5Insulin-sensitive Phospholipid Signaling Pathways Major Phospholipid effect of insulin:GPI and PC hydrolysis in membranede novo synthesis of PA in ERsynthesis of PI in ERactivation of PI3K in mambrane and ERSignaling substances: IPG, DAG, PI3,4,5P3DAG from 3 source can activate conventional and novel PKCs : PC hydrolysis account for most of the initial burst of DAG/PKC signaling in membraneInsulin-sensitive hydrolytic and synthetic phospholipid pathways are interrelated and integrated.
6Pertussis toxin-sensitive Gi protein Via Rho,ARFPertussis toxin-sensitive Gi protein
7Phospholipid pathway are adapted to provide for Rapid hydrolysis and resynthesis of GPI and PCGeneration of phospholipid-derived signal substance: IPG mediators, DAG, polyphosphoinositides, PI3,4,5P3, PI3,4P2
8Effects of Insulin on PC Hydrolysis Hydrolysis of PC DAG+phosphorylcholine orPA+cholineDAG activate PKCsInsulin-induced PC↓: very rapid, short-lived rapid resynthesis through the de novo pathwayIR/PC-PLD (?) : dependent PI3Ksmall G protein, Rho and ARF can activate PC-PLDInsulin via PI3K translocate or activate both Rho, ARF, GRP1(ARF exchange factor)
9Effects of Insulin On PI 3-kinase ↑D3 PO4 polyphosphoinositides(PI3,4,5P3/PI3,4P2)Activate PDK1, PKB, aPKCs, DAG-dep PKCs, PRK1/2/3Mambrane localizing factor to bind and coordinate signaling factorsTranslocate small G proteins,Rho ARFActivate Rac, Rab for cell ruffling and GLUT-4 translocationBy Tyrosin phospholylation of IRS familypYXXM of IRS / SH2 on p85 of PI3K
10Effects of Insulin On DAG Production Insulin provokes rapid increase in DAG massin DAG productionInitial burst : PC hydrolysis in membLater: de novo PA synthesis in ER
11General Aspects of PKC Activation 5 typesconventional or classic cPKCs(,1, 2,)Novel nPKCs(,,,)Atypical aPKC(,,)Membrane-anchored PKCs(PKC-m or PKD)PRK 1,2,3Activated by DAG, Ca++, PI3,4,5P3, PA
13DAG, Polyphosphoinositides, lipids activate PKCs Full activation of all PKCs: phosphorylation of Thr-, Ser- in “activation loop”/autoP in catalytic domainsbinding of DAG or PI3,4,5P3 molecular unfolding at V3allosteric dissociation of V1 autoinhibitory pseudosubstratephosphorylated/activated vulnable to proteasecleavage and release of regulatory/catalytic frag.Activation of all PKCs requires phospholylation of specific sites in activation loops by PDK1: cPKCs,nPKCs vs aPKCsPI3,4,5P3 activate PDK1 directly or unfold aPKCs to expose the loop site to PDK1
14Effects of Insulin On Activation of DAG-dependent PKC isoforms Insulin-induced DAGPKC activation? Membrane PKC enzyme activity? : cPKCs/nPKCsActivation of cPKCs/nPKCsPC hydrolysis or de novo PA synthesis? : wartmannin(PI3K inhibitor)Insulin strongly activate aPKCs: Activation of PKC may have been primarily reflective of activation of aPKCs rather than, or as well as, cPKC/nPKCs
15PKC downregulationThe biological effect of Insulin do not require DAG/PKC signaling?Acute phorbol ester-induced PKC depletion effects of insulin persistPKC-,- : relatively resistant to downregulationThe biological effect of Insulin require DAG/PKC signaling?Chronic phorbol ester Tx effects of insulinNonspecific? Activation of residual PKCs? Downregulation of insulin signaling mechanisms?
16Insulin-like effects of Phorbol esters Gluc transport↑Activation of enz in intracelluar gluc metabolismActivation of acetyl-CoA carboxylaseIon transport enz ↑: Na+/H+ transporter, Na+/K+ATPaseAA transport↑Activation of protein synthesis initiation and elongation factorsChange in gene expressionDNA synthesis ↑ in some cell typeInsulin effect: DAG-dependent PKCs?Phorbol ester may activate PI3K, Raf, ERK
17PKC InhibitorsInhibitors can be helpful in determining which metabolic processes are likely or not likely to be regulated by PKCInhibit cPKCs at low conc./nPKCs at intermediate conc./aPKCs at high conc.Not entirely specific for PKCsPhorbol ester + inhibitorGO6976 : Selectively inhibit PKC ,,
18The role of DAG-sensitive PKC in Glucose Transport ? Effect of Phorbol ester on glu transport are much less than insulinDAG itself increase glu transport ; PLC-induced, electrical/exercise-induced DAGDAG-dep PKCs are not required for activating gluc transport in rat adipocyte, myotube, myocytesPKC-, PKC- knockout mice : 오히려 insulin effect 증가 tonic inhibitory effect of cPKCs on activation of PI3K/aPKCsPhorbol ester-induced PKC downregulation of all cPKC(,): insulin effects on GLUT-4 translocation/glu transport 유지.Inhibitor study : selective inhibitor of cPKCsno effect: aPKCs rather than cPKCs or nPKCs may be required forglucose transport effect on insulin
19Glucose-induced Activation of DAG-sensitive PKC Extracellular Glu: de novo PA synthesis↑, activate DAG-sensitive PKCs diabetic Cx/ IR?Adipocyte vs skeletal m. with persistent hyperglycemia,hyperinsulinemiaDAG-sensitive PKCsGlucose-induced IR in tissue?DAG-dep PKCs may impair insulin-induced activation IR tyrosine kinase ?DAG-dep PKCs may inhibit IRS-dep PI3K activation by activation of MAP kinase ?Acute phorbol ester-induced PKC activationno effectDAG/PKC in IR, but normal tyrosin kinase/PI3K activation
20The role of Atypical PKCs in Insulin Action Activation of PKCζ and PKCλ : phosphorylation of PDK-1dep activation loop sites auto- or trans-phosphorylationBy PI3KInhibited by PI3K inhibitor or DN p85 subunits of PI3KActivated by phosphotyrosine-containing peptide(pYXXM) activators of PI3K and by direct addition of PI3,4,5P and PI3,4P2By PDK1(phosphorylate Thr-410, Thr-411)Overexpression of WT PDK1/kinase-inactive PDK1/activation resistant (Thr-410Ala) mutant PKCζPI3,4,5P3 may actby directly activating PDK1by interacting with PKCs to facilitate interaction with PDK1by increasing autophophorylation and allosterically relieving pseudosubstrate-dependent autoinhibition
21The role of PKCζ and PKCλ in Insulin-stimulated Glucose Transport Stable Transfection study: inactive PKCζ GLUT 4 translocation, glu uptake : wild type PKCζ GLUT4 translocation, glu uptake Transient transfection and gene transfer studyInsulin stimulation of HA-GLUT-4 translocation is inhibited by kinase-inactive form of PKCζ and PKCλ mutant : rescued interchangeably by wild-type of either PKCζ or PKCλKinase-inactive PDK1, mutant PKCζAdenoviral gene transfer studyPKCλ knock-out cellaPKCs and putative upstream activators are required for insulin-stimulated Glucose Transport in adipocyte and myocytes
22UpstreamaPKCs operate along with other signaling factors in specific intracellular sites : Cbl and Rho family member(TC10)Cbl-dep PI3K, IRS-1-dep PI3KdownstreamaPKCs phophorylate and regulate the SNARE protein VAMP2(translocation of GLUT-4 from ER to memb)Phosphorylation of insulin-responsive aminopeptidase(IRAP)(promote GLUT-4 translocation)
23Defects in Activation of PKCζ and PKCλ in Obesity,T2DM, and Other insulin-resistant states Defect in aPKC activation by insulin in muscle of obese, glucose intolerant, and T2DM humans and monkeysDefective activation of upstream regulators: IRS-1/2Defective responsiveness of aPKCs to PI3,4,5P3
24The role of PKCζ and PKCλ in ERK Activation by Insulin In some cells, inhibitors of PI3K inhibit insulin-induced ERK ½Transient cotransfection study: kinase inactive PKCζ/PDK1, mutant PKCζ inhibit ERK activationAdditional requirement of Grb2, SOS, Ras, Raf etc.: aPKC are needed to activate Raf
25The role of PKCζ and PKCλ in Activation of Protein Synthesis Initiation of Protein systhesis by insulinby phosphorylation-dependent inhibition of PHAS-1/2(constitutively inhibit initiation factor eIF-4E)Direct phosphorylation of eIF-4EActivation of p70 S6 kinasePI3K,PKB for PHAS, but aPKCs?PDK1 for phosphorylation of Thr-252 in p70 S6 kinaseaPKCs for phosphorylation of Thr-412 in p70 S6 kinase full activation of p70 S6 kinase?Role of aPKCs in gene expression of insulin?
26The role of Atypical PKC in PI3K-independent Activation of Glucose Transport by Noninsulin Agonists High glu/sorbitol activate PYK2 via osmotic sensorAICAR, DNP activate PYK2 via AMPKPYK2Grb2/SOS/Ras/Raf/MEK1/ERK pathway PLD activationPLD activation PAaPKC activationtranslocation GLUT4, glu uptakeExercise activate aPKCs & ERK via AMPKTZDs activate aPKCs via Cbl-dep PI3Kincrease IRS1/2 (Adipocyte: /Skeletal m : )