Glucose Exerts an Anti-Melanogenic Effect by Indirect Inactivation of Tyrosinase in Melanocytes and a Human Skin Equivalent

Abstract

Sugars are ubiquitous in organisms and well-known cosmetic ingredients for moisturizing skin with minimal side-effects. Glucose, a simple sugar used as an energy source by living cells, is often used in skin care products. Several reports have demonstrated that sugar and sugar-related compounds have anti-melanogenic effects on melanocytes. However, the underlying molecular mechanism by which glucose inhibits melanin synthesis is unknown, even though glucose is used as a whitening as well as moisturizing ingredient in cosmetics. Herein, we found that glucose significantly reduced the melanin content of α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells and darkly pigmented normal human melanocytes with no signs of cytotoxicity. Furthermore, topical treatment of glucose clearly demonstrated its whitening efficacy through photography, Fontana-Masson (F&M) staining, and multi-photon microscopy in a pigmented 3D human skin model, MelanoDerm. However, glucose did not alter the gene expression or protein levels of major melanogenic proteins in melanocytes. While glucose potently decreased intracellular tyrosinase activity in melanocytes, it did not reduce mushroom tyrosinase activity in a cell-free experimental system. However, glucose was metabolized into lactic acid, which can powerfully suppress tyrosinase activity. Thus, we concluded that glucose indirectly inhibits tyrosinase activity through conversion into lactic acid, explaining its anti-melanogenic effects in melanocytes.

Full text

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Int.J.Mol.Sci.2020,21,1736;doi:10.3390/ijms21051736www.mdpi.com/journal/ijms
Article
GlucoseExertsanAnti‐MelanogenicEffectby
IndirectInactivationofTyrosinaseinMelanocytes
andaHumanSkinEquivalent
SungHoonLee
1,2
,Il‐HongBae
1
,Eun‐SooLee
1
,Hyoung‐JuneKim
1
andJongsungLee
2,
*
andChangSeokLee
3,
*

1
AmorepacificCorporationR&DCenter,YonginCity,17074,Gyunggi‐do,Korea;
imstrong20@gmail.com(S.H.L.);baelong98@naver.com(I.H.B.);soopian@amorepacific.com(E.S.L.);
leojune@amorepacific.com(H.J.K.)
2
DepartmentofIntegrativeBiotechnology,CollegeofBiotechnologyandBioengineering,Sungkyunkwan
University,SuwonCity,16419,Gyunggi‐do,Korea
3
DepartmentofBeautyandCosmeticScience,EuljiUniversity,SeongnamCity,13135,Gyunggi‐do,Korea
*Correspondence:cslee2010@eulji.ac.kr(C.S.L.),Tel.:+82‐31‐740‐7549(C.S.L.);bioneer@skku.edu(J.L.),
Tel.:+82‐31‐290‐7861(J.L.)
Received:6February2020;Accepted:1March2020;Published:3March2020
Abstract:Sugarsareubiquitousinorganismsandwell‐knowncosmeticingredientsformoisturizing
skinwithminimalside‐effects.Glucose,asimplesugarusedasanenergysourcebylivingcells,is
oftenusedinskincareproducts.Severalreportshavedemonstratedthatsugarandsugar‐related
compoundshaveanti‐melanogeniceffectsonmelanocytes.However,theunderlyingmolecular
mechanismbywhichglucoseinhibitsmelaninsynthesisisunknown,eventhoughglucoseisused
asawhiteningaswellasmoisturizingingredientincosmetics.Herein,wefoundthatglucose
significantlyreducedthemelanincontentofα‐melanocyte‐stimulatinghormone(MSH)‐stimulated
B16cellsanddarklypigmentednormalhumanmelanocyteswithnosignsofcytotoxicity.
Furthermore,topicaltreatmentofglucoseclearlydemonstrateditswhiteningefficacythrough
photography,Fontana‐Masson(F&M)staining,andmulti‐photonmicroscopyinapigmented3D
humanskinmodel,MelanoDerm.However,glucosedidnotalterthegeneexpressionorprotein
levelsofmajormelanogenicproteinsinmelanocytes.Whileglucosepotentlydecreasedintracellular
tyrosinaseactivityinmelanocytes,itdidnotreducemushroomtyrosinaseactivityinacell‐free
experimentalsystem.However,glucosewasmetabolizedintolacticacid,whichcanpowerfully
suppresstyrosinaseactivity.Thus,weconcludedthatglucoseindirectlyinhibitstyrosinaseactivity
throughconversionintolacticacid,explainingitsanti‐melanogeniceffectsinmelanocytes.
Keywords:melanogenesis;sugar;glucose;tyrosinase;humanskinequivalent
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1.Introduction
Melanogenesisistheprocessofmelaninproductionandisessentialforskinprotection.Melanin
absorbsultraviolet(UV)lightandprotectstheskinfromthedamagingeffectsofUVlightandfree
radicals[1].However,becauseexcessiveproductionofmelanincauseshyperpigmentationsuchas
frecklesandlentigo,whichmaybeconsideredunaesthetic,muchresearchhasbeendedicated
towardsfindingeffectivedepigmentaryingredientsforcosmeticsormedicines[2–4].
Sugarisapowerfulhumectantforskinmoisturizationandisusedasacosmeticingredientfor
moisturizingskinwithminimalside‐effects.Inaddition,sugarsandsugar‐relatedagentsaffect
melanogenesis[5,6].Glycosylationoftyrosinase,akeyenzymeinvolvedinmelaninsynthesis,canbe

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altered,inhibititscatalyticactivityandacceleratingitsdegradation[7].Theroleofsugarsin
melanogenesishasbeenhighlightedbystudiesinvestigatingtheeffectofglycosylationonthe
pigmentationphenotypeofmelanocytesandtherolesofsugarresiduesonthecatalyticactivityof
tyrosinase[5–7].Somestudieshavereportedthatsugarderivativescaninhibittyrosinasematuration,
affectingitsglycosylation.Forexample,N‐acetylglucosamine(NAG),anaminohexoseproduced
physiologicallybyadditionofanaminogrouptoglucose,disruptstyrosinaseglycosylation,resulting
indepigmentingeffectsinguineapigskinandinhumanskin[8].Additionally,recentstudieshave
showedthatsugar‐relatedcompoundsinhibittyrosinaseexpressionoractivationaswellasalterits
glycosylation.Forexample,weevaluatedthewhiteningefficacyofgalacturonicacid(GA),asugar
acidthatisanoxidizedformofgalactoseandthemaincomponentofpectin.Galacturonicacidexerts
awhiteningeffectthroughregulationoftyrosinaseactivityandexpressioninB16murinemelanoma
cellsandahumanskinequivalent[9].Inanotherreport,anewtypeofcyclicoligosaccharide,known
ascyclicnigerosylnigerose(CNN),showedaweakbutsignificantdirectinhibitoryeffectonthe
enzymaticactivityoftyrosinase,suggestingonepossiblemechanismofhypopigmentation[10].
Similartotyrosinasematurationbyproperglycosylation,theexpressionoractivityofCNNcouldbe
atargetofanti‐melanogenicagents[11].However,numerousanti‐melanogenicagentshavesevere
side‐effects,suchasvitiligo[12,13].Thereisthereforegreatinterestinsaferdepigmentary
compounds.
Here,weinvestigatedtheanti‐melanogeniceffectsofglucoseonB16murinemelanomacellsand
normalhumanmelanocytes.Inaddition,weexaminedtissuecolorandepidermalstatususingtissue
sectionstainingofahumanskinequivalent.Basedonourfindings,weproposethatthewhitening
effectofglucoseisdependentonlacticacidproduction,resultingintyrosinaseinactivation.
2.Results
2.1.Anti‐MelanogenicEfficacyofGlucoseinB16andNHMs
Toinvestigatetheanti‐melanogeniceffectofglucose,weusedtwotypesofmelanocytes,B16
melanomacells(amurinemelanomacellsline)andnormalhumanmelanocytes(NHMs).First,we
determinedifglucosewastoxictoB16cells.Glucosedidnotshowanycytotoxicityatconcentrations
upto100mMinB16cells,asshowninFigure1A.Basedonthecytotoxicitydata,B16cellswere
treatedwithvariousconcentrationsofglucosefor72hinthepresenceofα‐melanocyte‐stimulating
hormone(MSH),aninducerofmelanogenesis.AsshowninFigure1B,glucoseclearlyand
significantlydown‐regulatedtheintracellularmelanincontentinadose‐dependentmanner.Kojic
acid(KA)wasusedasareferencecompoundforanti‐melanogenesisbecauseitisoftenusedasaskin
lighteningcosmeticingredient[1,3,4].Thecoloroflysatesinglucose‐treatedcellswaslighterthan
thecolorofcontrolcells(Figure1C).Inaddition,weconfirmedthattheamountofmelaninsecreted
intotheculturemediadecreasedandthecolorofthemediaclearlybrightened(Figure1D).Next,we
investigatedtheanti‐melanogeniceffectofglucoseondarklypigmentedNHMs.Glucoseat
concentrationsupto100mMwasnotcytotoxicforuptofor4days(Figure1E).Whenmelanincontent
wasdeterminedafterglucosetreatmentofNHMsfor4days,wefoundthatmelanincontent
decreasedinadose‐dependentmanner(Figure1F).Takentogether,theseresultsindicatethatglucose
suppressesmelaninsynthesisinmelanocytes.

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Figure1.EffectofglucoseonB16cellsandnormalhumanmelanocytes(NHMs).(A)Effectofglucose
ontheviabilityofB16cells.(B)Intracellularmelanincontentsinα‐MSH‐stimulatedB16cells.
Intracellularmelanincontentsweredeterminedusingcelllysates,asdescribedinthemethodssection.
(C)Thecolorofcelllysate.(D)Extracellularmelanincontentsweredeterminedusingculturedmedia
containingsecretedmelaninafterglucoseandα‐MSHco‐treatmentfor72h.KAindicateskojicacid
(100μg/mL),whichwasusedasareferencecompound.Thephotographshowsthecolorsofculture
media.(E)EffectofglucoseontheviabilityofNHMs.(F)Effectsofglucoseonmelaninsynthesisin
NHMs.NHMsweretreatedwiththeindicatedconcentrationsofglucosefor4d,washed,andlysed
withNaOHtodeterminetheintracellularmelanincontents.Themelanincontentswereestimatedby
absorbanceat405nmandnormalizedbythetotalproteincontents.Dataareexpressedasthemean±
SDofatleastthreeindependentmeasurements(*p<0.05,**p<0.01,***p<0.001).
2.2.WhiteningEffectofGlucoseona3DHumanSkinEquivalent
Tofurtherdefinetheanti‐melanogenicabilityofglucose,weusedapigmented3Dhumanskin
model,MelanoDerm.AsdescribedintheMaterialsandMethodssection,glucosewastopically
appliedtotheMelanoDermfor18days,andcellviabilitywasdeterminedbyCCK‐8assay.Asshown
inFigure2A,notissuecytotoxicitywasobservedafterglucosetreatmentfor18days.Tissuecolor
changeswereassessedbyphotography.AsshowninFigure2B,glucose‐treatedtissuewaslighterin
colorthanphosphate‐bufferedsaline(PBS)‐treatedtissue.Inaddition,hematoxylinandeosin(H&E)
stainingrevealedthatglucosedidnotinducetissuecollapse,whilefontana‐masson(F&M)staining
showedthatglucosedecreasedthenumberofhyperpigmentedmelanocyte(asindicatedbyarrows)
inthebasallayer(Figure2C).
Tofurtherinvestigatechangesinmelanincontent,wecomparedtheauto‐fluorescencesignals
ofmelanininthemelanocytelayersofPBS‐andglucose‐treatedtissuesusingtwo‐photonexcitation
fluorescence(TPEF)microscopy,asshowninFigure2D.Ananalysisoftheseimagesshowedthatthe
melaninvolumewasdecreasedbyapproximately36%andTPEFsignalintensityformelanininthe
melanocyte‐richareawasdecreasedbyapproximately38%inglucose‐treatedtissuescomparedwith
PBS‐treatedtissues.

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
Figure2.Effectofglucoseonhumanskinequivalent,MelanoDerm.(A)Viabilityofhumanskin
equivalentstreatedwithglucose.(B)Humanskinequivalents(MelanoDerm;n=3)weretopically
treatedwithglucosefor18d,andthenphotographed.TheΔLvalueindicatesthedegreeoflightness
comparedwithPBS‐treatedtissue.(C)H&EandF&Mstainingoftissuesections.Theslideswerefixed
informaldehydesolutionandembeddedinparaffinwaxforstaining(scalebar,50μm).Blackarrows
indicatepigmentedmelanocytes.(D)Melaninimaging(200×200×60μm
3
)ofhumanskinequivalents
wasperformedusingTPEFmicroscopy.Pseudocolored(red)signalsindicatemelanin(scalebar,50
μm).ThegraphsindicatethequantificationofmelaninvolumeandTPEFsignals.Dataareexpressed
asthemean±SDofatleastthreeindependentmeasurements(*p<0.05,***p<0.001).
2.3.EffectofGlucoseontheExpressionofMelanogenicProteinsinMelanocytes

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Next,toelucidatethedepigmentationmechanismofglucoseinmelanocytes,weassessedits
effectontheexpressionofmelanogenicenzymessuchastyrosinaseandTyrp‐1inB16cellsand
NHMs.B16cellsweretreatedwithglucosefortheindicatedtimesinthepresenceofα‐MSH.Then,
proteinlevelsoftyrosinaseandTyrp‐1weredeterminedbyWesternblotassay(Figure3A).
ExpressionleveloftyrosinaseandTyrp‐1werenotdecreasedbyglucoseatanytimepoint.
Furthermore,transcriptlevelsoftyrosinasewerenotaffectedbyglucoseinα‐MSH‐stimulatedB16
cells(Figure3B).SimilartoB16cells,proteinlevelsoftyrosinase,Tyrp‐1,andMITFwerenotinhibited
byglucose(Figure3C)inNHMcellstreatedwithglucose,andmRNAlevelsoftyrosinaseandTyrp‐
1werealsonotdecreased,butratherslightlyincreasedbyglucose(Figure3D).
2.4.EffectofGlucoseonTyrosinaseActivity
Tofurtherdefinetheactionmechanismofglucose,wetestedifithadaninhibitoryeffecton
mushroomtyrosinaseactivity.AsshowninFigure4A,wefoundthatglucosehadnoinhibitoryeffect
onmushroomtyrosinaseactivity,indicatingthatglucosedoesnotdirectlyaffecttyrosinaseactivity.
Wenextperformedatotalintracellulartyrosinaseactivityassayusingglucose‐treatedcelllysates
frombothB16cellandNHMs.Interestingly,intracellulartyrosinaseactivitywasclearlyinhibitedin
adose‐dependentmanneringlucose‐treatedB16cellsinthepresenceofα‐MSH(Figure4B).In
NHMs,glucosealsoinhibitedintracellulartyrosinaseactivity(Figure4C).Thesedatasupportthe
possibilitythatglucoseindirectlyinactivatestyrosinaseinmelanocytes.


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
Figure3.EffectofglucoseontheexpressionofmelanogenicproteinsinB16cellsandNHMs.(A,B)
B16cellsweretreatedwithα‐MSHfortheindicatedtimeinthepresenceorabsenceof20mMglucose.
Then,Westernblot(A)andqRT‐PCR(B)assayswereperformed.(C)NHMsweretreatedwiththe
indicatedconcentrationsofglucosefor48h.Then,theWesternblotassaywasperformed.(D)NHMs
weretreatedwiththeindicatedtimeinthepresenceof50mMglucose.Then,qRT‐PCRassayswere
performed.Dataareexpressedasthemean±SDofatleastthreeindependentmeasurements.

Figure4.Effectofglucoseonthetyrosinaseactivity.(A)Effectofglucoseonthemushroomtyrosinase
activityincell‐freesystem.(B–C)Cellulartyrosinaseactivityassay.(B)B16cellsweretreatedwiththe
indicatedconcentrationsofglucosefor24h.Then,thecellulartyrosinaseactivityassaywas
performed,asdescribedinthemethodssection.(C)NHMsweretreatedwith50mMglucoseforthe
indicatedtime.Then,thecellulartyrosinaseactivityassaywasperformed,asdescribedinthe
methodssection.Thecellulartyrosinaseactivitywasnormalizedbythetotalproteincontents.Data
areexpressedasthemean±SDofatleastthreeindependentmeasurements(*p<0.05,**p<0.01,***p
<0.001).

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2.5.TyrosinaseInactivationbytheProductionofLacticAcidinGlucose‐TreatedMelanocytes
Glucoseisconvertedintothecellularmetabolitelactate,whichislacticacidinsolutionand
whichhasbeenreportedtobeeffectiveintreatingpigmentarylesions[14,15].Therefore,we
hypothesizedthatglucoseisconvertedintolacticacidinmelanocytesandthatincreasedlevelsof
lacticacidinhibitmelanogenesisthroughtyrosinaseinactivation.Toevaluatethishypothesis,wefirst
assessedtheproductionoflacticacidinmediafromglucose‐treatedmelanocytes.Asexpected,
glucosesignificantlyup‐regulatedthelacticacidcontentinB16cells‐culturedmedia(Figure5A).In
addition,becauselacticacidisknowntodirectlyinhibittyrosinaseactivity[15],weevaluatediflactic
acidsuppressedmushroomtyrosinaseactivity.AsshowninFigure5B,lacticaciddramatically
inhibitedmushroomtyrosinaseactivity,unlikeglucose.Theseresultssuggestthatconversionof
glucosetolacticacidhasananti‐melanogeniceffectviatyrosinaseinactivationbythelacticacid.
3.Discussion
Sugarsorsugar‐derivedmaterialsareoftenusedascosmeticingredientsforskinprotectionand
physiologicalcontrol.Forexample,raffinoseincreasesmTOR‐independentautophagyandreduces
celldeathinUVB‐irradiatedkeratinocytes,indicatingthatthenaturalagentraffinosehaspotential
valueinlimitingphotodamage[16].Trehaloseandsucrosearenovelactivatorsofautophagyin
humankeratinocytesthroughanmTOR‐independentpathway[17].Thesefindingsprovidenew
insightintothesugar‐mediatedregulationofautophagyinkeratinocytes.Inthecaseofglucose,
topicalglucosewasshowntoinduceclaudin‐1andfilaggrinexpressioninamousemodelofatopic
dermatitisandinkeratinocyteculture,indicatingthatithasananti‐inflammatoryeffectbyrepairing
skinbarrierfunction[18].Inaddition,glucoseinhibitsproliferationandenhancesthedifferentiation
ofskinkeratinocytes[19].Therefore,glucoseregulatesvariousaspectsofepidermalphysiology,such
asskinbarrierfunctionsandkeratinocytehydrationlevels.

Figure5.Productionoflactatebyglucoseandeffectoflacticacidonthetyrosinaseactivity.(A)Lactate
productioninglucose‐treatedB16cells.(A)B16cellsweretreatedwiththeindicatedconcentrations
ofglucosefor3d.Then,alactateassaywasperformedusingtheculturedmedia,asdescribedinthe
methodssection.(B)Effectoflacticacidonthemushroomtyrosinaseactivityincell‐freesystem.Data

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areexpressedasthemean±SDofatleastthreeindependentmeasurements(*p<0.05,**p<0.01,***p
<0.001).
Sugarscanactasdepigmentaryagentsviaseveraldifferentmechanismsthatinvolvetyrosinase.
Forexample,someagentsinhibittyrosinasematuration,whileotheragentsinduceinhibitionof
tyrosinasegeneexpressionoractivity[5,8–10].However,fewstudieshaveinvestigatedthe
mechanismsunderlyingthedepigmentationeffectsofglucose.
Todeterminetheeffectofglucoseonmelanogenesis,wepreviouslyfocusedonliverXreceptor
(LXRs),whichareligand‐activatednuclearreceptorsthatplaypivotalrolesinlipidmetabolismand
cholesterolhomeostasis[20].WefoundthatliverXreceptoractivationinhibitsmelanogenesis
throughtheaccelerationofextracellularsignalregulatedkinase(ERK)‐mediatedmicrophthalmia‐
associatedtranscriptionfactor(MITF)degradation[21].Furthermore,glucoseisanendogenousLXR
ligand[22].Thus,wehypothesizedthatglucosehasanti‐melanogeniceffectsduetoactivationofan
LXR‐dependentpathway.However,asshowninFigure3C,glucosedidnotaltertheexpression
levelsoftyrosinaseorMITF,althoughLXRactivationreducedtheexpressionlevelsoftheseproteins
inmelanocytes.Therefore,weconcludedthatglucosehaddepigmentingeffectsonmelanocytes
independentofLXRactivation.
Glucoseistheprincipalsubstrateforenergyproduction,anditishydrolyzedviaserialreactions
ofseveralenzymes,knownasglycolysis.Numerousstudieshaveshownthatglycolysishasonlyone
endproduct,lacticacid,whetherunderaerobicoranaerobicconditions.Underaerobicconditions
(O2),lactateisutilizedasthesubstrateofmitochondriallactatedehydrogenase(mLDH).LDHs
convertittopyruvatethatentersthetricarboxylicacid(TCA)cycle.Underanaerobicconditions(N2),
lactateisaccumulatedinthecytosol.Therefore,theglycolysispathwaybeginswithglucoseasits
substrateandterminateswiththeproductionoflactateasitsmainendproduct[23].Inaddition,
Davidetal.measuredthefractionofglucosethatwasconvertedtolacticacidorpyruvateinthe
normalhumanmelanocyte[24].Mostofthemetabolitesofglucosewerelacticacidratherthan
pyruvate.
Lacticacidisanalphahydroxyacid(AHA);theseacidsareusedextensivelyincosmetic
formulationsassuperficialpeelingagents[25].Inaddition,lacticacidsuppressesmelaninformation
bydirectlyinhibitingtyrosinaseactivity,aneffectindependentofitsacidicnature,whichmeansthat
lacticacid’seffectsonpigmentarylesionsareduenotonlytoaccelerationofepidermalcellturnover,
butalsodirectinhibitionofmelaninformationinmelanocytes[15].Thus,wereasonedthatglucose
mayexertitsanti‐melanogeniceffectvialacticacidproduction,becauseglucosecanbeconvertedto
lacticacid.Asexpected,wefoundthatglucosetreatmentresultedinlacticacidproductionin
melanocytes(Figure5A).Inaddition,weconfirmedthatlacticacidpowerfullyanddirectlyinhibited
tyrosinaseactivity(Figure5B).Usukietal.alsodiscoveredthatlacticaciddecreasedintracellular
tyrosinaseactivityinB16cellsandhumanHM3KOcells[15].Inthereport,mRNAandproteinlevels
oftyrosinaseandTyrp‐1werenotaffectedbylacticacid.Together,thesedataindicatethatglucose
increaseslacticacidproductionandthatthislacticaciddirectlyinhibitstyrosinaseactivitywithout
affectinggeneexpressionlevels,indicatingthatglucosehasananti‐melanogeniceffectin
melanocytesviaindirecttyrosinaseinactivationdependentonlacticacidproduction.However,
furtherexperimentationsarenecessarytovalidatetheroleoflacticacidandglucosein
depigmentation.
Collectivedatafromthisstudyprovidepreliminaryevidencesupportingtheutilityoftopical
glucoseasaneffectivewhiteningaswellasmoisturizingreagentthatcanbesafelyusedincosmetics
andmedicinalformulations.
4.MaterialsandMethods
4.1.Materials
D‐glucose,α‐MSH,kojicacid(KA),L‐tyrosine,L‐DOPA,andlacticacidwerepurchasedfrom
Sigma‐Aldrich(St.Louis,MO,USA).Antibodiesagainsttyrosinaseandactinwerepurchasedfrom

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Abcam(Cambridge,UK).AntibodyagainstTyrp‐1waspurchasedfromSantaCruzBiotechnology
(CA,USA).AntibodyagainstMITFwaspurchasedfromProteintech(city,IL,USA).
4.2.CellCultureandViabilityAssay
WepurchasedB16murinemelanomacells,Dulbecco’smodifiedEagle’smedium(DMEM),and
fetalbovineserum(FBS)fromtheAmericanTypeCultureCollection(ATCC,Manassas,VA,USA).
B16cellswereculturedinDMEMcontaining4500mg/Lhighglucose(ATCC30‐2002)as
recommendedbythemanufacturer(ATCC)supplementedwith5%FBS,andincubatedat37°Cina
humidifiedatmospherecontaining95%airand10%CO2.DarklypigmentedprimaryNHMswere
purchasedfromThermoFisherScientific(#C2025C;Waltham,MA,USA).Cellswereculturedin
Medium254(#M254500)supplementedwithHumanMelanocyteGrowthSupplement(#S0025)and
incubatedat37°Cundera5%CO2atmosphere.Forexperiments,primaryNHMsbetweenpassages
4and7wereused.TheviabilityofculturedcellswasassessedusingaCellCountingKit‐8(CCK‐8)
asdescribedbythemanufacturer(DOJINDO,Tokyo,Japan).
4.3.MeasurementofMelaninContent
Melanincontentwasdeterminedasdescribedinpreviousreports[2–4].Briefly,B16cellswere
treatedwiththeindicatedconcentrationsofglucoseinthepresenceofα‐MSH(200nM)for72h.
NHMsweretreatedwiththeindicatedconcentrationsofglucosefor4d.Thereafter,allcellswere
washedwithphosphate‐bufferedsaline(PBS)anddissolvedin1NNaOHat60°Cfor1h.Celllysates
weretransferredtoa96‐wellplate,andabsorbancewasmeasuredat405nm.Thevalueswere
normalizedbasedontheproteinconcentrationsineachsamplewell.
4.4.MushroomTyrosinaseActivityAssay
Weinvestigatedthedirecteffectsoftheindicatedconcentrationsofglucoseonmushroom
tyrosinaseactivity.Briefly,100μLofphosphatebuffercontainingglucosewasmixedwithmushroom
tyrosinase(10units/well)andcombinedwith50μLof0.03%L‐tyrosineorL‐DOPAindistilledwater.
Then,themixturewasincubatedtogetherat37°Cfor10min,andabsorbancewasmeasuredat405
nm.Kojicacid(KA),awell‐knownanti‐tyrosinaseagent,wasusedasareferencecompound.
4.5.IntracellularTyrosinaseActivityAssay
Briefly,B16cellsorNHMsweretreatedwiththeindicatedconcentrationsofglucoseforthe
indicatedtimes.Then,cellswerewashedwithPBSandlysedbyincubationin50mMphosphate
buffer(pH6.8)containing1%TritonX‐100and0.1mMphenylmethyl‐sulfonylfluoride.Cellular
lysateswerethencentrifugedat12,000rpmat4°Cfor20min.Thesupernatantcontainingcellular
tyrosinasewascollectedandtheproteincontentwasdeterminedfornormalization.Thecellular
extractwasincubatedwithL‐DOPAinphosphatebufferanddopachromeformationwasmonitored
bymeasuringabsorbanceat405nmwithin30min.
4.6.RNAisolationandReal‐TimeQuantitativeReverseTranscription‐PolymeraseChainReaction(qRT‐
PCR)
TodeterminerelativemRNAexpressionofselectedgenes,totalRNAwasisolatedwithTRIzol
(Invitrogen,CA,USA),accordingtothemanufacturer’sinstructions,and4μgRNAwasreverse‐
transcribedintocDNAusingRT‐premix(Bioneer,Seoul,SouthKorea).QuantitativePCRwas
performedusinganABI7500FastReal‐TimePCRSystem(AppliedBiosystems,FosterCity,CA,
USA).TheqRT‐PCRprimersetsfortyrosinaseandTyrp‐1werepurchasedfromAppliedBiosystems,
andTaqManGeneExpressionAssaykits(AppliedBiosystems)wereusedforamplification.Target
geneexpressionwasnormalizedtothatofthehousekeepinggeneencodingribosomalproteinlateral
stalksubunitP0(RPLP0).Relativequantizationwasperformedusingthecomparative∆∆Ctmethod
accordingtothemanufacturer’sinstructions.

Int.J.Mol.Sci.2020,21,173611of13
4.7.WesternBlotting
CellswerewashedtwicewithcoldPBSandthenlysedinice‐coldmodifiedRIPAbuffer(Cell
SignalngTechnology,MA,USA)containingproteaseinhibitors(Calbiochem,LaJolla,CA,USA).
Totalproteinconcentrationwasdetermined,andtheproteinswereresolvedbySDS‐PAGEon4–12%
gradientBis‐Trisgels(ThermoFisherScientific,Waltham,MA,USA),transferredtonitrocellulose
membranes(ThermoFisherScientific).Aftertransfer,membraneswereblockedin5%blocking
solution.Membraneswereincubatedwithprimaryantibodiesat4°Cfor24h,washedwithTris‐
bufferedsalinecontaining0.1%Tween‐20(TBST),andexposedtoperoxidase‐conjugatedsecondary
antibodiesfor1hatroomtemperature.MembraneswererinsedthreetimeswithTBST.
ChemiuminescentsignalwasdevelopedusingWesternblottingECLreagent(GEHealthcare,
Hatfield,UK).
4.8.Three‐Dimensional(3D)HumanSkinEquivalent
WeusedMelanoDerm(MEL‐300‐B;MatTekCorp.,Ashland,MA,USA)asahumanskintissue
model.Thisviable,reconstituted,3Dhumanskinequivalentwasderivedfromblackdonorsand
containsnormalmelanocytesandkeratinocytes.MelanoDermwasgrownattheair‐liquidinterface
inEPI‐100‐NMM‐113medium(MatTekCorp,Ashland,MA,USA).Priortoglucosetreatment,tissues
werewashedwith1mLPBStoremoveresidualcompounds.GlucosewasdissolvedinPBS.Final
concentrationofglucosewas2%.ThecontrolsamplewastreatedonlywithPBS.Glucosewasapplied
toMelanoDermondays1,4,6,8,11,13,and15.After18days,MelanoDermtissueswerefixedin4%
bufferedformaldehyde,embeddedinparaffin,cuttoathicknessof3μm,andsubjectedtoH&Eand
F&Mstaining.TheviabilityofthetissuesampleswasassessedusingaCellCountingKit‐8(CCK‐8)
asdescribedbythemanufacturer(DOJINDO,Tokyo,Japan).PigmentationoftheMelanoDermwas
assessedbycomparingthechangeinL*value.
4.9.Two‐PhotonExcitationFluorescence(TPEF)Imaging
Tovisualizethedistributionofmelanininthe3Dhumanskinequivalent,weperformedTPEF
imaging,asdescribedinourpreviousreports[3,4].Briefly,eachMelanoDermpreparationwasfixed
in4%formalinfor24hat4°C,andthenwashedwithPBS/0.1%BSA(bovineserumalbumin,Merck,
Branchburg,NJ,USA).TheTPEFimageswereacquiredfromthebasallayertomeasureintracellular
melanininthemelanocytelayer.RelativeTPEFsignalintensitiesformelanininthemeasurement
volumewerequantifiedusingImage‐ProPremier3Dsoftware(MediaCybernetics,Inc.,Bethesda,
MD,USA).
4.10.L‐lactateAssay
B16cellswereseededat1.0 × 105cellsperwellina12‐wellplate.Aftertreatmentwithglucose
for3days,extracellularlactatelevelswerequantifiedusinganL‐lactatecolorimetricassaykit
(Abcam,ab65331,Cambridge,UK)accordingtothemanufacturer’sprotocol.
4.11.StatisticalAnalysis
Dataareexpressedasmeans±SDs(standarddeviations),andstatisticalsignificancewas
determinedbyStudent’st‐test.Ap‐value<0.05wasconsideredstatisticallysignificant.
AuthorContributions:Conceptualization,Hyoung‐JuneKim,JongsungLeeandChangSeokLee;Datacuration,
SungHoonLee;Investigation,SungHoonLee;Methodology,SungHoonLee,Il‐HongBaeandEun‐SooLee;
Supervision,JongsungLeeandChangSeokLee;Writing—originaldraft,SungHoonLeeandChangSeokLee;
Writing—reviewandediting,JongsungLee.Allauthorshavereadandagreedtothepublishedversionofthe
manuscript.
Funding:ThisresearchwasfundedbytheBasicScienceResearchProgramthroughtheNationalResearch
FoundationofKorea(NRF)fundedbytheMinistryofEducation(GrantNumber:2018R1D1A1B07049402).

Int.J.Mol.Sci.2020,21,173612of13
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
Abbreviations
α‐MSHα‐melanocyte‐stimulatinghormone
Tyrp‐1tyrosinase‐relatedprotein1
MITFmicrophthalmia‐associatedtranscriptionfactor
NHMsnormalhumanmelanocytes
TPEFtwo‐photonexcitationfluorescence
LXRliverXreceptor
AHAsalphahydroxyacids
H&Ehematoxylinandeosin
F&MFontana‐Masson
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