Acute effects of physical exercise with microcurrent in the adipose tissue of the abdominal region: A randomized controlled trial

Acute effects of physical exercise with microcurrent in the adipose tissue of the abdominal region: A randomized controlled trial

Andreia Noites, PhD

^a,

*, Anabela Moreira, BSc

^b

, Cristina Melo, PhD

^a

, Miriam Faria, PT

^a

, Rui Vilarinho, MSc

^a

, Carla Freitas, PT

^a

, Pedro R.R. Monteiro, PhD

^c

, Paulo Carvalho, PhD

^a

, Nuno Adubeiro, MSc

^d

, Manuela Amorim, MSc

^b

, Luísa Nogueira, PhD

^d

, Rubim Santos, PhD

^e

aDepartmentofPhysiotherapy,ActivityandHumanMovementStudyCenter,SchoolofAlliedHealthTechnologies—PolytechnicInstituteofPorto,Portugal

bDepartmentofClinicalAnalysisandPublicHealth,SchoolofAlliedHealthTechnologies—PolytechnicInstituteofPorto,Portugal

cDepartmentofPhysiology,ActivityandHumanMovementStudyCenter,SchoolofAlliedHealthTechnologies—PolytechnicInstituteofPorto,Portugal

dDepartmentofRadiology,SchoolofAlliedHealthTechnologies—PolytechnicInstituteofPorto,Portugal

eDepartmentofPhysicalandActivityandHumanMovementStudyCenter,SchoolofAlliedHealthTechnologies—PolytechnicInstituteofPorto,Portugal

Keywords:

Abdominalfat Microcurrent Aerobicexercise Adiposetissue Electricalstimulation Fatcelllipolysis

Randomisedcontrolledtrial

ABSTRACT

Introduction:Increasedabdominalfatandsedentarylifestylescontributetocardiovasculardiseaserisk.

Low-intensity electrical current (microcurrent) on theabdominal region, associated with physical exercise,appearstobeaninnovativemethodtoincreasethelipolyticrateofabdominaladipocytes,in ordertoreduceabdominalfat.Thisstudyaimedtoanalyzetheacuteeffectsofmicrocurrentassociated withanaerobicexerciseprograminhealthysubjectsinlipolysis.

Method: A double-blinded, randomized controlledtrial was developed and conducted in ahigher educationschool.Eighty-threehealthysubjects,agedbetween18and30yearsoldandwitha18.5to 29.9kg/m²bodymassindexwererandomlyassignedeithertoanexperimentalortoaplacebogroup.

Subjectsreceivedatrans-abdominalmicrocurrentstimulationfor40minwith(experimentalgroup)or without(placebogroup)electricalcurrent,followedbyasingleaerobicexercisesession(60minat45–

55% VO2max intensity). Lipolytic activity (serum glycerol), abdominal fat (waist circumference, abdominalskinfold,ultrasonography),andserumlipidprofile(serumtriglyceride,totalcholesterol,low- densitylipoproteincholesterolandhigh-densitylipoproteincholesterol)wereevaluatedinallsubjects.

Physical activity (International PhysicalActivityQuestionnaire) and dietaryintake (food-frequency questionnaire)questionnaireswereapplied.

Results:Aftertheintervention,lipolyticratewassignificantlyhigher(p=0.003)intheexperimental group (mean=0.15)thanintheplacebogroup (mean=0.09).Glycerol resultsshowedastatistically significantincreasebetweenbaselineandaftertheinterventionforbothexperimentalgroup(p=0.001) andtheplacebogroup(p=0.001).

Conclusion:Combineduseofmicrocurrentandphysicalaerobicexercisehadanacuteeffectenhancing lipolyticratecomparingtoexercisealone,inyounghealthysubjects.

1.Introduction

Abdominal obesity, namely visceral and deep subcutaneous adipose tissue, are associated with a greater risk for the developmentofmetabolicandcardiovascularcomplicationsthan in other regions of the body [1–3]. These tissues show higher metabolic(pro-inflammatory,lipogenic,andlipolytic)activityand

subsequentlyhigherproportionofsaturatedfattyacids[4,5].This enablesanincreasinginbloodtriglycerides,whichmayculminate in dyslipidemia, diabetes, hypertension and, thus, increased cardiovascular disease risk [3,6]. However it has been widely discussedintheliterature,thatnotonlyindividualswithabody mass index (BMI) considered high or excessive can present a dysfunction in adipocyte behavior, but also it can be found in adipocytesinindividualswithnormalBMI[7].

Amongothers,age,gender,genetics,hormonesandethnicity are broad etiological factors that contribute to a variation in adiposetissueaccumulationandplasmalipids[8,9].Ahypercaloric and fat-richdiet, togetherwith sedentarylifestyle, alsoplay a

majorroleinthedevelopmentofabdominalobesity,thuslifestyle modification programmes regarding nutrition, physical activity and weight control/loss are a major public health concern nowadays,showingpromisingresults[10,11].

Moderate aerobic exercise increases blood flowand enables fattyacid delivery from theadipose tissue to skeletal muscles duringmuscularactivity[12,13].Catecholamines(adrenalineand noradrenaline)regulatelipolysisduringexercise,stimulatingand inhibitinglipolysisthroughbeta-adrenergicandalpha-adrenergic pathwaysrespectively,andthusincreasingfattyacidsandglycerol secretionfrom the adipose tissue [12,14]. In addition, exercise promotes muscular cytokines secretion, which may induce systemic effects in the adipose tissue through IL-6 mediated lipolysis and fatty acids oxidation, and irisin-induced white adiposetissue to brown adiposetissue turnover [15].Also, the useoftranscutaneousmicrocurrenttogetherwithaerobicexercise appearsto havea cumulative effectin the mobilizationof the abdominaladiposetissue,providingmeanstolocalabdominalfat treatment[16].

A microcurrentis characterized by theapplication of a low frequencyandlow intensitycurrent,whichstimulatestriglycer- ideshydrolysis,hencereleasingfreefattyacidsandglycerolinto thebloodcirculation[17].Fattyacidsbecomethen availablefor oxidation and energy expenditure during physical exercise [18–21].Ontheotherhand,venousglycerolcanindicatelipolytic activity in adipose tissue, because is not reutilized for the resynthesisofnewtriglyceridesduetounavailabilityofglycerol kinaseinadiposetissue[7,14].

The present study aims to assess the effect of a single application of abdominal microcurrent, in addition to a single sessionofaerobicexercise,inacutelipolyticactivity,asevaluated by the serum glycerol levels variation in clinically healthy individuals.Itwasaimed,also,toevaluateifthegenderandthe amountofadiposetissuearerelatedtotheeffectsofmicrocurrent onthelipolyticactivity.

2. Methods

2.1.Studydesign,randomization,andimplementation

Thisstudywasadouble-blinded,randomizedcontrolledtrial (RCT)andperformedbetweenJanuary2013andMarch2014.

ThestudywasapprovedbytheEthicsBoard(nr.0325/2013and nr. 04131/2013) and Direction (n 000276) at the central coordinatingandparticipatinginstitution.Thetrialwasregistered at the US National Institutes of Health (ClinicalTrials.gov)

#NCT02110927. All participants provided informed consent in compliancewiththeprinciplesoftheDeclarationofHelsinki.

Following eligibility screening by the research coordinator, eligiblesubjectswererandomlyallocatedbysexintotheplacebo and experimental groups. The randomization schedule was stratified in mutable blocks of 4, through a unique random number designation (Microsoft Office Excel). Apart from the researcherresponsiblefortheelectrolipolysisprotocol,allother researchers and participants were blinded to the intervention allocation.

Participants

Thestudywasdevelopedandconductedinahighereducation school. All students (n=1966) were invited to participate by electronic email and answer a sample characterization and selection questionnaire. We assessed 196 participants who volunteered responding toan email, aged between 18 and 30 years old and with a 18.5 to 29.9kg/m² BMI. Students were excludedaccordingtothefollowingexclusioncriteria:pregnant,

pregnantinthepreviousyearorplanningtogetpregnantsoon;

clinicalhistoryofcancer;metabolic,renalordigestive dysfunc- tions; abdominal surgery; contraindications to microcurrent application,suchasabdominal,dermatologicortactilesensibility dysfunctions; pacemakers; intrauterine contraceptive device;

osteosynthesis material in the abdominal region; conditions limiting aerobic exercise suchas cardiovascular, respiratory or orthopaedicdiseases;involvementin otherfat reductionproce- dures/programmes; andhypocaloric diets.Atotalof 101volun- teerswereexcludedandthe95subjectseligibletoparticipatein thestudywererandomlyassignedtotheexperimentalgroup(EG) (n=47) or to the placebo group (PG) (n=48) (Figure I). Excel softwearwasusedtorandomizeparticipantsintothegroups.Five subjectsintheEGandsevensubjectswerelostinthePG(Fig.1).

Thus, attheend ofthestudy, theEGdatawas available for42 subjects(16males;26females)andforPG,41subjects(15males;

26females)

2.2.Measurements 2.2.1.Anthropometrics

Heightwasassessedbyastandard,wallmountedstadiometer, before intervention. Body mass and fat mass percentage were measured by bio-impedance using a Tanita InnerScan BC-522 (Tanita,Tokyo, Japan), withthe subjectswearing light clothing without shoes [22]. For the bio-impedance evaluation, the volunteerswereinstructednottodrinkalcohol nortoperform vigorousphysicalexercisebefore24h, aswellastoavoidheavy mealsandtoemptythebladderbeforethemeasurement.

The BMI was calculated according to the World Health Organizationguidelines[23].

2.3.Abdominalfat

Waistcircumferencewasmeasuredatthemidpointbetween thelowestribandtheiliaccrest.

Abdominal skinfold and suprailiac skinfold were measured usingan analogicCaliper Harpenden(BatyInternational,West Sussex,UnitedKingdom)[24].

UltrasonographywasperformedusingaViamoTM⁷ultrasound system(ToshibaMedical Systems,Tustin,California)tomeasure subcutaneous and visceral abdominal fat below the xiphoid apophysis,andnavelfatbelowthenavel[16].

Allthemeasurementswereevaluatedbeforeintervention.

2.4.Physicalactivity

Subjects’physicalactivitylevelswereassessed,onthedayof the intervention protocol, through the abridged form of the InternationalPhysical Activity Questionnaire(IPAQ) application, previously validated, which records the duration of physical activity reported as metabolic equivalent task (MET) minutes/

week, and categorizes the physical activity undertaken in the previous7daysonthreelevels(low,moderate,andhigh)[25,26].

2.5.Dietaryintake

Semi-quantitativeFoodFrequencyQuestionnaire,referringto 12monthspriortothestudy,wasusedtomonitordietaryintake.

All subjects answered to the questionnaire on the day of the interventionprotocol.TheFoodProcessorPlus¹(ESHAResearch, Salem,Oregon)wasusedtoconvertfoodintonutrient(consump- tion of caffeine, sugar, calcium, magnesium, alcohol, protein, carbohydrates, total fiber, sodium, total fat, saturated fat, monounsaturated fat, cholesterol, n-3 and n-6 fatty acids, and totalenergyintake)[27].

2.6.Serumglycerol,triglycerides,totalcholesterolLDLandHDL cholesterol

Bloodsamplewerecollectedbeforeandaftertheintervention.

Thebloodcollectionaftertheinterventionwasmade5minafter aerobicexercisewithsubjectinrest.

Blood samples from all subjects were collected through a standardantecubitalvenepunctureto8mlserum-separatortubes (BD Vacutainer¹ System, BD Diagnostics, Franklin Lakes, NJ), immediately prior to-Baseline (M0)and after theintervention protocol(M1).Serumwasseparatedbylowspeedcentrifugation (4500rpm,10min,22C)(Sigma¹3k15,SIGMALaborzentrifugen GmbH,OsterodeamHarz,Germany),transferredintoaliquots,and stored at 80C until the analysis. Serum triglyceride, total cholesterol,Low-densitylipoprotein(LDL)cholesteroland high- densitylipoprotein(HDL)cholesterol(P.Z.Cormay,Lublin,Poland) and glycerol (Randox Laboratories, Crumlim, United Kingdom) concentrationsweredeterminedbyaPrestige24iautoanalyzer(P.

Z.Cormay,Lublin,Poland)usingstandardenzymaticcolorimetric techniques.

Interventions

Subjects performed one microcurrent session with (experi- mental group) or without (placebo group) electrical current, followedbyasingleaerobicexercisesession(bothgroups).Blood samples from all subjects were collected before and after the intervention.

2.7.Microcurrentprotocol

Thenon-invasivemicrocurrentprotocol,appliedbeforephysi- calexercise,consistedintheapplicationoflowfrequencybipolar square-wave, alternated with electrical current to the lower abdominal region using transcutaneous band electrodes

(255,5cm;30,55,5cm;355,5cm)onconductivegel,with adistanceof5–10cmbetweenthem.A25Hzfrequency(impulse durationandrestingdurationof20ms)wasusedduringthefirst 20min, followed by another application of 20min at 10Hz frequency (impulseduration and resting duration of 50ms).In theexperimentalgroup,themicrocurrent’sintensitywas under each individual’s sensitivity threshold (registered values were superior to 700

m

^{A and inferior to 1000}

m

^A) [16,28,29]. In the placebogroup,alltheproceduresweresimilartotheEGbutthe microcuurentdevicewasoff.

Infactthemoststudiedmicrocurrentprotocols,usefrequencies of10Hzand25Hzinordertopotentiatetheeffectofmicrocurrent inlocalbloodflowstimulationandthroughstimulationoflipolytic mediatorsreleasedbythesympaticadrenergicsystem.[16,28,29].

Therewerenoharmfuloradverseeventsduringthestudy.As only mild electrical current was used, no perceptible muscle contractionormusclepainwasgeneratedinthesubjects.

2.8.Exerciseprotocol

Aerobic exercise of moderate intensity at 45–55% VO2max (Karvonen’s formula) was performed by both groups on a cycloergometerfor50min,rightafterthemicrocurrentprotocol, beingassessedbytheBorg’sscale(grade:12–13),togetherwitha Polar¹heartmonitorusedtocontrolheartrate[30,31].

Fatty acids oxidation increases progressively during aerobic exercisewithmoderateintensity,withintensitiesbetween45and 55%VO2max[21,32].

2.9.Samplesizecalculationandstatisticalanalysis

The powercalculationwas computedaprioribased ondata obtained from a pilotstudy (15 individuals divided by groups receivedtheprotocoltreatment,sincethereisnoeffectsizevalue in the literature). The sample size for the present study was Assessed for eligibility (n=196)

Excluded (n=101)

incomplete questionnaires (n=19) dermatological diseases (n=14) nutritional intervention (n=20) respiratory disease (n=9) vascular disease (n=2) cardiac disease (n=1) refused to participate (n=36)

Analysed(n=42) 16 males; 26 females Discontinued intervention (n=1) No show

Experimental group (n=47):

Received allocated intervention (n=43) Did not receive allocated intervention (n=4) lack availability

Discontinued intervention (n=2) No show

Placebo group (n=48)

Received allocated intervention (n=43) Did not receive allocated intervention (n=5) lack availability

Analysed (n=41) 15 males; 26 females Allocation

Analysis Follow-Up

Randomized (n=95) Enrollment

Fig.1.–Flowdiagramofpatients.

calculatedtoensurestatisticalpowerforatwo-groupanalysiswith thevariabledifference(M1–M0)ofglycerol,assumingapowerof 90%and5%significancelevel,revealinganeedfor40patientsto detectaneffectsizeof0.74.

Baselinecharacteristicsoftheparticipantsinbothplaceboand experimentalgroupswerereportedusingfrequencydistributions anddescriptivestatistics,includingmeasuresofcentraltendency (meanor median)and dispersion(standard deviationor inter- quartiledeviation).

Measuredvariableswerecomparedbetweengroups(atM0,M1 andM1-M0difference)andbetweenmoments(M0vsM1),within eachgroup,usinganindependenttwo-samplet-testand paired samplest-testrespectively,whennormaldistributionwasassured throughtheShapiro-Wilktest.Whennormalitywasnotverified, theMann-Whitneytestwasusedforbetween-groupcomparisons andtheWilcoxontestforwithin-groupcomparisons.

Pearson’scorrelationcoefficientwasappliedtoassesscorrela- tionbetweenglycerolmobilizationandanthropometricmeasures, BMI values, physical activity values, abdominal fat, serum triglyceridesandserumcholesterol.

StatisticalanalysiswereperformedusingaStatisticalPackage fortheSocialSciences(SPSS),version23(IBM),withasignificance levelof5%(p<0.05).

3. Results

A total of 83 randomized subjects completed the trial: 42 subjectsintheexperimentalgroup(16males;26females)and41 subjectsintheplacebogroup(15males;26females).

Themeanageofthetotalparticipantswas201.53years,and meanBMIwas22.862.97(80%ofthetotalparticipantspresented normalBMI,while20%hadanoverweightBMI)(WHO,2014)with nostatisticallysignificantdifferencesbetweengroups(Table1).

Baseline (M0) anthropometric characteristics (muscle mass, waistcircumference,waist-heightratioandskinfold)andphysical activity(totalMET’sforweek)weresimilarinbothgroups,withno statisticallysignificantdifferences(Table1).

Inabdominalfatmeasuredbyultrasonography,subcutaneous andvisceralwaistFATweresimilarinPGandEGatbaseline(M0) withoutanysignificantdifferences(Table2).

Regardingdietaryintake,food-frequencyquestionnaireresults showed no statistically significant differences between the experimental group and the placebo group at baseline (M0) (Table3).

Baseline(M0)lipidandglycerolserumvaluesweresimilarin bothgroups.

Serum glycerol results showed a statistically significant increase between M0 and M1 (after theintervention), both in theEG(p=0.001)andthePG(p=0.001)(Table4).AtM1,glycerol levelsintheEGweresignificantlyhigherthaninthePG(p=0.003), aswellasregardingthedifferencevariable(M1-M0)(p=0.004).

(TableIV).

Thelipidsprofile(totalcholesterol,TG,LDLandHDL)results betweengroupsshowednostatisticallysignificantdifferencesin M1 and in the difference variable. However, the resultsin PG showed a statistically significant increase in total cholesterol (p=0.001), triglycerides(p=0.023) andLDL (p=0.005)between M0andM1.(Table4).

Regardingtheglyceroldifference(M1-M0)andtotalbodyfat, weobservedamoderateandsignificantlypositivecorrelationin both groups (Table 5). On theother hand,for the visceraland subcutaneous fat measured by ultrasound, as well as for the abdominalcircumferenceandskinfolds,asignificantcorrelation between glycerol mobilization and these variables was only noticeableintheplacebogroup(Table5).Nosignificantcorrela- tionswereobservedregardingtriglycerides,cholesterol,BMIand IPAQresultsinbothgroups(Table5).

Inthegender-relatedanalysisofglycerolmobilization(Fig.2), atM1womenofbothexperimentalandplacebogroupsmobilized moreglycerolthanmen(p=0.014andp=0.024,respectively).Both gendersshowedhigherglycerollevelsaftertheinterventioninthe experimental group, in comparison with the placebo group (p=0.030andp=0.004,respectively).

4. Discussion

The groups showed similar characteristics in variables age, anthropometric measures, level of physicalactivityand dietary intake, which reforce the comparison between groups. This informationensurescontrolofthefactorsthatinfluencemetabo- lism[33].

We used serum glycerol levels as a lipolysis marker, as a measurementof triglycerideshydrolysisbecause,afterlipolysis, glycerol enters into the bloodstream and is not significantly reutilizedintotriglyceridessynthesisduetolowlevelsofglycerol kinaseintheadiposetissue[7],whilefreefattyacids(FFA)maybe oxidised during moderate exercise of long duration [34,35].

Althoughglycerolismostlymetabolizedinthelivertolaterre- jointheglycolysisandgluconeogenesispathwayasglyceraldehyde 3-phosphate- GA3P, a small part may be used in muscles to oxidationandtriglyceridessynthesis[14,34]

Table1

–Baselinedemographicandanthropometriccharacteristics.

Mean(SD)

EG PG pEGvs.PG

Age(years) 20.1 20.2 0.661

(1.5) (1.5)

Height(m) 1.69 1.70 0.513

(0.1) (0.1)

Bodymass(kg) 65.9 65.3 0.791

(11.8) (10.8)

BMI(kg/m²) 23.2 22.5 0.322

(3.3) (2.6)

MuscleMass(kg) 50.6 49.6 0.815

(12.2) (10.4)

Waistcircumference(cm) 79.7 79.3 0.798

(7.7) (7.1)

Waist-heightratio 0.47 0.46 0.445

(0.03) (0.03) Abdominalskinfold

(mm)

13.6 12.5 0.211

(4.3) (3.8)

Suprailiacskinfold(mm) 14.7 15.6 0.531

(5.7) (6.2)

IPAQ(MET-min/week) 2356.4 2010.1 0.142

(1394.3) (1099.5)

AbbreviationsBMI.BodyMassIndex;EG.experimentalgroup;PGplacebogroup;

IPAQ.internationalphysicalactivityquestionnaire.

pvalueforinitialmomentwitht-test.

Table2

–Baselineabdominalfatmeasuredbyultrasonography.

Mean(SD) Dif.Groups

EG PG pEGvsPG

SubcutaneousFat waist(mm) 14.41 14.39 0.994 (9.03) (8.79)

VisceralFat waist(mm) 5.54 5.58 0.963

(3.49) 4.62)

TotalFatiliaccrest(mm) 21.80 24.91 0.390

(12.12) (11.56) AbbreviationsEG.experimentalgroup;PGplacebogroup.

pvalueforinitialmomentwitht-test.

Throughtheanalysisofserumglycerolvariation,weobserved that a single session of abdominal microcurrent associated to aerobicexerciseincreasedsignificantlytheglycerolmobilization, compared toan isolated aerobic exercise session. According to Ramírez-Ponceetal.(1998,2002,2003),adipocytesaresensitiveto electric current,presenting voltage-dependent potassium chan- nels[36–38].Moreover,electrostimulationperseactivateslipolytic activityinthehumanadiposetissue[17].Arecentstudyshowed thatmildelectriccurrentwithheatshockmayreduceexcessive adiposetissueandmetabolicdysfunction[39],hencesupporting

that the application of abdominal microcurrent may play an significant role in lipolytic activity and sympathetic nervous systemstimulation.Theseresultsinanincreasedcatecholamine secretion, which inturnactivates theadenylylcyclasepathway leading to the conversion of ATP into cyclic adenosine mono- phosphate-cAMPandproteinkinaseA(PKA)activation[14,40,41].

PKAthenstimulateshormone-sensitivelipase,whichhydrolyses adipocyte-stored triglycerides into free fattyacids and glycerol [14,40,41].Inaddition,adipocytetriglyceridelipase(ATGL)maybe activatedsimultaneouslytohormone-sensitivelipase,resultingin approximately95%oftriglycerideshydrolysisinadipocytes[40].

Asexpected,glycerollevelsraisedbetweenM0andM1inthe placebogroup,whichcanbeexplainedbythefactthatexercise increasesenergeticdemand,promotingcatecholaminessecretion and, therefore, increasing triglycerides lipolysis rate and blood flowinadipose and musculartissues,enabling FFAuptake into muscles[13,42,43].Ontheotherhand,thehigherlevelsofglycerol observed in the experimental group show that microcurrent promotes ahigher mobilizationof abdominalfat,thusenabling higherFFAconsumptionduringmoderateaerobicexercise.

As microcurrent applicationmay activatelipolysis,we com- bined it witha singleexercise session inordertopromote FFA

b

^{-oxidationand}consequentlyreduceFFAre-esterificationinthe adiposetissue[7,44].Thus,alongersession(30minormore)of moderateaerobicexercise (Vo₂max25%to65%)wasappliedto promoteFFAmuscleuptake,inoppositiontoanaerobicexercise, which uptakes preferentially carbohydrates through glycolysis, beingmoreeffectiveonbodyfatreduction[13,43].Carbohydrates, FFAavailabilityandmuscleoxidationmaybeaffectedbyseveral factors other than exercise duration and intensity, including dietary intake, physical performance and body composition [33,43,45]. Even though we verified the homogeneityof these baselinevariablesbetweenexperimentalandcontrol

Theincreaseintriglyceride,totalcholesterolandLDLcholes- terolobservedintheplacebogroupafterexercisemaybeduetoa blood volume decrease [46]. Despite the significance of these results,theclinicalrelevanceofmedianvaluesislimited.

Thelipoproteinvaluescorrelationwiththeamountofglycerol released into bloodstream showed a non-significant and weak correlationinbothgroups,whichcanbeexplainedbythefactthat glycerol mobilizationis not associatedwith TGand cholesterol levels. However, participants in this sample were healthy individuals,normolipidicandnon-obese.

Regardinganthropometricmeasures, only theplacebogroup presentedapositivelysignificantcorrelationbetweentheamount Table3

Characterizationofdietaryintake,bygroup.

Md(IQR) Dif.Groups

EG PG pEGvsPG

Calories (Kcal)

2208.37(376.21) 2699.19(812.49) 0.297 Carbohydrates

(g)

266.80(61.35) 324.22(101.26) 0.545 Protein

(g)

100.43(14.03) 120.58(32.58) 0.199 TotalFat

(g)

81.50(14.20) 89.88(36.78) 0.287 SaturatedFat

(g)

25.41(4.61) 27.43(10.73) 0.410 MonounsaturatedFat

(g)

33.59(7.16) 36.73(14.07) 0.335 PolyunsaturatedFat

(g)

12.92(2.20) 15.53(5.73) 0.276 Cholesterol

(mg)

293.51(62.61) 364.63(108.06) 0.221 n-3FattyAcid

(g)

1.30(0.41) 1.62(0.53) 0.388 n-6FattyAcid

(g)

9.91(2.53) 10.80(4.53) 0.358 TotalFiber

(g)

23.23(5.24) 28.58(13.86) 0.171 Sugars

(g)

121.51(34.83) 134.56(58.75) 0.585 Alcohol

(g)

1.05(1.32) 1.43(1.32) 0.851 Calcium

(mg)

1055.05(338.15) 1144.05(386.73) 0.325 Sodium

(g)

1981.2(394.93) 2467.29(490.41) 0.253 Caffeine

(mg)

89.10(38.63) 72.51(42.14) 0.325

Abbreviations;EG.experimentalgroup;PGplacebogroup.

Dataareexpressedasmedian(Md)andInterquartiledeviation(IQR).

pvaluewithMann-Whitney.

Table4

Lipidsprofileandserumglycerol:Cholesterol,Triglycerides,Glycerol,LDLandHDL.

Mean(SD) Dif.Groups

Glycerol(mmol/l)

p^aM0VsM1

EG PG p^bEGvsPG

M0 M1 M0 M1 M1 M1-M0

0.04 0.15 0.04 0.09 0.003 0.004

(0.04) (0.09) (0.02) (0.07)

0.001 0.001

TotalCholesterol(mg/dl)

p^aM0VsM1

168.63 171.46 161.17 166.22 0.511 0.373

(40.93) (40.41) (29.42) (31.23)

0.174 0.001

Triglycerides(mg/dl)

p^aM0VsM1

88.58 87.67 84.07 90.05 0.791 0.078

(38.53) (36.02) (45.70) (45.01)

0.755 0.023

LDL(mg/dl)

p^aM0VsM1

69.75 80.17 76.24 77.69 0.684 0.999

(23.21) (21.05) (16.01) (16.04)

0.164 0.005

HDL(mg/dl)

p^aM0VsM1

61.00 60.94 55.67 56.55 0.179 0.596

(6.09) (10.51) (7.88) (8.96)

0.797 0.418

AbbreviationsEG.experimentalgroup;PGplacebogroup;M0,baselinemoment;M1,aftertheinterventionprotocol;M1-M0.differencevariable.

p^avaluewithpaired-samplest-test;p^bvaluewithindependentt-test.

ofglycerol releasedand waistcircumference, subcutaneousfat, visceralfatandabdominalandsuprailiacskinfold.Thissuggests that exercise increases lipolysis preferably in individuals with greater abdominal fat mass, as previously described, which suggeststhat individualswithhigher fathavehighermetabolic activity due to higher lipolytic response and lower insulin sensitivity [47]. On the other hand, we did not observe these results in the experimental group, which showed a weak correlation,suggestingthattheapplicationofexerciseassociated withmicrocurrentmaystimulatelipolysisinbothhighandlow abdominalfatmassindividuals.

Weregisteredapositivecorrelationbetweentotalbodyfatand glycerolmobilizationinbothgroupsalthoughttheapplicationof microcurrentwaslocal,abdominalregion,suggestingitcoulddue totheexerciseeffect.

Genderanalysisshowedsignificantlyhigherglycerollevelsin the experimental group than in the placebo group after the interventioninbothgenders.Moreover,intheexperimentalgroup andplacebogroupfemalespresentedgreaterglycerolmobilization thanmales.After theinterventionfemale glycerol mobilization was 140% and 60% compared to baseline level in EG and PG, respectively. The same profile was verified in male glycerol mobilization(90%and30%comparedtobaselinelevelinEGand PG,respectively).Infact,otherstudieshaveshowngreaterglycerol mobilizationinwomenduringandaftermoderateaerobicexercise [48,49]. This gender-dependent mobilization is yet to be completely explained. Glisezinski et al. (2007) suggested this couldbeduetothehigherquantityofadiposetissueinwomen [50]. Thus, higher fatmass maycorrelateto glycerol-measured lipolyticactivity [51],enhanced by exercicein associationwith

microcurrent.Thesediferencesbetweengendersmayberelated not only to adipocite’ssensitivity, but also to specific lipolytic properties [52,53] and balance between

b

^{- and}

a

-adrenergic receptors.Infact,thedistributionof

b

^-and

a

-adrenergicreceptors in the abdominal region is gender-dependent, with women’s adipocytespresenting lower number of

a

-adrenergic receptors andlowersensitivityintheabdominalregion[54,55].Considering that these receptors inhibit lipolysis [56] and determine the lipolyticresponse[52],theirloweractivityintheabdominalarea inwomenmayberelatedtotheresultsobtainedinthepresent study, and suggests that these may be independentof gender differences in catecholamine concentrations during physical exercise[52].

Hormonal factors furtherexplain these differences between genders,particularlyestrogenconcentrationandtheproportionof musclefiberswhichinterferewiththeeffectsofphysicalexercise, andevenmicrocurrent[9,57,58].Infact,womenhaveanincreased numberofoxidativemusclefibers(typeIfibers)[59]thatincrease theconcentrationofHSLcomparedtomusclesrichintypeIIfibers [34].

Somelimitationsofthepresentstudyshouldbeaddressed.On average, the subjects in our study showed most measured parametersnearorattherecommendedlevels,whichlimitsthe generalizationofourfindings tohigh-riskcsubjects.Inorderto complementthisstudy,wesuggesttheidentificationandcontrol ofparticipants’basalmetabolismthroughrespiratorycoefficient evaluation.Furtherstudiesshouldcoverbloodtestsbetweenthe microcurrent application and the exercise protocol, in order to identifytheexactamountofglycerol,triglycerides,totalcholes- terol,LDLandHDLreleasedbymicrocurrent,andalsoovertime (hoursafterprotocolintervention)toseehowlongtheeffectlasts.

5. Conclusion

A single abdominal microcurrent session seems tohave an additionaleffecttoaerobicexercise,increasingglycerolmobiliza- tion to blood stream, due to an increase in the hydrolysis of triglycerides.

Malesandfemalesbenefitfromtheapplicationofmicrocurrent withexercise,experiencinganincreaseinlipolyticactivity.

Theapplicationofmicrocurrentisadvantageousinindividuals withdifferentlevelsofabdominalfat.

Conflictofinterest

Theauthorshavenoconflictsofinteresttodeclare.Allauthors have reviewed the manuscript and take responsibility for its content.

Acknowledgments

TheauthorsaregratefultotheparticipantsandNunoDuartefor theEnglishReview.

Table5

Correlationbetweenglycerolmobilization(M1-M0).andabdominalfat.cholesterol.triglyceridesandanthropometricmeasurements.

TG(mg/

dl)

CT(mg/

dl)

TotalFat (%)

WaistCircum- ference(cm)

SAT-waist (mm)

VAT-waist (mm)

Abdomi-nal Skinfold(mm)

Supra-iliac Skinfold(mm)

BMI(Kg/

m²)

IPAQ(MET-min/

week) Glycerol

(mmol/l)

PG r= 0.239 r= 0.193 r=0.446 r=0.353 r=0.382 r=0.621 r=0.346 r=0.409 r=0.267 r= 0.086 p=0.133 p=0.228 p=0.004 p=0.023 p=0.015 p=0.002 p=0.027 p=0.008 p=0.092 p=0.601 EG r=0.114 r=0.260 r=0.398 r= 0.030 r=1.85 r=0.363 r=0.185 r=0.152 r=0.059 r=0.027 p=0.471 p=0.096 p=0.009 p=0.849 p=0.266 p=0.105 p=0.241 p=0.336 p=0.711 p=0.867 AbbreviationsEG.experimentalgroup;PGplacebogroup;CT.cholesterol;TG.triglycerides;SAT.subcutaneousadiposetissue;VAT.visceraladiposetissue;BMI.BodyMass Index;IPAQ.internationalphysicalactivityquestionnaire.

pvalueandrvaluefromPearsoncorrelation.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

M0 M1 M0 M1

GE GP

Glycerol (mmol/l)

Male Female p^a=0.014

p^b=0.030

p^a=0.024 p^b=0.004

EG PG

Fig.2.–Glycerolmobilizationatbaselineandfollowup,bygender.

Abbreviations:EG,experimentalgroup;PGplacebogroup;M0,baselinemoment;

M1,after theinterventionprotocol;Data are expressedasmedian(Md)and Interquartiledeviation(IQR)PavaluewithMann-WhitneyMalevsfemalePbvalue withMann-WhitneyGEvsGP.

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