[PDF] The biochemistry of diabetes

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Biochem.J;(1988)250,625-640(PrintedinGreatBritain)REVIEWARTICLEThebiochemistryofdiabetesRoyTAYLORandLoranneAGIUSDepartmentofMedicine,UniversityofNewcastleuponTyne,FramlingtonPlace,NewcastleuponTyneNE24HH,U.K.INTRODUCTIONWehaveattemptedtoreviewthisbroadsubjectnotbybeingcomprehensive,butratherbyemphasizingareasofrecentadvance.Thehallmarkofdiabetesmellitusisaninabilitytocontrolbloodglucose.Therearetwomajorclinicalsyndromes:onecharacterizedbyinsulindependenceandearlyageofonsetwithweightlossandketonuria,andthesecondcharacterizedbyrelativelylateronset,insensi-tivitytoinsulinandpartialinsulindeficiency.Dearthofpreciseknowledgeaboutpathogenesismakesaetiologicalclassificationhazardous,andinthisreviewthedescriptivetermsinsulin-dependentdiabetes(IDDM)andnon-insulindependentdiabetes(NIDDM)willbeused.METABOLICDISTURBANCEINMANCarbohydratemetabolismFastingstate.Innormalsubjects,fastingbloodglucoseismaintainedconstantbycontrolofhepaticglucoseoutput.Afteranovernightfast,approx.75%ofhepaticglucoseoutputisaccountedforbyglycogenolysisandtherestbygluconeogenesisfromlactate,alanine,glycerol,andpyruvateindecreasingorderofimportance(Hers&Hue,1983).Hepaticglucoseoutputiscontrolledbybasallevelsofinsulinandglucagon.Basalhepaticextractionofinsulinandglucagonisapprox.500(Waldhausletal.,1982);andhenceinsulinandglucagonconcentrationsintheperipheralcirculationarelowerthanintheportalvein.Atleast70%ofextrahepaticglucoseutilizationoccursininsulin-insensitivetissues(brain,redbloodcellsandrenalmedulla)(Vranic&Wrenshall,1968).InNIDDM,fastingbloodglucoseisraisedindirectproportiontohepaticglucoseoutput(Bogardusetal.,1984;Reversetal.,1984;DeFronzoetal.,1985),andappearsunlikelytobearesultofdecreasedinsulinactionattheperipheryasithasnotbeenshowntocorrelatecloselywithinsulin-stimulatedglucosedisposal(Bogardusetal.,1984;DeFronzoetal.,1982a).AsfastingplasmainsulinandC-peptideconcentrationsarenormalinNIDDM,theelevatedhepaticglucoseoutputislikelytoreflectadegreeofhepaticinsulininsensitivity.Studyoftheinsulindose-responseofsuppressionofhepaticglucoseoutputsupportsthisconcept(Reversetal.,1984).Postprandialstate.Innormalsubjects,first-passhepaticglucoseextractionislowandover90%ofanoralglucoseloadreachestheperipheralcirculation(Radziuketal.,1978;Pehlingetal.,1984).Overthesubsequentfewhoursapproximatelyone-thirdofglucoseisclearedbytheliver(Katzetal.,1983).Uncertaintypersistsabouttheproportionofglucosestoredbytheliverwhichisderivedfrom3-carbonprecursorsformedintheperiphery,butthisiscurrentlythoughttobeconsiderable(Katz&McGarry,1984).Thepost-mealhyperinsulinaemiainhibitslipolysisandstimulatesstorageofglucoseandfattyacidsasglycogeninmuscleandliverandastriacylglycerolinadiposetissue.InNIDDMsubjects,studyofplasmainsulinlevelsthroughoutthedayhasdemonstratedthat,althoughtheincrementalresponsestomealsaredecreasedanddelayed,theprolongedpostprandialpeaksensurethatmeandiurnalplasmainsulinlevelsareelevated(Liuetal.,1983).Postprandialratesofgluconeogenesisarenotsuppressed(Wahrenetal.,1972;Halletal.,1979)andtotalhepaticglucosereleaseasassessedisotopicallyisexcessive(Firthetal.,1986a).Gluconeogenesisfrommeal-derived3-carbonprecursorsissomewhatgreaterinNIDDMthaninnormalsubjects.Followingglucoseingestion,splanchniclactateuptakechangestonetoutputandforearmoxygenuptakeandlactatebalancedonotchange(Meistasetal.,1985;Jacksonetal.,1986).Thesefindingssuggestthatintestineorlivermetabolizesglucosetolactate,someofwhichiseventuallystoredbytheliverasglycogenandfattyacid.Mostoftheglucosetakenupbymusclemustthereforebestored.Jacksonetal.(1987b)demonstratedatransientperiodoflactateuptakeinforearmmuscleafterglucoseloading(probablyamassactioneffectrelatedtohyperlactataemia)andshowedthattherewasnomarkeddecreaseinskinlactateoutputoverthesameperiod.Fastingsuperficialvenouslactateconcentrationsaregreaterthaneitherarterialordeepvenousconcentrations,andthereforeskinmustbeoneofthemajororgansoflactateproduction(Jacksonetal.,1987b).Althoughtheinsulin-mediatedincrementinmuscleglucoseuptakeissubnormalinNIDDM,absolutepostprandialratesofglucoseuptakearenormalwhenmeasureddirectlybytheforearmtechniqueatprevailingbloodglucoseconcentrationsdespitethemarkedinsulininsensitivity(Jacksonetal.,1973;Firthetal.,1986a).Thisreflectsthemassactioneffectofglucoseontissueuptake(Reversetal.,1984).Relativelyfewstudiesonhumanmuscleenzymeshavebeencarriedout.Falholtetal.(1987)founddecreasedfastingactivityofhexokinaseandphosphofructokinase,suggestingapotentialdecreaseinglycolysisinNIDDMsubjects,butglucose-6-phosphatedehydrogenaseandmalicenzymeactivitieswereelevatedandsowasmuscletriacylglycerol.However,thepatientsstudiedwereremarkablyhyperinsulinaemic,fastingplasmainsulinlevelsbeing6-foldelevated,andthusnotrepresentativeofthemajorityofNIDDMsubjects.Vol.250625Abbreviationsused:IDDM,insulin-dependentdiabetesmellitus;NIDDM,non-insulin-dependentdiabetesmellitus;VLDL,very-low-densitylipoprotein;GH,growthhormone;IGF,insulin-likegrowthfactor;NEFA,non-esterifiedfattyacids.

R.TaylorandL.AgiusTable1.MeandiurnalplasmaconcentrationsofintermediarymetabolitesunderdifferingdegreesofglycaemiccontrolandinsulinaemiaDatarepresent12hday-timemeans,*24hmeans,ort4hmeans,comparedwithnormalsubjects.SU,sulphonylurea;SC,subcutaneousinsulin;N,normal;4,significantlyraised;4,significantlylow.Subjectsand3-Hydroxy-GlucoseInsulintherapyLactatePyruvateAlanineGlycerolNEFAbutyrate(mmol/l)(m-units/i)ReferenceNIDDMDietN444_N7.016Nattrass(1982)DietNNNN-N7.9-tSheppardetal.(1983)Diet4444N411.823tSamadetal.(1987)Diet----4418.47Nankervisetal.(1982)Diet----4-12.233tFrazeetal.(1985)Nil444N-413.2-tSheppardetal.(1983)SU44444N11.4-tI.R.Jones(unpublishedwork)SUNNN4NN5.220Nattrassetal.(1978)SU4441-N13.226Nattrass(1982)Metformin4444NN7.019Nattrassetal.(1977)IDDMSCN_N4-412.929Madsbadetal.(1981)SC4tNN-412.131*Capaldoetal.(1984)SCNN4N-48.516*Marshalletal.(1987)SC44N4NN8.352Nosadinietal.(1982)Biostator44N4NN5.681Nosadinietal.(1982)InsubjectswithIDDM,postprandialhyperglycaemiaistheconsequenceofthelackofanappropriatesharpincreaseincirculatinginsulinlevels,andreflectsin-adequaciesinthedynamicsofinsulindelivery.Hepaticreleaseofglucoseisrelativelyuninhibitedandthis,togetherwiththedemonstratedresistancetoinsulininperipheraltissues(Proiettoetal.,1983;Yki-Jarvinenetal.,1984),resultsinpostprandialhyperglycaemia.TheresultsofstudiesoncirculatingconcentrationsofintermediarymetabolitesaresummarizedinTable1.AstrikingfeatureisthehighlactateinIDDMandpoorlycontrolledNIDDM.Strongcorrelationshavebeenreportedbetweenmean24hplasmainsulinandlactateconcentrations(Capaldoetal.,1984;Albertietal.,1975).Thehighlactatepersistsduringestablishmentofnormoglycaemiaovera24hperiod,althoughinthissituationmeasuredCoricycleactivityissuppressed(Nosadinietal.,1982).Itispossiblethatthehyper-lactataemiamayrepresentanimbalanceofinsulinactiononperipheraltissuescomparedwithliver,assub-cutaneousorintravenousinsulinadministrationleadstoequallevelsofinsulinintheperipheralcirculationandportalvein,unlikethenormalsituation.Restorationoftheportal-peripheralinsulingradientreturnslactateconcentrationstowardsnormal(Jimenezetal.,1985;Stevensonetal.,1983).InpoorlycontrolledNIDDMsubjects,theportal-peripheralinsulingradientispre-served,butitispossiblethatperipheraltissuesbecomerelativelymoreresistanttoinsulinaction(seebelow).Proinsulinappearstohaveaslightlygreatereffectontheliverthanonperipheraltissuesinvivoandtheriseinplasmalactateusuallyassociatedwithinsulininfusionisnotseenwhendosesofproinsulinareinfusedtoproducesimilarincrementsinoverallglucosedisposal(Davisetal.1986).Changesincirculatingpyruvateandalaninetendtoreflectlactateconcentrationsandhencedegreeofmetaboliccontrolandhyperinsulinaemia(Table1).Thesorbitol(polyol)pathwayOneoftheconsequencesofhyperglycaemiainhumandiabetesmellitusisincreasedmetabolismofglucosebythesorbitolpathway.Thisinvolvesthereductionofglucosetosorbitolcatalysedbyaldosereductase(EC1.1.1.21)andtheoxidationofsorbitoltofructosebysorbitoldehydrogenase(EC1.1.1.14).Aldosereductaseispresentinhumanbrain,nerves,aorta,muscle,erythrocytesandocularlens(Srivastavaetal.,1984;Das&Srivastava,1985b).Althoughthepurifiedenzymehasalowaffinityforglucose(Kmapprox.100mM)(Moon-sammy&Stewart,1967),itcanbeactivatedbyglucose6-phosphate,NADPHandglucose(Das&Srivastava,1985a,b).Sorbitolisnotpermeabletocellmembranesandtendstoaccumulateinthecell.Athigh[glucose]thefluxthroughthesorbitolpathwayinrabbitlensmayaccountforone-thirdofglucosemetabolism(Gonzalezetal.,1984).ThishasimportantimplicationsintermsofredoxchangesofNADPandNADcouplesandmetabolismofglucosebyalternativepathways(Jeffrey&Jornvall,1983).ConversionofglucosetosorbitolbyaldosereductaserequiresNADPHandformsNADP+(Fig.1)andtherebycompeteswithotherNADPH-requiringreactions.NADPHisrequiredfortheconversionofoxidizedtoreducedglutathione,apowerfulantioxidantwhichprotectscellularcomponentsfromoxidativedamage,andforfattyacidandcholesterolbiosynthesis.ThepentosephosphatepathwayisthemajorsourceofNADPHinmosttissuesanditsfluxisgenerallydeterminedbytheNADP+/NADPHratio.ConversionofsorbitoltofructoseiscoupledtoreductionofNAD+toNADHandthiscompeteswithglycolysisattheglyceraldehydedehydrogenasestepforNAD+(Gonzalezetal.,1986).AnincreaseintheNADH/NAD+ratiofavoursincreasedconversionofdihydroxyacetone1988626

ThebiochemistryofdiabetesGlucoseHexokinaseGlucose6-phosphate*NADPHRibulose--/5-phosphate1PentosephosphateNADP+1pathwaySorbitolNADPHNADP+NAD+ise2GSSGSeGSSGGSHGlutathioneFreductaseFructose-----------------------------am-.xNADHGlyceraldehyde3-phosphate_NAD+-|NADH3-PhosphoglycerateDihydroxyacetonephosphateNADHNAD+Glycerol3-phosphatePyruvate-Acetyl-CoANADHiNAD+L-LactateNADPHNADP+&FattyacidFig.1.ThesorbitolpathwayanditslinkswiththepentosephosphatepathwayandglycolysisthroughtheNADP+andNADIredoxcouplesThesorbitolpathwayinvolves:conversionofglucosetosorbitolbyaldosereductaseandsorbitoltofructosebysorbitoldehydrogenase.ReactionscoupledtooxidationofNADPHareindicatedby*andreactionscoupledtoreductionofNADIareindicatedby**.AldosereductasecompeteswithglutathionereductaseforNADPHandsorbitoldehydrogenasecompeteswithglyceraldehyde-3-phosphatedehydrogenaseforNADI.(1)Anincreasedfluxthroughaldosereductasefavoursanincreasedactivityofthepentosephosphatepathway(Gonzalesetal.,1986)andincreasedfluxthroughsorbitoldehydrogenasefavoursincreasedconversionofdihydroxyacetonephosphatetoglycerol3-phosphate(Gonzalezetal.,1983)anddecreasedconversionofglyceraldehyde3-phosphateto3-phosphoglycerate.phosphatetoglycerol3-phosphate.Inratlens,increasedfluxthroughaldosereductaseisassociatedwithincreasedpentosephosphatepathwayactivity,decreasedglycolysis(Gonzalezetal.,1986),accumulationofglycerol3-phosphateanddepletionofreducedglutathione(Whikehart&Soppet,1981;Gonzalezetal.,1984).DisturbancesinlipidmetabolismFattyacidmobilizationandproductionandutilizationofketonebodies.Oneofthemostprominentfeaturesofinsulindeficiencyisrapidmobilizationoffattyacidsfromadiposetissue.InIDDM,excessivelipolysisduringinsulindeficiencyisthecombinedresultofinsulinlackandinsulinresistance(Singhetal.,1987).OneoftheconsequencesofexcessivemobilizationoffattyacidsinIDDMistheproductionofketonebodies(acetoacetate,3-hydroxybutyrateandacetone)inliver.Fattyacidstakenupbytheliver,afterconversiontotheirCoAesters,areeitheresterifiedtoglycerolipidoroxidizedtoacetyl-CoAinmitochondria.Ahighproportionoftheacetyl-CoAformedisconvertedtoacetoacetateand3-hydroxybutyrate.TherateoftransferoffattyacylunitstothemitochondriaisregulatedbytheactivityofcarnitinepalmitoyltransferaseI(EC2.3.1.21),whichfacestheintermitochondrialmembranespaceandcatalysesthefirststepspecifictomitochondrialfattyacidoxidation.CarnitinepalmitoyltransferaseIisregulatedbymalonyl-CoA,anintermediateinfattyacidsynthesis,anditisalsoregulatedbyaphosphorylationmechanism.Malonyl-CoAdecreasestheaffinityoftheenzymeforitsVol.250AldosereductasSorbitoldehydrogena627

R.TaylorandL.Agius*CH3COCH2CO2-+H+AcetoacetateabCO2*CH3COCH3Acetone*CH3COCH20HcAcetolh/CH3COCHOMethylglyoxalCH3COCH20PdVAcetolphosphateCH3CH(OH)CH20PL-Propane-1,2-diolphosphate*CH3CH(OH)CH20HL-Propane-1,2-diolfCH3CH(OH)CHO*CH3C02L-LactaldehydeAcetateQI+CH3CH(OH)C02D-Lactatek->CH3COC02Pyruvate-CH3CH(OH)CO2";zL-Lactate*D-GlucoseFig.2.PostulatedpathwaysforthemetabolismofacetonetoglucoseandacetateIntermediatesmarkedby*increaseinhumanplasmaduringketosis(Reichardetal.,1986).Enzymes:a,acetoacetatedecarboxylase;b,acetonemono-oxygenase(NADP+);c,acetolkinase;d,propanediolphosphatedehydrogenase(NADP+);e,phosphatase;f,lactaldehydereductase[NAD(P)+];g,lactaldehydedehydrogenase(NAD+);h,acetolmono-oxygenase(NADP+);i,2-oxoaldehydedehydrogenase[NAD(P)+];j,glyoxylaseIandII;k,D-2-hydroxyaciddehydrogenase(FAD);1,lactatedehydrogenase(NAD+).fattyacyl-CoAsubstrate(McGarryetal.,1977),whilephosphorylationincreasestheaffinityforsubstrate(Haranoetal.,1985).Ininsulindeficiency,therateoffattyacidsynthesisinliverdeclinesandconsequentlytheconcentrationofmalonyl-CoAalsodecreases,andtheaffinityofcarnitinepalmitoyltransferaseformalonyl-CoAalsodecreases(Gamble&Cook,1985),thusrelievingtheinhibitionofcarnitinepalmitoyltransferasebymalonyl-CoA.Changesinthekineticpropertiesofdetergent-solubilizedcarnitinepalmitoyltransferasefollowingexposureoflivercellstoglucagon(activation)orinsulin(inactivation)havealsobeenobserved(Haranoetal.,1985;Agiusetal.,1986b),indicatingthatadditionalmechanismscontributetoactivation(ordeinhibition)ofcarnitinepalmitoyltransferaseininsulindeficiency,there-byfavouringincreasedtransferoflong-chainfattyacidsintomitochondria.Theutilizationofacetoacetateand3-hydroxybutyrateasoxidativefuels(orlipogenicsubstrates)byavarietyoftissuesiswellestablished(Robinson&Williamson,1980)andincreaseswithbloodketonebodycon-centrationinthefed-to-fastedtransition(Milesetal.,1980).Inmuscle,atketonebodyconcentrationsattainedinprolongedstarvationordiabeticketosis,therateofuptakereachessaturation(Owen&Reichard,1971).Consequently,withincreasingketonebodyproductionandtherebyplasmaconcentration,thereisaprogressivedecreaseintotalfractionalclearance(Fery&Balasse,1985).Althoughthebiochemicalroutesofmetabolismofacetoacetateand3-hydroxybutyratearewellestablished,theconversionofacetoacetatetoacetoneanditssubsequentexcretionormetabolismhasonlyrecentlyreceivedattention.Inman,plasma[acetone]correlateswith,andisgenerallyhigherthan,[acetoacetate]infastinganddiabeticketosis(Owenetal.,1982)andtheproductionrateisestimatedtobeabouthalftherateofketogenesis(Reichardetal.,1986).Conversionofacetoacetatetoacetonecanoccureithernon-enzymicallyorcatalysedbyacetoacetatedecarboxylase(EC4.1.1.4)(Argiles,1986).Theoccurrenceofthisenzymehasbeendemonstratedinvariousrattissues,butonlyinhumanplasma(Koorevaar&vanStekelenburg,1976).Thehighacetoneproductionrateinmansuggeststheoccurrenceoftheenzymeinothertissues.Atlowplasmaacetoneconcentrations(1-2mM),asoccurinfastingketosis,about20%isexcreted(inbreathandurine)andtherestmetabolized,whereasathigherconcentrations,asindiabeticketosis(7-9mM),about80%isexcreted(Owenetal.,1982).Intherat,acetoneishydroxylatedtoacetolwhichiseitherconvertedtomethylglyoxalortopropane-1,2-diol(Fig.2).Theformerisconvertedtopyruvateeitherdirectlyorindirectly(Cassavaetal.,1984),whereaspropane-1,2-dioliseitheroxidizedtoL-lactateorconvertedtoacetateandformate(Kosugietal.,1986a,b)(Fig.2).Studiesontheincorporationof[2-'4Clacetoneintoglucoseintherathaveshownthatatlowplasma[acetone]itismetabolizedprimarilytolactateand1988HC02-Formate628

Thebiochemistryofdiabetespyruvate,butathighplasma[acetone],acetateformationpredominates(Kosugietal.,1986b).Recentevidencesuggeststhatinhumandiabeticketosisacetonemetabol-ismmaybeverysimilar(Reichardetal.,1986).[2-14C]-Acetonewasincorporatedintoglucoseviapyruvateandlactateinthemajorityofpatients,but,inonesubjectwithhighplasma[acetone],labelincorporationwaspredominantlyviaacetateformation(Reichardetal.,1986).Thesefindingssuggestthat,inmoderateketosisinman,acetoneisapotentialgluconeogenicsubstrate.Triacylglycerolsecretionandclearance.TheplasmatriacylglycerolconcentrationiselevatedinbothIDDMandNIDDM(Nikkila,1984).InIDDM,decreasedclearanceseemstobethemaincauseofthehightriacylglycerollevel(Bagdadeetal.,1968;Nikkilaetal.,1977;Taskinen&Nikkila,1979),whereasinNIDDMthetriacylglycerolaemiamaybeduetoincreasedproduc-tionbytheliver(Greenfieldetal.,1980;Kissebahetal.,1982;Dunnetal.,1984)anddecreasedclearance(Nikkilaetal.,1977;Taskinenetal.,1982;Pfeiferetal.,1983).Productionoftriacylglycerolbytheliverinvolvestheesterificationoffattyacid,eithersynthesizeddenovointheliverfromdietarycarbohydrateandaminoacid,orderivedfromadiposetissuereserves.ThetriacylglyceroliseitherstoredintracellularlyoritispackagedwithapoproteinsandsecretedintheformofVLDL.Undercertainconditionsfattyacidesterificationincreaseswithincreasingfattyacidconcentrationandappearstobenon-saturable(Ontko,1972).VLDLsecretion,incon-trast,maybelimitedbytheavailabilityofaproproteinsorothercomponentsofthelipoproteinparticle.WhenVLDLsecretionreachessaturation,triacylglycerolaccumulatesinsidetheliver.TheelevatedplasmatriacylglycerolindiabetesisgenerallyassociatedwithVLDL,althoughincreasesinotherlipoproteins(LDLandHDL)alsooccur(Gibbons,1986).Inabsoluteinsulindeficiency,theplasmaconcen-trationandturnoveroffattyacidsincreases,whereasfattyacidsynthesisdenovodecreasesandtheproportionoffattyacyl-CoAthatisesterifiedasopposedtooxidizedalsodecreases.Anyabsoluteincreaseinhepaticfattyacidesterificationdependsonwhethertheincreaseinfattyacidavailabilitycompensatesfortheincreasedfractionaldiversiontowardsmitochondrialoxidation.InpatientswithIDDM,intensiveinsulintherapy,whichisassociatedwithahighermeanplasmainsulincomparedwithconventionalinsulintherapy,resultsinadecreaseintriacylglycerolsecretionrate(Dunnetal.,1987,Pietrietal.,1983).Whethertheeffectoftheincreasedinsulin-izationwasprimarilyduetoadecreaseinfattyacidavailabilityortoinhibitionofVLDLsecretionisnotclear.TriacylglycerolsecretionratesaregenerallyhigherinNIDDMthaninIDDM(Greenfieldetal.,1980).InnormolipaemicNIDDM,triacylglycerolsecretionandclearancearebothincreased,whereasinhyperlipaemicNIDDM,fractionalturnoverrateisreducedsuchthatincreasedtriacylglycerolremovaldoesnotcompensatefortheincreaseinsecretionrate(Kissebahetal.,1982).InNIDDMwithdecreasedVLDLclearance,thecom-positionofVLDLisabnormal,withahightriacyl-glycerol/apoproteinBratio(Taskinenetal.,1986),indicatingmultipleabnormalitiesinVLDLmetabolism.Akeyissueiswhetherchangesinadiposetissuelipolysis,orintrahepaticmechanismsinvolvingeitherchangesinfractionalesterificationoffattyacidorintheassemblyandsecretionofVLDL,areresponsiblefortheincreaseintriacylglycerolsecretionrateinNIDDM.StudiesinvitroontheeffectsofinsulinonfattyacidesterificationandsecretionofVLDLhaveledtoconflictinghypotheses.Intheperfusedratliver,insulinacutelyincreasestriacylglycerolsecretion(Topping&Mayes,1982;Laker&Mayes,1984),butinrathepatocyteculturesincubatedunderconditionsofhighratesoffattyacidsynthesisdenovo,insulindecreasestherateoftriacylglycerolsecretionduringa16-18hincubation(Durringtonetal.,1982;Patschetal.,1983,1986).Onthebasisoftheformerstudies,elevatedtriacylglycerolsecretionratesinNIDDMaresuggestedtobeduetohyperinsulinizationoftheliver,increasingesterificationandVLDLsecretion,whereasonthebasisofthelatterstudiesitmightbearguedthathepaticinsulinresistancemayberesponsibleforthelackofinsulininhibitionoftriacylglycerolsecretion.ThelackofunderstandingofthephysiologicaleffectsofinsulinonhepaticfattyacidesterificationandVLDLsecretionrenderstheinterpretationofthelesionsindiabetesverydifficult.HORMONEACTIONSINMANInsulinMechanismofinsulinaction.Theextenttowhichprimaryorsecondarydefectsininsulinreceptoractivitycanexplaincellularinsensitivitytoinsulinisstillhotlydebated.Theinsulinreceptoriswellcharacterized(Kahn,1985)anditsgenehasbeencloned(Ullrichetal.,1985).Itcomprisestwo135kDaasubunitswhichareextracellularandcontaininsulin-bindingsites,andarelinkedbydisulphidebondstotwo95kDa/,subunits.The/3subunithasahydrophobictransmembraneregion,andanintracellulardomainwhichhasseveraltyrosineresidues,atyrosinekinaseandanATP-bindingsite.Insulinbindingtotheasubunitsactivatesthe/3subunittyrosineproteinkinaseandbringsaboutphosphorylationoftyrosineresiduesonthe/3subunit.Activationofthe/3subunitkinasemaybeinvolvedintransmissionoftheinsulinsignal,perhapsbyinitiatingaphosphorylation/dephosphorylationcascade(Dentonetal.,1981).KinaseactivityoftheinsulinreceptorcanbedecreasedbycyclicAMP-dependentproteinkinasephosphorylationofserineorthreoninesitesonthe,subunit,andthiscouldconceivablyunderliecatecholamine-inducedinsulinresistance(Roth&Beaudoin,1987).Instatesofextremeinsulinresistance(Grigorescuetal.,1987;Grunbergeretal.,1984;LeMarchand-Brusteletal.,1985)andNIDDM(Freidenbergetal.,1987;Caroetal.,1986)theprocessofsignaltransmissionfromthereceptorasubunitinsulin-bindingsitetoactivatethekinaseappearstobedefectiveatoneormoresites.AminoacidsubstitutionintheATP-bindingregionorthetyrosinekinaseregion(Chouetal.,1986;Ellisetal.,1986)abolishesinsulinaction.Whetherornotthekinaseisinvolvedinmediatingallactionsofinsulinremainsuncertain.Someantireceptorantibodiessimulateinsulinactionwithoutchangingkinaseactivity(Simpson&Hedo,1984;Forsayethetal.,1987).Notallactionsofinsulinaremodulatedinparallelbytreatmentofinsulin-resistantstates,suggestingthatdivergentpathwaysofintracellularinsulinsignaltransmissionmaybeseparatelyaffected(Bodenetal.,1983;Pedersen&Hjollund,1982).AremarkablearrayofsecondmessengersofinsulinVol.250629

R.TaylorandL.Agiusaction(Saltieletal.,1986;Gottschalk&Jarrett,1985)andsubstratesfortheinsulinreceptorkinase(Whiteetal.,1985;Accilietal.,1986)havebeenreported,butallawaitsubstantiation.Theactionofinsulininstimulatingglucosetransportinadiposetissueandmusclehasbeenintensivelystudied.Insulinbringsaboutrapidtranslocationofglucosetransportersfromanintracellularpooltotheplasmamembrane(Karnielietal.,198la;Cushman&Wardzala,1980).IgnoringpotentialtechnicaldifficultiesinassessingglucosetransporternumberbycytochalasinBbinding,insulinalsoenhancesintrinsicactivitypertransporterunit(Kahn&Cushman,1985).Instatesofinsulinopeniaorinsulininsensitivity,totalglucosetransporternumberdecreasesandthisislikelytoreflectlackofinsulineffectuponglucosetransportersynthesis(Karnielietal.,1981b;Hissinetal.,1982).Itmustbeconsidered,however,thatadecreaseintransporternumbercouldreflectacompensatorychangeinthefaceofhyper-glycaemia.Theroleoftheglucosetransportersysteminthepathogenesisofhyperglycaemiaremainsuncertain,asdothemechanismsoftranslocationtotheplasmamembraneandchangeinintrinsicactivity.StudiesinvivoinNIDDM.Whereasnormalsubjectsincreaseglucoseutilizationbyover300%0duringinsulininfusion,NIDDMsubjectsdosobyonly30%(Reavenetal.,1985;Donneretal.,1985;Swislockietal.,1987).ObesityandNIDDMfrequentlycoexist.HollenbeckandcolleagueshavedemonstratedthattheeffectofobesityuponmeasuredinsulinsensitivityissimilarinnormalandNIDDMsubjects,andthatthiseffectissmallincomparisonwiththeeffectofNIDDMperse(Hollenbecketal.,1984).Theparametermostcommonlyusedtoassessinsulinsensitivityistissueuptakeofglucoseadministeredintravenously,usuallyunderconditionsofhyper-insulinaemiasufficienttoinhibitnethepaticglucoseproduction.Henceuptakeandstorageofglucoseasglycogenpredominantlyinmuscleismeasured(Rizzaetal.,1980;Reversetal.,1984;DeFronzoetal.,1985).Asthisisincreasedbymassactionduringhyperglycaemia,theeffectofinsulininhyperglycaemicstatesmaybeoverestimated.Theexperimentalevidenceforinsulininsensitivity,gatheredinthehighlyartificialsettingofconstanthyperinsulinaemia,iscorroboratedbyobserva-tionsupondiurnalprofilesofbloodglucoseandplasmainsulin.Severalstudieshavedemonstratedtheco-existenceofhyperglycaemiaandrelativehyperinsulin-aemiainNIDDMsubjects(Swislockietal.,1987;Liuetal.,1983).Theoldertechniquesofassessmentofinsulinsensitivitywerenotabletodistinguishbetweentherelativecontri-butionsofmuscleandlivertotheapparentinsulinresistance.DeFronzoetal.(1985)demonstratedthatduringahyperinsulinaemiceuglycaemicclampapprox.8500oftheintravenouslyadministeredglucosewastakenupbyperipheraltissues(almostentirelymuscle).Ashepaticglucoseproductionappearedtobereducedtozeroundertheconditionsoftheclamp,itwasdeducedthatperipheraltissueinsulininsensitivitywasthepredominantlesioninNIDDM.Methodologicalprob-lemscauseoverestimationofthedegreeofsuppressionbyinsulinofhepaticglucoseproductioninNIDDMandnormalsubjects,butnonethelesshepaticinsulininsensi-tivityappearstocontributerelativelylittletooverallmeasuredinsulinsensitivity(Belletal.,1986;Firthetal.,1986a).StudiesinvitroinNIDDM.AdipocytesfromnewlydiagnosedNIDDMsubjectsdemonstrateminimalresponsivenesstoinsulinintermsofglucoseoxidationorlipogenesis(Bolinderetal.,1982;Hjollundetal.,1985).Interestingly,lipolysiswasfoundtobenormallyinhibitedbyinsulinincellsfromthesamesubjects(Bolinderetal.,1982).Insulinreceptornumberhasbeenshowntobenormalwhenmeasuredat37°C(Hjollundetal.,1985;Kashiwagietal.,1983).Onesiteofdefectmaybewithintheinsulinreceptoritself.Freidenbergetal.(1987)studiedinsulinreceptorspartiallypurifiedfromisolatedadipocytestakenfromabdominalsubcutaneoustissueofmoderatelyobeseNIDDM,moderatelyobesenormalandleannormalsubjects.Theconcentrationofinsulinrequiredforhalf-maximalstimulationofinsulinreceptorautophosphorylationwassimilarforallgroups,butthatforinsulinreceptorkinaseactivitytowardstheartificialsubstratepoly(Glu-Tyr)wasincreasedintheNIDDMgrouponly.Obesitypersehadnoeffectoninsulinstimulationofkinaseactivity.Althoughthesedataappeartoargueforadefectininsulinreceptorkinaseasanexplanationforthepost-bindingdefectcharacteristicofNIDDM,theresultsdependoncomparisonofequalnumbersofreceptors.ReceptornumberwasdeterminedbyextrapolationofaScatchardplottothex-axis,anotoriouslyinexactprocedurewhichdependslargelyonthebehaviourofthelow-affinitybinding.Thebiologicalsignificanceofthelatterisuncertain.Furthermore,quantificationofinsulinreceptorsbyonefunction,binding,inordertoassessasecondfunction,kinaseactivity,presupposesafixedrelationbetweenvariableswhichholdsforallinsulinreceptors.Sinhaandcolleagueswereunabletodemonstratedifferencesinauto-phosphorylationandkinaseactivitybetweenreceptorspreparedfromadiposetissueofmorbidlyobeseNIDDMandmorbidlyobesesubjectswithnormalglucosetolerance(Sinhaetal.,1987).Inthelatterstudy,datawerecorrectedtoequalbindingactivityofreceptorsuspensionsratherthanequalinsulinreceptornumber.Muscleinsulinreceptornumberhasbeenshowntobeabout30%decreasedinveryobesesubjectswithNIDDMornormalglucosetolerancecomparedwithleansubjects(Caroet'al.,1987).Insulinreceptorkinaseactivitywasdecreasedtothesameextentinthediabeticandnormalobesegroups,suggestingthatdiabetesdidnotfurtherexacerbatethekinasedefectofobesity.Instreptozotocindiabetesintherat,insulinreceptornumberwas607000increasedandtheinsulin-stimulatedautophosphorylationandkinaseactivityonexogenoussubstratesperunitreceptorwerediminished(Burantetal.,1986).Insulintreatmentfor3daysreversedthesechanges.NostudiesofviablehumanmuscleinvitrohaveexaminedNIDDM,althoughinsulinstimulationinviablehumanmusclestripshasrecentlybeendemonstratedwithrespecttoglucoseuptakeandglycogensynthesis(Dohmetal.,1987;Taylor&Argyraki,1987).Limitedinformationisavailableonsubstrateconcentrationsandenzymeactivitiesinneedlebiopsyspecimensofmuscle.ThestudiesofFalholtetal.(1985,1987)havebeendiscussedabove.SubnormalinsulinstimulationofglycogensynthaseinNIDDMsubjectswasnotrestoredbyan8weekperiodofgoodcontroloninsulintherapy(Y.T.Kruzsynska,unpublishedwork).1988630

ThebiochemistryofdiabetesTheisolationofviablehumanhepatocytespresentsanimportanttechnicaladvanceinmetabolicstudies(Caroetal.,1986).Hepatocytesfromanobesenon-diabeticgroupexhibitedamoderateright-shiftoftheinsulindose-response,buthepatocytesfromaNIDDMgroupfailedtoincreaseaminoaciduptakeratesevenatIO-'M-insulin.Insulinreceptornumberwassimilarinallthreegroupsasjudgedbyinsulinbindingexperimentsper-formedonintactcellsat4'C.EitherpatientselectionorexperimentaldifferencesmayaccountforthediscrepancywiththeresultsofAmerandcolleagues,whofounda2-foldincreaseininsulinbindingtotheliverplasmamembranesfromNIDDMsubjects(Ameretal.,1986).StudiesinIDDM.TheinsulinsensitivityofglucosedisposalinIDDM(DeFronzoetal.,1982b;Proiettoetal.,1983)impliesthatthedailydosageofinsulin(U.K.average52units/day)isinexcessofnormaldailysecretionrates.Thishasrecentlybeendemonstrated(Polanskyetal.,1986).AswithNIDDM,errorsininterpretationofradioactivetracermethodsleadtothesuppositionthathepaticglucoseproductionissuppressednormallybyinsulin.ItisnowapparentthattheliverofIDDMsubjectsismodestlyresistanttoinsulin(Belletal.,1986).IncontrastwiththefindingsinstudiesofadipocytesfromNIDDMsubjects,adipocytesfrompoorlycon-trolledIDDMsubjectsdissplayedincreasedinsulinreceptorbindingandnormalinsulinsensitivitywithrespecttoglucoseoxidationandlipogenesis(Pedersen&Hjolland,1982;Lonnrothet-al.,1983;Yki-Jarvinenetal.,1984).Ithasbeensuggestedthatconventionaltwice-dailyinjectionofinsulincauseshyperinsulinaemiawhichmaypartiallybeavoidedbycontinuoussubcutaneousinsulininfusion.Thelattertherapywasobservedtoincreasemaximally-stimulatedratesofglucosetransporttowardsnormalwithoutchangeineitheradipocyteinsulinbindingorinsulinsensitivity(Marshalletal.,1988).Thesedatasuggestthatsomeoftheobservedabnormalitiesinadiposetissuemaybesecondarytotheinsulintherapy.Hyperglycaemiaitselfdoesnotappeartocauseinsulinresistance,asmaintenanceofnear-normalglycaemiafor6weeksinagroupofpreviouslypoorly-controlleddiabeticsubjectsdidnotimproveinsulin-stimulatedglucosedisposalrates(Kruszynskaetal.,1986).Nochangesintheactivationstateofmuscleglycogensynthaseeitherbasallyorinresponsetoinsulinwereinduced,andtheresponsetoinsulinwaslessthaninnormalsubjects.GlucagonPlasmaglucagonlevelsarehighinuntreateddiabetes(Gerichetal.,1976a)andparticularlyduringepisodesofdiabeticketoacidosis(Mulleretal.,1973),butarerapidlysuppressedbyinsulintreatment(Gerichetal.,1976b;Starketal.,1987).InIDDM,glucagonsecretioninresponsetohypoglycaemiaisfrequentlybluntedorabsent.Thissubnormalresponsedevelopswithin1-5yearsafterdiagnosisandbecomesmoremarkedinlateryears(Gerichetal.,1973;Bensonetal.,1977;Bollietal.,1983).ImpairedglucagonsecretionisalsoobservedinNIDDM(Bollietal.,1984).Glucagonhasaprimaryroleinincreasinghepaticglucoseoutputbystimulatingglycogenolysisandgluco-neogenesis.Indiabeticsubjects,inhibitionofglucagonsecretionmarkedlysuppressesthedevelopmentofhyper-glycaemiaafterinsulinwithdrawal(Gerichetal.,1975).Thestimulatoryeffectofaglucagoninfusiononglucoseoutputappearstransient,becauseitreturnstonormalwithinabout90mindespitepersistentlyhighglucagonconcentrations,probablyduetofeedbackinhibitionviainsulinsecretion(Bratusch-Marrainetal.,1979).Thoughthestimulationofglycogenolysismaybetransient(Komjatietal.,1985),thatofgluconeogenesisisprobablysustainedbecausehighglucagonlevelsunaccompaniedbyappropriateincreasesininsulinsecretionresultinasustainedincreaseinhepaticglucoseoutput(Rizzaetal.,1979a,b).Atphysiologicallevels,glucagoninfusionhasbeenshowntohaveeithernoeffectonglucoseuptakeortocausemildglucoseintolerance(Bajorunasetal.,1986);whetherthelatterisduetoadirecteffectofglucagononextrahepatictissuesortostimulationofcatecholaminesecretionisnotclear.However,studiesinvitrousingisolatedadipocyteshaveshownthatpreincubationwithglucagondecreasessubsequentinsulinreceptorbindingandstimulatedglucosetransport(Yamauchi&Hashizume,1986).Glucagonalsohasadirectacutestimulatoryeffectonadipocytelipolysis(Lefebvre,1966;Honnor&Sagger-son,1980).Thereislittleevidenceforstimulationofketogenesisbyphysiologicallevelsofglucagoninnormalman(Liljenquistetal.,1974;Schade&Eaton,1976),althoughincreasedketonebodylevelsareobservedinsubjectsmadeinsulin-deficientwithsomatostatin(Gerichetal.,1976c).Ininsulin-depriveddiabetics,suppressionofglucagonsecretionwithsomatostatinimpairsketonebodyproduction(Gerichetal.,1975),suggestingthatendogenousglucagoncontributestothemaintenanceofketogenesis.CatecholaminesCatecholaminelevels(adrenalineandnoradrenaline)areelevatedininsulin-dependentdiabeticsduringketo-acidosisorpoormetaboliccontrol(Christensen,1974).Inman,adrenalinecausesatransientincreaseinhepaticglucoseproduction(Rizzaetal.,1980)mediatedthroughafl-receptormechanism,whilenoradrenalineisweaklyhyperglycaemicinnormalandinsulin-deficientman(Schade&Eaton,1978).Whenglucagonsecretionisnormal,blockadeoftheadrenergicreceptorsystemdoesnotmodifyglucosecounter-regulationfollowinginsulin-inducedhypoglycaemia(Rizzaetal.,1979a),suggestingthatcatecholaminesmaynotbeessentialforglucosehomoeostasisinhypoglycaemia.However,duringinfusionofinsulinandsomatostatin,toinhibitglucagonsecretion,adrenergicblockadepreventstheincreaseinhepaticglucoseproductioninresponsetohypoglycaemia(Hansenetal.,1986),suggestingthatcatecholaminesmightbeimportantforglucosecounter-regulationwhenglucagonsecretionisimpaired.Adrenaline,nor-adrenalineanddopaminehavebeenshowntobeketo-genicinman;theireffectislargelyduetostimulationoflipolysisinadiposetissue,buttheymayalsohaveadirectketogeniceffectontheliver(Kelleretal.,1984a;Keller,1986).Stimulationofthesympatheticnervesintheperfusedrat_liverincreasesglucoseproductionandketogenesis(Beuersetal.,1986),suggestingthatnor-adrenalinemayhavearoleinregulationthroughsympatheticnervestimulation.Vol.250631

R.TaylorandL.AgiusGrowthhormonePlasmaGHprofilesareelevatedindiabeticswithlong-termcomplicationsandpoormetaboliccontrol(Fineberg&Merimee,1974;Gerich,1984).ItisuncertainwhetherthehighGHlevelsarethecauseortheconsequenceofthemetabolicimbalance.InfusionofphysiologicallevelsofGHinnormalandhypophysectomizedsubjectsisassociatedwithanearlyinsulin-likeeffectonbloodglucosefollowedbyinsulinresistance.Theformerisduetoincreasedglucoseclearanceandsuppressedglucoseoutput;thelatterisduetoimpairedsuppressionofglucoseoutputanddecreasedglucoseclearance(MacGormanetal.,1981;Bratusch-Marrainetal.,1982,1984).PhysiologicaldosesofGHincreaseketonebodylevelsinman(Schadeetal.,1978;Metcalfeetal.,1981;Kelleretal.,1984b).Thisisduetoincreasedlipolysisandpossiblyalsoincreasedketogenesisatthehepaticlevel.AlthoughsomedirecteffectsofGHontheliverhavebeendemonstrated,includingstimulationofglucosetransportandoxidation(Fix&Moore,1981),increasedactivityofphosphatidatephosphohydrolase(Pittneretal.,1986)andacuteinactivationofacetyl-CoAcarboxylaseatmicromolarGHconcentrations(Born-steinetal.,1983),thereisasyetnoevidenceinvitroforadirectketogeniceffectofGHonlivercellsatphysiologicalhormonelevels.Inadiposetissueinvitro,acuteinsulin-likeeffectsofGHonglucosetransportandoxidation,leucineoxidationandglycogensynthesisandstimulationoflipolysisafterprolongedexposurearewelldocumented,althoughtheeffectsaremoreprominentinadiposetissuefromhypophysectomizedthanfromnormalanimals(Davidson,1987).ThehighGHlevelsinIDDMhavebeenimplicatedtobeinvolvedinthelong-termcomplicationsassociatedwithneovascularization(Gerich,1984).GHregulatestheproductionofinsulin-likegrowthfactors(IGF)bytheliver,althoughIGFlevelsdonotalwayscorrelatewithGH(Heringtonetal.,1983).IGFIlevelsareraisedinplasmaandocularvitreousindiabeticswithproliferativeretinopathy(Grantetal.,1986)andevidenceinvitrohasshownthatIGFIincreasestheproliferationofbovineretinalendothelialcellsinculture(Kingetal.,1985).GlucocorticoidsCortisolexcessincreaseshepaticglucoseoutputandketogenesisininsulin-depriveddiabetics(Barnesetal.,1978)andinnormalsubjectstreatedwithsomatostatintoinhibitinsulinsecretion,butnotwheninsulinsecretionisunimpaired(Johnstonetal.,1982).Highcortisollevelshavebeenobservedindiabeticketosisandhavebeenimplicatedtoaggravateincreasedlipolysisinadiposetissueandincreasedfractionaldiversionoffattyacidstowardsmitochondrialketogenesisatthehepaticlevel(Johnston&Alberti,1982).Glucocorticoidincreasedketogenesisandgluconeogenesisinrathepatocytesintheabsenceofinsulin,butnotinitspresence(Agiusetal.,1986a).Thestimulatoryeffectismuchsmallerthanthatofglucagon,supportingtheclinicalevidencethatcortisolhasarelativelyminorroleasregulatorofketogenesis.INSULINSECRETIONPhysiologically,plasmaglucoseconcentrationisamajorregulatorofinsulinsecretion.Thiseffectmaybemimickedandpotentiatedbyaminoacids(Floydetal.,1966),potentiatedbyglucoseinsulinotropicpeptide(Dupreetal.,1973;Jonesetal.,1987)andinhibitedbycatecholamineswhichactpredominantlyviathea-adrenergicreceptors(Robertsonetal.,1976).Whetherthesignalforinsulinreleaseisgeneratedbyaglucosereceptorsuchastheglucosetransporterorbyastepinthemetabolismofglucoseremainsuncertain.Ithasbeenarguedthatthecapacityofmoleculestostimulateinsulinsecretionisdirectlyproportionaltotheirsuitabilityas,cellfuelsforglycolysis(Ashcroft,1981;Sener&Malaisse,1984).Glyceraldehydeandpyruvatearerespectivelygoodandineffectiveinsulinsecretagogues(Zawalichetal.,1978;Seneretal.,1978).Whatevertheprecisenatureoftheinitiatingsignal,itisclearthatinsulinreleaseiscloselyassociatedwithariseinintracellularfreecalcium(Rorsmanetal.,1984).However,underanumberofexperimentalconditions,glucosebringsaboutaparadoxicaldecreaseinintracellularfreecalcium.GlucoseaffectscellularuptakeandeffluxofCa2"andmovementoffreeCa2"intoorganelle-boundcalcium,theseprocessesexhibitingdifferentlatencies(forreviewseeHellman,1986).Assessmentofpancreatic,cellcapacity/responsive-nessinNIDDMiscomplicatedbyprevailingbloodglucoseconcentrations.AlthoughNIDDMsubjectshavenoabsolutedeficiencyofinsulinasassessedbydiurnalplasmainsulinprofiles(Liuetal.,1983)andnormalorraisedfastingplasmainsulin(DeFronzo&Ferrannini,1982)thiscannotbeinterpretedasindicatingnormalityof,cellfunction.Whennormalsubjectsareexaminedduringhyperglycaemia,resultingratesofinsulinsecretionarefarhigherthaninequivalentlyhyperglycaemicNIDDMsubjects(Halteretal.,1979;Ferneretal.,1986).Conversely,whenNIDDMsubjectsarerenderednormoglycaemicbypriorinsulininfusion,steadystateinsulinlevelsafterawash-outperiodarelowerthaninweight-matchednormalsubjects(Turneretal.,1976).Followinganintravenousbolusofglucose,firstphaseinsulinreleaseisabsentinNIDDMsubjects,andindeedatemporarysharpdecreaseininsulinsecretionratesisoccasionallyobserved(Brunzelletal.,1976;Metzetal.,1979;Hellman,1986).AlthoughitisfrequentlyassumedthatsecondphaseinsulinreleaseisnotgrosslyabnormalinNIDDM,itisdependentuponthedegreeofhyper-glycaemia(Ferneretal.,1986).Initialassessmentoftheeffectofsuchnon-glucoseinsulinsecretagoguesasisoproterenolorargininesug-gestedthat,cellsofNIDDMsubjectsrespondednormally(Pfeiferetal.,1981).Examinationoftheslopeofglucosepotentiationdemonstratedthat,althoughtheinsulinresponsetoarginineinNIDDMsubjectsatabloodglucosearound22mmol/lwasequivalenttothatofnormalsubjects,thelatterexhibiteda5-foldenhance-mentofresponsewhenbloodglucosewasraisedto22mmol/l(Robertson&Chen,1977;Wardetal.,1984).Theglucoselevelrequiredforhalf-maximalresponsive-nesstoargininewasnormalinNIDDM.Thesefindingsimplyageneralizedlossof,cellcapacityratherthanaproblemofglucosesensingasthebasisoftheinsulinsecretoryabnormalityinNIDDM.Insupportofthis,postmortemstudieshavesuggesteda40-60%decreaseinmean,cellmassinNIDDM(Saitoetal.,1979;Gepts&Lecompte,1981).A70-90%pancreatectomydoesnotusuallybringaboutovertdiabetes(Brooks,1979),emphasizingtheimportanceofconcurrenceofamoderatedecreaseinboth,cellfunctionandtissue1988632

ThebiochemistryofdiabetessensitivityinthepathogenesisofNIDDM.Dysfunctionofanormalnumberof,cellsinducedbysomatostatininmanandpartialpancreatectomyintheratbothgiverisetoimpairedfirstandsecondphaseinsulinresponsestoglucoseandnormalresponsestonon-glucosestimuli(Wardetal.,1983;Leahyetal.,1984).EarlydefectsininsulinsecretioninIDDMItisnowrecognizedthatIDDMisadiseaseofslowonset,eventhoughclinicalpresentationmaybesuddenanddramatic(Tarnetal.,1987).Thepreclinicalphasemaylastfor5yearsormore,maybecharacterizedbyafluctuatingrateof,cellattrition(Spenceretal.,1984)andfirstphaseinsulinresponsemaydecreaseslowlybeforedisappearing(Srikantaetal.,1983).Itishighlylikelythatanimmunologicalattackcausesthepro-gressive,celldamage(Bottazzo,1986).However,immunological/?cellattackmaynotalwaysprogresstoIDDM,andmildabnormalitiesininsulinsecretionmayresult(Millwardetal.,1986;Heatonetal.,1987).ThepotentialforhaltingtheimmunologicaldamagehasledtoclinicalstudiesoftheeffectoftheimmunosuppressiveagentcyclosporinA.Althoughearlyresultsappearencouraging,itispossiblethatspontaneousfluctuationsindiseaseprogressionor'honeymoonphase'mayexplainthesedata(Stilleretal.,1984;Assanetal.,1985).THERAPEUTICASPECTSMechanismofactionofsulphonylureasSulphonylureashaveapronouncedacuteeffectinstimulatinginsulinrelease(Yalowetal.,1960).However,sulphonylureaspotentiateinsulinactioninrecentlypancreatectomizedanimals(Houssayetal.,1957;Caren&Corbo,1959)andtheyinhibitlipolysisandketogenesis,inadditiontoenhancingglycogenandnon-esterifiedfattyacidsynthesisinvitroapparentlyindependentlyofinsulin(Stone&Brown,1967;Boshelletal.,1960;Fleigetal.,1984,Salhanicketal.,1983).Chronicadministra-tionofsulphonylureasisnotassociatedwithpersistenceofinsulinotropiceffects(Duckworthetal.,1972).Adirectdrugeffectontissueinsulinsensitivityhasbeenclaimed(Koltermanetal.,1984;Wardetal.,1985),althoughcontraryevidenceexists(Marchandetal.,1985).Thepositivestudiesareopentointerpretation,asenhancedinsulinsensitivitymaybeasecondaryeffectofimprovedmetaboliccontrol,whetherachievedbydietarymeans(Pedersenetal.,1981),exercise(Yki-Yarvinenetal.,1984),orinsulintherapy(Andrewsetal.,1984;Garveyetal.,1985).Studiesonisolatedcellsinvitrohaveproducedsimilarlydiscordantresults(Vignerietal.,1982;Goldfineetal.,1984;Altanetal.,1985;Wardetal.,1985).StudiesinvivoofsulphonylureaeffectsinNIDDMaredifficulttointerpret,asanydirectdrugeffectcannotbeseparatedfromsecondaryeffectsmediatedviaincreasedinsulinsecretionorimprovedmetaboliccontrol.InIDDMsubjects,lackinganyinsulinsecretorycapacity,noeffectontissueinsulinsensitivitycouldbedetected(Grunbergeretal.,1982;Keller,1986).However,Pernetandcolleaguesdemonstratedthatatlowinsulininfusionratessufficienttoachievemoderatephysiologicallevelsofinsulin,glibenclamideappearedtoenhancetheinsulin-mediatedglucosedisposal(Pernetetal.,1985).Asthiseffectcouldnotbeseenathigherinsulinlevels,itispossiblethatitreflectsanalterationinhepaticratherthanperipheraltissuemetabolism.TheobservationofaraisedplasmainsulintoC-peptideratiohasledtothesuggestionthatdecreasedhepaticinsulinextractionisadirectdrugeffect(Beck-Nielsenetal.,1986;Scheenetal.,1984;Koltermanetal.,1983).Tolbutamidedecreasesinsulinextractionintheperfusedratliver(Marshalletal.,1970).Ifthisisthecase,thelesseramountofinsulininteractingwithandtakenupbyhepatocytesisrelativelymorepotentindecreasingnethepaticglucoseoutputinthepresenceofthedrug.MechanismofactionofmetforminMetformindoesnotincreaseplasmainsulincon-centrationsandithasbeenpostulatedtoactbydecreasinggluconeogenesis(Meyeretal.,1967;Jacksonetal.,1987b;Nosadinietal.,1987)orincreasinginsulin-mediatedglucosedisposal(Prageretal.,1986;Nosadinietal.,1987;Paganoetal.,1983).Invitro,metforminincreasesbasalbutnotmaximal,insulin-stimulatedratesofglucoseoxidationinratfatcells(Fantus&Brosseau,1986)andenhancesinsulin-mediatedglucoseuptakeinmuscleofdiabeticrodents(Bailey&Puah,1986;Frayn&Adnitt,1972).InNIDDMsubjects,metforminappearstoincreaseinsulin-mediatedglucoseuptake(Prageretal.,1986;Nosadinietal.,1987).Thisconcurswithpreviousstudiesonphenforminactioninthehumanforearm(Butterfieldetal.,1961),supportingadirectdrugeffect.MetforminadministrationtoIDDMsubjectsbroughtaboutaslightincreaseinmaximalinsulin-stimulatedglucoseuptakerates(Ginetal.,1985)andadecreaseindailyinsulinrequirements(Prageretal.,1986;Nosadinietal.,1987).However,Jacksonetal.(1987a),studyingagroupofNIDDMsubjectswhoexhibitedagoodresponsetometformintreatment,foundnoevidenceforanincreaseinmuscleglucoseuptakeafteranoralload.Rather,theimprovementinglycaemiccontrolappearedtobeduetodecreasedhepaticglucoseoutput(Jacksonetal.,1987a).Theglucosetolerancecurvewasnotchangedinshapebutstartedfromalowerpoint.Thereisevidenceforametformin-induceddecreaseingluconeogenesisbutnothepaticglycogenolysis(Meyeretal.,1967;Haeckel&Haeckel,1982).UnlessglycogenolysisaccountsforalesserproportionoffastinghepaticglucoseoutputinNIDDMthannormalsubjects,itmustbeinvolvedinanysubstantialreductioninhepaticglucoseoutput.Ageneralincreaseinhepaticinsulinsensitivitycouldaccountfortheobservations.Theeffectsofmetformininincreasingplasmaarteriallactateconcentrationsmaybeinterpretedasconfirmingthedrug-inducedinhibitionofgluconeogenesis(Jacksonetal.,1987a),sincemetformindoesnotincreaselactateproductionintheforearmofNIDDMsubjectsinvivo(Jacksonetal.,1987a).EffectsofdietarytherapyDietarycompositionaffectsmetaboliccontrolinsubjectswithimpairedglucosetoleranceanddiabetes.Prescriptionoflowcarbohydratedietsforalldiabeticsubjectswasthenormuntil1984,andthisinevitablyledtoconsumptionofahighfatdiet.Thiswasunsoundinviewofthepropensityofdiabeticsubjectstoischaemicheartdiseaseandhyperlipidaemia.Theobservationthatdietswithahighproportionofunrefinedcarbohydrateimprovedmetaboliccontrolwasofgreatinterest.SuchVol.250633

R.TaylorandL.Agiusdietshavebeenshowntoimproveinsulinsensitivityinvivoandinisolatedadipocytes(Hjollundetal.,1983,1987).AlthoughsomestudiessuggestthatadditionofsucrosetothedietofNIDDMsubjectsdoesnotaffectglycaemiccontrolunderexperimentalconditions(Bantleetal.,1983),thehyperlipidaemiceffectofaddedsucrose(Coulstonetal.,1985)hasbeendemonstrated.ManyNIDDMsubjectsareobese.Aweightlossaveraging6.7kgproducednochangesininsulin-mediatedglucoseuptakeatphysiologicalinsulincon-centrationsnorinadipocytemetabolisminvitro(Zawadzkietal.,1987).Inasimilargroup,weightlossof16.8kgaveragewasassociatedwithadecreaseinhepaticglucoseoutputdespitenochangeinfastingplasmainsulin,andincreasedglucoseuptakeathighphysio-logicalplasmainsulinlevelswasenhanced(Henryetal.,1986).Theunchangedfastingplasmainsulinlevelsdespiteapronounceddecreaseinbloodglucosesuggeststhat,cellsensitivitytoglucoseisimproved.EffectofinsulintreatmentAlthoughthetitlesoftheearlystudiesontheeffectofinsulintherapyoninsulinsensitivityinNIDDMsug-gestedthatitcouldbereturnedtonormal(Scarlettetal.,1982,1983),thedatawerelessimpressive.Hepaticsensitivitytoinsulinmaybeincreasedatonlymodestimprovementinglycaemiccontrolwhenperipheraltissueinsulinsensitivityisunaffected(Nankervisetal.,1982).Theimprovementseeninperipheraltissueinsulinsensitivityappearstobemostmarkedintheleastobeseindividuals(Andrewsetal.,1984).However,theminornatureofthechangesintissuesensitivityareemphasizedbytheobservationthatfastingplasmaglucoseandglucoseintolerancereturntopretreatmentlevelsshortlyafterinsulinwithdrawal,whereasthechangesintissuesensitivitypersistforupto6weeks(Gormleyetal.,1986).InsulintherapyinNIDDMimprovesinsulinsecretioninresponsetoglucagon(Garveyetal.,1985)andoralglucose(Andrewsetal.,1984;Gormleyetal.,1986;Hidakaetal.,1982).Unfortunately,fewofthesestudieslookedatlipidmetabolisminanattempttoestablishwhetherornotchangesininsulinactionmayberelatedtochangesinNEFAlevels(Randleetal.,1963).ImprovementofdiabeticcontrolfromabysmaltopoorwasassociatedwithareductioninfastingNEFAandketonebodies(Nankervisetal.,1982).Asimprovementofmetaboliccontrolbyexogenousinsulinandsulphonylureaadministrationresultsinsimilarchangesinhepaticglucoseoutputorperipheraltissueinsulinsensitivity,itisquitepossiblethatneithertherapyactsdirectlyoninsulinsensitivity,butthatsomeaspectofthemetabolicstateitselfisresponsible(Firthetal.,1986b).Indeed,dietarytherapyalonedecreasesfastingbloodglucoseandimprovesinsulin-stimulatedratesofglucoseoxidation(Bodenetal.,1983).DecreaseincirculatingNEFAimprovesglucoseutilization(Randleetal.,1963),butunfortunatelyNEFA,inter-mediarymetabolitesandcounter-regulatoryhormoneswerenotmeasuredinthemajorityofabovestudies.Itmaybehypothesizedthatonlyoneofseveralconcurrentdefectsismodifiedbyinsulintherapy.Conventionalsubcutaneousinsulinadministrationresultsinslowabsorptionfromthesiteofinjectionthroughoutthedayandhyperinsulinaemiainthesystemicbutnotportalcirculation.Hence,evenwhenbloodglucoseisalmostnormalizedonsucharegime,abnormalitiesinplasmafreeinsulinlevelsandinter-mediarymetabolitesremain.Achievementofstrictnormoglycaemiabyintravenousinsulinadministrationstillrequireshyperinsulinaemia(Nosadinietal.,1982).Administrationofinsulindirectlyintotheportalsystembyintraperitonealdeliverydoesindeedreducethehyperinsulinaemia(Jimenezetal.,1985;Husbandetal.,1984).Thenormalizationofcarbohydrateandlipidmetabolismindiabeticdogsbyintraportalbutnotintravenousinsulinadministrationhasbeenreported(Stevensonetal.,1983a,b).However,intraportalislettransplantationwithperipheralvenousdrainagenor-malizedglucosetolerance(Guyetal.,1987).Theresultsofstudiescurrentlyunderwayintothemetaboliceffectsofrelativelyhepatospecificinsulinswillbeofgreatinterest.NEWTHERAPIESFORDIABETESTetradecylglycidate(methylpalmoxirate)andEto-moxirareoxiranecarboxylicacidswhoseCoAestersinhibitcarnitinepalmitoyltransferaseI,therebypreventingmitochondriallongchainfattyacidoxidation.Thismightbeexpectedtoincreaseglucoseutilizationviatheglucose-fattyacidcycleandindeedthesecompoundsarehypoglycaemicinfastinganimals(Tutwileretal.,1978;Eistetter&Wolf,1982).Suggestionofanactionininhibitinggluconeogenesisinanimalsandmanisofgreatinterest(Selbyetal.,1987).Carnitinepalmitoyltransferaseinhibitiononlyinhibitsfattyacidoxidation.Theflux-generatingstepforfattyacidmetabolismislipolysis.NicotinicacidinhibitslipolysisinadiposetissueandthuslowersplasmaNEFAlevels.Oraladministrationofthenicotinicacidanalogue,Acipimox,isfollowedbyadecreaseinglycerol,NEFAand3-hydroxybutyratelevelsbyapprox.80%within2h,andanimprovementinglucosetoleranceandperipheralglucoseuptake(Piattietal.,1987).Thea-glucosidaseinhibitoracarboseanditsnewerderivativeBayml099havebeenshowntohavedramaticeffectsindecreasingpostprandialexcretionsofglucose,lactateandpyruvateinnormalanddiabeticsubjects.Despitethemarkedpostprandialchangesinbloodglucoseprofiles,overallindicesofglycaemiccontrolindiabeticsubjectshavebeenremarkablylittlechanged(Lardinoisetal.,1984;Samadetal.,1988).Theroleofsuchsubstancesinthemanagementofdiabeticpatientsremainsspeculative.CHRONICCOMPLICATIONSOFDIABETESLongtermdiabetesbringsaboutdistinctabnormalitiesinthemicrocirculationandinnervousfunctionwhichareclinicallymanifestedasnephropathy,retinopathyandneuropathy.Theriskofdevelopingsuchcom-plicationsisrelatedtolongtermaverageglycaemiccontrol(Tchobroutsky,1978;Pirart,1978),butacomponentofgeneticsusceptibilitytocomplicationsmeansthatnotallindividualswithrelativelygooddiabeticcontrolescapeproblemsandthatsomeindi-vidualscanenduremetabolicmayhemoverdecadeswithoutapparentproblems.Inaddition,butunrelatedtodegreeofglycaemiccontrol,arterial-diseaseismanifestedascoronaryheartdiseaseandperipheralvasculardisease.1988634

ThebiochemistryofdiabetesBasementmembranethickeningThickeningofbasementmembranesisauniversalfindinginlongstandingdiabetes.After5yearsofclinicaldiabetes,basementmembranesare25-30othickerthaninnormalsubjects(Osterbyetal.,1986).Thebasementmembranematrix,composedoftypeIVcollagenandlaminin,ismorepermeableindiabetes,andthickeningmayrepresentacompensatorychange(Rohrbachetal.,1982).Theroleofcollagenglycosylationinthepatho-genesisofthebasementmembraneabnormalityremainstobeelucidated(Cohenetal.,1980),butitisclearthatbasementmembranethickeningdoesnotantedatemetabolicdisturbance(Osterby,1975).Tightmetaboliccontrolcanpartiallyreversebasementmembranethickening(Raskinetal.,1983).Non-enzymicglycosylationEnzymicglycosylationisahighlyregulatedpost-translationalprocessresponsibleforconferringspecificstructuralandfunctionalchangesonproteins(Uy&Wold,1977).Incontrast,non-enzymicglycosylationisgovernedsolelybyprevailingglucoseconcentration.Lysineandvalineresiduesundergorapidaldimine(Schiffbase)andsubsequentlyketoamineformation.Advancedglycosylationendproductsultimatelyresult.Ifaffectedaminoacidsareclosetotheactivesitesoftheirmolecule,orifstereochemicalconfigurationisdisturbed,thenfunctionwillbealtered.Thusglycosylatedalbumininhibitshepaticuptakeofglycoproteins(Summerfieldetal.,1982).Glycosylatedfibrinislesssusceptibletofibrindigestion(Brownleeetal.,1983),thusmakinganythrombusmorelikelytoresultinpermanentvascularocclusion.GlycosylationofapoproteinBcausesareductionofaffinityforthelowdensitylipoproteinreceptor(Kesaniemietal.,1983).Glycosylatedcollagendisplaysincreasedintramolecularcross-linkings(Kohn&Schnider,1982)andthismayunderlythedecreasedsmalljointmobilityoflongstandingdiabetes.Thelatterhasbeenclaimedtobeanindicatorforthedevelopmentofmicrovascularcomplications(Rosenbloometal.,1983).Asredbloodcellshavearelativelyconstanthalf-life,estimationofthepercentageofhaemoglobinwhichhasbeenglycosylatedgivesareliableindexofaverageglycaemiccontroloverthepreceding2months(Bunn,1981).Thisindexisinvaluableforclinicalandresearchpurposes.RelevanceofthesorbitolpathwayIntheocularlensparticularly,sorbitolconcentrationsaregrosslyelevatedindiabeticrats,andthismayberelatedtoformationofcataracts,atleastoftheacutetype(Gabbay,1973).Glucose,sorbitolandfructoseconcentrationsareelevatedinthenervesofdiabeticanimalsandman(Wardetal.,1972;Greeneetal.,1975;Greene&Mackway,1986;Dycketal.,1980).Althoughinitiallyitwaspostulatedthattheosmoticeffectofthesechangescouldcausecellularoedema,Schwanncellsfromdiabeticratshaveadecreasedvolume(Jacobsen,1978)andcalculatedosmolarchangesareverysmall.Bothtreatmentwithaldosereductaseinhibitorsanddietarymyo-inositolsupplementationreversesneuraldepletionofmyo-inositolandcorrectsabnormalitiesinNa+/K+-ATPaseandaxonaltransport(Greene&Lattimer,1984;Tomlinson&Mayer,1984).Treatmentofhumandiabeticneuropathywithmyo-inositoloraldosereductaseinhibitorshasnotyieldedclearcutanswers.Clinicalandelectrophysiologicalimprovementshavebeendocumentedaftermyo-inositoltherapyinsome(Greeneetal.,1981;Salwayetal.,1978;Clementsetal.,1979)butnotall(Gregersenetal.,1978,1983;Greeneetal.,1981)studies.Aldosereductaseinhibitortherapybroughtaboutsmallbutsignificantimprove-mentsinmotor,sensoryandautonomicnervefunctioninthemajorityofstudies(Judzewitschetal.,1983;Fagiusetal.,1985;Jaspanetal.,1985).Otherstudiesfailedtodemonstrateanybenefit(Handlesman&Turtle,1981).Adegreeofsymptomaticimprovementinpainfulneuropathyfollowsaldosereductaseinhibition(Youngetal.,1983;Koglinetal.,1985).Asglucosecompeteswithmyo-inositolforactivetransportintonerves,hyperglycaemiaperseresultsinmyo-inositoldepletion.Maintenanceofnearnormoglycaemiafor4-8monthsresultsinsomeimprovementinsymptomaticandobjectivemeasuresofnervefunction(Boultonetal.,1982;Serviceetal.,1985).Thechronicneuropathyoflongstandingdiabetesisatleastinpartsecondarytoirreversiblemicrovasculardisease,andendoneurialoxygentensionislow(Lowetal.,1985).Insuchcircumstancesmetabolicmanipulationisobviouslytoolate.Glucoseuptakebytheretinaisinsulin-independent,andtheretinadependsforitsenergysupplyuponanaerobicglycolysis.Aldosereductaseinhibitorspreventlossofretinalcapillarypericytesandbasementmembranethickening,whicharethoughttobeassociatedwithpolyolpathwayactivation(Robisonetal.,1983,1985).Retinalpericytescontainaldosereductase(Akagietal.,1983).Lossofretinalpericytescouldinducesomeoftheretinalcapillarychangesobservedindiabetes.AtherogenesisDiabetesisamajorriskfactorforatherogenesisandarterialdisease.Elevatedlowdensitylipoproteincholes-terolmayresultfromglycosylationofthelysylresiduesofapoproteinB,decreasedaffinityforthelowdensitylipoproteinreceptorandhencedecreasedmetabolism(Kesaniemietal.,1983).Itremainstobeestablishedwhetherelevationofplasmalipidsisanecessarypathogenicstateormerelyamarkerforatherogenesis.Lipidsynthesisinsituinthearterialwallmayoccur,andincreasedactivityofglucose-6-phosphatedehydrogenasehasbeendemonstratedinmuscleandaortasofhyper-insulinaemicpigsandmuscleofNIDDMsubjects(Stout,1979;Falholtetal.,1985a,1987).TheperipheralhyperinsulinaemiaofNIDDMandofinsulin-treatedsubjectswouldstimulatelipogenesisinsituandinthiscontexttheepidemiologicalassociationbetweenraisedseruminsulinlevelsandcardiovasculardiseaseisofinterest.CONCLUSIONMorequestionsaboutthebiochemicalbasisofdiabetesremainthanhavebeenansweredtodate.Inparticular,hormoneinsensitivity,thereversalofsuchinsensitivity,metabolicinteractionsbetweentissuesandthepatho-genesisofchroniccomplicationsareareasofveryuncertainknowledge.Currentresearchonthesedifficultquestionswillprovideabasisforadvancesbothinunderstandingandtherapy.Vol.250635

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