Butanol Extract of 1:1 Mixture of PhellodendronCortex and Aralia Cortex Stimulates PI3-Kinaseand ERK2 with Increase of Glycogen Levels inHepG2 Cells

Sung-Jin Kim,1* You-Young Kim, 1 Kwang Ho Ko,2 Eun-Kyung Hong,3 Young-Bok Han,3

Bu-Hyun Kang4 and Harriet Kim 5

1Department of Pharmacology, School of Dentistry, Kyung-Hee University, Seoul, Korea 130-7012Department of Pharmacology, College of Pharmacy, Seoul National University3Taelim Pharmaceutical Company, Seoul, Korea4Toxicology Research Center, Korea Research Institute of Chemical Technology5Department of Food and Nutrition, College of Home Economics, Seoul National University

Extracts of Phellodendron amurenseRupr. cortex and Aralia cortex (P55A) have been used traditionallyby Koreans to treat diabetes. We tested the extracts of P55A to determine if they could mimic insulin ac-tions such as activation of ERK2, PI3-kinase and glycogen synthesis in liver cells. The butanol extract ofP55A activated ERK2 in the cytosol and nucleus of HepG2 cells; the extent of ERK2 activation wasmuch higher in the nucleus. More strikingly, the butanol extract significantly increased the level of glyco-gen in HepG2 cells. Association of PI3-kinase and IRS-1 in the cytoplasm was also stimulated by the bu-tanol extract of P55A. These results suggest that active component(s) of the butanol extract of P55Acould regulate blood glucose levels by activating ERK2 and PI3-kinase and by stimulation of glycogensynthesis in the liver.# 1998 John Wiley & Sons, Ltd.

Phytother. Res.12, 255–260 (1998)

Keywords: Phellodendroncortex;Aralia cortex; glycogen; ERK2; PI3-kinase.

INTRODUCTION

Aralia elata Seem. is a folk medicine of Korea used forthe treatment of diabetes, rheumatic arthritis andinflammation nephritis. The cortex ofAralia elatacontains a number of glycosides including elatoside Ethat has hypoglycaemic effects (Yoshikawaet al.,1993,elatoside F and eight known oleanolic acid glycosides(Sakai et al., 1994; Yoshikawaet al., 1996). It alsocontains elatoside A and B that exert a potent inhibitoryeffect on ethanol absorption (Yoshikawaet al., 1993).Phellodendron amurenseRupr. is another Korean folkmedicine used for antiinflammatory (Uchiyamaet al.,1989), antihypertensive, and blood glucose regulatingagents (Chen and Xie, 1987). The cortex ofPhelloden-dron amurenseRupr. contains several alkaloids includingberberine, palmatine (Wanget al., 1989), magnoflorine,phellodendrine (Mori et al., 1994) and limonoids(Miyake et al., 1992). Berberine has several biologicalactions such as a hypoglycaemic effect (Ni, 1988; Chenand Xie; 1987; Niet al.,1994), a bacteriocidal effect, an

anti-cholera toxin effect, an antiinflammatory effect andan anti-ulcer effect (Uchiyamaet al., 1989). ERK2(extracellular-regulated protein kinase or mitogen acti-vated protein kinase, MAP kinase) has been implicated inglycogen synthesis (Sturgillet al., 1988; Dentet al.,1990). It has been suggested that PI3-kinase is involved inthe regulation of glucose transporter (Cheathamet al.,1994) and glycogen synthesis (Yamamoto-Hondaet al.,1995). In the present study, we determined whether theextracts of a 1:1 mixture ofPhellodendron amurenseRupr. andAralia elataSeem. (named ‘P55A’) had effectson glycogen synthesis and ERK2 activity. The activity ofPI3-kinase in response to P55A extracts was alsoevaluated in terms of the association of IRS-1 (insulinreceptor substrate-1) and PI3-kinase since its activationrequires association with IRS-1.

MATERIALS AND METHODS

Materials. Pork insulin was obtained from ElancoProducts Co. (Indianapolis, IN). The reagents used forpolyacrylamide gel electrophoresis were from Bio-Rad.Nitrocellulose filters (0.45mm) were purchased fromSchleicher & Schuell. Antibodies to ERK2 werepurchased from Transduction Laboratories. Antibody tophospho-ERK2 was purchased from New EnglandBiolab. ECL kit was purchased from Amersham. HepG2

PHYTOTHERAPY RESEARCH, VOL.12, 255–260 (1998)

CCC 0951–418X/98/040255–06 $17.50# 1998 John Wiley & Sons, Ltd.

* Correspondence to: S.-J. Kim, Department of Pharmacology, School ofDentistry, Kyung-Hee University, Seoul, Korea 130-701. E-mail:kimsj@nms.kyunghee.ac.kr (Tel) 82-2-961-0868. (Fax) 82-2-957-5309.Contract/grant sponsor: Korean Ministry of Health and Welfare.Contract/grant sponsor: Korean Ministry of Education.Contract/grant sponsor: Korea Science and Engineering Foundation; Contract/grant number: 971-0704-026-2.

Accepted 16 January 1998

cells were purchasedfrom KCLB (Korean Cell LineBank).All otherchemicalswerepurchasedfrom SigmaChemicalCo.

Preparation of Plant extract. A mixture of Araliacortex (300g) and Phellodendroncortex (300g) wasgroundinto afinepowder,andstirredwith distilledwater(5,000mL) for 50h. The resultingfiltrate wassaturatedwith vapourpressure(121°C/15 pound/ina2). The pre-cipitatescontainingcoagulatedproteinswereremovedbycentrifugation.The supernatantwasmixed with chloro-form (2000mL) using a separatoryfunnel. The chloro-form fraction containingproteins,resin and fibres wasremovedandthewaterfractionwasfurthermixedwith n-hexane(2000mL). The n-hexane-solublefraction wasremoved.Talc was addedto the water-solublefractionand the mixture was subjected to filtration usingmembranefilter apparatus.The resulting filtrate wasfreeze-dried(named‘total extractof P55A’). Thefreeze-driedtotalextract(50g) wasresuspendedwith 250mL ofdistilled water and further mixed with n-butanol(250mL). The mixture was shakenfor 1 h using anorbital shaker.The butanol-solublefraction was sub-jectedto evaporationby theuseof a rotaryevaporatorat60°C. Thepelletwasresuspendedwith 100mL of waterandsubjectedto overnightlyophilization (named‘buta-nol extract of P55A’). The butanol-insoluble,water-soluble fractions were separatedand subjected tolyophilization (named‘water extractof P55A’).

Cell culture. HepG2cellsweregrownin DMEM mediawith 10%fetal calf serumin 5% humidifiedCO2 atmos-phereat 37°C.

Isolation of nuclei and cytoplasm.Nuclei wereisolatedas previously described(Chen et al., 1992). Confluentcells were serumstarvedfor 18h and stimulatedwithP55Aextracts(10mg/mL) for thetimesindicated.Mediawas removedand cultureswere washedwith ice-coldphosphatebufferedsaline.Thecells werescrapedinto abuffer containing20mM Hepes,50mM MgCl2, 25mMKCl, 2 mM PMSF, 0.1mg/mL aprotinin, 1 mM sodiumvanadate,1 mM sodium molybdate,10mM b-glycero-phosphate,5 mM sodium pyrophosphate,1 mM EDTA,1 mM EGTA and 0.25M sucrose.Cells were douncehomogenizedwith a tight-fitting glass-Teflonhomogeni-zer. The homogenatewas loaded onto 1 mL of 1Msucrosein lysis buffer and centrifugedat 1600� g for10min at 4°C to pellet nuclei. The nuclei werewashedwith 1M sucrose in lysis buffer and centrifuged at1600� g for 10min at 4°C. The resultingpelletswereresuspendedwith lysis buffer and subjectedto briefsonication.1600� g supernatantsweresavedandusedascytosolicfractions.

Western blot analysis.Electrotransferof proteinsfromthe gels to nitrocellulosepaper(Schleicher& Schuell)wascarriedout for 1 h at100V (constant)(Towbinetal.,1979). The filter paperswere preincubatedfor 1 h at23°C with PBScontaining0.1%Tween20 and5% skimmilk and washedwith PBS containing0.1% Tween20threetimesfor 10min each.Theblotswereprobedwithanti-ERK2antibodyor anti-PI3-kinaseantibodyfor 1 h at23°C. The blots were then incubated with HRP-conjugatedanti-rabbit IgG for 30min and washedwithPBScontainingTween20five timesfor 10min each.The

detectionof immobilized specific antigenswas carriedout by ECL (NEN). Quantitationof MAP kinasebandswerecarriedout by scanningdensitometry.

Immunoprecipitation. Immunoprecipitation was per-formed as describedearlier (Kim and Kahn 1994). Anequalamountof proteinsfrom nuclearand postnuclearpreparationwereincubatedwith IRS-1antibodyfor 1 h at4°C, followed by the addition of protein A-sepharose,andtheimmunecomplexwasprecipitatedby centrifuga-tion. Thepelletswerewashedsuccessivelywith 1 mL ofbuffer A (0.01M Tris, pH 7.4, 1M NaCl, 1% NonidetP-40),buffer B (0.01M Tris, pH 7.4, 0.1M NaCl, 0.01MEDTA, 1% Nonidet P-40, 0.3% SDS) and buffer C(0.01M Tris, pH 7.4 and 1% Nonidet P-40). The finalpelletsweresolubilizedwith Laemmli buffer containing100mM dithiothreitol, boiled for 5 min, centrifugedin amicrocentrifuge,and the supernatantwas subjectedtoSDS-PAGE and Western blot analysis with anti-PI3-kinaseantibody.

Measurementof ERK2 activity. Theactivitiesof ERK2weremeasuredasdescribedearlier(Kim andKim 1997).Following SDS-PAGE,Westernblotting analysiswithanti-phospho ERK2 antibody was carried out. ThephosphorylatedERK2 bandwasquantitatedby scanningdensitometry.

Determination of glycogenlevels.Following treatmentwith P55A extracts for 1 h, the HepG2 cells weresubjectedto lysis with sodiumlauryl sulphate.Fromtheresultingcell homogenate,glycogenwasprecipitatedbyethanoland the precipitatedglycogenwasdissolvedbyconcentratedsulphuricacid. The generatedglucosewasdetectedas a colouredproductby reactionwith phenol(Lo et al., 1970).

RESULTS

Effect of P55A on the associationof IRS-1 and PI3-kinase

The initial insulin signalling cascadefollowing insulinreceptoractivationis mediatedby thetyrosinephosphor-ylation of IRS-1. PhosphorylatedIRS-1 serves as adocking protein that recruits SH2 domain containingproteinssuch as PI3-kinaseand Grb2, resulting in theactivationof the SH2 domaincontainingproteins.Thisresultsin the activationof two major insulin signallingpathways:(1) activationof PI3-kinaseandp70S6kinaseand (2) activation of ERK2. We determinedif P55Aextracts(10mg/mL) had an effect on the associationofIRS-1 andPI3-kinase.Following incubationwith P55Aextracts for 5 min, HepG2 cells were subjected tosubcellular fractionation. Immunoprecipitation withanti-IRS-1 antibody and subsequentimmunoblot withantibody against the p85 subunit of PI3-kinasewerecarried out with the nuclear and postnuclearfractionsfrom cells treatedwith total extract,butanolextractorwaterextract.Thewaterextractof P55Ahadnoeffectontheassociationof PI3-kinaseandIRS-1in cytoplasmandnucleuswhile the butanolextractcausedan increaseinthe associationof IRS-1 and PI3-kinase in cytoplasm

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(Fig. 1A). Insulinstimulatedtheassociationof IRS-1andPI3-kinasein cytoplasmasexpected(Fig. 1B).

Effect of P55A on the ERK2 activity

We next testedif MAP kinase is regulatedby P55Aextracts.Following incubation with P55A extractsfor5 min, HepG2 cells were subjected to subcellularfractionation.Sinceactivationof ERK2requirestyrosinephosphorylationon the enzyme, the levels of phos-phorylationof ERK2,measuredby Westernblot analysiswith anti-phosphoERK2antibody,couldberegardedasameasure of ERK2 activation. The cytosolic ERK2activity was stimulatedby the total extract (10mg/mL)and butanolextract (10mg/mL) of P55A (Fig. 2, lower

panel). In contrast,the nuclearERK2 was much moregreatly stimulatedby the butanolextractas well as thewaterextractcomparedwith thecytosolicERK2 (Fig. 3,lower panel).The butanolextractof P55Ahada greatereffect on nuclearERK2 thandid thewaterextract.

The contentsof ERK2 in the cytoplasmand nucleuswere not changedin responseto P55A extracts(Fig. 2and3, upperpanels).

Effect of P55A on the glycogenlevels

ERK2 and PI3-kinase are associatedwith glycogensynthesis(Sturgill et al., 1988;Dent et al., 1990).Sincethe butanol extract of P55A stimulatescytosolic andnuclearERK2 aswell ascytosolicPI3-kinasein HepG2

Figure 1. Effect of P55A on the association of IRS-1 and PI3-kinase in HepG2 cells. Following treatmentwith P55A extracts (10 mg/mL) or insulin (10ÿ7

M) for 5 min, HepG2 cells were subjected to subcellularfractionation as described in Materials and Methods. The nuclear and postnuclear preparations wereincubated with anti-IRS-1 antibody, followed by the addition of protein A-sepharose. The immune-complex was centrifuged and subjected to SDS-PAGE and Western blot analysis with anti PI3-kinaseantibody.

Figure 2. Effect of P55A on the cytosolic ERK2 activity in HepG2 cells. Followingtreatment with P55A extracts (10 mg/mL) or insulin (10ÿ7

M) for 5 min, HepG2 cellswere subjected to subcellular fractionation as described in Materials andMethods. The postnuclear preparations were subjected to SDS-PAGE andWestern blot analysis with anti- ERK2 antibody or anti-phospho ERK2 antibody.

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cells, we determinedif it had an effect on the level ofglycogen.Treatmentof HepG2 cells with the butanolextractof P55A(10mg/mL) for 1 h causedanincreaseintheglycogenlevelsby 1.8-foldcomparedwith thecontrol(Fig.4).Theseresultssuggestthattheactivationof ERK2and PI3-kinase by the butanol extract of P55A couldcontributeto thesynthesisof glycogenin the liver.

DISCUSSION

Extracts of Phellodendronamurenseand Aralia elata

cortex(P55A)exertavarietyof biologicalactions.Thesetwo herbshave significant actionson glucosemetabo-lism. ERK2 andPI3-kinasehavebeenimplicatedin theregulationof glycogensynthesis,so we examinedtheeffect of extracts of P55A on ERK2 and PI3-kinaseactivitiesandglycogenlevelsin HepG2cells.

Oneof therationalesin developingantidiabeticagentsis to searchfor insulin mimicking agents.Consideringthis idea, we designed a series of experiments todetermineif P55Aextractshaveinsulin-like effectssuchas activation of ERK2, PI3-kinase and stimulation ofglycogenlevelsin HepG2cells.

Insulinactionsareinitiatedby theactivationof insulinreceptortyrosinekinase,followed by tyrosinephosphor-ylation of insulin receptorsubstrate,IRS-1.Next insulinsignallingis mediatedby the associationof IRS-1andanumberof SH2-domaincontainingproteins(Myers andWhite, 1996).Consequently,insulin signallingcascadesdivergeto two majorpathways:(1) sequentialactivationof PI3-kinase which is supposedto regulate glucosetransporter(Katagiri et al., 1996) and P70 S6 kinase(Cheathamet al., 1994), (2) activationof MAP kinasewhich is involvedin theactivationof glycogensynthesis(Sturgill et al., 1988;Dentet al., 1990).Recently,it hasbeensuggestedthatregulationof glycogensynthesismaybe mediatedby p70 S6 kinasethat is regulatedby PI3-kinase(Azpiazu et al., 1996). Of the abovedescribedinsulin signalling cascades,P55A extracts have asignificanteffectontheactivationof ERK2andglycogensynthesiswith little effect on insulin receptoractivation(data not shown). The butanol extract of P55A alsocausedan increasein the associationof IRS-1 andPI3-kinase. Since activation of PI3-kinase require theassociationof a regulatory subunit of PI3-kinase withtyrosine phosphorylatedIRS-1, the increase in theassociationof IRS-1 and PI3-kinase by the butanolextractof P55A could be regardedas the activationofPI3-kinase.In termsof mechanism,activecomponentsofthebutanolextractof P55Acrosstheplasmamembrane,activating PI3-kinaseand ERK2 in cytoplasmand alsocrossthe nuclearmembrane,stimulatingnuclearERK2.The water extract stimulates only nuclear ERK2,suggestingthe active componentsin the water extractfavour the nuclear environmentrather than the cyto-plasm.The activatedERK2 in cytoplasmin responseto

Figure 3. Effect of P55A on the nuclear ERK2 activity in HepG2 cells. Following treatmentwith P55A extracts (10 mg/mL) or insulin (10ÿ7

M) for 5 min, HepG2 cells were subjected tosubcellular fractionation as described in Materials and Methods. The nuclear prepara-tions were subjected to SDS-PAGE and Western blot analysis with anti-ERK2 antibody oranti-phospho ERK2 antibody.

Figure 4. Effect of P55A on the glycogen level in HepG2 cells.Following treatment with P55A extracts (10 mg/mL) or insulin(10ÿ7

M) for 5 min, HepG2 cells were subjected to lysis in thepresence of 0.1% sodium lauryl sulphate. Glycogen contentsin the cell lysates were measured as described in Materialsand Methods. (** p< 0.01; *** p<0.001)

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thebutanolextractof P55Amayplayarolein stimulatingglycogen synthesis: glycogen synthase is activatedthroughdephosphorylationof the enzymeby glycogensynthasephosphatasethat is phosphorylatedandstimu-latedby ERK2 (LawrenceandRoach,1997).Consider-ing PI3-kinaseis anupstreamkinasethatregulatesp70S6kinaseactivity (Cheathametal., 1994)andp70S6kinaseis involved in glycogensynthesis(Azpiazuet al., 1996),the activated PI3-kinase in cytoplasm by the butanolextractof P55Amay alsoplay a role in the synthesisofglycogen.The humanglycogensynthasegenecontainsseveral AP-1 binding sites (TPA-responsiveelement,TRE) in the5'-flankingregionof thegene(Nakayamaetal., 1994). AP-1 is a transcriptionfactor consistingofc-Jun, c-Fos and several related proteins that arephosphorylatedby ERK2, therebyits DNA binding andtranscriptionalactivitiesarestimulated(Kim andKahn,1994; Kim and Kahn, 1995; Whitmarsh and Davis,1996). ERK2 activated in the nucleus in responsetoP55A extractscould stimulate the transcriptionof theglycogen synthasegene possibly by phosphorylatingAP-1 transcriptionfactorsthatbind to AP-1bindingsites

in the 5'-flanking regionof the gene.The recentfindingthat PI3-kinase is involved in TRE-dependentgeneexpressionsupport the hypothesisthat activated PI3-kinasein responseto the butanolextractof P55A mayplay a role in the expressionof the glycogensynthasegene(Nishioka et al., 1995).TheseresultssuggestthatP55Aextractscouldbeusedto lower high bloodglucoselevels in diabetesby the activationof ERK2, PI3-kinaseand glycogen synthesis. Isolation, purification andcharacterizationof active principlescausingthe activa-tion of ERK2 andPI3-kinaseandglycogensynthesisinthebutanolextractof P55Aarecurrentlyunderstudy.

Acknowledgements

This work wassupportedin part by grantsfrom KoreanMinistry ofHealthandWelfare(SJKim), KoreanMinistry of Educationthroughresearch fund (SJ Kim) and Korea Science and EngineeringFoundation(Projectno.971-0704-026-2)(SJKim). TheauthorsthankDrs YS Chung(CentralResearchInstitute,Tae-Rim Pharm.Co.) andHS Choi (Animal ResourcesResearchCenter,Kon-Kuk University)for helpful discussions.SJ Kim greatly acknowledgessupportfromKyung-HeeUniversity.

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