Mangiferin Accelerates Glycolysis and Enhances Mitochondrial Bioenergetics

16 Pages·2017·1.3 MB·English

International Journal o f
Molecular Sciences
Article
Mangiferin Accelerates Glycolysis and Enhances
Mitochondrial Bioenergetics
ZhongboLiu1,PashaApontes1,EkaterinaV.Fomenko1,NanChi1,VictorL.Schuster1,2,
IrwinJ.Kurland1,3,JeffreyE.Pessin1,4andYulingChi1,*
1 DepartmentofMedicine,AlbertEinsteinCollegeofMedicine,Bronx,NY10461,USA;
[email protected](Z.L.);[email protected](P.A.);[email protected](E.V.F.);
[email protected](N.C.);[email protected](V.L.S.);
[email protected](I.J.K.);[email protected](J.E.P.)
2 DepartmentofPhysiologyandBiophysics,AlbertEinsteinCollegeofMedicine,Bronx,NY10461,USA
3 EinsteinStableIsotopeandMetabolomicsCore,AlbertEinsteinCollegeofMedicine,Bronx,NY10461,USA
4 DepartmentofMolecularPharmacology,AlbertEinsteinCollegeofMedicine,Bronx,NY10461,USA
* Correspondence:[email protected];Tel.:[phone]
Received:1December2017;Accepted:27December2017;Published:9January2018
Abstract: One of the main causes of hyperglycemia is inefficient or impaired glucose utilization
by skeletal muscle, which can be exacerbated by chronic high caloric intake. Previously, we
identified a natural compound, mangiferin (MGF) that improved glucose utilization in high fat
diet(HFD)-inducedinsulinresistantmice. TofurtheridentifythemolecularmechanismsofMGF
actiononglucosemetabolism, weconductedtargetedmetabolomicsandtranscriptomicsstudies
of glycolyic and mitochondrial bioenergetics pathways in skeletal muscle. These data revealed
that MGF increased glycolytic metabolites that were further augmented as glycolysis proceeded
fromtheearlytothelatesteps. ConsistentwithanMGF-stimulationofglycolyticfluxtherewas
a concomitant increase in the expression of enzymes catalyzing glycolysis. MGF also increased
importantmetabolitesinthetricarboxylicacid(TCA)cycle,suchasα-ketoglutarateandfumarate.
Interestinglyhowever,therewasareductioninsuccinate,ametabolitethatalsofeedsintotheelectron
transportchaintoproduceenergy. MGFincreasedsuccinateclearancebyenhancingtheexpression
andactivityofsuccinatedehydrogenase,leadingtoincreasedATPproduction. Atthetranscriptional
level, MGF induced mRNAs of mitochondrial genes and their transcriptional factors. Together,
these data suggest that MGF upregulates mitochondrial oxidative capacity that likely drives the
accelerationofglycolysisflux.
Keywords: mangiferin;glucose;glycolysis;TCAcycle;mitochondrialbioenergetics;metabolomics;
transcriptomics
1. Introduction
Thehallmarkofdiabetesishyperglycemia. Oneofthemaincausesofhyperglycemiaisinefficient
orimpairedglucoseutilizationinskeletalmuscle,whichcanbeexacerbatedbychronichighcaloric
intake. Followinguptake,glucosemetabolismtakesplaceinthreepathways,glycogensynthesis,the
pentosephosphateshuntandglycolysis. Thelastonegeneratespyruvate,akeymetabolicbranching
pointatwhichcarbohydratemetabolismeithergoesthroughtheanaerobicpathwaytoformlactateor
theaerobicpathwaytogenerateenergyinmitochondria. Whileformationoflactateisarelativelyfast
wayforglucosemetabolism,theoxidativemetabolisminmitochondriaproducesmuchmoreenergy
andisamoreeffectivepathwaytoutilizecarbohydratenutrientsandeliminateenergystorageinform
of,forinstance,fat.
Int.J.Mol.Sci. 2018,19,201;doi:10.3390/ijms19010201 www.mdpi.com/journal/ijms
Int.J.Mol.Sci. 2018,19,201 2of16
Theoxidativemetabolismofcarbohydratesinmitochondriaiscomposedoftwoprocesses,the
tricarboxylicacid(TCA)cycleandtheelectrontransportchain(ETC).PyruvateenterstheTCAcycle
mainlyviatwopossiblepathways,pyruvatedecarboxylationbypyruvatedehydrogenase(PDH)to
formacetyl-CoA,whosesubsequentmetabolitesfeedintotheETCformitochondrialrespirationto
produceenergy,orcarboxylationbypyruvatecarboxylasetoformoxaloacetate,whichparticipates
in TCA cycle anaplerosis [1,2]. The majority of pyruvate enters the TCA cycle via the irreversible
pyruvatedecarboxylationbyPDH[2]. Inaddition,therearetwootherirreversiblereactionsinthe
TCAcycle,citratesynthase(CS)catalyzedformationofcitratefromacetyl-CoAandα-ketoglutarate
dehydrogenase(α-KGDH)catalyzedformationofsuccinyl-CoAfromα-KG.Basedonthereversibility
ofcatabolicreactions,theTCAcycleisthoughttooperateintwosequentialsegments,thefirstbeing
fromacetyl-CoAuntilα-KGandthesecondbeingfromα-KGtooxaloacetate[3].
Impaired glycolysis and mitochondrial bioenergetics have been reported under pathological
conditionssuchasobesityanddiabetes[4]. Previousstudieshaveidentifiedseveralenzymaticsteps
thataredysregulatedindiabetesinvivoorunderhyperglycemicconditionsinvitro. Inparticular,
phosphofructose kinase (PFK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are
down-regulatedintermsofbothexpressionandactivity[5–12].Significantlyreducedrestingmetabolic
fluxthroughtheTCAcycleandoxidativephosphorylationwerereportedinskeletalmuscleofpatients
with type 2 diabetes and insulin resistant offspring of type 2 diabetic patients [13–15]. Reduced
metabolitesintheTCAcyclewerealsoreportedinhighfatdiet(HFD)-fedmice[2,16]. Inparallel
withreducedmetabolicflux,mitochondrialDNA(mtDNA)andthetranscriptionofmitochondrial
genes are also suppressed in skeletal muscle in insulin-resistant individuals [17,18]. It has been
proposed that correction of impaired catalytic steps in glycolysis and mitochondrial bioenergetics
couldmitigatehyperglycemia. Thus, therehasbeengreatinterestinidentifyingmechanismsthat
increase glycolysis and mitochondrial bioenergetics, and based on such mechanisms identifying
nutraceuticals/pharmaceuticalsthatcanimproveglucosehomeostasis.
MGF is a C-glucosyl xanthone (C2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) that is
apredominantconstituentinextractsofthemangoplantMangiferaindicaL.Recently,wereported
thatMGFcanenhanceinsulinsensitivityandmitigateglucoseintoleranceinHFD-inducedinsulin
resistantmice[19]. Inaddition,MGFenhancedglucoseoxidationbothinculturedC2C12myotubes
and,particularlyinterestingly,inskeletalmuscleisolatedfromobeseandinsulinresistantmiceinduced
byHFDfeeding. WehavealsodemonstratedthatMGFactivatesPDH,whichpresumablydirects
glucose metabolism to the mitochondrial oxidative processes. However, detailed information on
howMGFaffectseachstepofglycolysisandmitochondrialoxidativemetabolismremainsunknown.
Inthisstudy, we investigated the molecular mechanisms responsible for the metabolic beneficial
effectsofMGFbyusingtargetedmetabolomicsandtranscriptomics.
2. Results
2.1. ProfilingofMetabolitesinGlucoseMetabolism
Inourpreviousstudy,wefoundthatMGForallyadministeredtomiceonHFDwasabletoreduce
plasmaglucoseandinsulinlevels[19]. WealsofoundthatMGFenhancedcarbohydrateoxidationin
skeletalmuscleofmicefedeitherchoworHFD[19]. Thus,weproposedthattheMGF’sabilitiesof
sensitizinginsulinsignalingandloweringglucoseis,atleastpartially,duetoMGFenhancedoxidative
utilizationofglucoseinskeletalmuscle.Theprocessesofoxidativeutilizationofcarbohydratesinclude
glycolysis,andsubsequententryintotheTCAcycleandtheETC.TounderstandhowMGFaffected
thesemetabolicprocesses,weisolatedquadricepsskeletalmusclefrommicemaintainedonanormal
lowfatorHFDandweretreatedwithorwithoutMGFfor16weeks,aspreviousreported[19]and
analyzedmetabolitesinthoseskeletalmuscletissues.
Quantitative targeted metabolomics of several metabolic intermediates are listed in
Table 1. Among all metabolites listed in Table 1, HFD significantly increased one metabolite,
Int.J.Mol.Sci. 2018,19,201 3of16
glucose-6-phosphate. Conversely,itsignificantlyreducedseveralothermetabolitesintheglycolysis
andtheTCAcycleincludingphosphoenolpyruvate(PEP),pyruvate,α-KG,succinate,fumarateand
aspartate. OverallMGFsignificantlyaffectedmoremetabolitesinHFD-fedmicethaninchowfedmice.
2.2. MGFAcceleratesGlycolysis
2.2.1. MGFIncreasesGlucoseMetabolismviaGlycolysis
TomoreclearlydemonstratetheeffectsofMGFinthecontextofmetabolicpathways,wepresent
significantly altered metabolites in bar graphs in the pathways (Figure 1). The first metabolite of
glucoseisglucose-6-phosphate,whichcanbemetabolizedbythreepathways,glycogensynthesis,the
pentosephosphateshunt,andglycolysis. Urindinediphosphateglucose(UDPG)istheproductof
glucose-6-phosphateandprecursorofglycogen. MGFdidnotsignificantlyaffectUDPGineitherchow
orHFD-fedmice(Table1),suggestingthatMGFprobablydidnotaffectglycogensynthesispathway
(Figure1). Astothepentosephosphateshunt,MGFdidnotsignificantlyaffect6-phosphogluconate
(animportantintermediatemetabolite),norribose-5-phosphate(animportantterminalmetabolite)
(Table1),suggestingthatMGFprobablydidnotexertanysignificanteffectsonthispathway(Figure1).
Incontrast,MGFincreasedglucose6-phosphate,F-1,6-bisphosphate,3-phosphoglycerateandPEP
inchowand/orHFD-fedmice. Themagnitudeofthesedifferencesbecamegreater, i.e., theratios
(MGF/Ctl)increased,asglycolysisproceededfromtheearlystepstothelatesteps,indicatingthatMGF
acceleratedmetabolicfluxviatheglycolysispathway. Theeffectsaremoresignificantandprofoundin
HFD-fedmicethaninchowmice(Table1,Figure1).
2.2.2. MGFInducesSomeEnzymesinGlycolysis
TofurtherunderstandhowMGFmightalterthemetabolitesinglycolysis,weprofiledmRNAs
ofthoseenzymesinmuscletissuesfromthosechoworHFD-fedmicetreatedwithorwithoutMGF.
HFDsignificantlyreducedthemRNAofGAPDH,whereasHFDhadnosignificanteffectontheother
glycolyticenzymemRNAsexamined(Figure2). However,MGFtreatmentinducedthemRNAsof
three enzymes, HK, PFK, and GAPDH, in both chow and HFD-fed mice. Among these enzymes
GAPDHistheoneonwhichMGFexertedthegreatesteffect(Figure2).
Int.J.Mol.Sci. 2018,19,201 4of16
Table1.Profilesofmetabolitesinquadricepsofmice.
Chow HFD HFD/Chow(Ctl)
Metabolite Ratio *p(MGF Ratio *p(MGF
Ctl MGF Ctl MGF Ratio *p
(MGF/Ctl) VersusCtl) (MGF/Ctl) VersusCtl)
UDPG 7.95 7.63 0.96 0.115 6.40 5.89 0.92 0.316 0.80 0.098
Glucose-6-phosphate 6.93 8.90 1.28 0.272 13.19 18.84 1.43 0.018 1.90 0.034
Fructose-1,6-bisphosphate 44.17 58.90 1.33 0.099 54.28 76.40 1.41 0.043 1.23 0.451
3-Phosphoglycerate 26.03 34.09 1.31 0.089 28.39 46.18 1.63 0.030 1.09 0.445
Phosphoeolpyruate 1.58 2.18 1.38 0.054 0.89 2.27 2.54 0.024 0.57 0.025
Pyruvate 20.48 25.11 1.23 0.175 15.41 18.94 1.23 0.083 0.75 0.038
Lactate 350.59 272.19 0.78 0.046 313.57 213.15 0.68 0.029 0.89 0.144
Glycerol-3-phosphate 120.48 131.00 1.09 0.329 103.34 133.68 1.29 0.152 0.86 0.274
Ribose-5-phosphate 0.39 0.43 1.09 0.216 0.44 0.47 1.05 0.688 1.13 0.406
6-Phosphogluconate 1.40 1.58 1.13 0.193 1.19 1.07 0.89 0.217 0.85 0.130
α-Ketoglutarate 0.45 0.67 1.48 0.043 0.28 0.62 2.20 0.041 0.63 0.035
Succinate 50.81 31.23 0.61 0.038 36.96 21.85 0.59 0.032 0.73 0.050
Fumarate 8.17 11.24 1.38 0.049 6.01 9.09 1.51 0.041 0.74 0.049
Malate 43.89 48.31 1.10 0.121 35.64 40.45 1.13 0.322 0.81 0.084
Aspartate 26.48 29.53 1.12 0.090 19.45 20.82 1.07 0.210 0.73 0.042
C57BL6/JmicewerefedchoworHFDandtreatedwithMGForwithoutMGFfor16weeks.Micewerethensacrificedafterfastingfor12hfrom10p.m.to10a.m.Theirquadricepswere
collectedandsnapfrozeninliquidN2.Metaboliteswereanalyzedbythemetabolomicsstudy.Valuesareaverage,n=6.*p<0.05isconsideredsignificant.HFD,highfatdiet;MGF,
mangiferin;Ctl,control;UDPG,urindinediphosphateglucose.
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Int. J. Mol. Sci. 2018, 19, 201 5 of 17

Figure 1. MGF accelerates glycolysis. Glucose metabolic pathways include glycogen synthesis,
Figure 1. MGF accelerates glycolysis. Glucose metabolic pathways include glycogen synthesis,
pentose phosphate shunt and glycolysis. Arrows in the glycolysis pathway indicate mangiferin
pentose phodspirehcatetde msehtaubonltic aflunxd frogmly ocnoel pyrsoidsu.ct Ato rthreo nwexst. iAnrrtohwes ign loythceor lpyasthiswapyas tmhewana cyhaninged oifc oantee mangiferin
directedmetambetoalbiocliflc purxodfurcot mto tohne eothperro. Sdigunciftictaonttlhy ealtnereexdt m.Aetarbrooliwtess airne portehseenrtepda inth bwar agyrasphms. eVaanluecsh angeofone
are average ± SEM (n = 6). * p < 0.05. HK, Hexose kinase; PGI, Phosphoglucose isomerase; PFK,
metabolicproducttotheother.Significantlyalteredmetabolitesarepresentedinbargraphs.Values
Phosphofructokinase; FBA, Fructose-1,6-bisphosphate aldolase; GAPDH, Glyceraldehyde 3-
areaverage ±phoSspEhMate (dneh=yd6r)o.ge*napse<; P0G.K0,5 .PhHospKh,ogHlyecxeroatsee kkiniansae;s PeG;MPG, PIh,oPsphhoosgplyhceoragtel umcuotsaese;i sPoKm, erase; PFK,
PhosphofructPoykruivnaates e;kiFnBasAe; ,FPrEuP,c tophsoes-p1h,6oe-nboislppyhruovsape;h aLtDeHa, ldlaocltaatsee ;dGehAydPrDogHen,aGse;l yPcDerHa, ldpeyhruyvdatee 3-phosphate
dehydrogenase; UDPG, urindine diphosphate glucose.
dehydrogenase;PGK,Phosphoglyceratekinase;PGM,Phosphoglyceratemutase;PK,Pyruvatekinase;
PEP, phosphoenolpyruvae; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; UDPG,
urindinediphosphateglucose.
Int. J. Mol. Sci. 2018, 19, 201 6 of 17

Figure2. MGFFigiunrde 2u. cMeGsFm inRdNuceAs smoRNfsAos mof esoemnez eynmzymesesp paarrttiiccipipataintgi ning gilnycoglylysics.o Plryosfiilses. oPf rmoRfiNleAss ooff mRNAsof
glycolyticenzgylymcoelystiicn enqzuymadesr iinc eqpuasdfrircoepms fmromic meitcree taretaetdedw wiitthh chcohwo wor HoFrDH ±F MDGF± foMr 1G6 wFefeokrs. 1V6aluwese eks.Values
are average ± SEM (n = 6/group). * p < 0.05, ** p < 0.01.
areaverage±SEM(n=6/group).*p<0.05,**p<0.01.
2.3. MGF Enhances Mitochondrial Bioenergetics
2.3.1. MGF Enhances Metabolism in the TCA Cycle
Accelerated glycolysis by itself would lead to an increase in the end product, pyruvate.
However, there was no significant increase in pyruvate levels in the MGF treated mice, suggesting a
more rapid clearance of pyruvate. Pyruvate can be catabolized to several products via different
pathways. Pyruvate can be converted to lactate by lactate dehydrogenase (LDH) via the anaerobic
pathway or enter the TCA cycle to be metabolized via the aerobic pathway. Figure 1 shows that there
is significantly less lactate produced in MGF treated mice, suggesting that the MGF-increased
clearance of pyruvate was not via the anaerobic pathway in the cytosol, but rather via the TCA cycle
occurring in mitochondria.
In mitochondria, the majority of pyruvate is catabolized to acetyl CoA by PDH [2]. Acetyl CoA
is then further metabolized to α-KG, which is a marker of the TCA flux during the first segment. We
have previously reported that MGF activates PDH [19], and therefore we hypothesized that MGF
accelerated the first segment of the TCA cycle. Indeed, MGF significantly increased α-KG, by about
150% in chow mice and by 220% in HFD muscle (Table 1, Figure 3). Interestingly, MGF elevated α-
KG did not result in any increase in the subsequent metabolite of α-KG, succinate. Rather, MGF
significantly reduced succinate by about 40% in chow and HFD muscles. On the other hand, MGF
significantly increased fumarate formed from succinate by SDH by 138% in chow mice and by 151%
in HFD muscle (Table 1, Figure 3). Fumarate is in rapid exchange with other four-carbon
intermediates, malate and oxaloacetate, which can be reversibly converted to aspartate. MGF
increased all these metabolites in the second segment of the TCA cycle, albeit some increases were
insignificant.
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2.3. MGFEnhancesMitochondrialBioenergetics
2.3.1. MGFEnhancesMetabolismintheTCACycle
Acceleratedglycolysisbyitselfwouldleadtoanincreaseintheendproduct,pyruvate. However,
there was no significant increase in pyruvate levels in the MGF treated mice, suggesting a more
rapidclearanceofpyruvate. Pyruvatecanbecatabolizedtoseveralproductsviadifferentpathways.
Pyruvate can be converted to lactate by lactate dehydrogenase (LDH) via the anaerobic pathway
or enter the TCA cycle to be metabolized via the aerobic pathway. Figure 1 shows that there is
significantlylesslactateproducedinMGFtreatedmice,suggestingthattheMGF-increasedclearance
ofpyruvatewasnotviatheanaerobicpathwayinthecytosol,butratherviatheTCAcycleoccurring
inmitochondria.
Inmitochondria,themajorityofpyruvateiscatabolizedtoacetylCoAbyPDH[2]. AcetylCoAis
thenfurthermetabolizedtoα-KG,whichisamarkeroftheTCAfluxduringthefirstsegment. We
have previously reported that MGF activates PDH [19], and therefore we hypothesized that MGF
acceleratedthefirstsegmentoftheTCAcycle. Indeed,MGFsignificantlyincreasedα-KG,byabout
150% in chow mice and by 220% in HFD muscle (Table 1, Figure 3). Interestingly, MGF elevated
α-KGdidnotresultinanyincreaseinthesubsequentmetaboliteofα-KG,succinate. Rather,MGF
significantlyreducedsuccinatebyabout40%inchowandHFDmuscles. Ontheotherhand,MGF
significantlyincreasedfumarateformedfromsuccinatebySDHby138%inchowmiceandby151%in
HFDmuscle(Table1,Figure3). Fumarateisinrapidexchangewithotherfour-carbonintermediates,
malate andoxaloacetate, which can bereversiblyconverted to aspartate. MGF increased allthese
metabolitesinthesecondsegmentoftheTCAcycle,albeitsomeincreaseswereinsignificant.
Int. J. Mol. Sci. 2018, 19, 201 7 of 17

Figure 3. MGF modulates metabolite profiles in the TCA cycle. One-end arrows mean unidirectional
Figure3.MGFmodulatesmetaboliteprofilesintheTCAcycle.One-endarrowsmeanunidirectional
and two-end arrows mean bidirectional. Significantly altered metabolites are presented in bar graphs.
andtwo-endarVroalwuess amre eavaenragbei d± SiEreMc (tni o= n6)a. *l p. <S 0i.g05n. iPfiCc, paynrutlvyatea clatrebroexdylamse;e PtDaHb,o plyirtuevsatae rdeehpydrerosgeennatseed; inbargraphs.
ValuesareaveraCgSe, c±itraSteE Msyn(thnas=e; 6A).C,* Apc<on0ita.0se5; .IDPHC, ,ipsoycirturavtea tdeehcyadrrobgoenxaysela; sαe-K;GPDDHH, ,α-pkyetrouglvutaatraeted ehydrogenase;
dehydrogenase; Suclg, succinyl coenzyme A synthetase; SDH, succinate dehydrogenase; FH,
CS, citrate synftuhmaasraes;e; MADCH,, mAaclaoten diethaysdero;geInDasHe; α,-KisGo, cαi-ktretaotgelutadraethe.y drogenase; α-KGDH, α-ketoglutarate
dehydrogenase;Suclg,succinylcoenzymeAsynthetase;SDH,succinatedehydrogenase;FH,fumarase;
2.3.2. MGF Enhances SDH Activity and Overall Energy Production in Mitochondria
MDH,malatedehydrogenase;α-KG,α-ketoglutarate.
Succinate is an intermediate between α-KG and fumarate that were both increased, suggesting
that the decrease in succinate levels was due to enhanced succinate consumption. MGF significantly
increased the activity of SDH in mitochondrial isolated from quadriceps by 63% and 86% in mice fed
with chow and HFD, respectively (Figure 4a). To confirm these in vivo effects, we treated C2C12
myotubes with various concentrations of MGF for about 4 h and isolated mitochondria from those
cells and measured SDH activity. MGF also significantly increased SDH activity in those cells in a
dose-dependent manner (Figure 4b).
Among the metabolites in the TCA cycle, succinate is the only one that enters the ETC to
participate in a chain of oxidative reactions to ultimately produce energy, ATP. SDH is also the
mitochondrial complex II in the ETC. SDH activity is a marker of mitochondrial oxidative capacity
and positively related to energy production [20]. Thus, we measured ATP production in freshly
isolated mitochondria from snap frozen quadriceps. HFD significantly reduced ATP production
(Figure 4c). MGF significantly increased ATP production by about 150% in chow mice. Moreover,
MGF reversed the reduction in ATP in HFD mice and increased the level of ATP slightly higher than
that in chow control mice.
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2.3.2. MGFEnhancesSDHActivityandOverallEnergyProductioninMitochondria
Succinateisanintermediatebetweenα-KGandfumaratethatwerebothincreased,suggesting
thatthedecreaseinsuccinatelevelswasduetoenhancedsuccinateconsumption. MGFsignificantly
increased the activity of SDH in mitochondrial isolated from quadriceps by 63% and 86% in mice
fedwithchowandHFD,respectively(Figure4a). Toconfirmtheseinvivoeffects,wetreatedC2C12
myotubeswithvariousconcentrationsofMGFforabout4handisolatedmitochondriafromthose
cellsandmeasuredSDHactivity. MGFalsosignificantlyincreasedSDHactivityinthosecellsina
dose-Indte. Jp. Menold. Secni. 2t0m18,a 1n9,n 2e0r1 ( Figure4b). 8 of 17

Figure 4. MGF enhances SDH activity and increases ATP production. Mitochondria were isolated
Figure4. MGFenhancesSDHactivityandincreasesATPproduction. Mitochondriawereisolated
from quadriceps of mice treated with chow or HFD ± MGF for 16 weeks (n = 6/group) (a) or C2C12
fromquadricepsofmicetreatedwithchoworHFD±MGFfor16weeks(n=6/group)(a)orC2C12
myotubes treated with various concentrations of MGF for 4 h (b). In vitro experiment presented in (b)
myotubestreatedwithvariousconcentrationsofMGFfor4h(b).Invitroexperimentpresentedin(b)
was repeated for three times. The comparisons are between the rate at each MGF concentration with
wasrepeatedforthreetimes.ThecomparisonsarebetweentherateateachMGFconcentrationwith
the rate when [MGF] = 0; (c) ATP production in freshly isolated mitochondria from quadriceps of
theratewhen[MGF]=0;(c)ATPproductioninfreshlyisolatedmitochondriafromquadricepsofmice
mice treated with chow or HFD ± MGF for 16 weeks (n = 6/group). Values are average ± SEM. * p <
treate0d.05w, *it*h p c<h 0o.0w1. orHFD±MGFfor16weeks(n=6/group).Valuesareaverage±SEM.*p<0.05,
**p<0.01.
2.3.3. MGF Induces Mitochondrial Biogenesis
AmoTnog itnhveemstiegtaatbe owlitheesthinert hMeGTFC Ahacsy acnley, seuffceccitns aotne itshteh eexopnrleyssoionnest hoaf tmenittoecrhsotnhderEiaTl Cgetnoepsa, rwtiec ipate
inacphraoifnileodf moRxiNdAatsi vofe arlel aecntziyomnsest oinu thlteim TCatAe lcyycplero adnudc tehee nEeTrCg yfr,oAmT tPh.eS qDuHadrisiceaplsso sktheleetmal imtoucshcolen. drial
compHleFxDI fIeienditnhge cEauTsCe.dS rDedHucaticotniv iint ymiossat omf amriktoecrhoofnmdriitaol cghenoensd, railathloouxgihd amtiovset ocfa tphaecsiet ychaanndgeps odsidit ively
not reach statistical significance (Figure 5a,b). On the other hand, MGF significantly induced several
relatedtoenergyproduction[20]. Thus,wemeasuredATPproductioninfreshlyisolatedmitochondria
genes in the TCA cycle and the ETC in chow and/or HFD-fed mice (Figure 5a,b). These genes included
fromsnapfrozenquadriceps.HFDsignificantlyreducedATPproduction(Figure4c).MGFsignificantly
IDH, αKGDH, SDH, CoxI, Cyt-c, COXIV and ATP5g1. The greatest effects occur in SDH, which could
increasedATPproductionbyabout150%inchowmice. Moreover,MGFreversedthereductionin
be a factor contributing MGF enhanced catabolism of succinate to fumarate.
ATPinHFDmiceandincreasedthelevelofATPslightlyhigherthanthatinchowcontrolmice.
Mitochondrial genes are tightly regulated by several transcription factors including
transcriptional factor A (TFAM), nuclear respiratory factor 1 (NRF1) and estrogen-related receptor α
2.3.3. MGFInducesMitochondrialBiogenesis
(ERRα), and the co-factors of these transcriptional factors, peroxisome proliferator-activated receptor-
Tγo coinavcteivstaitgoart (ePwGChe)-t1hαe ranMdG PFGhCa-1sβa [n2y1–e2f6fe].c MtsGonF sthigeneifxicparnetslsyi oinncsreoafsmedi taolcl hoof nthdorsiea ltrgaennsecsri,pwtieonparlo filed
mRNfAacstoorsf, aelxlceepntz EyRmReαs, iinn cthhoewT CorA HcFyDc-lfeeda nmdiceth (eFiEguTrCe 5fcr)o, mindthiceatqinuga tdhraitc MepGsFs kupelreetgaullamteus stchloes.e HFD
enzymes in the TCA cycle and the ETC at the transcriptional level. PGC-1α is positively correlated to
feedingcausedreductioninmostofmitochondrialgenes, althoughmostofthesechangesdidnot
mtDNA. Therefore, we measured mtDNA contents in mitochondria isolated from those muscle
reachstatisticalsignificance(Figure5a,b). Ontheotherhand,MGFsignificantlyinducedseveralgenes
tissues. MGF almost doubled mtDNA in both chow and HFD-fed mice (Figure 5d). Together, these
intheTCAcycleandtheETCinchowand/orHFD-fedmice(Figure5a,b). ThesegenesincludedIDH,
results indicate that MGF induces mitochondrial biogenesis.
αKGDH,SDH,CoxI,Cyt-c,COXIVandATP5g1. ThegreatesteffectsoccurinSDH,whichcouldbea
factorcontributingMGFenhancedcatabolismofsuccinatetofumarate.
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Int. J. Mol. Sci. 2018, 19, 201 9 of 17

FiguFrieg5u.reM 5G. MFiGnFd uincdeuscmesi tmocithoocnhdonridarliabli obgioegneenseissi.s(. a(–ac–)c)P Prorofiflielesso offm mRRNNAAss ooff mmiittoocchhoonnddrriiaal lggeenneess (a,b)
and(tar,abn) sacnrdip ttriaonnsaclrifpatciotonrasl f(acc)tionrsq (uc)a dinr iqcueapdsroicfepmsi coef mtreicaet etrdeawteidth wcihtho wchoowr HorF DHF±D M± MGFGFfo fror1 616w eeks
(n=w6/egekros u(np =) ;6(/gdr)oRupel)a; (tdiv) eRemlaittiovceh mointodcrhioanDdrNiaA DNcoAn cteonnttesnitns iqnu qaudardircieceppsso off mmiiccee trtreeaatetde dwwithi tchhocwho wor
HFDor± HMFDG ±F MfoGrF1 6fowr 1e6e kwse.eVksa.l uVeasluaerse aarev earvaegraeg±e ±S SEEMM ((nn == 66)). .* *pp < <0.005.0, 5**, *p* <p 0<.010.. 01.
3. Discussion
Mitochondrialgenesaretightlyregulatedbyseveraltranscriptionfactorsincludingtranscriptional
Obese, insulin resistant and diabetic patients display impaired glycolysis and dysfunctional
factorA(TFAM),nuclearrespiratoryfactor1(NRF1)andestrogen-relatedreceptorα(ERRα),andthe
mitochondrial oxidation [4,13–15,17,18], which are major contributors to impaired glucose
co-factorsofthesetranscriptionalfactors, peroxisomeproliferator-activatedreceptor-γcoactivator
disposal/utilization and hence to hyperglycemia. Pharmaceutical and/or nutraceutical agents that can
(PGC)-1αandPGC-1β[21–26]. MGFsignificantlyincreasedallofthosetranscriptionalfactors,except
improve glycolysis and mitochondrial oxidative capacity have the potential for therapeutic value in
ERRα, in chow or HFD-fed mice (Figure 5c), indicating that MGF upregulates those enzymes in
treating diet induced obesity and insulin resistance. Using a HFD mouse model of obesity and insulin
theTreCsAistacnyccel,e waen pdretvhieouEsTlyC idaetntthifeietdr aa nnsacturirpalt icoonmaploleuvnedl,. MPGGFC, t-h1aαt iwsaps oasbiltei vtoe lcyorcroecrtr eHlFaDte dindtoucmedt DNA.
Thergelfuocroes,ew inetomleeraansucer eadndm intDsuNlinA recsoisnttaennctes biyn emnhiatoncchinogn gdluricaosies oultailtiezdatiforno m[19t]h. Uosseinmg musectalebotilsitseu aensd. MGF
almotsratndsocruipbtloedmimc tpDroNfiAlinign wbeo thhavche ofowunadn dthHat FMDG-fFe dgrmadiuceal(lyF iignucrreea5sedd). mToetgaebtohliecr ,fltuhxe sine rgelyscuolltyssiins,d icate
thatwMhGicFh iwnadsu fcaecislimtatietodc bhyo MndGrFia inldbuiocgeden eensziysm. es in glycolysis. In mitochondria, MGF directs pyruvate
metabolism in sequence from the first segment to the second. In such manner MGF enhances
3. Diosxciudastsiivoen metabolism, resulting in increased production of energy, ATP. MGF does so by
upregulating transcription of mitochondrial genes and enhancing the activity of SDH.
Obese, insulin resistant and diabetic patients display impaired glycolysis and dysfunctional
Glucose-6-phosphate is the first metabolite in glucose metabolism. HFD feeding almost doubled
mitogclhuoconsder-6ia-plhoosxpidhaattei opnrob[a4b,1ly3 –d1u5e, 1to7 ,H18F]D, bwluhntiecdh maerteabomlisamjo rat tchoen sturbibseuqtuoernst mtoetaibmoplica isrteedps, gaslu cose
disptohsearle/ wutaisl igzraatdiounallayn aduhgmenecnetetdo rheydpucetrigonly icne mmeiata.bPohliatersm fraocmeu tthiec aelaarlnyd s/teoprs ntou tthraec leautetri.c Naleaagr ethnet sthat
caniemndp roofv gelygcloylcyosilsy, sHisFaDn cdaumseitdo csihgonnifdicrainatl roexdiudcattiiovne inca PpEaPc iatyndh payvreutvhaetep (oTtaebnleti a1,l Ffoigrutrhee 1r)a. pOenu tthice value
intrecoantitnragryd, iMetGiFn dguracdeudalolyb eansidty siagnnidficiannstulyli inncrreesaissetdan mceet.abUosliitnegs aanHd tFhDe imncoreuasseesm woedree lauogfmoebnetseidty and
insulfirnomre sthiset aenacrley, wsteepps retov itohue sllayteird e(Tnatibfilee d1,a Fnigauturer a1l).c oTmhepseo urnesdu,ltMs GinFd,ictahtaet twhaats MabGleF tios caobrler etcot HFD
counteract the inhibitory effects of HFD on glycolysis.
induced glucose intolerance and insulin resistance by enhancing glucose utilization [19]. Using
The conversion of glucose to glucose-6-phosphate is catalyzed by HK. Previously Sellamuthu et
metaboliteandtranscriptomicprofilingwehavefoundthatMGFgraduallyincreasedmetabolicflux
al. [27] showed that MGF increased HK activity in liver of streptozotocin induced diabetic rats. Here
inglycolysis,whichwasfacilitatedbyMGFinducedenzymesinglycolysis. Inmitochondria,MGF
we show that MGF induces HK expression in muscle of both chow and HFD-fed mice (Figure 2). In
directspyruvatemetabolisminsequencefromthefirstsegmenttothesecond. InsuchmannerMGF
glycolysis, glucose-6-phosphate is subsequently converted to fructose-1,6-bisphosphate by PFK. While
enhatnhec eesffoecxtisd oaft iHveFDm oenta tbhoe liascmtiv,itrye souf lPtiFnKg iisn uinnccerretaaisne d[28p–r3o0d],u dcitaiboenteos freednuecrgesy ,thAeT ePx.pMresGsiFond oanesd soby
upre gulatingtranscriptionofmitochondrialgenesandenhancingtheactivityofSDH.
Glucose-6-phosphateisthefirstmetaboliteinglucosemetabolism. HFDfeedingalmostdoubled
glucose-6-phosphateprobablyduetoHFDbluntedmetabolismatthesubsequentmetabolicsteps,as
Int.J.Mol.Sci. 2018,19,201 9of16
therewasgraduallyaugmentedreductioninmetabolitesfromtheearlystepstothelater. Nearthe
endofglycolysis,HFDcausedsignificantreductioninPEPandpyruvate(Table1,Figure1). Onthe
contrary,MGFgraduallyandsignificantlyincreasedmetabolitesandtheincreaseswereaugmented
fromtheearlystepstothelater(Table1,Figure1). TheseresultsindicatethatMGFisabletocounteract
theinhibitoryeffectsofHFDonglycolysis.
Theconversionofglucosetoglucose-6-phosphateiscatalyzedbyHK.PreviouslySellamuthu
et al. [27] showed that MGF increased HK activity in liver of streptozotocin induced diabetic rats.
HereweshowthatMGFinducesHKexpressioninmuscleofbothchowandHFD-fedmice(Figure2).
Inglycolysis,glucose-6-phosphateissubsequentlyconvertedtofructose-1,6-bisphosphatebyPFK.
WhiletheeffectsofHFDontheactivityofPFKisuncertain[28–30],diabetesreducestheexpression
andactivityofPFK[31]. DeficiencyinPFKisassociatedwithimpairedinsulinsecretionandinsulin
resistanceinhuman[5,6,8,32]. MGFinducesPFK(Figure2)andthereforehasthepossiblecapability
ofmitigatingPFKdeficiencyindiabetes. MGFalsosignificantlyinducedthemRNAofGAPDH,which
isakeyenzymeintheglycolyticconversionofglucosetopyruvateandhasbeenconsideredtoplay
aregulatoryroleinsettingtheglycolyticrate[33,34]. ThereportsontheeffectsofHFDfeedingon
expressionofGAPDHareinconsistentprobablyduetotissuespecificity[35–37]. Hereweshowthat
HFD significantly reduced GAPDH mRNA in skeletal muscle (Figure 2). More importantly, MGF
treatmentcausedmorethan2-foldinductioninmRNAofGAPDHinbothchowandHFD-fedmice
(Figure2).
Theendproductofglycolysisispyruvate,whichcanbeconvertedtolactateviatheanaerobic
processorentermitochondriatobemetabolizedbytheaerobicprocess. WhileHFDdidnotcause
significantchangeinlactate,itsignificantlyreducedseveralkeymetabolitesintheTCAcycle(Table1,
Figure3),whichwasalsoreportedbyothers[2,16]. MGFmodulatedthosemetabolitesinabiologically
importantmanner. PyruvateisconvertedfromPEPbyPK.MGFincreasedPEP,butdidnotsuppress
PK.OnewouldexpectthattherecouldbesimilarmagnitudeofincreaseinpyruvateinMGFtreated
mice. However,MGFonlycausedmildandinsignificantincreaseinpyruvate,suggestingthatMGF
probablyenhancedpyruvateconsumption,eitherbyincreasinglactateproductionorbyenhancing
oxidative metabolic flux in the TCA cycle. MGF significantly reduced lactate in muscle tissues of
bothchowandHFD-fedmice(Table1,Figure1),whichrecapitulatestheeffectsofMGFonlactate
inculturedC2C12myotubesshowninourpreviousstudy[19]. WefurtherdemonstratedthatMGF
reducedlactateproductionnotbyinhibitingpyruvatecarboxylase(lactatedehydrogenase),butby
enhancing PDH activity, shuttling pyruvate to mitochondria. In mitochondria PDH catalyzes the
irreversibleconversionofpyruvatetoacetyl-CoA,whichwillbeirreversiblyconvertedtocitrateby
citratesynthase(CS).TheseunidirectionalreactionsmakecommitmenttotheflowoftheTCAcycleto
producetheendproductofthefirstsegmentoftheTCAcycle,α-KG.ThefactthatMGFincreased
α-KG,asshowninthisstudy(Table1,Figure3),confirmsthatMGFenhancesPDHandaccelerates
themetabolisminthefirstsegmentoftheTCAcycle. ThisenhancementisalsosupportedbyMGF
increasedCSand,moresignificantly,IDH(Figure5a).
Subsequently,α-KGisirreversiblyconvertedtosuccinyl-CoAbyα-KGDH.MGFsignificantly
increasedα-KGDH,presumablyincreasingsuccinyl-CoA,thestartingpointofthesecondsegmentof
theTCAcycle,leadingtotheproductionofoxaloacetate. Allofthereactionsinthesecondsegmentof
theTCAcyclearereversibleandsomeofthemetabolitesareinrapidexchangewitheachother. MGF
increasedmostmetabolitesinthesecondsegmentoftheTCAcycle,exceptsuccinate. Succinateisa
uniquemetabolite. ItistheonlymetaboliteintheTCAcyclethatalsofeedsintheETCandparticipates
inoxidativephosphorylation.Accumulationofsuccinateisamarkerofanaerobicmetabolism[3].MGF
significantlyreducedsuccinate,whichsupportstheideathatMGFenhancesoxidativemetabolism.
MGFeliminatessuccinateprobablybyenhancingSDH,asMGFupregulatesSDHatboththe
expressionandtheactivitylevels(Figures4a,band5a). SDHistheonlyenzymethatisacomponent
ofboththeTCAcycleandtheETC.IntheETC,itisnamedcomplexII.SDHcatalyzestheoxidation
ofsuccinatetofumarateandusestheelectronsderivedfromthisoxidationtocatalyzethereduction
Int.J.Mol.Sci. 2018,19,201 10of16
ofubiquinionetoubiquinol. TheseelectronsarepassedtocomplexIIIandthencomplexIV,thereby
contributing to the establishment of the electrochemical gradient across the mitochondria inner
membrane for ATP synthesis [38]. Among all mitochondrial complexes, complex II loses activity
mostrapidlyafterdeathandoninappropriatestorage,andthereforeitsactivityisconsideredasa
markeroftheintegrityofthetissueandthereliabilityofrespiratorychainenzymology[20]. Mutation
ofSDHgeneresultsinmitochondrialrespiratorychaindeficiency[39]. ReducedSDHexpressionand
activitywerereportedinobeseanddiabeticpatients[18]. HereweshowthatMGFupregulatesSHD
atbothexpressionandactivitylevelsinquadricepsofnotonlywildtypemicebutalso,importantly,in
obeseandinsulinresistantmice(Figures4a,band5a). MGFwasalsoshowntoenhanceSDHactivity
insoleusandtibialisofobeserats,andtoincreaseSDHactivityinheartofexperimentallyinduced
myocardialinfarctedrats[40,41].
TheeffectsofMGFonmitochondriaarenotlimitedtoSDH.InadditiontoSDH,MGFwasshown
toincreasetheactivitiesofotherimportantenzymesintheTCAcycleandtheETC,includingα-KGDH,
MDH and cytochrome c [41]. At the expression level, here we found that MGF induced mRNAs
ofmostenzymesintheTCAcycleandtheETC(Figure5a,b). Furthermore, MGFinducedseveral
importantmitochondrialtranscriptionalfactorsandtheirco-factorsincludingTFAM,NRF1,PGC-1α
andPGC-1β(Figure5c),aswellasincreasingmitochondrialDNAcontent(Figure5d). Takingtogether,
theseresultsindicatethatMGFenhancedoverallmitochondrialoxidativecapacity,whichprobably
drivestheacceleratedmetabolicfluxinglycolysis(Figure1).
Inadditiontoacceleratingglycolysis,MGFhasbeenshowntoreduceglucose6-phosphateand
fructosebisphosphataseandthereforeinhibitgluconeogenesis[27]. Theseeffects,together,suggest
thatMGFcouldbeaneffectiveanti-hyperglycemiaandanti-diabetesagent. Furthermore, weand
othershaveshownthatMGFhasanti-hyperlipidemiaeffectsinrodents[19,42–47],andimpressivelyin
humans[48].Asanaturalcompound,MGFhasbeenshowntouniquelypossessmulti-beneficialeffects
inmitigatingmetabolicdisorders. Thedrawbackisthatithasverylowbioavailability(1.2%)[49–51]
andthereforethedoserequiredforMGFtoexertsignificanteffectsishigh. Nevertheless,thestructural
featuresofMGFallowchemicalmodificationsandMGFhasthepotentialtobedevelopedintoan
effectivenutraceutical/pharmaceuticalforthetreatmentofcomplexmetabolicsyndrome.
4. MaterialsandMethods
ChemicalreagentswerepurchasedfromSigma-Aldrich(St. Louis,MO,USA)unlessspecified.
4.1. CellCulture
C2C12 myoblast cells were maintained in DMEM High Glucose (Hyclone, Logan, UT, USA),
supplementedwith1%penicillinandstreptomycin(GIBCO,Carlsbad,CA,USA)and10%FBS.C2C12
cells were differentiated into myotubes in the above DMEM medium containing 2% horse serum
(replacing10%FBS)for4–6daysaftercellsreachedconfluence.
4.2. AnimalExperiments
Wild type C57BL6/J mice at age of 5–6 weeks were purchased from Jackson laboratory
(BarHarbor,ME,USA).Allstudieswereapprovedbyandperformedincompliancewiththeguidelines
oftheEinsteinInstitutionalAnimalCareandUseCommittee. Chowdiet(CD)(providing28.5%(Kcal)
protein,13.5%fatand58%carbohydrates)wasobtainedfromPurinaLabDiet,Framingham,MA,USA.
HFD(providing20%(Kcal)protein,60%fatand20%carbohydrates)wasobtainedfromResearchDiets,
NewBrunswick,NJ,USA.MGFwasobtainedfromCTMedChem(Bronx,NY,USA)andadministered
orallyviafood. CDandHFDweregroundintofinepowderandmixedwithpureMGF.Themixtures
werere-pelletedandadministeredtoindividuallyhousedmice. MicewerefedadlibitumwithCDor
HFD±MGF(MGFdose,400mg/kgbodyweight)andwaterfor16weeks. Micewerethensacrificed
after12-hfastingfrom10p.m. to10a.m. Quadricepswerecollected,snapfrozeninliquidN and
2
storedat−80◦C.