GLUCONEOGENESIS HANDOUT.pdf

1. UniversityofIllinoisatChicago MedicalBiochemistry531 Lecture19 Gluconeogenesis GlucoseMetabolismintheLiver(continued): “GlycolysisversusGluconeogenesis” thematchofthecentury! It’snevertoearlyto startreviewing biochemistryfor the 2nd exam! http://www.uic.edu/depts/mcmi/biochem/curriculum/531/ 1 • SuggestedreadingforLectures18&19 • Marks’-Chapters22(Glycolysis)and26 (BasicConceptsinFuelMetabolism)and31 (Gluconeogenesis) • Devlin-Chapter14.1-14.5(evenbetterthanMarks’) • VoetandVoet-Chapters16(Glycolysis)and21 (Gluconeogenesis (muchgreater detail) • Stryer-Chapter19(verygood) • ChampeandHarvey-Chapters8and10(forreview only) • 2 9/15/2004

2. UniversityofIllinoisatChicago • Glycolysisvs. • Gluconeogenesis intheliver • gluconeogenesis is almostthereverseof glycolysis,savefor threekey reactions whicharecatalyzed by differentenzymes • theenergy requirementsdiffer • onepathwayis activatedwhilethe otherisinhibited 3 • Pathwayof Gluconeogenesis • thedistinctivereactionsare shownintheredboxes • theotherreactionsarecommon toglycolysis • theenzymesofgluconeogenesis arelocatedinthecytosol,except for: • pyruvatecarboxylase(in mitochondria) • glucose6-phosphatase(membrane boundinsideendoplasmic reticulum) • entrypointsforlactate,glycerol andaminoacidsareshown 4 9/15/2004

3. UniversityofIllinoisatChicago Enzymaticdifferencesbetween glycolysisandgluconeogenesis 6 9/15/2004

4. UniversityofIllinoisatChicago PhysiologicActionsofInsulinand InsulinCounterregulatoryHormones YoucanfindthistableMarks’-Table26.1inChapter26 SuggestyoureadChapter26on“BasicConceptsintheRegulationofFuelMetabolism”7 • RegulationofGlycolysisbyPyruvateKinase • liverglycolysisisregulatedviatheactionofinsulin versusglucagonatthepyruvatekinasestep • Duringfasting-glucagoninducesproteinkinaseA (PKA),whichphosphorylatespyruvatekinasetoa less activeform • Afterahighcarbohydratemeal,highinsulinand low glucagondecreasePKAactivityandinducephosphatase activity,dephosphorylatingpyruvatekinase,which becomesmoreactive • principalroleofthisregulatorymechanism: • –inhibitionofglycolysisduringfasting,whenthe reversepathway-gluconeogenesis-isactivated • 8 9/15/2004

5. UniversityofIllinoisatChicago Regulationofpyruvatekinaseintheliver bychangesintheinsulin/glucagonratio 9 Fasting State insulin glucagon insulin glucagon FedState RegulationofGlycolysis by • PyruvateKinase • activation by fructose 1,6 bisphosphate • “feed forward” mechanism= product of earlier step in pathway activates an enzyme catalyzingalaterstep • ATPandalaninefunctionas allostericinhibitorswhenthe reversepathway- gluconeogenesis-isactivated Glycolysis 10 9/15/2004

6. UniversityofIllinoisatChicago • GLUCONEOGENESIS • theprocessbywhichglucoseissynthesized from non-carbohydrateprecursors • occurs mainly in the liver (~90%) under • fastingconditions • underextremestarvation,italsooccursin thekidneycortex(~10%)forutilization primarily by kidney (medulla) • –butglucosefromkidneycortexcanalsoenterthe peripheralcirculationandtissuestosomeextent • 11 12 9/15/2004

7. UniversityofIllinoisatChicago • Sourceofcytosolic oxaloacetatefor gluconeogenesis • oxaloacetateutilizedinthecytosol for gluconeogenesis isformedfirst inthe mitochondrialmatrixby carboxylationofpyruvate • oxaloacetateleavesthe mitochondrionintheformof malate,whichisreoxidizedback tooxaloacetate inthecytosol • 13 14 9/15/2004

8. UniversityofIllinoisatChicago 15 • PrecursorsofGluconeogenesis • Fourmajorcarbonsourcesinhumansare: • lactate:producedbyanaerobicglycolysisin tissuessuchasworkingmuscleorRBC’s • glycerol:releasedfromadipose(fat)stores of triacylglycerols • aminoacids:principallyalanine,fromamino acid poolsderivedfromdegradationofmuscleproteins • »alanineisproducedinmusclefromotheramino acidsandfrom glucose • propionatefromoddchainlengthfattyacids • carbonsources forgluconeogenesis are frompyruvate, TCAcycleintermediatesorcommonintermediatesfor glycolysisand gluconeogenesis16 9/15/2004

9. UniversityofIllinoisatChicago 9/15/2004

10. UniversityofIllinoisatChicago Pathwayofgluconeogenesisfrompropionate 19 GlucogenicAminoAcids (groupedby“site”ofentry) 20 9/15/2004

11. UniversityofIllinoisatChicago 22 9/15/2004

12. UniversityofIllinoisatChicago • ConversionofpyruvatetoPEP • theCO2thatwasaddedtopyruvatetoformOAAisthen released by phosphoenolpyruvatecarboxykinase(PEPCK) • GTPprovidestheenergysource • theseenzymesarelocatedintwodifferentsubcellular compartments • pyruvatecarboxylaseisinthemitochondria • PEPCKisinthecytosolandmitochondria • OAAformedinthemitochondriabytheTCAcycleorpyruvate carboxylasedoesnotreadilycrossthemembrane • itiseitherdecarboxylatedbyPEPCKorconvertedtomalate or aspartateinthemitochondria transamination 23 24 9/15/2004

13. UniversityofIllinoisatChicago Mitochondrial andcytosolic pathwaysfor conversionof lactate and pyruvatetoPEP 25 9/15/2004

14. UniversityofIllinoisatChicago ConversionofOAAtoPEPviaPEPCK Oxaloacetatederivedfrommalateoraspartateinthecytosol isconvertedbydecarboxylationandkinasereactionsto PEP Thisisanenergyrequiringreactionwhichconvertsahigh energyphosphatebondofGTPtothatofPEP,ahighenergy phosphointermediate 27 • ConversionofPEPtoFructose1,6 bisphosphate • theremaining stepsofgluconeogenesis occur in thecytosol • PEPreversesthestepsofglycolysistoform glyceraldehyde-3-phosphate • foreverytwoglyceraldehyde-3-phosphate,1 isconvertedtoDHAP • thesetwotriosephosphatescondensetoform fructose1,6-bisphosphateviareversalofthe aldolasereactionof glycolysis • 28 9/15/2004

15. UniversityofIllinoisatChicago 29 30 9/15/2004

16. UniversityofIllinoisatChicago • Schematicoftheendoplasmicreticulum(ER) proteinsthatparticipateinthegenerationof glucosefromcytosolicglucose-6-phosphate • T1transportsglucose-6-phosphateinto theER lumen • T2andT3transportPiandglucose,respectively,backintothe cytosol • Glucose-6-phosphataseisstabilizedbyaCa2+bindingprotein(SP) • 31 • Energyisrequiredforsynthesisofglucose • 6 moles of high energy phosphate bonds • 2molespyruvatearerequiredforsynthesisof1mole of glucose • pyruvatedecarboxylase reaction hydrolyzes 2 moles ATP • PEPCKrequires2molesGTPforOAAtoPEP • 2molesATPareusedtophosphorylate2molesof3- phosphoglycerate to1,3bisphosphoglycerate • NADHreducingequivalentsareusedtoconvert1,3, bisphosphoglyceratetoglyceraldehyde3-phosphate • 32 9/15/2004

17. UniversityofIllinoisatChicago • Energyisrequiredforsynthesisofglucose • thenetequationforgluconeogenesis: • 2pyruvate+4ATP+2GTP+2NADH+2H++6H2Oglucose+2NAD++4ADP+2GDP+6Pi+6H+ • rememberthenetequationforglycolysis: glucose+2NAD+2Pi+2ADP • 2pyruvate +2NADH+4H++2ATP+2H2O • 33 34 9/15/2004

18. UniversityofIllinoisatChicago • RegulationofGluconeogenesis • thispathwayfunctionsduringfasting, prolongedexercise,highprotein/low carbohydratediet,conditionsof“stress” • the overall flow of carbon frompyruvateto glucoseis controlledby factorsincluding: • substrateavailability • activityleveloramountofkeyglycolyticor gluconeogenicenzymes • 35 • RegulationofGluconeogenesis • -substrateavailability- • stimulatedbytheflowofmajorsubstratesfromperipheral tissuestotheliver,e.g.fromskeletalmuscle • Glycerol:releasedfromadiposetissuewhenlevelsof insulinarelow,andglucagonor“stress”hormones (epinephrineandcortisol)areelevatedintheblood • Lactate:producedbymuscleduringprolonged excerciseandbyerythrocytes(CoriCycle) • Aminoacids:releasedfrommusclewheninsulin levelsarelowandcortisolishigh,ordietaryintakeof proteinishighandcarbohydrateislow(alaninecycle) • 36 9/15/2004

19. UniversityofIllinoisatChicago 9/15/2004

20. UniversityofIllinoisatChicago • RegulationofGluconeogenesis • -conversionofpyruvatetoPEP- • pyruvateisnotconvertedtoacetylCoAunder conditions favoring gluconeogenesis • i.e.pyruvatedehydrogenaserelativelyinactive • pyruvateisinsteadconvertedtoOAAbypyruvate carboxylaseandtoPEPby PEPCK • sincepyruvatekinaseisinactivatedby phosphorylation andalanine,PEPisnotre-convertedtopyruvate(asin glycolysis) • otherwise,the situation wouldlead toanon-productive substratecycle(called “afutilecycle”) • so,PEPreversesthestepsofglycolysis • 39 • RegulationofGluconeogenesis • -pyruvatedehydrogenaseisinactive - • duringfasting,insulinislowandglucagonishigh • fattyacidsandglycerolarereleasedfromtriacylglycerol storesinadiposetissue • fattyacidsintheliver,via-oxidation,produce: acetylCoA,NADH+ATP([ADP]decreases) • thenetresult: • phosphorylation ofpyruvatedehydrogenase toits inactiveform • pyruvateisnotconvertedtoacetylCoAforentryinto theTCAcycle,butusedforgluconeogenesis • acetylCoAforTCAcyclecomesfirstfrommuscle- derivedaminoacidsandthenFA-oxidation 40 9/15/2004

21. UniversityofIllinoisatChicago • RegulationofGluconeogenesis • -pyruvatecarboxylaseisactive • acetylCoAfrom-oxidationoffatty acidsactivates pyruvatecarboxylase • pyruvatederived from lactate or alanine (from muscle) (Corioralanine cycles)isconverted toOAA • OAAconvertedtoPEPviaPEPCK enzyme • 41 • RegulationofGluconeogenesis • -PEP carboxykinaseisinduced- • PEPCKisaninducibleenzyme,i.e.transcriptionof itsgeneandtranslationofmRNAtoproteinare upregulated • cAMP,amajorinducer,isincreasedviaactivationof adenylatecyclasefromsignalingbyglucagon(during fasting)orepinephrine(duringexerciseorstress) • cAMPactivatesproteinkinaseAwhich phosphorylatestranscriptionfactorswhichactivate thePEPCKgene,whichisalsoinducedbyincreased cortisol(glucocorticoid) • 42 9/15/2004

22. UniversityofIllinoisatChicago • RegulationofGluconeogenesis • -pyruvatekinaseisinactive- • elevatedglucagoninduces phosphorylation/inactivationofpyruvatekinase byacAMP/proteinkinaseA-dependent mechanism • PEPisnotreconvertedtopyruvateand continuesalongthegluconeogenesispathway toglucose • inactivationofpyruvatekinaseprevents: “futilecycling”,netlossofenergy,nonet generationofuseful products • 43 • RegulationofGluconeogenesis • -preventionofafutilecycle- • conversionoffructose1,6-bisphosphateto fructose6-phosphate • afutilecycleispreventedatthisstepsince theactivators(fructose2,6-bisphosphateand cAMP)of PFK-1are low • PFK-1islessactive,fructose1,6- bisphosphataseismoreactive • fastinginducesexpressionofthisenzyme • 44 9/15/2004

23. UniversityofIllinoisatChicago • RegulationofGluconeogenesis • conversionofglucose 6-phosphatetoglucose by glucose 6-phosphatase • glucokinase(glycolyticenzyme)isrelatively inactiveduetoitshighKmforglucoseand low bloodglucoselevels(~5mM)duringfasting • glucokinaseexpressionisinducible,i.e. • »increasesduringfedstate(elevatedglucose andinsulinlevels) • »decreasesduringfasting(lowglucoseand insulinlevels) • 45 Reactionsthatinhibitglucosesynthesis viagluconeogenesis or • Forcingthesereactionsinthedirectionsshowninhibitsglucose synthesisbylimitingtheamountsofpyruvateandoxaloacetate availableforthereactionscatalyzedbypyruvatecarboxylase and PEPcarboxykinase,respectively. 46 9/15/2004

24. UniversityofIllinoisatChicago Comparative Regulationof Glycolyticand Gluconeogenic Enzymesinthe Liver  - -       -   -    47 9/15/2004