increases REDD1 gene and proteinexpression in skeletal muscle with a paradoxically greaterprotein synthesis in myotubes expressing more REDD1 Theexpression of REDD2 in this same study was decreased byIGF-1 administration In our study REDD1 expression didnot show consistent changes however REDD2 expression


increased significantly in the DX group compared to thecontrol group

The reduction inMyogenin levels observed in theDX+n-3 group is consistent with results from other studies showingthat dexamethasone induces many unfavorable conditions tomyogenesis partially through Myogenin inhibition [54 55]but we did not observe a similar increase in DX groupAnother study showed that dexamethasone prevents the for-mation of TNF-alpha and the release of lipopolysaccharide-stimulated IL-6 which influences myoblast proliferation[56] However no change was demonstrated in transcriptionfactors related to myogenesis in DX group a decrease wasobserved only in DX + n-3 group compared to the CT groupThe increased MyoD expression observed in the n-3 groupis consistent with results reported by Castillero et al (2009)[57] which showed an increasedMyoD expression in normaland arthritic rats after EPA administration without changesinMyogenin levels

In conclusion this study confirms the deleterious effectsof corticosteroids on skeletal muscle As this type ofmedicineis commonly used to treat several medical conditions theidentification of drugs or nutritional supplements that couldovercome these deleterious effects would be extremely usefulin clinical practice Furthermore this study highlights thatn-3 fatty acids (EPADHA) supplementation in rats did notattenuate the negative effects of dexamethasone on skeletalmuscle showing in addition atrophy on type 1 and 2Amuscle fibers increased atrogene expression and reducedMyogenin levels Thus food supplements such as n-3 usuallyconsidered healthful may potentiate some of the side effectsof glucocorticoids

Conflict of Interests

The authors declare that there is no conflict of interestsfinancial or otherwise regarding the publication of this paper

Authorsrsquo Contribution

Alan Fappi Jessica R Maximino and Edmar Zanoteli partic-ipated in the conception and design of research Alan FappiTiago S Godoy Vanessa R Rizzato and Juliana de C Nevesperformed experiments Alan Fappi Jessica R Maximinoand Edmar Zanoteli analyzed data and interpreted resultsof experiments Alan Fappi drafted the paper Alan FappiJessica R Maximino Gerson Chadi Juliana de C Neves andEdmar Zanoteli edited and revised the paper Alan Fappi andEdmar Zanoteli approved the final version of the paper

Acknowledgments

The authors thank Dr Chin Jia Lin and colleagues for theirassistance in the real-time PCR assays Alan Fappi wassponsored by Fundacao de Amparo a Pesquisa do Estado deSao Paulo (FAPESP 201103862-8) and CAPES (Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior)

References

[1] S Schiaffino K A Dyar S Ciciliot B Blaauw and M SandrildquoMechanisms regulating skeletal muscle growth and atrophyrdquoThe FEBS Journal vol 280 no 17 pp 4294ndash4314 2013

[2] S C Kandarian and R W Jackman ldquoIntracellular signalingduring skeletal muscle atrophyrdquo Muscle and Nerve vol 33 no2 pp 155ndash165 2006

[3] L Voisin D Breuille and L Combaret C Pouyet D TaillandierE Aurousseau C Obled D Attaix ldquoMuscle wasting in a ratmodel of long-lasting sepsis results from the activation of lyso-somal Ca2+-activated and ubiquitin-proteasome proteolyticpathwaysrdquo Journal of Clinical Investigation vol 97 no 7 pp1610ndash1617 1996

[4] J K Clark W T Schrader and B W OrsquoMalley ldquoMechanismof steroid hormonesrdquo in Willians Textbook of EndocrinologyD Wilson and D W Foster Eds pp 35ndash90 WB SandersPhiladelphia Pa USA 1992

[5] U C Reed ldquoNeuromuscular disordersrdquo Journal of Pediatricsvol 78 supplement 1 pp S89ndashS103 2002

[6] F J Kelly and D F Goldspink ldquoThe differing responses offour muscle types to dexamethasone treatment in the ratrdquoBiochemical Journal vol 208 no 1 pp 147ndash151 1982

[7] H Gilson O Schakman L Combaret et al ldquoMyostatingene deletion prevents glucocorticoid-inducedmuscle atrophyrdquoEndocrinology vol 148 no 1 pp 452ndash460 2007

[8] O Schakman H Gilson S Kalista and J P Thissen ldquoMecha-nisms of muscle atrophy induced by glucocorticoidsrdquoHormoneResearch vol 72 supplement 1 pp 36ndash41 2009

[9] K Komamura H Shirotani-Ikejima R Tatsumi et al ldquoDif-ferential gene expression in the rat skeletal and heart musclein glucocorticoid-induced myopathy analysis by microarrayrdquoCardiovascular Drugs and Therapy vol 17 no 4 pp 303ndash3102003

[10] H Wang N Kubica L W Ellisen L S Jefferson and SR Kimball ldquoDexamethasone represses signaling through themammalian target of rapamycin in muscle cells by enhancingexpression of REDD1rdquo Journal of Biological Chemistry vol 281no 51 pp 39128ndash39134 2006

[11] K J P Verhees A M W J Schols M C J M Kelders C MH O den Kamp J L J van der Velden and R C J LangenldquoGlycogen synthase kinase-3120573 is required for the induction ofskeletal muscle atrophyrdquo The American Journal of PhysiologyCell Physiology vol 301 no 5 pp C995ndashC1007 2011

[12] S Ciciliot A C Rossi K A Dyar B Blaauw and S SchiaffinoldquoMuscle type and fiber type specificity in muscle wastingrdquo TheInternational Journal of Biochemistry and Cell Biology vol 5 no10 pp 2191ndash2199 2013

[13] M Sandri C Sandri A Gilbert et al ldquoFoxo transcriptionfactors induce the atrophy-related ubiquitin ligase atrogin-1 andcause skeletal muscle atrophyrdquo Cell vol 117 no 3 pp 399ndash4122004

[14] O Schakman H Gilson and J P Thissen ldquoMechanisms ofglucocorticoid-induced myopathyrdquo Journal of Endocrinologyvol 197 no 1 pp 1ndash10 2008

[15] T N Stitt D Drujan B A Clarke et al ldquoThe IGF-1PI3KAktpathway prevents expression of muscle atrophy-induced ubiq-uitin ligases by inhibiting FOXO transcription factorsrdquoMolecu-lar Cell vol 14 no 3 pp 395ndash403 2004

[16] A Brunet A Bonni M J Zigmond et al ldquoAkt promotescell survival by phosphorylating and inhibiting a forkheadtranscription factorrdquo Cell vol 96 no 6 pp 857ndash868 1999


[17] Y Wu W Zhao J Zhao et al ldquoREDD1 is a major targetof testosterone action in preventing dexamethasone-inducedmuscle lossrdquo Endocrinology vol 151 no 3 pp 1050ndash1059 2010

[18] J Delgado-Lista P Perez-Martinez J Lopez-Miranda and FPerez-Jimenez ldquoLong chain omega-3 fatty acids and cardiovas-cular disease a systematic reviewrdquo British Journal of Nutritionvol 107 supplement 2 pp 201ndash213 2012

[19] A P Simopoulos ldquoOmega-6omega-3 essential fatty acidsbiological effectsrdquo World review of nutrition and dietetics vol99 pp 1ndash16 2009

[20] A P Simopoulos ldquoOmega-3 fatty acids in inflammation andautoimmune diseasesrdquo Journal of the American College ofNutrition vol 21 no 6 pp 495ndash505 2002

[21] World Health Organization ldquoInterim Summary of Conclusionsand Dietary Recommendations on Total Fatrdquo httpwwwwhointnutritiontopicsFFA summary rec conclusionpdf

[22] FDA ldquoFDA Announces Qualified Health Claims for Omega-3Fatty Acidsrdquo 2004 httpwwwfdagovnewseventsnewsroompressannouncements2004ucm108351htm

[23] American Heart Association ldquoFish 101rdquo 2014 httpwwwheartorgHEARTORGGettingHealthyNutritionCenterFish-101 UCM 305986 Articlejsp

[24] M E Surette ldquoThe science behind dietary omega-3 fatty acidsrdquoCanadian Medical Association Journal vol 178 no 2 pp 177ndash180 2008

[25] A S Whitehouse H J Smith J L Drake and M J Tis-dale ldquoMechanism of attenuation of skeletal muscle proteincatabolism in cancer cachexia by eicosapentaenoic acidrdquoCancerResearch vol 61 no 9 pp 3604ndash3609 2001

[26] E G Bligh and W J Dyer ldquoA rapid method of total lipidextraction and purificationrdquo Canadian Journal of Biochemistryand Physiology vol 37 no 8 pp 911ndash917 1959

[27] S J Iverson S L C Lang and M H Cooper ldquoComparison ofthe bligh and dyer and folch methods for total lipid determina-tion in a broad range of marine tissuerdquo Lipids vol 36 no 11 pp1283ndash1287 2001

[28] N Shirai H Suzuki and S Wada ldquoDirect methylation frommouse plasma and from liver and brain homogenatesrdquo Analyt-ical Biochemistry vol 343 no 1 pp 48ndash53 2005

[29] R W Ogilvie and D L Feeback ldquoA metachromatic dye-ATPasemethod for the simultaneous identification of skeletal musclefiber types I IIA IIB ands IICrdquo Stain Technology vol 65 no 5pp 231ndash241 1990

[30] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[31] S Dasarathy M Dodig S M Muc S C Kalhan and AJ McCullough ldquoSkeletal muscle atrophy is associated withan increased expression of myostatin and impaired satellitecell function in the portacaval anastamosis ratrdquo The AmericanJournal of Physiology Gastrointestinal and Liver Physiology vol287 no 6 pp G1124ndashG1130 2004

[32] D Chen S Chen W Wang F Liu C Zhang and H ZhengldquoModulation of satellite cells in rat facial muscle following den-ervation and delayed reinnervationrdquo Acta Oto-Laryngologicavol 130 no 12 pp 1411ndash1420 2010

[33] T Murakami K Hasegawa and M Yoshinaga ldquoRapid induc-tion of REDD1 expression by endurance exercise in rat skeletalmusclerdquoBiochemical andBiophysical ResearchCommunicationsvol 405 no 4 pp 615ndash619 2011

[34] M Pires-Oliveira A L G C Maragno L T Parreiras-e-SilvaT Chiavegatti M D Gomes and R O Godinho ldquoTestosteronerepresses ubiquitin ligases atrogin-1 and Murf-1 expression inan androgen-sensitive rat skeletal muscle in vivordquo Journal ofApplied Physiology vol 108 no 2 pp 266ndash273 2010

[35] L Janjoppi M H Katayama F A Scorza et al ldquoExpressionof vitamin D receptor mRNA in the hippocampal formation ofrats submitted to a model of temporal lobe epilepsy induced bypilocarpinerdquo Brain Research Bulletin vol 76 no 5 pp 480ndash4842008

[36] H J Smith P Mukerji and M J Tisdale ldquoAttenuationof proteasome-induced proteolysis in skeletal muscle by 120573-hydroxy-120573-methylbutyrate in cancer-induced muscle lossrdquoCancer Research vol 65 no 1 pp 277ndash283 2005

[37] J Khal and M J Tisdale ldquoDownregulation of muscle proteindegradation in sepsis by eicosapentaenoic acid (EPA)rdquo Bio-chemical andBiophysical ResearchCommunications vol 375 no2 pp 238ndash240 2008

[38] A S Whitehouse and M J Tisdale ldquoDownregulation ofubiquitin-dependent proteolysis by eicosapentaenoic acid inacute starvationrdquo Biochemical and Biophysical Research Com-munications vol 285 no 3 pp 598ndash602 2001

[39] F J Soriguer F J Tinahones A Monzon et al ldquoVaryingincorporation of fatty acids into phospholipids from muscleadipose and pancreatic exocrine tissues and thymocytes in adultrats fed with diets rich in different fatty acidsrdquo European Journalof Epidemiology vol 16 no 6 pp 585ndash594 2000

[40] C Le Foll C Corporeau V Le Guen J-P Gouygou J-P Bergeand J Delarue ldquoLong-chain n-3 polyunsaturated fatty acidsdissociate phosphorylation of Akt from phosphatidylinositol31015840-kinase activity in ratsrdquo The American Journal of PhysiologyEndocrinology andMetabolism vol 292 no 4 pp E1223ndashE12302007

[41] S K Abbott P L Else and A J Hulbert ldquoMembrane fattyacid composition of rat skeletal muscle is most responsive tothe balance of dietary n-3 and n-6 PUFArdquo British Journal ofNutrition vol 103 no 4 pp 522ndash529 2010

[42] J M Bando M Fournier X Da and M I Lewis ldquoEffectsof malnutrition with or without eicosapentaenoic acid onproteolytic pathways in diaphragmrdquo Respiratory Physiology andNeurobiology vol 180 no 1 pp 14ndash24 2012

[43] D J Prezant M L Karwa B Richner D Maggiore E IGentry and J Cahill ldquoGender-specific effects of dexamethasonetreatment on rat diaphragm structure and functionrdquo Journal ofApplied Physiology vol 82 no 1 pp 125ndash133 1997

[44] M Sandri J Lin C Handschin et al ldquoPGC-1120572 protectsskeletal muscle from atrophy by suppressing FoxO3 action andatrophy-specific gene transcriptionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no44 pp 16260ndash16265 2006

[45] R M Palmer and K W J Wahle ldquoProtein synthesis anddegradation in isolated muscle Effect of 1205963 and 1205966 fatty acidsrdquoBiochemical Journal vol 242 no 2 pp 615ndash618 1987

[46] P S Sohal V E Baracos and M T Clandinin ldquoDietary 1205963fatty acid alters prostaglandin synthesis glucose transport andprotein turnover in skeletal muscle of healthy and diabetic ratsrdquoBiochemical Journal vol 286 no 2 pp 405ndash411 1992

[47] M L Garg E Sebokova A B R Thomson and M TClandinin ldquoΔ6-Desaturase activity in liver microsomes of ratsfed diets enriched with cholesterol andor 1205963 fatty acidsrdquoBiochemical Journal vol 249 no 2 pp 351ndash356 1988


[48] M J Jackson J Roberts and R H T Edwards ldquoEffects ofdietary-fish-oil feeding on muscle growth and damage in theratrdquo British Journal of Nutrition vol 60 no 2 pp 217ndash224 1988

[49] 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

[50] A Jones D-J Hwang R Narayanan D D Miller and J TDalton ldquoEffects of a novel selective androgen receptor modu-lator on dexamethasone-induced and hypogonadism-inducedmuscle atrophyrdquo Endocrinology vol 151 no 8 pp 3706ndash37192010

[51] A P Simopoulos ldquoOmega-3 fatty acids in health and diseaseand in growth and developmentrdquo The American Journal ofClinical Nutrition vol 54 no 3 pp 438ndash463 1991

[52] D J Glass ldquoMolecular mechanisms modulating muscle massrdquoTrends in Molecular Medicine vol 9 no 8 pp 344ndash350 2003

[53] R A Frost D Huber A Pruznak and C H Lang ldquoRegulationof REDD1 by insulin-like growth factor-I in skeletal muscle andmyotubesrdquo Journal of Cellular Biochemistry vol 108 no 5 pp1192ndash1202 2009

[54] R M R Pereira and J Freire de Carvalho ldquoGlucocorticoid-induced myopathyrdquo Joint Bone Spine vol 78 no 1 pp 41ndash442011

[55] M F W te Pas P R de Jong and F J Verburg ldquoGlucocorti-coid inhibition of C2C12 proliferation rate and differentiationcapacity in relation to mRNA levels of the MRF gene familyrdquoMolecular Biology Reports vol 27 no 2 pp 87ndash98 2000

[56] O Prelovsek T Mars M Jevsek M Podbregar and Z Gru-bic ldquoHigh dexamethasone concentration prevents stimulatoryeffects of TNF-120572 and LPS on IL-6 secretion from the precursorsof human muscle regenerationrdquoThe American Journal of Phys-iology Regulatory Integrative and Comparative Physiology vol291 no 6 pp R1651ndashR1656 2006

[57] E Castillero A I Martın M Lopez-Menduina M A Villanuaand A Lopez-Calderon ldquoEicosapentaenoic acid attenuatesarthritis-induced muscle wasting acting on atrogin-1 and onmyogenic regulatory factorsrdquo The American Journal of Physiol-ogy Regulatory Integrative andComparative Physiology vol 297no 5 pp R1322ndashR1331 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Polyunsaturated fatty acids (PUFAs) are considered ben-eficial food supplements to human health particularly effec-tive in the cardiovascular and central nervous systems [18ndash20] Two particular PUFAs families the Omega-3 (n-3) andOmega-6 (n-6) fatty acids are the most consumed worldwideand are linked to effects widely discussed in the scien-tific community The n-3 as supplement is commercializedworldwide with free access of the population in capsulesranging from 500ndash2000mg of fish oil extract per capsuleTheWorld Health Organization (WHO) considers 025 to 2 g ofEPADHA per day as part of a healthy diet [21] and the USFood and Drug Administration (FDA) recommends a maxi-mum intake of 3 g per day of n-3 (EPADHA) [22] equivalentto 50mgkgday in a 60 kg person The American HeartAssociation (AHA) recommends the intake of 2 to 4 g ofEPADHA per day to patients with increased levels of serumtriglycerides provided as capsules under a physicianrsquos care[23] The n-3 PUFAs such as EPA (eicosapentaenoic acid)DHA (docosahexaenoic acid) andALA (120572-linolenic acid) areincorporated into the cell membrane after absorption andcan modulate various cellular functions such as signalingand gene expression [24] and decrease the activity of certainUPS components as seen in cancer cachexia [25] Howeverto our knowledge the effects of n-3 PUFA on glucocorticoid-induced muscle atrophy have not been tested

This study evaluated the effects of n-3 PUFA (capsuleformulation) on the development of dexamethasone-inducedmuscle atrophy through histological analysis expression ofgenes involved in protein synthesis and degradation andassessment of expression of proteins involved in the IGF-1PI-3kAktmTOR pathway

2 Methods

21 Animal Treatment We used in this study 24 maleWistarrats aged between 10 to 12 weeks with body weights from 330to 370 grams All experiments were performed according tothe National Institutes of Health (NIH) guidelines on carehandling and use of laboratory animals and approved bythe local ethics committee Animals were housed in fourcages (6 animals each) and placed under controlled light (12-hour lightdark cycle) and temperature (25∘C) conditionsAnimals were fed ad libitum with a standard commercialdiet (Nuvilab CR1) with an approximated composition of220 crude protein 45 ether extract 80 fibrous matter14 calcium and 08 phosphorus and amino acids (DL-Methionine 300mg lysine 100mg)

In the first part of the study 24 animals were divided intotwo initial groups 12 animals were supplemented daily for 40days via gavage (vg) with n-3 capsules (Proepa Ache labo-ratories) diluted in vehicle solution (tween-20 and ultrapurewater) and the other 12 animals received the same volumeof vehicle solution for the same period of time According tothe manufacturer each n-3 capsule contained energetic value(94 Kcal = 395 KJ) total fats (10 g) monounsaturated fats(03 g) polyunsaturated fats (04 g) eicosapentaenoic acid(180mg) and docosahexaenoic acid (120mg) The amount

of EPA and DHA administrated was adjusted in vehiclesolution to the dosage of 100mgkgday per animal Afterday 30 six animals from each group received 5mgkgdayof subcutaneous (sc) dexamethasone (Decadron Ache) for10 days to induce muscle atrophy along with n-3 supple-mentationThe six remaining animals in each group receivedsaline solution as a control The experimental protocolsutilized in these four groups are summarized as DX +n-3 group [n-3 PUFA vg (40 days) + dexamethasone sc (last10 days)] n-3 group [n-3 PUFAvg (40 days) + saline solutionsc (last 10 days)] DX group [vehicle solution vg (40 days)+ dexamethasone sc (last 10 days)] and CT group [vehiclesolution vg (40 days) + saline solution sc (last 10 days)]Theanimals were weighed on days 1 10 20 30 32 34 36 and40

Animals were euthanized after 40 days by intraperitonealinjection with sodium pentobarbital (30mgkg) Bilateralgastrocnemius (GA) and tibialis anterior (TA) muscles werecollected and weighed Right-side samples were mounted inTissue-Tek immersed in isopentane and cooled in liquidnitrogen for histological analysis The remaining muscleswere frozen in liquid nitrogen and stored atminus80∘C for subseq-uent gas chromatography and extraction of protein andRNA

22 Analysis of PUFAs and Fat Content by Gas Chromatogra-phy GAmuscle samples were analyzed by gas chromatogra-phy to evaluate PUFAs and fat content Fat extractionwas car-ried out by the Bligh and Dyer methodology [26] accordingto Iverson et al (2001) [27] Initially 125mg of samples washomogenized (Ultra-turrax IKA T10 Basic) with a mixturecontaining chloroform (125 120583L) and methanol (200120583L) Thesolution was homogenized again with chloroform (125 120583L)and saline (125 120583L of 088 NaCl) The final emulsion wascentrifuged at 13000 g for 5minutes at 25∘CThe upper phasewas removed and the lower phase evaporated under nitrogenstream Fat samples were derivatized using the techniqueof direct esterification described by Shirai et al (2005) [28]and their compositionwas determined by gas chromatograph(Agilent 7890 AGC System Agilent Technologies Inc SantaClara USA) A fused silica capillary column (JampW DB-23Agilent 122-236 60m times 250mm inner diameter) was usedfor injection High-purity helium was used as the carrier gasat a flow rate of 1mLminute and a split injection of 50 1The programming of column temperature was starting at80∘C a gradient step at a heating rate of 5∘Cminute to reach175∘C a gradient step at a heating rate of 3∘Cminute to reach230∘C and maintenance of 230∘C for 5 minutes The injectorand detector (FID) temperatures were 250∘C and 280∘Crespectively The fatty acids were identified by comparingtheir retention times to those of four purified standardmixtures of fatty-acid methyl esters (Sigma Chemical Co4-7801 47085-U 49453-U and 47885-U) The results wereexpressed as percentages of total fatty acids Muscle tissuecontents of total PUFAs and fat were determined DX groupmuscles were not evaluated because this group did not receiven-3 supplementation








3 Results



450

400

350

300

250

1 10 20 30 32 34 36 40

(days)

(g)

Body weight

CTDX

n-3DX + n-3




10

05

00

(g)

CT DX n-3 DX + n-3


(a)


(g)

30

25

20

15

10

05



(b)






lowastlowastlowast


lowast

lowast lowast


1500

1000

500

0

1 2A 2BFiber type

Are

a (120583

m2)

CTDX

n-3DX + n-3

(a)

lowast

lowast

(b)








lowast

lowast

80

60

40

20

0

(g100

g)

+917

minus1263 minus821

+600

Fat



(a)


CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


15

10

5

0

(b)

lowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


10

05

00

(c)







lowastt

20

15

10

5

0

CT DX n-3 DX + n-3

MyoD

Rela

tive f

old

chan

ge

(a)

lowast

20

15

10

05

00

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Myogenin

(b)

lowast

lowastlowast

lowastlowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Atrogin-1

120

90

60

30

0

P = 011

(c)

lowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

80

60

40

20

0

MURF-1

(d)

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

REDD-28

6

4

2

0

(e)



tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








3 Results



450

400

350

300

250

1 10 20 30 32 34 36 40

(days)

(g)

Body weight

CTDX

n-3DX + n-3




10

05

00

(g)

CT DX n-3 DX + n-3


(a)


(g)

30

25

20

15

10

05



(b)






lowastlowastlowast


lowast

lowast lowast


1500

1000

500

0

1 2A 2BFiber type

Are

a (120583

m2)

CTDX

n-3DX + n-3

(a)

lowast

lowast

(b)








lowast

lowast

80

60

40

20

0

(g100

g)

+917

minus1263 minus821

+600

Fat



(a)


CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


15

10

5

0

(b)

lowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


10

05

00

(c)







lowastt

20

15

10

5

0

CT DX n-3 DX + n-3

MyoD

Rela

tive f

old

chan

ge

(a)

lowast

20

15

10

05

00

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Myogenin

(b)

lowast

lowastlowast

lowastlowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Atrogin-1

120

90

60

30

0

P = 011

(c)

lowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

80

60

40

20

0

MURF-1

(d)

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

REDD-28

6

4

2

0

(e)



tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


450

400

350

300

250

1 10 20 30 32 34 36 40

(days)

(g)

Body weight

CTDX

n-3DX + n-3




10

05

00

(g)

CT DX n-3 DX + n-3


(a)


(g)

30

25

20

15

10

05



(b)






lowastlowastlowast


lowast

lowast lowast


1500

1000

500

0

1 2A 2BFiber type

Are

a (120583

m2)

CTDX

n-3DX + n-3

(a)

lowast

lowast

(b)








lowast

lowast

80

60

40

20

0

(g100

g)

+917

minus1263 minus821

+600

Fat



(a)


CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


15

10

5

0

(b)

lowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


10

05

00

(c)







lowastt

20

15

10

5

0

CT DX n-3 DX + n-3

MyoD

Rela

tive f

old

chan

ge

(a)

lowast

20

15

10

05

00

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Myogenin

(b)

lowast

lowastlowast

lowastlowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Atrogin-1

120

90

60

30

0

P = 011

(c)

lowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

80

60

40

20

0

MURF-1

(d)

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

REDD-28

6

4

2

0

(e)



tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowastlowastlowast


lowast

lowast lowast


1500

1000

500

0

1 2A 2BFiber type

Are

a (120583

m2)

CTDX

n-3DX + n-3

(a)

lowast

lowast

(b)








lowast

lowast

80

60

40

20

0

(g100

g)

+917

minus1263 minus821

+600

Fat



(a)


CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


15

10

5

0

(b)

lowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


10

05

00

(c)







lowastt

20

15

10

5

0

CT DX n-3 DX + n-3

MyoD

Rela

tive f

old

chan

ge

(a)

lowast

20

15

10

05

00

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Myogenin

(b)

lowast

lowastlowast

lowastlowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Atrogin-1

120

90

60

30

0

P = 011

(c)

lowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

80

60

40

20

0

MURF-1

(d)

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

REDD-28

6

4

2

0

(e)



tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast

lowast

80

60

40

20

0

(g100

g)

+917

minus1263 minus821

+600

Fat



(a)


CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


15

10

5

0

(b)

lowastlowast

CTn-3DX + n-3

CT n-3 DX + n-3

(g100

g)


10

05

00

(c)







lowastt

20

15

10

5

0

CT DX n-3 DX + n-3

MyoD

Rela

tive f

old

chan

ge

(a)

lowast

20

15

10

05

00

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Myogenin

(b)

lowast

lowastlowast

lowastlowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Atrogin-1

120

90

60

30

0

P = 011

(c)

lowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

80

60

40

20

0

MURF-1

(d)

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

REDD-28

6

4

2

0

(e)



tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowastt

20

15

10

5

0

CT DX n-3 DX + n-3

MyoD

Rela

tive f

old

chan

ge

(a)

lowast

20

15

10

05

00

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Myogenin

(b)

lowast

lowastlowast

lowastlowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

Atrogin-1

120

90

60

30

0

P = 011

(c)

lowast

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

80

60

40

20

0

MURF-1

(d)

CT DX n-3 DX + n-3

Rela

tive f

old

chan

ge

REDD-28

6

4

2

0

(e)



tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


tAkt20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tGSK312057325

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-Akt

25

20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-GSK312057330

20

10

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(d)

P-AkttAkt25

20

15

10

00

05

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-GSK3120573tGSK312057325

20

15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total Akt

P-Akt

Total GSK3120573

P-GSK3b

120572-Tubulin

60

60

48

48

55

CT DX n-3 DX + n-3

(kD

a)

(g)



tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


tFOXO3a20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(a)

tmTOR20

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(b)

P-FOXO3a

15

10

5

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(c)

P-mTOR

6

4

10

8

2

0A

rbitr

ary

valu

e

CT DX n-3 DX + n-3

(d)

P-FOXO3atFOXO3a

3

2

4

1

0

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(e)

P-mTORtmTOR15

10

05

00

Arb

itrar

y va

lue

CT DX n-3 DX + n-3

(f)

Total FOXO3a

P-FOXO3a

Total mTOR

P-mTOR

120572-Tubulin

82

97

248

248

55

CT DX n-3 DX + n-3

(kD

a)

(g)






4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Discussion
















Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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Research Article The Effects of Omega-3 Fatty Acid …downloads.hindawi.com/journals/bmri/2014/961438.pdf · 2019-07-31 · Research Article The Effects of Omega-3 Fatty Acid Supplementation