Palmitic acid mediates hypothalamic insulin resistance by altering
Transcript of Palmitic acid mediates hypothalamic insulin resistance by altering
Research article
TheJournalofClinicalInvestigationâ â â http://www.jci.orgâ â â Volumeâ119â â â Numberâ9â â â Septemberâ2009â 2577
Palmitic acid mediates hypothalamic insulin resistance by altering PKC-Ξ subcellular
localization in rodentsStephen C. Benoit,1 Christopher J. Kemp,1 Carol F. Elias,2 William Abplanalp,1 James P. Herman,1 Stephanie Migrenne,3 Anne-Laure Lefevre,3 CĂ©line Cruciani-Guglielmacci,3 Christophe Magnan,3
Fang Yu,4,5 Kevin Niswender,4,5 Boman G. Irani,2 William L. Holland,2 and Deborah J. Clegg2
1Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio, USA. 2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA. 3CNRS, University of Paris 7, Paris, France. 4Department of Veterans Affairs, Tennessee Valley Healthcare System,
Nashville, Tennessee, USA. 5Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
InsulinsignalingcanbemodulatedbyseveralisoformsofPKCinperipheraltissues.Here,weassessedwhetheronespecificisoform,PKC-Ξ,wasexpressedincriticalCNSregionsthatregulateenergybalanceandwhetheritmediatedthedeleteriouseffectsofdietshighinfat,specificallypalmiticacid,onhypothalamicinsulinactivityinratsandmice.Usingacombinationofinsituhybridizationandimmunohistochemistry,wefoundthatPKC-Ξwasexpressedindiscreteneuronalpopulationsofthearcuatenucleus,specificallytheneuropep-tideY/agouti-relatedproteinneuronsandthedorsalmedialnucleusinthehypothalamus.CNSexposuretopalmiticacidviadirectinfusionorbyoralgavageincreasedthelocalizationofPKC-Ξtocellmembranesinthehypothalamus,whichwasassociatedwithimpairedhypothalamicinsulinandleptinsignaling.Thisfind-ingwasspecificforpalmiticacid,asthemonounsaturatedfattyacid,oleicacid,neitherincreasedmembranelocalizationofPKC-Ξnorinducedinsulinresistance.Finally,arcuate-specificknockdownofPKC-Ξattenu-ateddiet-inducedobesityandimprovedinsulinsignaling.Theseresultssuggestthatmanyofthedeleteriouseffectsofhigh-fatdiets,specificallythoseenrichedwithpalmiticacid,areCNSmediatedviaPKC-Ξactivation,resultinginreducedinsulinactivity.
IntroductionTheâregulationâofâbodyâweightâoccursâthroughâtheâintegrationâofâperipheralâandâcentralâsignalsâreflectingâavailableâenergy.âNutri-tionalâstatusâisâaâkeyâcomponentâofâthisâprocessâandâisâsensedâwithinâtheâhypothalamusâandâotherâareasâofâtheâbrainâasâwellâasâthroughoutâtheâbody.âNutritionalâstatus,âinâturn,âcontributesâtoâtheâcontrolâofâfoodâintake,âenergyâexpenditure,âandâenergyâfluxesâthroughoutâtheâbody.âUnderstandingâtheâmechanismsâbyâwhichâspecificânutrients,âsuchâasâfattyâacids,âinfluenceâsignalsâthatâinter-actâwithinâtheâbrainâhasâbecomeâaâgoalâinâtheâdevelopmentâofâeffec-tiveâtreatmentsâforâobesityâandâotherâmetabolicâdisturbances.
Considerableâevidenceâimplicatesâtheâpancreaticâhormoneâinsu-linâandâtheâadipocyteâhormoneâleptinâasâkeyâsignalsâthatâactâinâtheâhypothalamusâtoâregulateâbodyâweightâandâhepaticâglucoseâproduction.âBothâhormonesâexertâaânetâcatabolicâeffectâ(1,â2),âandâtheyâshareâcommonâintracellularâsignalingâpathways,âeachârequir-ingâactivationâofâPI3Kâforâitsâcatabolicâactionâ(3,â4).âManyâperiph-eralâtissuesâbecomeâresistantâtoâinsulinâandâleptinâinâtheâpres-enceâofâcertainâfattyâacidsâ(5â9).âWeârecentlyâdemonstratedâthatâsaturatedâfattyâacidsâand,âmoreâspecifically,âpalmiticâacidâcauseâbrainâinsulinâresistanceâ(10).âCNSâresistanceâtoâleptinâandâinsu-linâcompromisesâtheâabilityâofâbothâhormonesâtoâregulateâfoodâintakeâandâbodyâweightâinâtheâpresenceâofâdietsâhighâinâsaturatedâfat/palmiticâacid,âsubsequentlyâresultingâinâobesity.âAdditionally,âweâfoundâthatâpalmiticâacidâimpairsâtheâabilityâofâinsulinâtoâacti-
vateâitsâintracellularâsignalingâpathwaysâ(10).âImportantly,âtheseâeffectsâofâpalmiticâacidâareâuniqueâforâthisâtheâtypeâofâfattyâacid.âOleicâacid,âforâexample,âhasâbeenâdemonstratedâtoâactâasâaâcentralâinsulinâmimeticâandâtoâhaveâbeneficialâeffectsâonâhepaticâglucoseâhomeostasisâandâbodyâweightâregulationâ(11).âConsistently,âitâhasâbeenâdemonstratedâthatâdietsâhighâinâmonounsaturatedâfattyâacidsâ(oleicâacid)âpreventâdietaryâfatâinducedâinsulinâresistanceâ(11,â12),âwhereasâdietsâhighâinâpalmiticâacid accelerateâobesityâ(10).âPotentialâmechanismsâbyâwhichâtheseâfattyâacidsâdifferen-tiallyâinfluenceâinsulinâsignalingâasâwellâasâbodyâweightâregulationâhaveâbeenâposited.âHere,âweâfocusâonâtheâCNSâandâprovideâdataâsuggestingâwhatâweâbelieveâtoâbeâaânovelâmechanismâbyâwhichâspe-cificâfattyâacidsâimpairâcentralâinsulinâ(andâleptin)âsignalingâbyâactivatingâaânovelâfamilyâmemberâofâPKC.
Inâtheâperiphery,âisoformsâofâtheânovelâclassâofâPKCâserine-threo-nineâkinasesâareâinvolvedâinâsignalâtransductionâpathwaysâthatâgov-ernâaâwideârangeâofâphysiologicalâprocesses,âincludingâdifferentia-tion,âproliferation,âgeneâexpression,âmembraneâtransport,âandâtheâorganizationâofâcytoskeletalâandâextracellularâmatrixâproteinsâ(13).âTheseâPKCsâareâthoughtâtoâbecomeâactiveâwhenâtranslocatedâfromâintracellularâpoolsâtoâaâsiteâatâtheâinnerâsurfaceâofâtheâcellâmem-brane,âinâwhichâtheyâinteractâwithâreceptorsâandâotherâproteinsâtoâmodifyâcellâsignaling.âMoreâspecifically,âPKCsâhaveâbeenâproposedâtoâmediateâdietaryâfatâinducedâmetabolicâdiseaseâ(14â16).âTranslo-cationâofâPKC-Ξâtoâtheâcellâmembraneâinhibitsâinsulinâsignalingâ(17,â18)âbyâenhancingâSer/ThrâphosphorylationâofâtheâIRâandâmodulat-ingâIRâinternalizationâ(19).âBecauseâofâthis,âaâconsiderableâamountâofâresearchâhasâfocusedâonâtheâroleâofânovelâPKCsâinâfattyâacidâinducedâperipheralâinsulinâresistanceâ(5â7,â20,â21).âUntilârecently,â
Authorshipnote:âChristopherâJ.âKempâandâStephenâC.âBenoitâareâcoâfirstâauthors.
Conflictofinterest:âTheâauthorsâhaveâdeclaredâthatânoâconflictâofâinterestâexists.
Citationforthisarticle:âJ. Clin. Invest.â119:2577â2589â(2009).âdoi:10.1172/JCI36714.
research article
2578 TheJournalofClinicalInvestigationâ â â http://www.jci.orgâ â â Volumeâ119â â â Numberâ9â â â Septemberâ2009
veryâlittleâresearchâhadâaddressedâwhetherâPKCsâinâtheâCNSâmediateâtheâsameâeffectsâasâtheyâdoâinâtheâperiphery.âImportantly,âRossâetâal.ârecentlyâreportedâthatâaâmemberâofâtheânovelâclassâofâPKCâisoforms,âPKC-ÎŽ,âactuallyâfacilitatesâinsulinâsignalingâinâtheâCNSâ(22).
Inâmuscle,âaâshort-termâinfusionâofâIntralipidâ(aâfattyâacidâemul-sionâcomprisedâofâaâmixtureâofâfattyâacids,âincludingâlinoleic,âoleic,âandâpalmiticâacid)âimpairsâinsulin-stimulatedâglucoseâdisposal,âandâthisâisâthoughtâtoâbeâdueâtoâincreasedâactivationâofâPKC-Ξ,âtheâmostâabundantâofâtheânovelâclassâofâPKCâisoformsâinâskeletalâmuscleâ(23â27),âwithâaâconsequentâdecreaseâinâinsulin-stimulatedâIRS-1âtyrosineâphosphorylationâandâPI3Kâactivityâ(5â7,â28).âBasedâonâtheseâdataâfromâperipheralâtissues,âweâhypothesizeâthatâPKC-Ξâisâaâcriticalâmediatorâofâfattyâacidâinducedâcen-tralâinsulinâandâleptinâresistance.âFurthermore,âweâhypothesizeâthatâexposureâtoâpalmiticâacidâ(asâopposedâtoâoleicâacid)â increasesâhypotha-lamicâPKC-Ξâactivationâandâtranslocationâwith-inâcriticalâbrainâregionsâandâthatâthisâinterferesâwithâtheâabilityâofâcentralâinsulinâandâleptinâtoâactivateâPI3K.âThatâis,âincreasedâhypothalamicâPKC-Ξâ activationâ andâ translocationâ contrib-utesâtoâleptinâandâinsulinâresistanceâwithinâtheâbrainâandâtheâdevelopmentâofâobesityâandâdys-regulationâofâhepaticâglucoseâproduction.âTheseâhypothesesâareâbasedâonâourârecentâfindingsâthatâeitherâaâsaturatedâhigh-fatâdietâ(highâinâpalmiticâacid)âorâdirectâCNSâinfusionâofâpalmiticâacidâconfersâinsulinâresistanceâwithinâtheâbrainâ(10).
ResultsPalmitic acid, regardless of obesity, impairs leptin and insulinâs ability to regulate food intake and body weight and decreases activation of PI3K.âInâagreementâwithâpreviousâreportsâ(10,â29,â30),âweâfirstâconfirmedâthatâmaintenanceâonâaâhigh-fatâsaturatedâ(HFS)âdietâreducesâhypothalamicâinsulinâandâleptinâsen-sitivity.âRatsâfedâaâdietâhighâinâsaturatedâfatâ(40%âofâcaloriesâfromâHFSâdiet)âorâaâdietâhighâinâoleicâacidâ(40%âofâcaloriesâfromâoleicâacidâdiet)âforâ3âmonthsâweighedâmoreâandâhadâmoreâbodyâfatâ(Tableâ1)âthanâratsâfedâaâmatchedâlow-fatâcontrolâdietâ(forânutrientâinformation,âseeâref.â31âandâTableâ2).âTheâfattyâacidâcompositionâofâtheâHFSâdietâwasâ10%âmyristate/27%âpalmitate/12%âstearate/25%âoleate.âTheâcompositionâofâtheâoleicâacidâdietâ
wasâ11%âpalmitate/â70%âoleate/13%âlinoleate.âItâisâimportantâtoânoteâtheâHFSâcontainsâaâhighâamountâofâsaturatedâfattyâacids,âbutâitâalsoâcontainsâunsatu-ratedâfattyâacids,âincludingâoleicâacid.âTheâcriticalâvariableâweâareâtestingâisâtheâroleâofâpalmitate,âandâtheâHFSâdietâhasâsubstantiallyâmoreâpalmitateâthanâtheâoleicâacidâenrichedâdiet.âTheâHFSâdietâfedâratsâhaveâanâattenuatedâanorexicâresponseâtoâthird-ven-tricularâ(i.c.v.)âinsulinâ(Figureâ1A)âcomparedâwithâtheâanimalsâmaintainedâonâtheâlow-fatâorâtheâhigh-fatâoleicâacidâdiets.âToâassessâwhetherâtheâfailureâofâinsulinâtoâreduceâbodyâweightâinâHFSâdietâfedâratsâwasâassociatedâwithâreducedâinsulin-inducedâphos-phorylationâofâAKTâ(p-AKT),âinsulinâwasâinfusedâi.c.v.âandâratsâwereâsacrificedâ10âminutesâlater.âInsu-
lin-inducedâp-AKTâwasâassessedâinâmedialâbasalâhypothalamicâtissueâ(theâbrainâwasârapidlyâremoved,âsnapâfrozen,âandâaâ~70-mgâwedgeâofâhypothalamusâwasâexcisedâcaudalâtoâtheâmammillaryâbodies,ârostralâtoâtheâopticâchiasm,âlateralâtoâtheâopticâtract,âandâsuperiorâtoâtheâapexâofâtheâhypothalamicâthirdâventricle).âAnimalsâmaintainedâonâtheâHFSâdietâhaveâreducedâinsulin-inducedâp-AKTârelativeâtoâthoseâonâtheâlow-fatâandâtheâoleicâacidâdietsâ(Figureâ1,âBâandâC),âimplyingâthatâmaintenanceâonâaâsaturatedâfattyâacidâdiet,âwhichâisâhighâinâpalmiticâacid,âbutânotâaâdietâhighâinâoleicâacid,âattenuatesâinsulinâactivityâandâinducesâinsulinâresistance.
Leptinâ(3)âandâinsulinâ(4)âexertâtheirâcatabolicâactionâthroughâPI3K,âandâweâfoundâreductionsâinâleptinâsâabilityâtoâreduceâfoodâ
Table 1Characteristics of rats
Low fat Oleic HFS-R HFSWeight (g) 466 ± 6.1 478 ± 7.8A 458 ± 4.6 529 ± 9B
Adiposity (g) 111 ± 4.75 128.8 ± 8.8A 113 ± 3.6 148.4 ± 8.4B
Fasting glucose (mg/dl) 117 ± 3.6 129 ± 4A 112 ± 1.7 129 ± 3.8A
Fasting insulin (pM) 56 ± 7.7 86 ± 10.4A 67 ± 9.4 122 ± 22.5B
Body weight (g), carcass fat (g fat/100 g carcass), plasma insulin, and plasma glucose of rats maintained on either the low-fat, oleic acid, HFS-R, or HFS diets for 3 months (n = 8â10/group). In each column, values with different superscripts are statistically differ-ent (P < 0.05; mean ± SEM). Low fat, low-fat dietâfed rats; Oleic, oleic acid dietâfed rats; HFS-R, HFS-R dietâfed rats HFS, HFS dietâfed rats.
Table 2Composition (g/kg of diet) of the low-fat, HFS, and oleic acid diets
Product Low-fat diet HFS diet Oleic acid diet g kcal g kcal g kcalProtein (%) 14.2 15 16.6 15 16.6 15Carbohydrate (%) 72.1 76 52.0 46 52.0 46Fat (%) 4.0 9 20.0 40 20.0 40Total (%) 100 100 100Total (kcal/g) 3.81 4.54 4.54Ingredient Casein, 80 Mesh 140 560 164 656 164 656 l-Cystine 1.8 7 2.1 8 2.1 8 Corn starch 485.7 194.3 303.1 121.2 303.1 121.2 Maltodextrin 10 125 500 115 460 115 460 Sucrose 100 400 89.9 360 89.9 360 Cellulose, BW200 50 0 58.6 0 58.6 0 Soybean oil 10 90 10 90 10 90 Butter, anhydrous 30 270 190 1,710 0 0 Olive oil 0 0 0 0 190 1,710 AIN-93G-MX 35 0 41 0 41 0 AIN-93-VX 10 0 11.7 0 11.7 0 AIN-93M-MX 10 40 11.7 47 11.7 47 Choline bitartrate 2.5 0 2.9 0 2.9 0 FD&C Yellow Dye no. 5 0 0 0.025 0 0.025 0 FD&C Red Dye no. 40 0.025 0 0 0 0.025 0 FD&C Blue Dye no. 1 0.025 0 0.025 0 0 0Total 1,000.05 3,810 1,000.05 4,543 1,000.05 4,543Vitamin E (IU/kg) 77.1 93.4 112.0Vitamin E (IU/3810 kcal) 77.1 78.3 93.9
AIN-93G-MX, AIN93 Growth Mineral Mix; AIN-93-VX, AIN93 Vitamin Mix; AIN-93M-MX, AIN93 Maintenance Mineral Mix.
research article
TheJournalofClinicalInvestigationâ â â http://www.jci.orgâ â â Volumeâ119â â â Numberâ9â â â Septemberâ2009â 2579
intakeâinâHFSâdietâfedâversusâlow-fatââorâtheâoleicâacidâfedâratsâ(Figureâ1D).âAdditionally,âweâfoundâthatâconsumptionâofâtheâHFSâdietâreducedâleptin-inducedâp-STAT3âimmunoreactivity,âaâmarkerâofâleptinâreceptorâactivationâ(32,â33)â(dataânotâshown).âTheseâdataâimplyâthatâtheâHFSâdietâ(withâelevatedâpalmiticâlevels),âimpairsâbothâsignalingâpathways.
Toâconfirmâthatâtheâobservedâinsulinâresistanceâisâtheâresultâofâdietaryâfattyâacids,âratherâthanâobesityâperâse,âweâperformedâanâadditionalâstudyâinâwhichâ1âgroupâofâratsâhadârestrictedâaccessâtoâtheâHFSâdietâandâwereânotâallowedâtoâbecomeâobese,ârelativeâtoâcontrols.âTheseâanimalsâwereâgivenâtheâsameânumberâofâcaloriesâconsumedâbyâtheâlow-fatâdietâfedâcontrolâanimalsâ(HFS-restrictedâdietâ[HFS-Râdiet]).âHFS-Râdietâfedâratsâdevelopedâinsulinâresis-tanceârelativeâtoâtheâlow-fatâdietâfedâcontrolsâ(Figureâ1E),âimply-ingâthatâmaintenanceâonâtheâhigh-saturated-fatâdiet,âratherâthanâincreasedâbodyâfatâperâseâ(Tableâ1),âisâsufficientâtoâreduceâcentralâinsulinâsensitivityâandâconfirmingâourârecentâresults.
Palmitic acid inhibits CNS insulin regulation of hepatic glucose produc-tion.âInsulinâregulatesâglucoseâuseâthroughoutâtheâbody,âincludingâtheâbrainâ(1,â34).âToâtestâtheâhypothesisâthatâaâcomponentâofâsatu-ratedâfattyâacidsâdiets,âspecificallyâpalmiticâacid,âresultsâinâimpairedâinsulinâregulationâofâhepaticâglucoseâproduction,âweâadministeredâlowâdosesâofâpalmiticâacid,âoleicâacid,âorâvehicleâdirectlyâintoâtheâbrain,âfollowedâbyâanâintracarotidâinfusionâofâinsulin.âIntracarotidâinsulinâinfusionsâmimicâtheârouteâbyâwhichâendogenousâinsulinâphysiologicallyâaccessesâtheâbrainâandâactâatâcentralâsitesâwithoutâalteringâplasmaâinsulin,âglucose,âorâfattyâacidâlevelsâ(35).âi.c.v.âinfu-
sionsâofâpalmiticâacid,âbutânotâoleicâacidâorâvehicleâalone,âattenu-ateâinsulin-inducedâsuppressionâofâhepaticâglucoseâproduction,âasâdemonstratedâbyâaâeuglycemicâclampâinâawake,âunrestrainedâratsâmaintainedâonâtheâlow-fatâdietâ(Figureâ2).âImportantly,âtheâcentralâinfusionsâofâfattyâacidsâdidânotâsignificantlyâchangeâperipheralâFFAâconcentrationsâ(vehicle,â384â±â30âÎŒmol/l;âpalmiticâacid,â419â±â39ââÎŒmol/l;âoleicâacid,â392â±â51âÎŒmol/l),âimplyingâthatâchangesâinâpal-miticâacidâconcentrationsâinâtheâCNSâhaveâprofoundâeffectsâonâCNSâactionsâandâdoânotâincreaseâcirculatingâFFA.
Palmitic acid inhibits CNS insulin signaling in vitro.âToâfurtherâtestâtheâspecificâeffectsâofâpalmiticâacidâonâinsulinâsignaling,âweâexposedâhypothalamicâneuronalâcellsâinâcultureâtoâpalmiticâacidâorâoleicâacid,âusingâaâcellâlineâthatâexpressesâbothâleptinâandâIRsâ(36)âandâisâresponsiveâtoâbothâleptinâandâinsulinâ(dataânotâshown).âSimilarâtoâourâinâvivoâfindings,âpalmiticâacid,âbutânotâoleicâacid,âattenuatedâinsulin-inducedâp-AKTâ(Figureâ3,âAâandâB)âinâvitro.âBecauseâfattyâacidsâcanâbeâtoxicâtoâcellsâinâculture,âweâalsoâassessedâtheâtoxicityâofâtheâconcentrationâofâpalmiticâacidâusedâinâourâexperiments,âwithâtheâTrypanâblueâexclusionâtestâofâcellâviabilityâ(Figureâ3,âCâandâD).âFollowingâexposureâtoâ100âÎŒmol/lâpalmiticâacid,âcellsâwereânormalâandâviableâ(Figureâ3C;âasâusedâinâFigureâ3,âAâandâB),âwhereasâtoxic-ityâandâcellâdeathâwasâdemonstratedâfollowingâexposureâtoâaâ300âÎŒmol/lâdoseâ(Figureâ3D).âAdditionally,âweâfound,âfollowingâexpo-sureâtoâ100âÎŒmol/lâpalmiticâacid,âlevelsâofâpJNKâorâotherâinflamma-toryâintermediatesâdidânotâchange.âTheseâdataâareâconsistentâwithâourâhypothesisâthatâpalmiticâacidâdirectlyâimpairsâinsulin-inducedâp-AKT,âratherâthanâexertingânonspecificâtoxicâeffects.
Figure 1Diets high in saturated fats impair insulin- and leptin-induced anorexia and hypothalamic insulin-signaling. Rats (n = 8â10/group) were main-tained on a low-fat diet (low fat), a HFS diet, or diet high in oleic acid (Oleic; see Table 2 for nutrient composition). Rats received i.c.v. infusions of 8 mU insulin or saline and food intake was measured at 24 hours. (A) Food intake after drug and saline (vehicle) control injections (*P < 0.05 compared with saline intakes). Rats maintained on low-fat, oleic acid, or HFS diet received an i.c.v. infusion of 8 mU insulin or saline and were sacrificed after 10 minutes. (B) Representative Western blots for p-AKT and GAPDH. (C) Densitometry analysis for all rats. ANOVA and post-hoc tests confirmed that rats maintained on HFS diet had lower p-AKT (P < 0.05 compared with other groups) and that only rats consuming low-fat and oleic acid diets exhibited insulin-induced p-AKT (*P < 0.05 compared with saline). (D) A separate group of rats were maintained on the same diets and received 10 ÎŒg i.c.v. leptin or saline (*P < 0.05 compared with saline). (E) Animals on the HFS-R diet (Table 1) were insulin resistant relative to the low-fatâfed animals, implying that the HFS diet, rather than increased body fat per se, is sufficient to reduce central insulin sensitivity. ANOVA and post-hoc tests confirmed only rats maintained on low-fat diet exhibited insulin-induced anorexia (data are mean ± SEM; *P < 0.05 compared with saline injection).
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2580 TheJournalofClinicalInvestigationâ â â http://www.jci.orgâ â â Volumeâ119â â â Numberâ9â â â Septemberâ2009
Palmitic acid increases CNS diacylglycerol levels.âOurâworkingâhypothesisâisâthatâfattyâacidsâincreaseâintracellularâdiacylglycerolâ(DAG),âwhichâfacilitatesâtheâtranslocationâofâPKC-Ξâandâreducesâinâinsulinâactivity.âWeâassessedâCNSâDAGâlevelsâfollowingâpalmiticâandâoleicâacidâexpo-sure,âbothâinâvitroâandâinâvivo.âHere,âweâreportâanimalsâexposedâtoâtheâHFSâdietâhaveâsignificantlyâ(Pâ<â0.05)âelevatedâDAGâlevelsârelativeâtoâtheâoleicâacidââandâlow-fatâfedâanimalsâ(lowâfat,â549.2â±â22.5âpmol/mg;âHFS,â863.7â±â121.1âpmol/mg;âoleicâacid,â599.1â±â45.5âpmol/mg).âAdditionally,âfollowingâtheâgavageâofâtheâfattyâacids,âweâagainâfoundâsignificantlyâ(Pâ<â0.05)âelevatedâDAGâlevelsâinâpalmitateâacidâgavagedâanimalsârelativeâtoâtheâoleicâacidâgavagedâorâcontrolâgavagedâanimalsâ(control,â320â±â25.6âpmol/mg;âpalmitate,â449â±â28.9âpmol/mg;âoleicâacid,â338â±â49.7âpmol/mg).âInâvitro,âweâobservedâthatâcellsâexposedâtoâpalmiticâacidâexhibitedâaâsignificantâ(Pâ<â0.05)âincreaseâinâDAGâlevels,âwhichâdidânotâoccurâinâtheâcellsâexposedâtoâoleicâacidâ(con-trol,â1.0â±â0.04âpmol/mg;âpalmitate,â3.2â±â0.9âpmol/mg;âoleicâacid,â1.1â±â0.05âpmol/mg).âTherefore,âexposureâtoâdietsâelevatedâinâpalmiticâacidâresultsâinâincreasedâCNSâDAGâlevels,âprovidingâaâmechanismâbyâwhichâPKC-Ξâtranslocationâisâactivated.
PKC-Ξ is expressed in hypothalamic nuclei critical for hepatic glucose production and body weight regulation.âInâorderâtoâdetermineâtheâmechanismâbyâwhichâfattyâacidsâwereâinhibitingâinsulinâsignaling,âweânextâsoughtâtoâdetermineâwhetherâPKC-Ξâisâexpressedâinâtheâbrainâandâwhetherâitâisâaâcriticalâmediatorâinâreducingâhypotha-lamicâinsulinâsensitivity.âWeâdesignedâprimersâagainstâratâPrkcqâmRNAâ(GenBankâaccessionânumberâAB020614)âandâperformedâ
RT-PCRâonâmuscleâandâhypothalamicâtissueâfromâuntreatedâcon-trolârats.âTheâresultingâproductâsequenceâwasâidenticalâtoâbothâratâandâhumanâPKC-Ξâwithâonlyâ10%â(nonprimer)âhomologyâtoâtheâreportedâPKC-ÎŽâsequencesâ(seeâMethods).âConsistentâwithâaârecentâreportâbyâDewingâetâal.â(37),âPKC-Ξâwasâexpressedâinâtheâmedialâbasalâhypothalamusâandâmuscleâofâratsâ(Figureâ4A).âToâdetermineâwhichâregionsâwithinâtheâhypothalamusâexpressâPKC-Ξ,ââweâperformedâimmunocytochemistryâforâPKC-ΞâandâfoundâPKC-Ξââlikeâimmunoreactivityâinâdiscreteâneuronalâpopulationsâinâtheâarcuateânucleusâ(Figureâ4,âBâandâC).âWeâconfirmedâtheâspecificityâofâourâantibodyâbyâcomparingâPKC-ΞâlabelingâinâWTâmiceâandâmiceâwithâaâtargetedâdeletionâofâtheâPrkcqâgeneâ(Prkcqâ/â;âFigureâ4,âDâandâE).âTheseâresultsâdemonstrateâthatâtheâPKC-ΞâlabelingâinâFigureâ4,âBâandâC,âisâhighlyâspecific.âToâassessâtheâphenotypeâofâhypothalamicâcellsâexpressingâPKC-Ξ,âweâusedâ2âdifferentâtrans-genicâmouseâlinesâthatâexpressâGFPâeitherâinâneuropeptideâYâ(NPY)âorâpro-opiomelanocortinâ(POMC)âneurons.âWeâperformedâdualâimmunocytochemistryâtoâcolocalizeâPKC-ΞâexpressionâwithâPOMCâ(Figureâ4,âFâH)âandâNPYâ(Figureâ4,âIâK).âThereâwasâ20%âcolocalizationâofâPKC-ΞâwithâNPYâ(Figureâ4K),âandânoâcolocal-izationâwithâPOMCâ(Figureâ4H).âAdditionally,âasâdemonstratedâinâFigureâ4,âLâandâM,âPKC-ΞâimmunoreactivityâcolocalizedâwithâNeuN,âaâmarkerâofâneuronalâcells,âbutânotâtheâglial-cellâmarkerâGFAP.âToâassessâwhetherâPKC-Ξâ isâexpressedâinâneuronsâthatârespondâtoâleptin,âweâusedâaâtransgenicâlineâthatâexpressesâGFPâonlyâinâcellsâthatâalsoâexpressâtheâleptinâreceptorâ(38).âWeâidenti-
Figure 2i.c.v. palmitic acid (but not oleic acid) attenuates insulin-induced sup-pression of hepatic glucose production. Rats (6â8/group) received a 3-day i.c.v. infusion of palmitic acid, oleic acid, or control (cont) (PBS), before a carotid-artery insulin infusion under euglycemic clamp condi-tions. Comparable glucose use rates (~100 mg/dl) were achieved in all groups, beginning 90 minutes after the start of the experiment. All groups had plasma insulin values of approximately 900 pmol/l (data not shown). Carotid insulin infusions reduced hepatic glucose production in control and oleic acidâinfused rats but not in rats infused with palmitic acid (data are mean ± SEM; ***P < 0.001 compared with control infusions).
Figure 3Palmitic acid attenuates insulin signaling in vitro. Hypothalamic IVB cells (36) were exposed to 100 ÎŒmol/l palmitic acid, 100 ÎŒmol/l oleic acid, or vehicle (10% fatty acidâfree BSA in PBS) for 4 hours and then tested with insulin (50 ng/ml in PBS) or vehicle (PBS) for 10 minutes. (A) Quanti-fied data (mean ± SEM) from 2 separate Western blot analyses for p-AKT, using GAPDH as a control (n = 4â6/treatment). Insulin increased p-AKT relative to vehicle (*P < 0.05 compared with vehicle), though this increase was significantly reduced by palmitic acid infusion (P < 0.05). (B) A representative Western blot is shown (the top bands depict p-AKT, vehicle, vehicle/insulin, palmitic acid, and palmitic acid/insulin lanes were run on the same gel but were noncontiguous, and oleic acid and oleic acid/insulin were run on a separate gel but under the same conditions; and the bottom band depicts GAPDH, lanes were run on a different gel). To test for the viability of the cells following exposure to 100 ÎŒmol/l pal-mitic acid, we used the Trypan blue exclusion test of cell viability. (C) Cells exposed to 100 ÎŒmol/l palmitic acid. (D) Cells exposed to 300 ÎŒmol/l palmitic acid. Viable cells have a clear cytoplasm, whereas nonviable cells have a blue cytoplasm. Original magnification, Ă10 (C and D).
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fiedâcellsâinâtheâarcuateânucleusâthatâhadâ22%âcolocalizationâforâleptinâreceptorâandâPKC-Ξâ(Figureâ4,âNâandâO).âTogether,âourâdataâdemonstrateâforâtheâfirstâtimeâtoâourâknowledgeâthatâPKC-ΞâisâlocatedâinâarcuateâNPYâandâleptin-responsiveâneurons,âprovid-ingâevidenceâthatâPKC-Ξâisâcriticallyâpositionedâtoâbeâresponsiveâtoâfattyâacidsâandâtoâmodulateâhepaticâglucoseâproductionâandâbodyâweightâregulation.
Palmitic acid induces translocation and activation of CNS PKC-Ξ.âSatu-ratedâfatâdiets,âspecificallyâthoseâwithâelevatedâlevelsâofâpalmiticâacid,âinduceâtranslocationâofâPKC-Ξâtoâtheâmembraneâinâperiph-eralâtissuesâ(5â7,â14,â28).âWeâthereforeâassessedâwhetherâpalmiticâacidâinducesâtranslocationâofâPKC-Ξâinâbrain.âToâthisâend,âweâusedâultracentrifugationâtoâaccuratelyâassessâmembraneâandâcytosolicâproteinâcontentâtoâdetermineâtranslocationâofâPKC-Ξ.âValidationâ
Figure 4PKC-Ξ is expressed in the hypothalamus. (A) PKC-Ξ expression, using RT-PCR in the hypothalamus and muscle of rats (con, water con-trol). To determine regions within in the hypothalamus that express PKC-Ξ, we assessed PKC-Ξâlike immunoreactivity in cells by DAB and fluorescent immunocytochemistry. (B and C) Hypothalamic PKC-Ξâlike immunoreactivity in the arcuate assessed by fluorescent and DAB immunocytochemistry, respectively. (D and E) The specificity of the PKC-Ξ antibody is confirmed by comparing PKC-Ξ labeling in the hypothala-mus of WT mice and PKC-Ξâknockout mice (Prkcqâ/â) respectively. We used 2 different transgenic mouse lines that express GFP either in NPY or POMC neurons. In the arcuate, we did not observe colocalization of PKC-Ξ with POMC neurons, (F) in POMC neurons (green), (G) with PKC-Ξ (red), or (H) PKC-Ξ/POMC colocalization. We did observe 20% colocalization of (I) NPY neurons (green) (arrow identifies an NPY neuron), (J) with PKC-Ξ (red) (arrow identifies a PKC neuron), and (K) NPY/PKC-Ξ colocalization (arrow identifies a colocalized NPY/PKC neuron). The mice express humanized renilla GFP (hrGFP) on the NPY promoter. To determine whether PKC-Ξ is expressed in neurons or on glia, (L) we colocalized PKC-Ξ (green) with NeuN (red) but not with (M) GFAP (red), a glial-cell marker. To assess whether PKC-Ξ is expressed in neurons that respond to leptin, we used a transgenic line that expresses GFP in only cells that also express the leptin receptor (LepR-GFP). We identi-fied 22% colocalization of cells in the arcuate nucleus that were positive for (N) leptin receptor (green) or (O) both leptin receptor and PKC-Ξ (yellow) (arrow identifies a PKC/LepR-GFP colocalized neuron). Original magnification, Ă10 (B, D, and E); Ă20 (C and N); Ă40 (L and M); Ă120 (O). Scale bar: 120 ÎŒm (FâH); 100 ÎŒm (IâK); 300 ÎŒm (N); 50 ÎŒm (O). ARH, arcuate; ME, median eminence; PKC-ir, PKC immunoreactivity; 3v, third ventricle; VMH, ventral medial hypothalamic nucleus.
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ofâthisâtechniqueâisâdemonstratedâinâFigureâ5A,âshowingâimmuno-labelingâofâmembrane-specificâfragmentâmarkers,âSNAP25âandâIR,âandâlabelingâforâaânuclearâprotein,âHistoneâ3.
Ratsâreceivedâintragastricâgavageâofânutritionallyâmatched,âfattyâacidâenrichedâemulsionsâcontainingâpalmiticâorâoleicâacidâforâ3âdays.âToâourâknowledge,âthisâisâaânovelâmethod,âwhichâtestsâtheâroleâofâfattyâacidsâindependentâofâdifferencesâinâpalatabilityâ(hedonics),âcaloricâconsumption,âorâmixturesâofâfattyâacids.âTheâ3-dayâgavageâofâfattyâacidsâelicitedâsignificantâ(Pâ<â0.05)âdifferencesâinâCNSâfattyâacidâcontent.âTheâbrainâuptakeâindexâ(BUI)â(39)âforâanimalsâgavagedâwithâpalmiticâacidâwasâ25.45%âofâthatâforâtheâtotalâfattyâacidsâ(oleicâacidâcontentâinâtheseâanimalsâwasâ15.98%)âandâtheâoleicâ
acidâgavagedâanimalsâhadâaâBUIâofâ23.19%âofâthatâforâoleicâacidâ(palmiticâacidâcontentâinâtheseâanimalsâwasâ15.56%).âAdditionally,âweârecentlyâpublishedâelevationsâinâhypothalamicâlong-chainâfattyâacyl-CoAs,âfollowingâexposureâtoâaâhigh-fatâdietâorâdirectâinfusionâofâpalmitateâ(10).âHere,âweâextendâthoseâfindingsâandâdemonstrateâsignificantlyâ(Pâ<â0.05)âelevatedâlevelsâofâpalmitoylâ(16:0)âlong-chainâfattyâacyl-CoAâlevelsâfollowingâtheâpalmitateâacidâgavageârelativeâtoâtheâcontrolâorâoleicâacidâgavageâ(controlâ=â20â±â3.4âpmol/gâtissue;âpalmitate,â35â±â4.9âpmol/gâtissue;âoleicâacid,â23â±â3.2âpmol/gâtis-sue).âPalmiticâacidâgavagedâanimalsâhaveâincreasedâhypothalamicâmembrane-boundâPKC-Ξârelativeâtoâcontrolâorâoleicâacidâgavagedâanimalsâ(Figureâ5,âBâandâC),âandâthisâisâassociatedâwithâconcomi-
Figure 5Saturated fatty acids increased PKC-Ξ transloca-tion to the membrane. To confirm our protocol to isolate membrane from cytosolic fractions, medial basal hypothalamic cell lysate was ultracentri-fuged. (A) The membrane fraction was stained with membrane-associated proteins, IR, SNAP 25, and Histone 3, a nuclear protein. Rats (n = 8â10/group), gavaged 3 times per day for 3 days with palmitic acid, oleic acid, or vehicle had no change in body weight (data not shown). Western blots were performed in 3 replications. There was a sig-nificant (P < 0.05) increase in medial basal hypo-thalamic membrane translocation of PKC-Ξ in the palmitic acidâgavaged animals but not the oleic acidâ or vehicle-gavaged animals. Cyt, cytosolic; NUC, nuclear; TOT, total. (B) The GAPDH control was run on the same blot as AKT. Quantification (mean ± SEM) of 3 representative blots (n = 3/group) in C (vehicle and palmitic acid lanes were run on the same gel but were noncontiguous; oleic acid was run on a separate gel but under the same conditions; GAPDH lanes were run on separate gels). There was a significant decrease in medial basal hypothalamic cytosolic location of PKC-Ξ. (D) Quantified data (mean ± SEM) from 2 separate Western blots (n = 4/group), of which E is a representative blot. Cell membrane content of medial basal hypothalamic PKC-Ξ was also increased by i.c.v. osmotic minipump infusion of palmitic acid relative to oleic acid or vehicle. (F) Quantification (mean ± SEM) from 2 separate Western blots (n = 4â5/group), with a representa-tive of the blot shown in G (vehicle, palmitic acid, and oleic acid lanes were run on the same gel but were noncontiguous). No significant change in cytosolic PKC-Ξ was found. (H) Quantification (mean ± SEM) from 2 separate Western blots (n = 4â5/group), representative of the blot shown in I (vehicle, palmitic acid, and oleic acid lanes were run on the same gel but were noncontigu-ous). (J) Represents increased serine phosphory-lation of IRS-1, which is associated with reductions in insulin signaling in the animals i.c.v. infused the palmitic acid but not the oleic acid or vehicle infu-sion. (*P < 0.05 compared with vehicle treatment; quantification [mean ± SEM] from 3 separate Western blots [n = 5â6/group]).
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tantâreductionsâinâcytosolicâcontentâ(Figureâ5,âDâandâE).âToâtestâtheâabilityâofâfattyâacids,âlocallyâwithinâtheâhypothalamus,âtoâtranslo-cateâPKC-Ξâtoâtheâmembrane,âindependentâofâperipheralâeffects,âweâinfusedâpalmiticâacid,âoleicâacid,âorâvehicleâi.c.v.âSimilarâtoâgavage,âi.c.v.âpalmiticâacidâincreasedâtheâmembraneâcontentâofâPKC-Ξâ(Fig-ureâ5,âFâandâG),âwithâconsequentâreductionsâinâcytosolicâcontentâ(Figureâ5,âHâandâI),âwhileâoleicâacidâinfusionâhadânoâeffect.âConsis-tentâwithâtheâhypothesisâthatâincreasedâmembraneâtranslocationâofâPKC-Ξâinducesâinsulinâresistance,âonlyâtheâpalmiticâacidâinfusedâanimalsâexhibitedâanâincreaseâinâhypothalamicâserineâ307âphos-phorylationâofâIRS-1â(Figureâ5J).âToâconfirmâthatâtheseâeffectsâwereâuniqueâforâPKC-Ξ,âweâfoundâthatâneitherâPKC-ÎŽânorâPKC-Δâwereâtranslocatedâfollowingâfattyâacidâexposureâ(dataânotâshown).
PKCsâalsoâengageâaânumberâofâdownstreamâsignalingâsystemsâthatâcanâbeâmeasuredâasâsurrogateâ indicatorsâofâPKCâactivity.âCytosolicâmyristoylatedâalanine-richâCâkinaseâsubstrateâ(MARCKS)âisâphosphorylatedâbyâactivationâandâtranslocationâofâmanyâmem-bersâofâtheâPKCâfamily,âincludingâPKC-Ξâ(40).âTherefore,âtoâspecifi-callyâtestâwhetherâtheâfattyâacidsâareâimpactingâdownstreamâtargetsâofâPKC-Ξ,âweâdeterminedâthatâi.c.v.âpalmiticâacidâbutânotâoleicâacidâincreasedâp-MARCKsâinâtheâcytosolâ(Figureâ6,âAâandâB).âTheseâdataâtogetherâdemonstrateâthatâexposureâtoâpalmiticâbutânotâoleicâacidâresultsâinâmembraneâlocalizationâandâactivationâofâPKC-Ξâinâtheâhypothalamus,âresultingâinâattenuationâofâinsulinâsignaling.
Adeno-associated virus PKC-Ξ shRNA reduces PKC-Ξ expression and improves insulin sensitivity.âInâaâfinalâseriesâofâexperiments,âweâassessedâwhetherâselectiveâknockdownâofâhypothalamicâPKC-ΞâbyâadministrationâofâshRNA-expressingâadeno-associatedâvirusâ(AAV)âwouldâimproveâglucoseâhomeostasis,âdecreaseâbodyâweightâgain,âandâimproveâhypothalamicâinsulinâsignalingâfollowingâexposureâtoâtheâHFSâdietâelevatedâinâpalmiticâacid.âToâaccomplishâthis,âweâallowedâadâlibitumâconsumptionâofâtheâdiet,âspecificallyâtoâtestâwhetherâknockdownâofâPKC-Ξâattenuatedâdiet-inducedâobesity.âWhereasâtheâpreviousâexperimentsâattemptedâtoâavoidâtheâcon-foundâofâobesity,âhereâweâspecificallyâsoughtâtoâtestâwhetherâreduc-tionsâinâPKC-ΞâinâtheâarcuateâhadâanyâinfluenceâonâreducingâbodyâweightâgainâandâimprovingâCNSâinsulinâsensitivityâwhenâanimalsâareâexposedâtoâaâHFSâdiet.âWeâgeneratedâshRNAâsequences,âwhichâareâannealedâintoâaâhairpinâAAVâvector.âSequencesâofâtheseâoligosâareâidenticalâtoâtheâpubliclyâavailableâmouseâPKC-Ξâsequences:âshRNAâ1â(5âČ-AAACCACCGTGGAGCTCTACT-3âČ)âandâshRNAâ2â(5âČ-AAGAGCCCGACCTTCTGTGAA-3âČ).âToâtestâtheâefficacyâofâthisâsequence,âPKC-ΞâshRNAâknockedâdownâexpressionâofâPKC-Ξâ
inâneuronalâcultureâ(Figureâ7A).âNext,âweâsiteâspecificallyâinfusedâtheâPKC-ΞâshRNAâbilaterallyâintoâtheâarcuateânucleusâofâmiceâ(orâperformedâcontrolâsiteâspecificâinjectionsâofâscrambledâoligoâcontrolâAAV).âFollowingâtheâinjections,âmiceâwereâplacedâonâHFSâdietâ(withâelevatedâlevelsâofâpalmiticâacid)âorâlow-fatâdietâforâ2.5âweeks.âMiceâthatâreceivedâtheâPKC-ΞâshRNAâgainedâlessâweightâonâHFSâdietâthanâdidâmiceâwithâtheâcontrolâsiteâspecificâinjectionsâofâscrambledâoligoâAAV.âHowever,âthereâwereânoâdifferencesâinâweightâgainâbetweenâPKC-ΞâshRNAâandâscrambledâAAVâinjectedâmiceâmaintainedâonâtheâlow-fatâdietâ(Figureâ7B),âprovidingâfur-therâevidenceâthatâtheâPKC-Ξâinducedâeffectsâareâdirectlyâdown-streamâofâelevatedâhypothalamicâsaturatedâfattyâacids.âUsingâanâi.p.âglucoseâtoleranceâtestâ(0.75âg/kgâbodyâweightâofâ20%âd-glucoseâ[PhoenixâPharmaceuticalâSt.]),âweâcomparedâglucoseâsensitivityâinâmiceâmaintainedâonâtheâHFSâdietâthatâwereâeitherâinjectedâPKC-ΞâshRNAâorâscrambledâoligoâcontrolâAAV,âandâfoundâaâsignificantâimprovementâinâglucoseâtoleranceâinâtheâmiceâthatâreceivedâtheâPKC-ΞâshRNAâAAVâinjectionsâ(Figureâ7C),âdemonstratingâtheâperipheralâeffectsâofâHFSâdietâinducedâhypothalamicâPKC-Ξâacti-vation.âMoreover,âtoâfurtherâshowâthatâHFSâdietâinducedâPKC-Ξâactivationâinâtheâhypothalamusâdirectlyâattenuatesâhypothalam-icâinsulinâsignaling,âweâassayedâp-AKTâasâaâmeasureâofâinsulinâresponsiveness.âTwoâweeksâfollowingâPKC-ΞâshRNAâorâscrambledâoligoâcontrolâAAVâadministration,â1âcohortâofâmiceâwasâperipher-allyâinjectedâwithâinsulinâ(5âU/kg),âsacrificedâ30âminutesâlater,âandâhypothalamicâtissueâwasâprocessedâforâp-AKTâimmunoreactivityâ(Figureâ7D).âTheseâresultsâclearlyâdemonstrateâanâimprovementâinâinsulinâsignalingâ(asâmeasuredâbyâp-AKT)âinâthoseâmiceâwithâPKC-Ξâknockâdown.âInâagreementâwithâourâoverallâhypothesis,âweâfoundâthatâmiceâinfusedâwithâPKC-ΞâshRNAâandânotâtheâcontrolâscrambledâoligoâAAV,âhaveâsignificantlyâ(Pâ<â0.05)âhigherâlevelsâofâinsulin-inducedâarcuateâp-AKT.âToâconfirmâtheâspecificityâofâtheâinjections,âtheâPKC-ΞâshRNAâandâtheâscrambledâoligoâAAVâcontrolâwereâtaggedâwithâGFP.âAsâshownâinâFigureâ7E,â2.5âweeksâfollowingâi.c.v.âadministrationâofâ0.5âÎŒlâofâPKC-ΞâshRNA,âGFPâimmunoreac-tivityâwasâpresentâinâtheâarcuateânucleus,âdemonstratingâsuccessfulâtransfectionâandâexpressionâofâviralâproteins.âInâaâsecondâcohortâofâmice,âtheâarcuateâwasâdissectedâandâWesternâblotâanalysisâdemon-stratedâaâsignificantâreductionâinâPKC-Ξâproteinâlevelsâonlyâinâtheâarcuate,âfollowingâPKC-ΞâshRNAâandânotâtheâcontrolâscrambledâoligoâAAVâadministrationâ(Figureâ7F),âwithânoâchangesâinâPKC-Ξââinâtheârestâofâtheâhypothalamusâ(Figureâ7G).âAdditionally,âthisâreductionâwasâspecificâforâPKC-ΞâandânotâforâPKC-ÎŽ,âaâPKCâisoformâ
Figure 6Translocation of PKC-Ξ is associated with increased cytosolic p-MARCKS. A 3-day i.c.v. infusion of palmitic acid but not oleic acid or vehicle (n = 8â10/group) increased cytosolic p-MARCKS. (A) Quantifi-cation (mean ± SEM) from 3 separate Western blots (n = 8â10/ group) representative of the blot shown in B (vehicle and palmitic acid lanes were run on the same gel but were noncontiguous; oleic acid was run on a separate gel but under the same conditions; the bottom band depicts GAPDH from a separate blot). *P < 0.05.
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withâhighâsequenceâhomologyâtoâPKC-Ξâ(Figureâ7,âHâandâI).âAsâanâadditionalâcontrol,âtheâPKC-ΞâshRNAâandâtheâcontrolâscrambledâoligoâAAVâadministrationâhadânoâeffectâonâmuscleâlevelsâofâPKC-Ξâ(Figureâ7J).âTheseâdataâconfirmâourâhypothesisâthatâarcuate-spe-cificâshRNA-inducedâknockâdownâofâPKC-ΞââprotectsââagainstâtheâdeleteriousâeffectsâofâtheâHFSâdietâ(elevatedâwithâpalmiticâacid)âonâCNS-mediatedâinsulinâresistanceâandâtheâresultantâobesity.
DiscussionWeâdemonstrateâforâwhatâweâbelieveâtoâbeâtheâfirstâtimeâthatâPKC-Ξââisâexpressedâinâtheâhypothalamus,âisâcolocalizedâwithâneuronalâpopulationsâpreviouslyâshownâtoâbeâcriticalâforânutrientâsensingâandâbodyâweightâregulation,âandâisâresponsiveâtoâpalmiticâacid,âwhichâinducesâtranslocationâandâactivationâofâPKC-Ξ,âresultingâinâinhibitionâofâPI3Kâsignaling.âFurthermore,âweâprovideâevidenceâ
Figure 7Arcuate-specific knockdown of PKC-Ξ attenuates diet-induced obesity. (A) AAV PKC-Ξ shRNA knocked down expression of PKC-Ξ in neuronal cul-ture (lanes 1 and 3). (B) Attenuated diet-induced obesity in mice (n = 8â10/ group), following bilateral arcuate infusion of 0.5 ÎŒl AAV PKC-Ξ shRNA or scrambled oligo AAV administra-tion (data are mean ± SEM; *P < 0.05 relative to scrambled oligo control for body weight change). (C) Improved response to an i.p. glucose tolerance test (mean ± SEM; n = 8â10/group) in mice on the HFS diet, following AAV PKC-Ξ shRNA versus scrambled oligo AAV administration (*P < 0.05 relative to scrambled oligo AAV injections). (D) Improved insulin signaling (enhanced p-AKT 30 minutes following a peripher-al injection of 5 U/kg) in mice (n = 4â5/ group) on the HFS diet, following AAV PKC-Ξ shRNA versus scrambled oligo AAV administration (data are mean ± SEM; *P < 0.05 relative to control). (E) GFP immunoreactivity in the arcuate nucleus demonstrates transfection and expression of viral proteins at the site of injection. Original magnification, Ă10 (left panel); Ă20 (right panel). (F) Western blot analysis demonstrates a significant reduction (*P < 0.05) in PKC-Ξ protein levels, 2.5 weeks after AAV PKC-Ξ shRNA administration, only in the arcuate after AAV PKC-Ξ shRNA versus scrambled oligo AAV administration (quantification [mean ± SEM] from 2 separate Western blots; n = 4â5/group), (G) with no changes in PKC-Ξ in the remaining hypothalamus (quantification [mean ± SEM] from 2 separate Western blots; n = 4â5/ group). (H and I) PKC-ÎŽ, a PKC isoform with high sequence homology to PKC-Ξ was not affected by viral injections (quantification [mean ± SEM] from 2 separate Western blots; n = 4â5/ group). (J) The AAV PKC-Ξ shRNA versus scrambled oligo AAV admin-istration had no effect on muscle lev-els of PKC-Ξ (quantification [mean ± SEM] from 2 separate Western blots; n = 4â5/group).
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toâsuggestâthatâinâtheâbrain,âpalmiticâandânotâoleicâacid,âinducesâtranslocationâofâPKC-Ξ.âTakenâtogether,âtheseâfindingsâsuggestâthatâdietsâhighâinâpalmiticâacidâconferâcentralâinsulinâresistanceâthroughâPKC-Ξâinâaâmechanismâsimilarâtoâthatâwhichâhasâbeenâdescribedâinâperipheralâtissues.
Theâprevalenceâofâobesityâcontinuesâtoâriseâatâanâalarmingârate.âThereâisâincreasingâsupportâforâaâroleâofâelevatedâcirculatingâfattyâacidsâasâaâcriticalâfactorâtoâexplainâtheâconnectionâbetweenâobesityâandâinsulinâresistanceâ(41,â42).âAnâacuteâincreaseâinâplasmaâfattyâacidsâbyâlipidâinfusionâcausesâinsulinâresistanceâinâanimalsâ(43)âasâwellâasâinâhealthyâandâdiabeticâhumansâ(44â47),âpotentiallyâbyâalteringâinsulinâsignalingâdownstreamâofâPI3Kâ(28,â43).âRecently,âweâextendedâtheseâfindingsâtoâincludeâfattyâacidâinhibitionâofâinsu-linâsignalingâinâhypothalamicâcircuitsâthatâcontrolâenergyâhomeo-stasisâ(10).âHere,âweâprovideâaâpotentialâmechanismâbyâwhichâdietsâhighâinâpalmiticâacidâinterfereâwithâhypothalamicâinsulinâandâleptinâsignalingâthroughâmembraneâtranslocationâandâactivationâofâhypothalamicâPKC-Ξ.
Weâfurtherâdemonstrateâthatâthisâphenomenonâisâspecificâforâpalmiticâacidâandâdoesânotâoccurâfollowingâexposureâtoâoleicâacid.âMoreâthanâotherâfattyâacids,âpalmiticâacidâisâimplicatedâinâfattyâacidâinducedâ insulinâresistanceâ (48,â49)â inâ theâperipheryâandârecentlyâinâtheâCNSâ(10).âPicinatoâetâal.âdemonstratedâthatâratsâfedâaâdietâenrichedâwithâoliveâoilâforâ6âweeksâ(10%âofâenergy,â90%âoleateâacid)âhaveâimprovedâglucoseâtoleranceâandâinsulinâsecretionâcom-paredâwithâratsâfedâaâdietâhighâinâanimalâfatâ(10%âofâenergy,â30%âpalmitateâacid,â50%âoleateâacid)âdietâ(50).âImportantly,âhereâweâpro-videâdataâtoâsuggestâthatâtheârelative ratiosâofâtheâfattyâacidsâmayâbeâcritical.âDirectâinfusionsâorâgavagesâofâpalmiticâbutânotâoleicâacidâinducedâarcuateâmembraneâtranslocationâofâhypothalamicâPKC-ΞâandâimpairedâtyrosineâphosphorylationâofâIRSâproteinsâbyâincreas-ingâserineâphosphorylation.âMoreover,âtheâspecificityâofâpalmiticâacidâisâdemonstratedâbyâtheâdataâshowingâthatâoleicâacidâhasânoâeffectâonâtheâtranslocationâofâPKC-Ξ,âconsistentâwithâmaintenanceâofâinsulinâsignalingâinâanimalsâmaintainedâonâaâdietâhighâinâoleicâacid.âTherefore,âtheseâdataâsuggestâthatâtheâbrainâisâresponsiveâtoâchangesâinâfattyâacidsâandâoneâmechanismâbyâwhichâtheâfattyâacidsâimpairâinsulinâandâleptinâsignalingâandâhepaticâglucoseâproduc-tionâisâthroughâfattyâacidâmediatedâactivationâandâtranslocationâofâPKC-Ξâinâtheâhypothalamus.
Insulinârapidlyâ lowersâbloodâglucoseâbyâpromotingâglucoseâuptakeâ andâ suppressingâ glucoseâ productionâ byâ bothâ directâactionsâonâtheâliverâ(51)âandâbyâinsulinâsignalingâinâextrahepaticâtissuesâthatâinâturnâinhibitâglucoseâproductionâviaâneuralâand/orâhumoralâmediatorsâ(2).âInhibitionâofâarcuateâinsulinâactionâviaâantibodies,âantisenseâoligonucleotides,âorâblockadeâofâsignalingâintermediatesâ(PI3K)âleadsâtoâanâattenuationâofâglucoseâproduc-tionâbyâcirculatingâinsulinâ(52).âThus,âinsulinâactionâinâtheâhypo-thalamusâisârequiredâforâtheâfullâinhibitoryâeffectâofâsystemicâinsu-linâonâglucoseâproductionâandârequiresâanâintactâinsulinâsignalingâcascade,âinvolvingâtheâactivationâofâtheâIR,âIRS,âandâPI3Kâ(2,â52).âTheâarcuateâhasâbeenârepeatedlyâimplicatedâasâtheâcrucialâsiteâforâregulatingâperipheralâglucoseâhomeostasisâ(53,â54).âDespiteâtheâfactâinâtheâarcuateâinsulinâandâleptinâreceptorsâareâfoundâonâNPY/agouti-relatedâproteinâ(NPY/AgRP)âandâPOMC/CARTâneuronsâ(55),âthereâisânoâchangeâinâhepaticâglucoseâproductionâinâmiceâlackingâIRsâinâPOMCâneuronsâ(56).âHowever,âmiceâlackingâIRsâonlyâonâNPY/AgRPâneuronsâfailâtoâfullyâsuppressâhepaticâglucoseâproductionâ(56).âHence,âitâappearsâthatâsuppressionâofâhepaticâglucoseâproductionâbyâCNSâinsulinâisâmediatedâprimarilyâbyâitsâ
effectsâonâtheâNPY/AgRPâneurons.âHere,âweâconfirmâtheâfindingsâofâDewingâetâal.âthatâPKC-Ξâisâexpressedâinâneuronsâinâtheâarcu-ateâ(37)âandâextendâthoseâfindingsâtoâalsoâdemonstrateâPKC-ΞâisâcolocalizedâwithâAgRP/NPYâneuronsâandâitsâtranslocationâandâactivityâisâmodulatedâbyâfattyâacids.âAdditionally,âinsulinâeffectsâinâtheâCNSâareâmediatedâbyâactivationâofâK+ATPâchannelsâinâarcuateâneuronsâthroughâinsulin-inducedâactivationâofâPI3K,âresultingâinâsubsequentâmembraneâhyperpolarization.âOurâdataâsuggestâthatâpalmiticâacidâexposureâinducesâtranslocationâofâPKC-ΞâexclusivelyâinâarcuateâNPY/AgRPâneurons,âwhichâwouldâleadâtoâpreventionâofâtheâactivationâofâK+ATPâchannels,âreducedâhyperpolarization,âandâeventuallyâdisinhibitionâofâhepaticâglucoseâproduction.
ItâhasâpreviouslyâbeenâdemonstratedâthatâPKCâisoformsâinfluenceâCNSâregulationâofâhepaticâglucoseâproductionâ(2).âRecently,âLamâandâcolleaguesâ(22)âreportedâactivationâofâhypothalamicâPKC-ÎŽ,ââviaâdirectâdeliveryâofâtheâPKCâactivatorâ1-oleoyl-2-acetyl-sn-glyc-erolâ(OAG),âsuppressedâhepaticâglucoseâproduction.âTheseâauthorsâconcludedâhypothalamicâPKC-ÎŽâactivationâisâsufficientâandâneces-saryâtoâlowerâglucoseâproductionâ(22).âWhereâtheseâfindingsâmightâseemâcontradictoryâtoâourâfindings,âweânoteâthatâtheyâaddressedâtheâroleâofâPKC-ÎŽ,âwhereasâweâprovideâdataâonâtheâeffectsâofâPKC-Ξ.ââItâ isâwellâestablishedâthatâdifferentâPKCâisoformsâcanâandâdoâexertâopposingâactionsâonâphysiologicalâprocesses.âSpecificallyâinâperipheralâtissues,âPKC-ÎŽâandâ-Ξâexertâopposingâactionsâonâinsulinâsignaling.âThus,âbothâisoforms,âlocalizedâtoâtheâarcuateânucleus,âmayâoperateâunderâdifferentâmechanismsâthatâcoordinateâtheâeffectsâofâcentralâinsulinâ(22).
Importantly,âfattyâacidsâinduceâinsulinâresistanceâthroughâaccu-mulationâofâintracellularâtriglycerideâandâDAG,âwhichâinducesârecruitmentâandâtranslocationâofâPKCs.âHere,âweâdemonstrateâinâtheâCNS,âsignificantâ(Pâ<â0.05)âincreasesâinâDAGâlevelsâfollowingâexposureâtoâtheâHFSâdietâandâfollowingâtheâ3-dayâgavageâofâpal-miticâacidârelativeâtoâtheâoleicâacidâandâcontrolâdiets/emulsions.âOurâfindingsâprovideâfurtherâevidenceâthatâinâtheâCNSâelevatedâlevelsâofâDAGâresultedâinâreducedâinsulin-stimulatedâIRS-1âtyro-sineâphosphorylation,âincreasedâserineâ307âphosphorylationâofâIRS-1,âandâreducedâPI3Kâactivity,âcollectivelyâconsistentâwithâfattyâacidâmediatedâreductionsâinâinsulinâsignaling.
Inâaâseminalâpaper,âShulmanâandâcolleaguesâ(15)âdemonstratedâthatâmiceâlackingâPKC-Ξâareâresistantâtoâfattyâacidâinducedâinsulinâresistance.âImportantly,âtheyâfoundâthatâexposureâtoâIntralipidâ(aâmixtureâofâfattyâacids)âincreasesâtranslocationâofâPKC-Ξâfromâtheâcytosolâtoâtheâcellâmembraneâinâskeletalâmuscleâandâconsequentlyâcausedâinsulinâresistance.âHowever,âaâmoreârecentâpaperâreportedâtheâoppositeâeffectsâ(57).âWeâbelieveâthatâtheseâapparentlyâcontra-dictoryâfindingsâmayâbeâexplainedâbyâdifferencesâinâtheâroute,âtype,âandâdurationâofâfattyâacidâadministration.âHere,âweâdemonstrateâthatâshort-termâinfusionsâofâfattyâacidsâviaâgastricâgavage,âorâdirectlyââintoâtheâCNS,âwereâsufficientâtoâinduceâtranslocationâofâPKC-Ξâandâproduceâcentralâinsulinâresistance.âInterestingly,âinâtheâShul-manâpaperâ(15),âthereâwasânoâmodulationâofâglucoseâproductionâduringâtheâclampâafterâlipidâinfusionâbetweenâtheâPKC-ΞâknockoutâandâWTâmice.âHere,âweâreportâthatâdirectâinfusionâofâfattyâacidsâintoâtheâCNSâinducedâtranslocationâofâPKC-Ξ,âreductionsâinâinsu-linâsignaling,âandâmodulationâofâhepaticâglucoseâproduction.âOurâdataâsuggestâthatâactivationâandâmovementâofâthisâisozymeâinâtheâCNSâisânecessaryâandâsufficientâforâHFSâdietâinducedâhypotha-lamicâinsulinâresistanceâandâdysregulationâofâglucoseâhomeostasis.âTheâdifferenceâbetweenâourâfindingsâandâthoseâofâGaoâandâcol-leaguesâ(57)âmayâbeâdueâtoâtheâdirectâinfusionâofâtheâfattyâacidsâ
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intoâtheâCNSâasâwellâasâourâisolationâofâtheâspecificâtypeâofâfattyâacidsâimportantâforâdysregulationâofâglucoseâhomeostasis.
Attentionâhasâbeenâfocusedâonâunderstandingâtheâroleâofâhigh-fatâdietsâinâmediatingâinsulinâresistanceâandâobesity.âImportantly,âinâourâexperiments,âweâdemonstrateâthatâaâshortâexposureâtoâfattyâacidsâresultedâinâsignificantâ(Pâ<â0.05)âreductionsâinâinsulinâsignal-ingâinâtheâCNS.âHowever,âinâlightâofârecentâdataâfromâShulmanâetâal.ââ(58),âtheâliverâalsoâappearsâtoâbecomeâinsulinâresistantâfollowingâ3-dayâexposureâtoâfattyâacids.âTherefore,âweâcanânotâconcludeâthatâtheâinfluenceâofâtheâfattyâacidsâisâsolelyâonâtheâCNS.âManyârecentâreportsâhaveâlinkedâinsulinâresistanceâwithâotherâdisordersâofâtheâCNS,âincludingâAlzheimerâdiseaseâandâmildâcognitiveâimpairmentâ(59â61).âHowever,âperhapsâbecauseâofâtheirârelativeâubiquityâinâtheâCNS,âinvestigationâintoâtheâroleâofâspecificâCNSâPKCsâinâbodyâweightâregulationâorâCNSâinsulinâsignalingâwasâpreviouslyâunex-plored.âWeâdemonstrateâforâtheâfirstâtimeâtoâourâknowledgeâthatâshort-termâexposureâtoâpalmiticâacidâinducedâmembraneâtranslo-cationâandâactivationâofâPKC-Ξâandâthatâthisâwasâassociatedâwithâreductionsâinâhypothalamicâinsulinâsignaling.âFinally,âweâdemon-strateâCNSâfattyâacidâinducedâtranslocationâofâPKC-Ξâisâcriticalâforâmediatingâinsulinâresistanceâandâobesity.âOurâdataâshowâthatâsite-specificâknockdownâofâPKC-Ξâinâtheâarcuateâattenuatedâdiet-inducedâobesityâandâimprovedâperipheralâglucoseâhomeostasisâandâinsulinâsignalingâinâtheâarcuateânucleus.âTheseâdataâsuggestâthatâthisâspecificâPKCâisozymeâmayârepresentâanâimportantâyetâunex-ploredâmechanisticâlinkâbetweenâperipheralâmetabolicâdiseaseâandâimpairedâCNSâinsulinâsignaling.
MethodsSubjects.âSubjectsâforâratâexperimentsâwereâmaleâLong-Evansâratsâ(250â300âg;ââHarlan)âorâmaleâC57BL/6âmiceâ(7âweeksâold,â25â30âg;âTheâJacksonâLabo-ratory).âPKC-ΞâknockoutâandâWTâmiceâwereâaâgiftâfromâGeraldâShulmanâ(YaleâUniversity,âNewâHaven,âConnecticut,âUSA)âandâwereâbredâin-house.âExceptâasânoted,âallâanimalsâwereâindividuallyâhousedâinâPlexiglasâtubsâwithâsterileâbedding,âgivenâadâlibitumâaccessâtoâpelletedâlow-fatâdietâ(10%âkcalâfromâfat)âorâpurifiedâhigh-fatâdietâ(40%âkcalâfromâfat,âeitherâasâsaturatedâfattyâacidsâorâoleicâacid;âResearchâDietsâInc.;âseeâTableâ2)âandâtapâwater,âandâmaintainedâonâaâ12-hourâlight/12-hourâdarkâcycleâinâaâtemperature-controlled,âAssociationâforâAssessmentâandâAccreditationâofâLaboratoryâAnimalâCareâInternationalâaccreditedâvivarium.âOneâcohortâofâratsâwasâgivenâtheâcaloriesâconsumedâbyâtheâadâlibitum low-fatâdietâfedâanimalsâasâtheâHFSâdietâ(HFS-R)âatâtheâonsetâofâtheâdarkâcycleâforâeachâdayâoverâ3âmonths.âAllâproceduresâwereâapprovedâbyâtheâInstitutionalâAnimalâCareâandâUseâCommitteeâofâtheâUniversityâofâCincinnati.
i.c.v. surgeries. Surgeriesâwereâperformedâafterâtheâratsâhadâbeenâonâtheâdietsâforâ2âmonths.âRatsâwereâanesthetizedâwithâ1âmg/kgâi.p.âinjectionsâofâaâmixtureâofâ70âmg/kgâketamineâ(FortâDodgeâAnimalâHealth)âandâ2âmg/kgâxylazineâ(LloydâLaboratories).âForâi.c.v.âcannulationâ(thirdâventricle),âratsâwereâplacedâinâaâstereotaxicâinstrumentâwithâtheâskullâheldâhorizontally.âTheâsagittalâsinusâwasâdisplacedâlaterally,âandâaâ21-gaugeâstainless-steelâguideâcannulaâ(PlasticsâOne)âwasâloweredâdirectlyâonâtheâmidline,â2.2-mmâposteriorâtoâbregmaâandâ7.5-mmâventralâtoâtheâdura,âandâfixedâtoâtheâskullâwithâanchorâscrewsâandâdentalâacrylic.âObturatorsâthatâextendedâ0.5âmmâbeyondâtheâcannulaâtipsâwereâinserted.âRatsâwereâmaintainedâonâtheirârespectiveâdiets,âandâwhenâratsâregainedâtheirâpreoperativeâbodyâweightsâfollowingâsurgery,âplacementâofâi.c.v.âcannulasâwasâconfirmedâfunction-ally,âbyâverifyingâthatâi.c.v.âinjectionâofâ10ângâangiotensinâIIâ(AmericanâPeptides)âinâ1âÎŒlânormalâsalineâinâwater-repleteâratsâelicitedâanâintakeâofâatâleastâ5âmlâwaterâwithinâ30âminutesâ(62â64).âAnimalsâthatâdidânotâmeetâthisâcriterionâwereânotâused.
i.c.v. infusions. Insulinâ(8âmU/1âÎŒl,âHumulin;âEliâLilly)âorâleptinâ(10âÎŒg/1âÎŒl;ââR&DâSystems)âwereâdissolvedâinâphysiologicalâsalineâandâinfusedâ(1âÎŒlâoverâ30âseconds)â4âhoursâpriorâtoâtheâbeginningâofâtheâdarkâphase.âFoodâintakeâwasâmeasuredâafterâ4âandâ24âhoursâ(onlyâ24-hourâdataâareâshown).âToâassessâwhetherâtheâfattyâacidsâimpairedâinsulinâsignaling,âp-AKTâwasâmeasuredâafterâcentralâinsulinâinjections.âForâassessmentâofâp-AKT,âratsâwereâtreatedâwithâi.c.v.âinsulinâ(8âmU)âandâsacrificedâafterâ10âminutesâbasedâonâpreviousâworkâ(4).âTheâbrainâwasârapidlyâremoved,âandâaâ70-mgâwedgeâofâmedialâbasalâhypothalamusâ(definedâcaudallyâbyâtheâmammillaryâbodies,ârostrallyâbyâtheâopticâchiasm,âlaterallyâbyâtheâopticâtract,âandâsuperiorlyâbyâtheâapexâofâtheâhypothalamicâthirdâventricle)âwasâdissectedâandâtissueâwasâprocessedâforâWesternâblotâanalysis.
Fatty acid infusion.âRatsâwereâimplantedâwithâaâcannulaâaimedâintoâtheâthirdâventricleâasâdescribedâabove.âTheâcannulaâwasâconnectedâviaâaâpoly-ethyleneâcatheterâtoâaâsubcutaneousâosmoticâminipumpâ(AlzaâCorpora-tion)âfilledâwithâeitherâpalmiticâorâoleicâacidâ(equimolarâconcentrations,â10âÎŒmol/l;âSigma-Aldrich)âorâvehicleâ(PBS)âforâcontinuousâinfusionâoverâ3âdays.âTheâfattyâacidsâwereâinfusedâatâaârateâofâ12âÎŒl/dayâ(orâ8.3ânl/min);âthus,âweâinfusedâaâtotalâvolumeâofâ36âÎŒl/3âdays,âwhichârepresentsâ1.8ânmol/3âdaysâ(i.e.,â0.41âpmol/min).
Gavage feeding.âRatsâ(nâ=â8â10/group)âreceivedâanâintragastricâgavageâofânutritionallyâcomplete,âfattyâacidâenriched,âsemipurifiedâdietsâpreparedâbyâDyetsâ(madeâaccordingâtoâtheâguidelinesârecommendedâbyâtheâNutri-ent requirements for laboratory animalsâ[NationalâResearchâCouncil,â1995]).âTheâpalmiticâacidâdietâcontainsâ20âgâfat/100âgâdietâ(19âgâofâethylâpalmitateâdissolvedâinâmedium-chainâtriglycerideâandâ1âgâofâsoybeanâoilâtoâprovideâessentialâfattyâacids)âandâprovidesâ19.34âkJ/gâofâdiet,âincludingâ7.74âkJ/gâasâfat.âTheâoleicâacidâdietâhasâexactlyâtheâsameâcompositionâbutâdiffersâonlyâinâtheâtypeâofâfatâused.âTheâcontrolâdietâ(control)âcontainsâ3âgâethylâpalmitateâandâ1âgâsoybeanâoil/100âgâdietâandâprovidesâ16.12âkJ/gâofâdiet,âincludingâ1.29âkJâasâfat.âWeâequalizedâtheâamountâofâproteinâandâallâofâtheâessentialâmineralsâandâvitaminsârequiredâperâkilojoule.âTheâaboveânutritionallyâcom-plete,âsemipurifiedâdietâwasâthoroughlyâmixedâ4:3âwithâtapâwaterâtoâyieldâaâliquidâdietâcontainingâ0.71âgramsâofâdietâperâmilliliterâofâliquid.âThisâliquidâdietâwasâslowlyâdeliveredâintoâtheâstomachâofâtheâratsâfromâaâgavageâfeedingâneedleâ(ScienceâTools).âRatsâwereâgivenâ3âequallyâsizedâfeedingsâdailyâatâ8:00âAM,â1:00âPM,âandâ6:00âPM.âGavage-fedâanimalsâwereâgraduallyâadaptedâtoâthisâfeedingâregimenâforâ3âdays.âAtâtheâendâofâdayâ3,âratsâwereâsacrificedââ3âhoursâafterâtheâfinalâgavage.âBrainsâwereâremoved,âandâtheâmedialâbasalâportionâofâtheâhypothalamusâwasâisolatedâandâfrozenâinâliquidânitrogenâinâorderâtoâestimateâtranslocationâofâPKC-Ξâandâassessâp-MARCKs.
Arterial infusions of insulin and hepatic glucose production.â Inâaâseparateâcohort,âratsâwereâanesthetizedâwithâketamineâ(125âmg/100âgâofâbodyâweightâi.p.)âforâsurgicalâplacementâofâaâcatheterâinâtheâcarotidâarteryâ(cen-tralâinfusion)âandâcannulaâimplantation,âasâdescribedâabove.âFollowingâtheâ3-dayâinfusionâofâfattyâacids,âinsulinâwasâinfusedâthroughâtheâintraca-rotidâcatheterâatâaârateâofâ2.5âpmol/min.âTheâflowârateâforâcarotidâinfusionâwasâ7âÎŒl/minâforâaâtotalâofâ3.6ânmolesâoverâ24âhours.âAâvariableârateâofâglucoseâinfusionâwasâinitiatedâafterâtheâstartâofâinsulinâinfusion.âBloodâ(0.1âml)âwasâsampledâatâ5-âtoâ10-minuteâintervalsâtoâmonitorâandâestablishâsteady-stateâconditions.âBloodâwithdrawnâduringâtheâeuglycemicâclampsâwasâreplacedâwithâanâequalâvolumeâofâsaline.âWhole-bodyâglucoseâkineticsâwasâestimatedâbyâuseâofâprimed,âcontinuousâ(40âÎŒCiâbolus,â0.4âÎŒCi/min)âi.v.âinfusionâofâ[3-3H]âglucose.âBloodâsamplesâ(0.15âml)âwereâobtainedâatâ60,â75,âandâ90âminutesâafterâtheâinitiationâofâ[3-3H]âglucoseâinfusionâinâtheâbasalâstateâandâatâ90,â105,âandâ120âminutesâafterâtheâinitiationâofâinfusionâofâ[3-3H]âglucoseâandâinsulinâinâtheâhyperinsulinemicâstate.âEndogenousâglucoseâproductionâwasâcalculatedâusingâSteeleâsâequationsâforâsteady-stateâconditions.âForâtheâassayâofâ[3-3H]âglucoseâradioactiv-ity,âbloodâsamplesâwereâdeproteinizedâwithâBa(OH)2âandâZnSO4,âandâtheâ
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supernatantâwasâevaporatedâtoâdrynessâatâ50°Câtoâremoveâtritiatedâwater.âTheâdryâresidueâwasâdissolvedâinâ0.5âmlâwater,âtoâwhichâ10âmlâscintillationâsolutionâwasâaddedâ(AqualumaâPlus).âRadioactivityâwasâdeterminedâinâaâPackardâTri-Carbâ460CâLiquidâScintillationâSystem.
Plasma analysis.âPlasmaâglucoseâwasâmeasuredâbyâtheâglucoseâoxidaseâmethodâ(GlucoseâAnalyzerâII;âBeckmanâInstrumentsâInc.).âPlasmaâinsulinâwasâdeterminedâbyâradioimmunoassayâ(RatâInsulinâRadioimmunoassayâKit;âLincoâResearchâInc.).âPlasmaâFFAâconcentrationsâwereâdeterminedâfollowingâanâenzymaticâmethod,âusingâanâautomatedâkitâaccordingâtoâtheâmanufacturerâsâspecificationsâ(NEFA-Câtest;âWako).
CNS DAG levels.âDAGâconcentrationsâwereâenzymaticallyâdetermined,âasâpreviouslyâdescribedâ(65).
Long-chain acyl-CoA analysis.âAnimalsâwereâeuthanizedâafterâaâ4-hourâgavageâofâeitherâpalmitate,âoleicâacid,âorâcontrol,âandâmediobasalâhypo-thalamusâwasârapidlyâremovedâandâfrozen.âLong-chainâfattyâacylâCoAsâwereâquantifiedâbyâliquidâchromatography/electrosprayâionizationâtan-demâmassâspectrometryâ(LC/ESI/MS/MS),âasâdescribedâpreviouslyâ(66,â67).âBriefly,âsamplesâwereâderivatizedâwithân-butylamineâandâanalyzedâusingâanâAgilentâ6410âTripleâQuadrupoleâLC/MSâSystemâequippedâwithâanâESIâsource.âTheâneutralâlossâofâm/zâ507âisâtheâmostâintenseâproductâionâforâeachâcompound.âWeâdetectedâtheâsubspeciesâbyâcharacteristicâLCâreten-tionâtimeâinâtheâmultipleâreactionâmonitoringâ(MRM)âmode,âfollowingâESI.ââAâknownâamountâofâC17:0âwasâspikedâintoâbiologicalâsamplesâtoâquantifyâC16:0â(palmitylâCoA)âbyâcomparingâtheârelativeâpeakâareaâinâtheârecon-structedâionâchromatogramâinâMRMâmode.
Arcuate administration of AAV.âWeâgeneratedâshRNAâsequences,âwhichâareâannealedâintoâaâhairpinâAAVâvector.âHumanâH1âpromoterâwasâamplifiedâfromâgenomicâDNA.âTheâPCRâproductsâwereâclonedâintoâanâAAVâcis-plas-midâtoâgenerateâpAAV.H1.âDNAâoligosâencodingâforâsmallâhairpinâRNAsâ(shRNAs)âtargetingâmouseâPKC-ΞâwereâannealedâandâclonedâimmediatelyâdownstreamâfromâtheâH1âpromoterâintoâBglIIâandâXbaIâsites.âVirusâstocksâwereâpreparedâbyâpackagingâtheâcis-plasmidsâintoâAAV-2âparticles,âusingâaâhelper-freeâplasmidâtransfectionâsystem.âTheâvectorsâwereâpurifiedâusingâheparinâaffinityâchromatographyâandâdialyzedâagainstâPBS.ârAAVâtitersâwereâdeterminedâbyâquantitativeâPCRâandâadjustedâtoâ1012âgenomicâpar-ticlesâperâmicroliter.âSequencesâofâtheseâoligosâwereâidenticalâtoâtheâpubliclyâavailableâmouseâPKC-Ξâsequences:âshRNAâ1â(5âČ-AAACCACCGTGGAGCTC-TACT-3âČ)âandâshRNAâ2â(5âČ-AAGAGCCCGACCTTCTGTGAA-3âČ).âForâourâcontrolâsite-specificâinjections,âweâusedâaâscrambledâoligoâAAV.
Miceâwereâplacedâinâaâstereotaxicâframeâ(previouslyâdescribed),âandâ0.5âÎŒlââofâeachâvectorâinâPBSâ(PKC-ΞâshRNAâandâorâcontrolâscrambledâoligoâAAV)âwasâinjectedâbilaterallyâintoâtheâarcuateâ(2.3âmmâanteroposteriorâfromâbregma,â0.3âmmâmediolateralâfromâmidline,â5.6âmmâdorsoventralâfromâdura)âoverâ10âminutesâusingâaâ5-mlâHamiltonâsyringeâandâanâinfusionâpumpâ(WorldâPrecisionâInstruments;âcontrolâinjectionsâwereâmadeâwithâaâscrambledâsequence).âTheâneedleâwasâleftâforâanâadditionalâ5âminutesâandâthenâslowlyâwithdrawn.âWeâdemonstratedâthatâtheseâvectorsâsuccessfullyâknockedâdownâmouseâPrkcqâgeneâexpressionâbyâ75%â80%â(Figureâ7F).
Glucose tolerance test.âi.p.âglucoseâtoleranceâtestâwasâperformedâaccordingâtoâpreviouslyâestablishedâproceduresâ(68).âMiceâwereâfastedâforâ16âhours,âandâallâbloodâsamplesâwereâobtainedâfromâtheâtipâofâtheâtailâvein.âAfterâaâbaselineâbloodâsampleâwasâtakenâ(0âminutes),â0.75âg/kgâbodyâweightâofâ20%âd-glucoseâ(PhoenixâPharmaceutical)âwasâinjectedâi.p.âAâlowerâdoseâofâglucoseâwasâadministeredâtoâtheâHFDâdietâfedâmiceâtoâensureâthatâtheâresultingâglucoseâlevelsâwereâinâtheâsensitivityârangeâofâtheâglucometers;âi.e.,â20â500âmg/dl.âSubsequentâbloodâsamplesâwereâtakenâatâ15,â30,â45,â60,âandâ120âminutesâafterâglucoseâadministration.âGlucoseâwasâmeasuredâonâduplicateâsamplesâusingâFreeStyleâglucometersâandâtestâstripsâ(Free-Style).âTheâcoefficientsâofâvariationâofâintra-assayâandâinterassayâwereâ5.5%âandâ6.1%,ârespectively.
Cell culture.âIVBâcells,âaâderivedâhypothalamicâcellâlineâ(36),âwereâculturedâinâDMEMâwithâglucoseâ(4,500âmg/l),â10%â(v/v)âFBS,âpenicillinâ(100âIU/ml),âstreptomycinâ(100âÎŒg/ml),âandâFungizoneâ(250âng/ml)â(LifeâTechnolo-gies).âApproximatelyâ5âĂâ106âcells/wellâwereâplatedâinâ100-mmâĂâ20-mmâcellâcultureâdishesâ(Corning)âandâgrownâtoâconfluence,âatâwhichâtimeâcellsâwereâserumâstarvedâinâmediaâwithâlowâglucoseâ(1,000âmg/l)âforâ24âhoursâbeforeâreceivingâtreatments.âTheâmediumâwasâthenâreplacedâwithâserum-freeâDMEMâcontainingâeitherâ100âÎŒmol/lâpalmiticâacid,â100âÎŒmol/lâoleicâacid,âorâvehicleâcontainingâBSAâforâ4âhours.âAâratioâofâ2âmolesâfattyâacidâtoâoneâmoleâBSAâwasâusedâatâaâpHâofâ7.3.âTheâvehicleâwasâ10%âfattyâacidâfreeâBSAâinâPBS.âForâinsulinâsignalingâexperiments,âcellsâwereâtreatedâasâabove,âonlyâtheyâwereâgrownâinâ24-wellâtissueâcultureâplatesâandâsubjectedâtoâ1âmlâofâinsulinâ(50âng/ml;âSigma-Aldrich;âinâPBS)âorâPBSâaloneâforâ10âminutes,âatâwhichâtimeâcellsâwereâharvestedâforâproteinâassayâ(BCAâProteinâAssayâKit;âPierce)âandâWesternâblotâanalysis.
Protein extraction and quantification.âForâproteinâassaysâonâhypothalamicâtissue,âratsâwereâeuthanizedâbyâCO2âandâquicklyâdecapitated,âandâtheâbrainsâwereâremoved.âUsingâaâbrainâblockâguide,âtheâtissueârostralâandâcaudalâtoâtheâhypothalamusâwasâdissected.âMedialâbasalâhypothalamusâwasâthenârap-idlyâexcisedâandâplacedâinâice-coldâhomogenizationâbuffer.âForâmembraneâtranslocationâstudies,âtheâtissueâwasâhomogenizedâonâiceâinâaâbufferâcon-tainingâ20âmMâTris-HClâ(pHâ7.5),â0.32âmMâsucrose,â2âmMâEGTA,â2âmMâETA,â0.2âmMâNaâorthovanadate,â50âmMâNa+âfluoride,â0.3âmMâPMSF,â5âÎŒg/mlâaprotinin,â5âÎŒg/mlâleupeptinâandâspunâatâ1,000âgâforâ10âminutes,âandâtheâpelletâwasâdiscarded.âTheâsupernatantâwasâcollectedâinâaâfreshâtubeâandâspunâatâ20,000âgâforâ60âminutesâatâ4°C.âTheâresultingâsupernatantâwasâthenâcollectedâasâtheâcytosolicâfraction.âTheâremainingâpelletâwasâsolubilizedâinâtheâaboveâbufferâwithâ1%âTritonâX-100âforâ30âminutesâandâspunâatâ20,000âgââforâ30âminutes,âandâtheâfinalâsupernatantâwasâcollectedâasâtheâmembraneâfraction.âAliquotsâforâproteinâassayâwereâtakenâandâaâ4Xâloadingâbuffer,âcontainingâ0.25âMâTrisâ(pHâ6.8),â8%âSDS,â40%âglycerol,â0.5%âbromophenolâblue,â50âmMâDTT,âwasâadded,âandâsamplesâwereâboiledâforâ2âminutes.
Western blots. Tissuesânotâsubjectedâforâtranslocationâstudiesâwereâhomog-enizedâinâice-coldâRIPAâbufferâ(1XâPBS,â1%âNonidetâP-40,â0.5%âsodiumâdeoxycholate,â0.1%âSDS,â0.2âmMâNaâorthovanadate,â50âmMâNa+âfluoride,â0.3âmMâPMSF,â5âÎŒg/mlâaprotinin,â5âÎŒg/mlâleupeptin).âSamplesâwereâthenâcentrifugedâatâ1,000âgâforâ10âminutesâatâ4°C,âandâsupernatantâwasâtrans-ferredâtoânewâtubes.âAliquotsâforâproteinâassayâareâtaken,âandâsamplesâwereâboiledâinâ4Xâloadingâbufferâasâmentionedâabove.âProteinâ(50âÎŒg)âwasâresolvedâonâ9%âPAGEâ(w/v)âandâtransferredâtoâaâHybondânitrocellu-loseâmembraneâ(AmershamâBiosciences).âMembranesâwereâthenâblockedâwithâ5%ânonfatâmilkâinâ0.1âTweenâinâTris-bufferedâsalineâ(TTBS),âaddingâappropriateâantibodyâovernight.âMembranesâwereâwashedâandâplacedâinâHRP-conjugatedâanti-rabbitâIgGâinâmilk/TTBSâforâ1âhour.âTheyâwereâthenâwashedâ4âtimesâinâTTBS,âandâbandsâwereâdetectedâusingâLuminol/EnhancerâSuperSignalâreagentâ(Pierce)âforâ1âminuteâandâvisualizedâusingâCL-XPosureâfilmâ(Pierce).âAntibodiesâusedâforâp-AKTâ(catalogâno.â7985-R),âAKTâ(catalogâno.â8312),âGAPDHâ(catalogâno.â25778),âPKC-Ξâ(catalogâno.â212),âtotalâPKCâ(catalogâno.â10800),âPKC-ÎŽâ(catalogâno.â937),âPKC-Δâ(cata-logâno.â214),âSNAPâ25â(catalogâno.â20038),âandâIRâ(ÎČ-subunit,âcatalogâno.â711)âareâfromâSantaâCruz;âantibodiesâforâserineâphosphorylationâofâIRS-1â(catalogâno.â07-247)âareâfromâUpstate;âp-MARCKSâ(Serâ152/156;âcatalogâno.2741)âandâHistoneâH3â(catalogâno.â9715)âareâfromâCellâSignalingâTech-nology.âSecondaryâantibodyâHRP-conjugatedâanti-rabbitâIgGâ(catalogâno.â170-6515)âisâfromâBio-Rad.âDataâwereâexpressedâandâanalyzedâasârelativeâtoânonphosphorylatedâproteinâand/orâGAPDH.
Trypan blue exclusion test of cell viability.âToâdetermineâviabilityâofâcellsâafterâexposureâtoâpalmiticâacid,âcellsâwereâgrownâtoâconfluenceâinâaâ24-wellâcul-tureâplateâandâexposedâtoâ100âÎŒmol/lâorâ300âÎŒmol/lâpalmiticâacidâforâtheâstatedâtimeâinâserum-freeâmedium.âCellsâwereâwashedâ2âtimesâwithâPBS.ââ
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2588 TheJournalofClinicalInvestigationâ â â http://www.jci.orgâ â â Volumeâ119â â â Numberâ9â â â Septemberâ2009
Aâ0.2%âtrypanâblueâsolutionâ(final)âwasâmadeâinâPBSâandâaddedâtoâcellsâforâ3âminutesâatâroomâtemperature.âAfterâwhichâtimeâcellsâwereâagainâwashedâwithâPBSâ(2X)âandâevidenceâforâdyeâexclusionâwasâdetermined.âAâviableâcellâhadâclearâcytoplasm,âwhereasâaânonviableâcellâhadâblueâcytoplasm.
RT-PCR.âAdultâmaleâratsâmaintainedâonâtheâlow-fatâdietâwereâsacrificedâbyâexposureâtoâcarbonâdioxideâandâsubsequentâdecapitation.âTissuesâwereâremovedâandâstoredâatââ80°CâforâmeasurementâofâPrkcqâmRNAâexpres-sionâinâmuscleâandâhypothalamus.âTotalâRNAâwasâextractedâfromâsamplesâusingâtheâTri-Reagentâmethodâ(catalogâno.âTR118;âMolecularâResearchâCenterâInc.),âaccordingâtoâtheâmanufacturerâsâinstructions.âSubsequently,ââcDNAâtemplatesâforâRT-PCRâwereâgeneratedâfromâ5âÎŒgâofâtotalâRNA,âusingâtheâSuperScriptâmethodâ(SuperScriptâIIIâkit;âcatalogâno.â18080-051;âInvitrogen),âaccordingâtoâtheâmanufacturerâsâinstructions.âPCRâwasâper-formedâforâPrkcqâmRNAâusingâtheâfollowingâprimers:âPKC-Ξ-A:â5âČ-TGT-GATGGGATGTTCTCCAA-3âČ;âandâPCK-Ξ-B:â5âČ-CGTCCACGTACGTCAT-CATC-3âČ.âPCRâproductsâwereârunâonâaâ1%âagaroseâgelâandâassessedâforâtheâpresenceâofâaâ530âbaseâpairâsizedâband.
Immunocytochemistry.âAnimalsâ(experimentalâseriesâ4âconsistedâofâ5ârats,â5âWTâmice,â3âPrkcqâ/ââmice,â5âNPYGFPâmice,â5âPOMCGFPâmice,âandâ3âLep-RGFPââmiceâ[providedâbyâMartinâG.âMyersâJr.];âexperimentalâseriesâ7âconsistedâofâ5âscrambledâoligoâAAVâandâ5âAAVâPKC-ΞâshRNA-infusedâmice)âwereâgivenâanâoverdoseâofâpentobarbitalâ(60âmg/kg)âandâperfusedâtranscardi-allyâwithâ4%âparaformaldehydeâinâ0.1âMâsodiumâphosphateâbufferâ(PB).âBrainsâwereâremoved,âpostfixedâ(1.5âhoursâatâroomâtemperature),âandâstoredâinâ20%âsucroseâinâPB.âCoronalâsectionsâwereâcutâ(40-ÎŒmâthick)âwithâaâCryostatâ(Leica)âandâstoredâinâcryoprotectantâ(30%âsucrose,â30%âethyleneâglycol,âinâ0.1âMâPB)âatââ20°CâuntilâfurtherâprocessingâforâeitherâPKC-Ξâorâp-AKT.âAvailableâantibodiesârecognizingâtheseâantigensâwereâallâraisedâinârabbits.âFree-floatingâsectionsâwereâincubatedâovernightâatâ4°C,âwithâpolyclonalâantiserumârecognizingâPKC-Ξâ(SantaâCruzâBiotechnol-ogyâInc.)âdilutedâ1:1,000âinâincubationâsolutionâ(PBSâcontainingâ4%ânor-malâdonkeyâserumâandâ0.1%âtritonâX-100).âSectionsâwereâsubsequentlyâexposedâtoâbiotin-conjugatedâdonkeyâanti-rabbitâIgGâ(1:400âinâincuba-tionâsolution;â60âminutes;âJacksonâImmunoResearchâLaboratoriesâInc.),âavidin-biotinâhorseradish-conjugatedâperoxidaseâ(ABCâElite,â1:1,500âinâ
PBS;â60âminutes;âVectorâLaboratories),âbiotinylatedâtyramideâ(1:250;â10âminutes;âTyramideâSignalâAmplification;âNEN).âAdditionalâtissueâwasâprocessedâasâaboveâandâcolocalizedâwithâPOMCâ(catalogâno.âH-029-30;âChemicon),âGFAPâ(catalogâno.âGFP-1020;âAvesâLabs),âorâp-AKTâ(cata-logâno.â7985-R;âSantaâCruzâBiotechnologyâInc.).âMouseâsectionsâwereâthenâprocessedâwithâDAB;âratâandâmouseâsectionsâwereâthenâprocessedâwithâCY3-conjugatedâstreptavidinâ(1:400âinâPBS;â30âminutes;âJacksonâImmunoResearchâLaboratoriesâInc.)âorâAlexaâFluorâ488âandâ594âconju-gatedâsecondaryâantiseraâ(Invitrogen).âSectionsâwereâmountedâonâglassâslidesâandâcover-slippedâwithâGelvatol,âcontainingâanâantifadingâagentââ[1,4-diazabicycloâ(2,2)âoctane].
Statistics.âFoodâintakeâandâWesternâblotâdensitometryâratiosâwereâana-lyzedâbyâfactorialâANOVAâ(Statisticaâ6.0;âStatsoftâInc.),âusingâdietâ(fattyâacid)âandâdrugâasâfactorsâwhereâappropriate.âInâtheâcaseâofâfoodâintakeâmeasuresâwithâsalineâbaseline,âweâusedârepeatedâmeasuresâANOVA,âwithâdietâ(fattyâacid)âasâaâbetweenâsubjectsâfactorâandâdrugâasâaârepeated-mea-suresâfactor.âTheâsourceâofâsignificantâmainâeffectsâandâinteractionsâwasâconfirmedâbyâTukeyâsâhonestlyâsignificantâdifferencesâpost-hocâtests.ââPâvaluesâofâlessâthanâ0.00âwereâconsideredâstatisticallyâsignificant.âDataâareâdepictedâasâmeanâ±âSEMâunlessânotedâotherwise.
AcknowledgmentsThisâworkâwasâsupportedâbyâgrantsâfromâNationalâInstituteâofâDia-betesâandâDigestiveâandâKidneyâDiseasesâ(NIDDK):âNIH-NIDDKâ056863âandâNIH-NIDDKâ17844â(toâtheâUniversityâofâCincinnati).âLep-RGFPâreporterâmiceâwereâprovidedâbyâMartinâG.âMyersâJr.
ReceivedâforâpublicationâJulyâ7,â2008,âandâacceptedâinârevisedâformâMayâ20,â2009.
Addressâcorrespondenceâto:âDeborahâJ.âClegg,âDepartmentâofâInter-nalâMedicine,âTouchstoneâDiabetesâCenter,âUniversityâofâTexasâSouthwesternâMedicalâCenter,â5323âHarryâHinesâBlvd.,âK5.252,âDallas,âTexasâ75390-8854,âUSA.âPhone:â(214)â648-3401;âFax:â(214)â648-8720;âE-mail:â[email protected].
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