Folia Biologica (Praha) 60, 168-179 (2014)

Original Article

Adipose Cells Induce Phospho-Thr-172 AMPK Production by Epinephrine or CL316243 in Mouse 3T3-L1 Adipocytes or MAPK Activation and G Protein-Associated PI3K Responses Induced by CL316243 or Aluminum Fluoride in Rat White Adipocytes(aluminumfluoride/AMP-activatedproteinkinase/CL316243/epinephrine/G-proteinsubunits/mitogen-activatedproteinkinase/mouse3T3-L1adipocytes/phosphatidylinositol3-kinase/ratwhiteadipocytes)

Y. OHSAKA1,3,5, H. NISHINO2,3, Y. NOMURA4,5

1Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Choshi, Japan2Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, Kusatsu, Japan3Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan4Yokohama College of Pharmacy, Yokohama, Japan5Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Abstract. Responses of adipose cells to adrenoceptor regulation, including that of β-adrenoceptor (AR), and the signalling machinery involved in these re-sponses are not sufficiently understood; information that is helpful for elucidating the adrenoceptor (adren-ergic and β-AR)-responsive machinery is insuffi-cient. We examined phospho-Thr-172 AMPK pro-duction in mouse-derived 3T3-L1 adipocytes treated with epinephrine or CL316243 (a β3-AR agonist) for 15 min. We also examined MAPK activation or G protein-associated PI3K activation or -associated PI3K p85 complex formation in rat epididymal

ReceivedJune17,2013.AcceptedMarch27,2014.

This work was supported in part by GrantsinAid from the Ministry of Education, Science and Culture of Japan.

Correspondingauthor:yasuhitoOhsaka,departmentofPharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan. E-mails:y-ohsaka@live.jp;y-ohsaka@cis.ac.jp

Abbreviations: AMP – adenosine monophosphate, AMPK –AMPactivated protein kinase, ATP – adenosine triphosphate, BRL37344–sodium4-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxyacetate, cAMP – adenosine3’,5’-cyclicmonophosphate,CL316243–disodium(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate,ERK–extracellular signal-regulatedkinase, Gi – inhibitory G protein, GLUT – glucose transporter, Gs – stimulatory G protein, GTP – guanosine triphosphate, GTPγS – guanosine 5’-[γ-thio]triphosphate, HSL – hormone-sensitive lipase, ISO – isoproterenol, LKB – liver kinase B, MAPK –mitogen-activated protein kinase, PI3K – phosphatidylinositol3-kinase,PTX–pertussistoxin.

(white) adipocytes treated with CL316243 for 15 min or aluminum fluoride (a G-protein signalling activa-tor) for 20 min. Furthermore, we examined the effect of PTX (a trimeric G-protein inactivator) on p85 complex formation induced by aluminum fluoride treatment. Western blot analysis revealed that epi-nephrine or CL316243 treatment increased the phos-pho-Thr-172 AMPK (an active form of AMPK) level in 3T3-L1 adipocytes. Activated kinase analysis with a specific substrate showed that CL316243 or alu-minum fluoride treatment activated MAPK in rat adipocytes. Immunoprecipitation experiments with a G-protein β subunit (Gβ) antibody showed that treatment of rat adipocytes with CL316243 activated PI3K and increased the PI3K p85 level in the Gβ an-tibody immunoprecipitates. Such an increase in the p85 level was similarly elicited by aluminum fluoride treatment in a PTX-sensitive manner. Our results provide possible clues for clarifying the signalling machinery involved in adrenoceptor responses, in-cluding those of β3-AR, in mouse-derived adipocytes and rat white adipocytes. Our findings advance the understanding of responses to adrenoceptor regula-tion in adipose cells and of the cellular signalling ma-chinery present in the cells.

IntroductionExposure of adipose cells to adrenergic stimulation

induces cellular responses, including molecular phosphorylation, lipolysis and glucose transport. Adrenoceptor signalling is implicated in the development of adipocyte disorders and can be targeted to improve adi

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pocytedysregulation(ArnerandHoffstedt,1999).How-ever, adrenergic responses in adipocytes and the signalling machinery present in the cells, including signalling machinery that induces adrenoceptor responses, have not been fully elucidated.Treatmentofratadipocytes,whichmainlyexpressβ1

andβ3-adrenoceptors (ARs),with adrenaline, a stress-responsive hormone, for 10 min activates AMPactivated protein kinase (AMPK) in a β-AR-dependentmanner(Kohetal.,2007).AMPKtargetingexperimentsshowedthat on treatment of rat or mouse adipocytes with a nonselectiveβ-ARactivator (for20min to1h), the lipo-lytic and antilipolytic effects involved activation of AMPK(yinetal.,2003;davaletal.,2005).However,the function of AMPK in response to adrenaline in adipocytes and adrenalineresponsive signalling to AMPK in the cells are not well understood, although an AMPK inhibitor partially blunts lipolysis induced by adrenaline in rat adipocytes (Koh et al., 2007). Treatment of ratadipocyteswithaβ3-ARagonist,BRL37344,for1hactivatesAMPK(davaletal.,2005),butthisagonistwasreportedlymore effective than anotherβ3AR agonist, CL316243 (CL), in tissues expressing β1- or β2AR (Bloometal.,1992).Mouse-derived3T3-L1(L1)adipocytes expressmainlyβ2- andβ3ARs (Monjo et al., 2005) and preferentially bind to epinephrine (EPI,adrenaline)(Laietal.,1982),and15-mintreatmentwithCL induced a response similar to that induced by EPI in L1adipocytes(OhsakaandNishino,2013).Long-termtreatmentofC57BL/6micewithCLactivatesAMPKinadipose tissues (Mulliganet al., 2007).However, it isnot clear whether shortterm treatment with CL or adrenaline activates AMPK in mouse adipocytes, although the effect of 1h EPI treatment on AMPK activation (phospho-Thr-172AMPKproduction)inL1adipocyteshasbeenexamined(Wattetal.,2006).

Treatment of mouse L1 adipocytes with CL for 15 min activatesERK(MAPK)(Robidouxetal.,2006).MAPKinactivation partially suppressed CLinduced lipolysis inL1adipocytes(Greenbergetal.,2001;Robidouxetal., 2006), and GTP-binding (G) protein regulatedMAPK activation in mouse adipocytes (Soeder et al., 1999;Caoetal.,2000).Mouseadipocytesexpresstwotypesofβ3-AR(β3a-andβ3b-ARs).TheC-terminalcytoplasmictailofthemouseβ3aAR is similar to that of the ratβ3-AR(Satoetal.,2005).ExaminationofMAPKactivationfollowingβ3AR or Gprotein signalling in rat adipocytes provides information that is helpful for clarifyingwhich typeofβ3AR mediates mouse adipocyte responsestoβ3AR activation and what signalling machineryactivatesMAPKinratadipocytes.Suchexamination is also helpful for clarifying the signalling machineryinvolvedinratβ3ARmediated lipolysis and rat MAPKdependent receptor responses. The effects of the selectiveβ3AR agonist CL or Gprotein signalling on MAPK activation in rat adipocytes remain unclear.

MAPK phosphorylates a lowmolecularweight protein in vitro(Haysteadetal.,1994).Inrat(epididymal)adipocytes, this lowmolecularweight protein is phos

phorylatedviaβ-ARfollowing10-minadrenergicstimulation(diggleanddenton,1992);suchphosphorylation induced by β-AR activation is induced in cellstreated with insulin, a hormone that activates MAPK (Sevetson et al., 1993). The signalling machinery involved in phosphorylation of the lowmolecularweight proteinfollowingβ-AR(non-selectiveβ-AR)activationin rat adipocytes is poorly understood. It is not known whetherMAPKactivationisregulatedbyβ-ARsignalling in rat white adipocytes.Treatmentof rat (white) adipocyteswithCL for10

min induces protein kinase activation in a wortmannin (WT)-sensitive manner (ZmudaTrzebiatowska et al., 2007).WTinactivatesseveralmolecules(Knightetal.,2006),includingphosphatidylinositolkinase.However,previous research has not investigated the rat adipocyte molecules involved in the WTsensitive response to CL. TheCL-responsivekinase isactivatedby3-phosphoinositide-related molecules (Walker et al., 1998), andWTwasshowntodirectlybindtosomePI3KsthatareactivatedbytheG-proteinβγ(Gβγ)subunits(Kurosuetal., 1995). Additionally, when rat adipocytes express3-phosphoinositide-dependentproteinkinase-1,MAPKisactivated(Sajanetal.,1999).TheeffectofCLonthePI3Kpathwayandthesignallingthatinducesthiseffectin rat adipocytes are poorly understood.PI3Kp85/p110activityissensitivetoWTandisalso

increased by Gβγ in vitro(Hazekietal.,1998).ThePI3Kp85/p110dimercanbeimmunoprecipitatedwithanantibodytoinsulinreceptorsubstrate-1(IRS-1)andactivated following treatment of rat adipocytes with insulin (KellyandRuderman,1993).Expressionofaconstitutively active form of PI3K p110 in rat adipocytes inducestranslocationofaglucosetransporter(GLUT)tothe plasma membrane (PM) (Tanti et al., 1996). Thenumber of PM GLUT molecules is increased by a nonselective β-AR activator for 30min in rat adipocytes(Shirakuraetal.,1990).PMGLUTtranslocationisalsoinducedbytreatmentofthecellsfor20minwithaGTPanalogue that activates both trimeric and small G proteins(Baldinietal.,1991).Thistreatment(for15min)activatedmembranePI3Kin ratadipocytes (Standaertetal.,1998).Themachinerythatmediatestheβ-ARactivationcaused PM GLUT induction and the GTP analogue-regulated PI3K activation and subsequent response in the cells is not well understood. A 10min treatmentofratadipocyteswithafluorideion(thestimulatory effects of which are blocked by a chelator of the aluminumion)(Suzukietal.,1992),whichisatrimericGprotein signalling activator, induces a response (glucosetransport)similartothatinducedbyβ-ARactivators,includingCL(Ohsakaetal.,1998).Treatmentwitha trimeric G-protein inactivator, PTX, alters GTP-re-gulatory signalling in rat adipocytes (Chaudhry et al., 1994).ItisunclearwhetherPI3Kp85/p110andGβγare involvedinβ-ARortrimericG-proteinsignallinginratadipocytes.To investigate adrenoceptor (adrenergic and β-AR)

responses in adipocytes and to obtain information help

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ful for elucidating the adrenoceptorresponsive machinery in the cells, we treated mouse L1 adipocytes with EPI or CL for 15 min or rat epididymal white adipocytes with CL or Gprotein signalling activator aluminum fluoride for15or20min andexamined thephospho-Thr-172AMPKlevelinL1adipocytesorMAPKactivation in rat adipocytes. In addition, rat adipocytes were treatedwithCLfor15min,andactivationofPI3KandassociationofPI3Kp85/p110withG-proteinsubunitswere examined by immunoprecipitation with an antibodytotheG-proteinβ(Gβ)subunitandbydetectionofPI3Kp85.Wealsoexamined,byimmunoprecipitation,theeffectoftrimericG-proteininactivationbyPTXonthe20-minaluminumfluoridetreatment-inducedassociationofPI3Kp85withtheGβ subunit.

Material and Methods

Materials

dulbecco’smodifiedEagle’smedium(dMEM)waspurchased from Nissui Pharmaceutical Co. (Tokyo, Japan).Bovine serum albumin (BSA, fatty-acid free),CL,EPI,insulin,3-isobutyl-1-methyxanthine(IBMX),leupeptin,Nonidet P (NP)-40, phenylmethylsulphonylfluoride(PMSF),sodiumdodecylsulphate(SdS),andTris were purchased from SigmaAldrich Co. (St. Louis, MO).AprotininwasobtainedfromRochediagnostics(Indianapolis, IN). Collagenase type I and PTXwereobtained from Worthington Biochemical Co. (Freehold, NJ)andKakenPharmaceuticalCo.(Tokyo,Japan),respectively.Anti-phospho-AMPK(Thr-172)rabbitantibody and anti-PI3K (p85) and anti-IRS-1 rabbit antibodies were purchased from Cell Signaling Technology Inc. (danvers, MA) and Upstate Biotechnology Inc.(LakePlacid,Ny),respectively.Anti-Gβ rabbit antibody (KT, MS, RA or SW, for a different Gβ peptide;Mu-rakamietal.,1992)wasakindgiftfromdr.TMurakami(Department of Clinical Biochemistry, Hokkaido University School ofMedicine, Japan). ProteinG-Sepha-rose 4 Fast Flowwas purchased fromGEHealthcareLife Sciences (Piscataway, NJ). Phosphatidylinositol(PI),Silicagel60plate,and[γ-32P]ATPwerepurchasedfromSerdaryResearchLaboratories(Ontario,Canada),Merck Millipore (darmstadt, Germany), and dupontNEN (Boston,MA), respectively.Ethylenediaminetet-raacetic acid (EdTA), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonicacid(HEPES),oilredO,polyacryl-amide, sodium chloride, and sucrose were pur chased fromNacalai Tesque (Kyoto, Japan). Ethylene glycoltetraacetic acid (EGTA)was fromdojindoMolecularTechnologies,Inc.(Kumamoto,Japan).Aluminumchloride, dithiothreitol (dTT), sodium fluoride, and otherchemicals (unless otherwise stated) were purchasedfromWakoChemicals(Osaka,Japan).

Treatment of 3T3-L1 adipocytes3T3-L1fibroblastswereplatedatadensityof3.5×

104cells/cm2 in DMEM supplemented with 10% heat

inactivated foetal calf serum (HyClone, South Logan, UT)inahumidifiedatmosphereof5%CO2and95%airat37°Candweredifferentiatedintoadipocytesasdescribed previously (Ohsaka and Nishino, 2013).Adi-pocytedifferentiationwasconfirmedbystainingtriacylglycerol droplets in the cells with oil red O and also by examiningthesignallingpathwaydownstreamofβ3AR whoseexpressionisinducedafterdifferentiation(Mon-joetal.,2005);cAMPaccumulationwasdeterminedbyradioimmunoassay using a cAMP assay kit (Yamasa ShoyuCo.,Chiba,Japan)followingCLtreatmentofL1adipocytes. Seven to nine days following the onset of differentiation, L1 adipocytes were treated with or withoutEPIorCL inKrebs-RingerHEPES (KRH)buffercontaining1%BSAat37°C.Beforeexposuretoagonists,differentiationofcellsineachdishwasexaminedby spectrophotometric measurement of lipid dye absorbanceat510nm(Ramírez-Zacaríasetal.,1992).Theagonisttreated and untreated L1 adipocytes were homogenizedwithabuffercontaining10μg/mlaprotinin,1mMEdTA,20mMHEPES(pH7.4),1mMPMSF,and0.25Msucroseandthenanalysedbywesternblotting.

Treatment of rat adipocytesEpididymal adipose tissues were isolated from a

Wistar-derivedstrainweighingbetween200and300g.Adipocytes were isolated by digesting adipose tissues in a Krebs-Ringer bicarbonate (KRB) buffer containing3%BSAand250U/mlcollagenase,asdescribedpreviously(Ohsakaetal.1998).CellssuspendedintheKRBbuffercontaining3%BSAatafinalconcentrationof106 cells/mlwereincubatedwithorwithoutCL,aluminumfluoride,orinsulinat37°Corpre-treatedwithPTXbeforethealuminumfluoridetreatment.

Measurement of MAPK activationMAPK activation was determined by measuring 32P

incorporationinaMAPK-specificsubstratebyusingaMAPK enzyme assay system (GE Healthcare Life Sciences),accordingtothemanufacturer’sinstructions.Rat adipocytes treated or untreated with agents were lysedusingabuffercontaining10μg/mlaprotinin,2mMdTT,2mMEGTA,10μg/mlleupeptin,150mMNaCl,1%NP-40,1mMPMSF,and10mMTris(pH7.4),andthelysateswereincubatedwith[γ-32P]ATPandtheassay substrate at 32 °C for 30 min. The reaction wasstopped by transferring the reactionmixture onto p81filterpaper(Whatmanplc,GEHealthcareLifeSciences).32P radioactivity incorporated in the substrate was quantifiedbyliquidscintillationcounting.

Immunoprecipitation and measurement of PI3K activation

Rat adipocytes were lysed as described previously (Ohsaka et al., 1997). Aliquots of cell lysates weremixedwith the indicated antibody that had been pre-adsorbed on protein GSepharose and then stirred over

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nightat4°C.Followingcentrifugation, immunecomplexesrecoveredinthepelletwereincubatedinabuffercontaining PI and [γ-32P]ATP as described previously(Giorgetti et al., 1992) or were analysed by westernblotting.Reactionmixturescontaining radiolabeledPIwere separated by thinlayer chromatography on a Silica Gel 60 plate in a chloroform/methanol/25% NH4OH/water(100:70:25:15,v/v)mixture.32Plabled PI was detected and quantified by using a Fuji BAS2000 Bio-imagingAnalyzer(FujiPhotoFilmCo.,Tokyo,Japan).

Western blot analysisAliquots of L1 adipocyte homogenates or rat adipo

cyte-derived immune complexes were separated by10%or8%SdSpolyacrylamidegelelectrophoresisandtransferred onto a Hybondenhanced chemiluminescence(ECL)filter(GEHealthcareLifeSciences).Theseparatedproteinstransferredontothefilterwereincubatedfor1−1.5hwiththeindicatedantibody(1 : 1000).After incubatingwith a horseradish peroxidase-conjugatedanti-rabbitIgGantibody(1 : 2500;CellSignalingTechnologyInc.),theimmunecomplexesweredetectedon X-ray film (Kodak X-Omat AR; Eastman-KodakCo.,Rochester,Ny)byusinganECLdetectionsystem(GEHealthcare Life Sciences). The blots on the filmwerequantifiedusing the ImageJprogram(http://rsb.info.nih.gov/ij/index.html).

Statistical analysisThe results were evaluated by using analysis of vari

ance (ANOVA) with Scheffe’s test (for comparisonsamongmultiple groups) and using unpaired Student’st-test (for differences between twogroups). Statisticalsignificancewas defined as P < 0.05 and is indicated withanasteriskmark(*).Valuesarepresentedasmeans±Sdofthreeorfourexperiments.

Results

Increases in the level of phospho-Thr-172 AMPK in L1 adipocytes treated with EPI or CLWeexamined the phospho-Thr-172AMPK level in

L1 adipocytes treated with EPI or CL. The level of phospho-Thr-172AMPKincreasedfollowingtreatmentwithEPIorCL (0.01−1μM) for15min (Fig. 1a, b);therewasnosignificantincreaseinthephospho-AMPKlevel after treatment with 1 or 0.1 nM of EPI or CL. Prior to treatment with agonists, cells plated on each dishdidnotexhibitquantitativedifferencesinthelevelsofcellularlipids(Fig.1d),whichserveasamarkerforadipocyte differentiation. Additionally, no variation in thephospho-Thr-172AMPKlevelwasobservedinL1adipocytes after incubation for 15 min in the absence of agonists (Fig. 1c).Results showing that the levels remained unchanged without agonists were also obtained in other experimental replicates (data not shown).Increased production of cAMP, a key signalling molecule in differentiated L1 adipocytes, as well as induction

oflipiddroplets(datanotshown),wasobservedintheadipocytes(Fig.1e).

Induced MAPK activation in rat adipocytes treated with CL

We treated rat adipocytes with CL and examinedMAPK activation. Treatment of cells for 15 min with CL(0.01and0.1μM)activatedMAPK(Fig.2a).How-ever,theincreaseinMAPKactivationwasnotsignificant in the treatment with 1 nM CL and was lost by increasingtheCLconcentrationto1μM(Fig.2a).MAPKwas activated by treatment with insulin (0.7 μM) for15min(Fig.2b).MAPKactivationdidnotvaryincellsuspensionsincubatedwithoutagonists(Fig.2c).

Induced PI3K activation and PI3K p85 immunoprecipitation with Gβ RA antibody in rat adipocytes treated with CL

PI3K was immunoprecipitated with some Gβ antibodies in rat adipocytes following treatment with CL for 15 min. Following immunoprecipitation with the Gβ RA antibody, treatmentofcellswith0.1μMCLactivatedPI3K(Fig.3a,b).However,PI3Kactivationwasnotinduced following immunoprecipitation with the Gβ MS, KT,orSWantibody (Fig.3a). Inaccordancewith results of previous studies (Giorgetti et al., 1992;KellyandRuderman,1993),ratadipocytesinducedPI3Kactivation following immunoprecipitation with an antiIRS-1antibody(Fig.3b)andthelevelofPI3Kp85increasedinIRS-1antibodyimmunoprecipitates(Fig.3c)inresponseto insulin.PI3Kactivationdidnotvaryinexperimentalreplicatesincubatedintheabsenceofagonists(Fig.3b,inset).WeexaminedtheeffectofCLonthelevelofPI3Kp85intheGβ RA antibody immunoprecipitates.Treatment of rat adipocyteswith 0.1 μMCLfor15minincreasedthePI3Kp85levelinGβ RA antibody immunoprecipitates (Fig.3d); treatmentwith1or10nMCLdidnotsignificantlyincreasethePI3Kp85level.ThePI3Kp85level inGβ RA antibody immunoprecipitates did not change in cells incubated in theabsenceofCL(datanotshown).

Induced MAPK activation and PTX-sensitive immunoprecipitation of PI3K p85 with Gβ RA antibody in aluminum fluoride-treated rat adipocytes

WetreatedratadipocyteswithaluminumfluorideandexaminedMAPKactivationandPI3Kp85level inGβ RA antibody immunoprecipitates. We also pretreated ratadipocyteswithPTXbeforealuminumfluoridetreatmentandexaminedthelevelofPI3Kp85intheimmunoprecipitates. Following treatment of cells with 10 mM aluminum fluoride for 20 min, MAPK was activated(Fig.4a)andthelevelofPI3Kp85inGβ RA antibody immunoprecipitatesincreased(Fig.4b);theseresponseswere not observed significantly after treatment with

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Fig. 1.EffectsofepinephrineorCL316243onphospho-Thr-172AMPKlevelinL1adipocytesa) L1 adipocytes were treated with the indicated concentrations of a)epinephrine(EPI)orb)CL316243(CL)for15min.Celllysateswerepreparedfromtreatedoruntreatedcells,andphospho-Thr-172AMPKlevelsweredeterminedbywesternblottingusingaphospho-AMPK(Thr-172)antibody.c)andinsets1−5showthephospho-Thr-172AMPKlevelsinthe cells treated for 15 min without agonists. d) shows the amount of oil red O dye staining in each dish of cells prior to treatmentwithagonists;dataareexpressedaspercentagesofvaluesobtainedfromcellstreatedfor15minwithoutagonists. e)showscAMPaccumulationinL1adipocytesoritsprecursorcells(L1fibroblasts)treatedwith0.1μMCLfor10mininthepresenceofIBMX(aphoshodiesteraseinhibitor,0.3mM).Blotsandinsets1−5representtypicalresults(3or4experiments).*P < 0.05 vs. L1 adipocytes not treated with EPI (a)orCL(b),orvs.L1fibroblasts(e)

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Fig. 2. Effects ofCL316243onMAPKactivation in ratadipocytes. Rat adipocytes were treated with the indicated concentrations of CL316243 (CL, a) or insulin (b) for15 min. Cell lysates were prepared from cells treated with or without agonists, and MAPK activation was measured. C shows MAPK activation in cells treated for 15 min without agents.*P < 0.05 vs. cells not treated with CL (a)orinsulin(b)

Fig. 3.EffectsofCL316243onPI3Kactivationfollowingimmunoprecipitation with a Gβ antibody and on immunoprecipitationofPI3Kp85withtheGβ antibody. Rat adipocytes were treated with the indicated concentrations of CL316243(CL;a, b, and d)orinsulin(b and c)for15min.Cell lysates were prepared and then immunoprecipitated (IP)withtheindicatedGβ antibody (MS (a),KT(a),SW(a),orRA(a, b and d))orananti-IRS-1antibody(b and c).ImmunoprecipitatedproteinswereusedtoexaminePI3Kactivation (a and b)anddetectionofPI3KbywesternblottingusingaPI3Kp85antibody(c and d).Insets1−4showPI3K activation in cells treated for 15minwithout agonists. Autoradiographic images and blots represent typical results(3or4experiments).*P < 0.05 vs. cells not treated with agonists (b and d)

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5mMaluminumfluoride.TherewasnochangeineithertheMAPKactivationlevel(datanotshown)orthePI3Kp85levelinGβRAimmunoprecipitates(Fig.4b,inset)in cell suspensions incubated in the absence of aluminumfluoride.After pre-treatmentwith PTX for 60min,thePI3Kp85levelinGβ RA immunoprecipitates didnotchangeincellstreatedfor20minwithaluminumfluoride(Fig.4c)andwithoutaluminumfluoride(datanotshown).

DiscussionIn this study, we observed responses to EPI,

CL316243,oraluminumfluorideinL1adipocytesandrat adipocytes. Adipocyte responses induced by the β3AR agonist CL and the Gprotein signalling activator aluminum fluoride were similar to those induced byadrenergicstimulation inL1adipocytesandbyβ3AR stimulation in rat adipocytes. These adipocytes possess cellular machinery, including that mediating adrenergic orβ-ARstimulation-inducedlipolytic(Chernicketal.,1986) or anti-lipolytic effects, low-molecular-weightprotein phosphorylation, PM GLUT induction and glucosetransport(Ludvigsenetal.,1980;Shirakuraetal.1990),andG-proteinactivatortreatment-inducedPI3Kactivation and glucose transportrelated responses. Aluminumfluoridetreatmentmimicsthesignallingeventsthatfollowreceptoractivation;itseffectsincludeinduction of trimeric Gprotein activation and adenylyl cyclase modulation (Sternweise and Gilman, 1982).Phospho-Thr-172AMPKproduction is induced inL1adipocytes by treatment with aluminum fluoride or anon-selective β-AR activator, isoproterenol (ISO), for15min(yinetal.,2003;Ohsakaetal.,2010).Molecularresponses to adrenergic stimulation in adipocytes and the signalling machinery included in these responses (machinery present in the cells), including those ofβ-ARs,arenotfullyunderstood.AMPKisactivatedinrat adipocytes treated with adrenaline or BRL37344.ERK phosphorylation (phospho-tyrosine/threonineERK(s)production)isinducedinmouseadipocytesby5or15-mintreatmentwithCL(Soederetal.,1999;Caoetal.,2000;Robidouxetal.,2006)andinmousebrownadipocytesby5-mintreatmentwithBRL37344(Lind-quistetal.,2000).Inratadipocytes,CLtreatmentactivatedproteinkinaseB(PKB)inaWT-sensitivemanner(Zmuda-Trzebiatowskaetal.,2007)andPTXtreatmentaltered trimeric Gi or Gs signalling (Chaudhry et al., 1994).Wefound thatEPIorCLtreatment for15mininducesphospho-Thr-172AMPKproductioninL1adipocytesandthatCLoraluminumfluoridetreatmentfor15or20mininducesMAPKactivationinratwhiteadipocytes. We also found that treatment of the rat white adipocyteswithCLfor15minactivatesPI3Kinimmunoprecipitates with a Gβ antibody and increases the PI3Kp85levelinGβ antibody immunoprecipitates and thatthisincreasedp85levelissimilarlyelicitedbyaluminumfluoride treatment in ratwhite adipocytes in aPTX-sensitivemanner.

Fig. 4.EffectsofaluminumfluorideonMAPKactivationandonPI3Kp85immunoprecipitationwithaGβ antibody followingpre-treatmentwithorwithoutPTX.Ratadipocytes were treated with the indicated concentrations of aluminumfluoridefor20min(a and b)orwerepre-treatedwithPTXfor60minat37°Cbeforethe20-minaluminumfluoride treatment (c). Cell lysates were prepared, andMAPK activation was measured (a).Preparedcelllysateswerealsoimmunoprecipitated(IP)withaGβ RA antibody, andPI3KwasdetectedbywesternblottingusingaPI3Kp85antibody(b and c).Insets1−3showthelevelofPI3Kp85 inGβRAantibody immunoprecipitates after20-mintreatmentwithoutaluminumfluoride.Blotsrepresenttypicalresults(3or4experiments).*P<0.05vs. cellsnot treatedwithaluminumfluoride(a and b)

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cAMPdependent kinase was activated and cAMP was produced in L1 adipocytes after CL treatment for 5 (Robidoux et al., 2006) or 10 min (Fig. 1e). CL isthought to activate the adenylyl cyclase system. AMPK isphosphorylatedattheThr-172residueandactivatedfollowing treatment with an adenylyl cyclase activator, forskolin,oracAMPanaloguefor5−30mininL1adipocytes (yin et al., 2003).These agents can alter theAMP/ATPratioandmodifyanAMPKkinase,LKB1,inthe cells (Gauthier et al., 2008). Experiments usingLKB1 hypomorphic mice showed that AMPK kinase in adipocytes regulates AMPK phosphorylation at the Thr172 residue and activation of the AMPK isoformsAMPKα1andα2(Gormandetal.,2011).L1adipocytesexpressedtheseisoforms(Saltetal.,2000).ThecAMPcontent in the cells increases in response to EPI treatmentfor≤10min(Elksetal.,1984).EPIorCLtreatment may induce production of a phospho-Thr-172AMPKisoform(s)byalteringtheAMPKkinaseactivityand/ortheAMP/ATPratioandactivateAMPK.AMPKα2 knockout (KO) mice develop adiposity

(Villenaetal.,2004).ThesizeofadipocytesisreducedinAMPKα1KOmice(davaletal.,2005).Theuseofanantagonist has shown that the response to EPI is mediated by signalling through β-ARs in L1 adipocytes(Mulder et al., 2005). Previous studies using ISO inmouse adipocytes have shown both a lipolytic function and an anti-lipolytic function ofAMPK; ISO-inducedlipolysisisdecreasedbytheexpressionofadominant-negativeformofAMPKα2inL1adipocytes(yinetal.,2003)andisincreasedbytheexpressionofadominant-negativeformofAMPKα1inisolatedmouseadipocytes(daval et al., 2005). EPI-induced phospho-Thr-172AMPK production would be helpful for working toward elucidation of the function of AMPK in adrenergic stimulation in mousederived adipocytes.β3AR mediates an L1 adipocyte response to EPI

(10-min treatment) (Mulder et al., 2005). Fifteen-mintreatment with ISO induces phospho-Thr-172AMPKproduction in L1 adipocytes. EPI may induce phosphoThr-172AMPKproductionviaβ-ARs,includingβ3AR. TreatmentwithEPIorISOfor15−20mininduceslipo-lysisinL1adipocytes(Chernicketal.,1986;yinetal.,2003),andpre-treatmentofthecellswithanAMPKactivator inhibits lipolysis and translocation of the enzyme hormone-sensitivelipase(HSL)tolipiddroplets,whichareinducedbyISO(davaletal.,2005).Thesignallingmachinery that mediates these adrenoceptor responses is not sufficiently understood. CL-induced phospho-Thr-172 AMPK production may be helpful for exa-mining the signalling machinery involved in EPI or ISOinduced phosphoAMPK production and also in lipolytic or antilipolytic responses to adrenoceptor (adrenergicorβ-AR)regulationinL1adipocytes.PhosphorylationofERK(s)occursinβ3AR agonist

treated mouse adipocytes and is required for activation ofthesekinases(Roskoski,2012).3-PhosphoinositidesareproduceduponPI3KactivationinaPI3Kp85-containingimmunocomplex(KellyandRuderman,1993).

Expressionof3-phosphoinositide-dependentproteinkinase-1orexpressionofanother3-phosphoinositide-responsivekinase(Standaertetal.,1997)activatesERK2inratadipocytes(Sajanetal.,1999).ERK1activationinduced by Gβγ is impaired in a cell line expressing adominant-negativemutantofPI3Kp85 (Haweset al.,1996).RatadipocytesexpressedtheisoformsERK1and2(Sevetsonetal.,1993);wedetectedMAPKactivationin the cells by insulin treatment, which produces phosphoproteins and activates these ERK isoforms. CL or aluminum fluoride treatment may activate a MAPKisoform(s)throughp85signallingandphosphorylationofMAPK(s)inratadipocytes.

A receptorkinase inhibitor of the epidermal growth factor(EGF)receptorimpairstheERKphosphorylation(activation)inducedbytreatmentwith0.1μMCLfor15min(non-kinasetypereceptorstimulation)inL1adipocytes(Robidouxetal.,2006).TheERKactivationinratadipocytes treated with EGF or insulin (which activates itsownreceptorkinase)isinhibitedbyforskolintreatment(Sevetsonetal.,1993).PI3Kactivationininsulin-treated rat adipocytes is suppressed by exposure to ahigh concentration of cAMP (Ohsaka et al., 1997).Induction of MAPK activation by treatment with low concentrations(0.01and0.1μM)ofCLwasimpairedwhentheCLconcentrationwasincreasedto1μM.Suchinhibition of activation by a high concentration of CL wasalsoobservedinanotherstudy(Ohsakaetal.,1998)andexaminationofPI3Kactivation(datanotshown)in≥1μMCL-treatedratadipocytes.AdecreasedMAPKresponse to increasing concentrations of CL in rat adipocytes may be due to negative regulation of the low CL concentrationsensitive signalling to MAPK.

MAPK was activated by CL treatment in rat adipocytes expressing β3AR with a Cterminal tail that is similartothatofthemouseβ3aAR. A peptide derived fromtheC-terminaltailofmouseβ3a-AR(butnotβ3bAR)altersitsownreceptorresponseinacellline(Satoetal.,2005),whichismodulatedbyPTXpre-treatment.SuchaPTX-sensitiveresponseissimilarlyobservedinratadipocytes(Chaudhryetal.,1994)andmouse3T3-F442 adipocytes (Soeder et al., 1999);PTX treatmentaltersCL-inducedMAPKactivation in3T3-F442adipocytesandmouseC3H10T1/2adipocytes(Caoetal.,2000). The receptors that mediate the CL-inducedMAPK activation in mousederived adipocytes may includemouseβ3aAR.Rat adipocytes express G proteins including PTX-

sensitive G proteins, such as Gi and its subtypes (Gi1 and 2) (Mitchell et al., 1989) and Gs (Hinsch et al.,1988).β3AR can be coupled to Gi and Gs in rat adipocytes(Chaudhryetal.,1994).Aluminumfluoridetreatment appears to activate MAPK by these G proteins in rat adipocytes.Aluminum fluoride-induced effects onMAPK activation may be useful for clarifying the signalling machinery involved in CLinduced MAPK activation in rat adipocytes and the signalling machinery thatisexpressedinthecells.

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Lipolysis is induced by CL treatment and also adrenergicstimulationinratadipocytes(Ohsakaetal.,1998)as well as in L1 adipocytes. Lipolysis induced by adrenaline treatment was not completely suppressed by an AMPK inhibitor in rat adipocytes. Activated MAPK phosphorylated HSL in vitro(Greenbergetal.,2001).A22-kdaprotein(PHAS-I,alow-molecular-weightprotein)thatisphosphorylatedbyMAPK(Haysteadetal.,1994) isphosphorylatedfollowingtreatmentwithISOor adrenergic stimulation (diggle anddenton, 1992).The machinery that mediates the adrenergic stimulationinduced lipolysis and PHASI phoshorylation in rat adipocytes isnotwellunderstood.A selectiveβ3AR antagonist impairs adrenergic responses in rat adipocytes (Nisolietal.,1996).CLandaluminumfluoride-inducedMAPKactivationmaybehelpfulforexaminingthesignalling machinery involved in the lipolytic and phosphorylative responses to adrenoceptor regulation in rat adipocytes. PTX-catalysedAdP-ribosylationoftheαsubunitof

trimeric G protein, which inactivates this protein, does not occur when the G protein is activated (when the Gα and Gβγsubunitsaredissociated)followingactivationoftheGprotein-coupledreceptor(Katadaetal.,1984).Inthe presence of Gβγ, ADPribosylation of the Gα subunit inducedbyPTXis inhibited in vitro with increases in the ratio of a Gβ antibody to Gβγ(Murakamietal.,1992);the inhibitory potential of the Gβ antibody is ranked as follows:RA>>SW=MS=KT,andtheGβ RA antibody interferes with the interaction between the Gα and Gβγ subunits.ActivationofPI3Korimmunoprecipitationofap85complexbytheGβ RA antibody and disruption of thiscomplexbyPTXinthetreatedcellsseemtoimplythatCLoraluminumfluoridetreatmentinducesdissociation of the Gα and Gβγ subunits of G proteins, includingthosesensitivetoPTX,andimmunoprecipitatestheGβγ subunits dissociated from these G proteins, and also thatcomplexesarenot formed through theGβ subunit peptide that is recognized by the Gβ RA antibody. p85/p110αandp85/p110βPI3Kisoformsareactivat

edbyaphosphotyrosylpeptide(thatcontainsayMXMmotifthatinteractswithPI3Kp85)orbyGβγ or both in vitro(Hazekietal.,1998).Ratadipocytesexpressbothoftheseisoforms(Ozanneetal.,1997);wedetectedap85 subunit-containing complex by immunoprecipitationwithIRS-I(whichhastheyMXMmotif)orGβ RA antibody. Previous work showed that 10min adrenergic stimulationinratadipocytesdoesnotactivatePI3Kinimmunoprecipitates with a phosphotyrosine antibody (Kellyetal.,1992).CLoraluminumfluoridetreatmentmayregulateaPI3Kp85/p110isoformviaGβγ in order toproduce3-phosphoinositidesoractivatePI3Kinratadipocytes.

The Gβderived peptide recognized by the Gβ RA antibody shows sequence similarity with the peptides of other types of Gβ (Gβ1-5,Watsonetal.,1994).Thep85subunitofPI3Kformsadimerwiththep110catalyticsubunit of PI3K. CL and aluminum fluoride-formedcomplexesinimmunoprecipitateswiththeGβ RA anti

body seem to contain a type of Gβsubunit and the p110 subunitofPI3Kandmaybeuseful forexamining themachineryinvolvedintheCL-inducedPI3Kresponsesassociated with Gβ.Ratadipocytesexpressnotonlythep85subunitofPI3Kbutalso,atalowerlevel,thep55subunitofPI3K(Inukaietal.,1997)andaresensitivetoaninhibitorofanothertypeofPI3K(PI3Kγ)thatisactivated by Gβγ(Baraglietal.,2011).WecannotexcludethepossibilitythatothertypesofPI3KareinvolvedinCL- and aluminum fluoride-induced responses in ratadipocytes. Expression of a 3-phosphoinositide-responsive ki

nase in rat adipocytes activates an ERK isoform or PKB (activationthatisinducedbyCL)(Grilloetal.,1999).CLmayactivateaPI3Kactivation-dependentmolecularmachinery via a Gβγ type in order to activate the PKB or ERK pathway. The number of PM GLUT molecules in rat adipocytes is increased by expression of an activeformofPI3KorPKB(Tantietal.,1997)andbyexpressionofthe3-phosphoinositide-responsivekinase(Stan-daertetal.,1997;Grilloetal.,1999).TreatmentofratadipocyteswithISOortheGTPanalogueGTPγS,whichactivates both trimeric and small G proteins, induces increasedPMGLUTmolecules (Shirakura et al., 1990;Baldinietal.,1991)orPI3Kactivation(Standaertetal.,1998). CL-induced PI3K activation, CL-induced p85complex formation, or aluminum fluoride-inducedPTX-sensitive complex formationmay be helpful forclarifying the machinery involved in the PKB or MAPK response induced by CL, induction of PM GLUT caused byβ-ARactivation,andGTPγS-regulatedPI3Kactivation and subsequent response.

Glucose transport increases in rat white adipocytes exposed to ISO or CL treatment and also adrenergicstimulation for 15 or 30min (Ludvigsen et al., 1980;Shirakuraetal.,1990;Ohsakaetal.,1998)orexposedtoanactivatorofGproteins (that includePTX-sensitiveGproteins) for 10 or 30 min (Suzuki et al., 1992;Standaertetal.,1998);ISOorGTPγStreatmentincreases glucose transport together with PM GLUT translocationorafterPI3Kactivation.Thesignallingmechanismsunderlying these responses of increased glucose transport in rat white adipocytes are not well understood. CL and aluminum fluoride-induced responses related toPI3Kmay also be helpful for elucidating themechanisms involved in glucose transport responses, which are increased by adrenoceptor regulation and by Gprotein signalling activators in rat white adipocytes.

AMPK positively or negatively regulates lipolysis and adipose cell size. Activated MAPK modulates HSL in vitro. InL1adipocytes, the activityofAMPKα1 isseveral-fold higher than that ofAMPKα2 (Salt et al.,2000).AnAMPKactivatorphosphorylatesacetyl-CoAcarboxylase (inhibits fatty acid synthesis activation)(Gormandetal.,2011)andinducesPMGLUTtranslocationandglucosetransport(yamaguchietal.,2005)inL1adipocytes.Ap85-containingimmunocomplexactivatesPI3Kandproduces3-phosphoinositides.ActivatedPI3KisinducedbyGβγ in vitro and induces PM GLUT

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translocation in rat adipocytes. The kinaserelated responses observed in this study may play a regulatory role in lipolytic and antilipolytic processes, lipogenic inhibition,Gprotein-associatedsignallingtoPI3Kandthe subsequent responses, and glucose transportrelated responses. Further analysis is needed to clarify the events that are related to the responses induced in this study and to determine the role of these responses.Moleculartargetingexperiments,whicharedesigned

on the basis of similar responses to separate signals, make it possible to work toward elucidation of the machinery involvedinreceptorsignalling.InhibitoryexperimentstargetingCL-andaluminumfluoride-responsivemolecules are needed to determine the signalling machinery involved in adrenoceptor responses. We provide possible clues for clarifying the signalling machinery involved in adrenergic and β-AR responses, includingthose of β3AR, in mousederived adipocytes and rat whiteadipocytes.Ourfindingsadvancetheunderstanding of adrenoceptor responses in adipose cells and of the cellular signalling machinery present in the cells.

AcknowledgementsWe thank Dr. T. Murakami and Dr. Y. Tokumitsu

(Department of Pharmacology, Faculty of Pharma ceuticalSciences,HokkaidoUniversity,Japan)fortheiradvice on the antiGβ antibody.

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