mechanismofactionofinsulin11-190623063339.pdf

Introduction to Insulin

1. AkashMahadevIyer
S4 M Sc Biochemistry
Department of Biochemistry
Karyavattomcampus
Mechanism of Action
of Insulin

2. Outline…
•IntroductiontoInsulin
•History
•StructureofInsulin
•BiosynthesisofInsulin
•DegradationofInsulin
•Regulationofsecretion
•Biologicalactions
EffectonCarbohydrate,lipidandproteinmetabolism
•InsulinReceptor
•Mechanismofaction
•Insulinsignalingpathways
•Disorders

3. Insulinisapeptidehormonesecretedbyβ-cellsinthe
pancreaticisletsofLangerhans.
Themainfunctionofinsulinistolowerserumglucoseand
promoteanabolism.
Insulinisanessentialgrowthfactorrequiredfornormal
development.

4. In1869,aGermanmedicalstudent,PaulLangerhans,notedthatthepancreas
containstwodistinctgroupsofcellstheacinarcells,whichsecretedigestive
enzymes,andcellsthatareclusteredinislands,orislets,whichhesuggested
servedasecondfunction.
In1889,OskarMinkowskiandJosephvonMeringobservedthattotal
pancreatectomyinexperimentalanimalsleadstothedevelopmentofsevere
diabetesmellitus
•In1920,Frederickbantingmanagedtoisolatethefluidextract-thathecalled
‘isletin’-fromIsletsofLangerhansandinjectedintodiabeticdogs.Thedogs
abnormallyhighsugarlevelinthebloodloweredandtheysurvivedforlonga
theyreceivedtheextract.TheNobelPrizeinmedicineandphysiologywas
awardedtoBantingandMacleodwithin1923.
•InsulinissequencedbyBritishbiochemistFrederickSanger,andisthefirst
proteintobefullysequenced.In1958SangerreceivestheNobelPrizein
Chemistry.
•DorothyHodgkinwonthe1964NobelPrizeinChemistryforherworkinprotein
crystallography/x-raycrystallography.
•1978,BiotechnologyfirmGenentech(Califor.)usedrDNAtechnologytoproduce
synthetic“human”insulin-firsthumanproteintobemanufacturedthrough
biotechnology.

6. Insulin should have been named
“Protein of the 20
th
century”
•Insulin was:
•the first protein shown to have hormonal action
•the first protein to be crystallized in pure form (Abel, 1926)
•the first protein to be fully sequenced (Sanger et al, 1955)
•the first protein to be synthesized chemically (Du et al, Zahn, 1964)
•the first protein to be synthesized as a large precursor molecule (Steiner
et al, 1967)
•the first protein synthesized for commercial use by
DNArecombinanttechnology (1979)

7. Inadulthumans,thepancreasweighsabout80g.
Locatedbehindthestomach
Derivedfromendoderm
Thepancreasisaretroperitonealorgananddoesnothaveacapsule.
Thespleenisadjacenttothepancreatictail.Theregionsofthepancreas
arethehead,body,tailanduncinateprocess.
Thedistalendofthecommonbileductpassesthroughtheheadofthe
pancreasandjoinsthepancreaticductenteringtheduodenum.
Forthisreason,pathologicprocessesofthepancreas,suchasacancer
attheheadofthepancreasorswellingand/orscarringoftheheadofthe
pancreasduetopancreatitis,canleadtobiliarysystemobstructionand
injury.Becauseofitsposteriorposition,thepancreasisusuallyprotected
fromtrauma
Pancreas; a dual gland

8. Pancreas-bothanendocrineglandandanexocrinegland
The pancreas provides both
Theenzymesthatdigestthefoodinthegut(Theductalandacinar
cellsoftheexocrinecompartment-80%)and
1.Thehormonesthatcontrolutilizationofthenutrientssuppliedbythat
digestedfood(theisletsofLangerhansoftheendocrineportionare
scatteredthroughouttheexocrinematrix-2%)

9. Thehumanpancreashas1to2millionisletsofLangerhans,each
onlyabout0.3millimeterindiameterandorganizedaroundsmall
capillariesintowhichitscellssecretetheirhormones.
Theisletscontainthreemajortypesofcells,
alpha,
beta,and
deltacells,whicharedistinguishedfromoneanotherbytheir
morphologicalandstainingcharacteristics.

11. Structure of Insulin
Humaninsulin-51aa
Mw;5.7kDa
Comprisesof2polypeptidechains
I.A(with21aminoacidresidues)and
II.B(with30aminoacidresidues)
ThetwochainsarelinkedbytwointerchaindisulfidebridgesthatconnectA7toB7
andA20toB19
Athirdintrachaindisulfidebridgeconnectsresidues6and11ofAchain
-

12. •Insulinexistsprimarilyasamonomeratlowconcentrations(~10–6M)
andformsadimerathigherconcentrationsatneutralpH.
•Athighconcentrationsandinthepresenceofzincions,insulin
aggregatesfurthertoformhexamericcomplexes.
•Monomersanddimersreadilydiffuseintoblood,whereashexamers
diffusepoorly.
•Hence,absorptionofinsulinpreparationscontainingahighproportionof
hexamersisdelayedandslow.
•Thethreeconservedregionsininsulin—
•aminoterminalA-chain(GlyA1-IleA2-ValA3-GluA4orAspA4),
•carboxylterminalA-chain(TyrA19-CysA20-AsnA21),and
•carboxylterminalBchain(GlyB23-PheB24-PheB25-TyrB26)—are
locatedatornearthesurfaceofinsulinandthereforemayinteractwith
theinsulinreceptor.

13. •Insulinmoleculeshaveatendencytoformdimersinsolutiondueto
hydrogen-bondingbetweentheC-terminiofBchains.
•Additionally,inthepresenceofzincions,insulindimersassociateinto
hexamers.
•Monomersanddimersreadilydiffuseintoblood,whereashexamers
diffusepoorly.
•Hence,absorptionofinsulinpreparationscontainingahighproportionof
hexamersisdelayedandsomewhatslow.Thisphenomenon,among
others,hasstimulateddevelopmentofanumberofrecombinantinsulin
analogs.
•Thefirstofthesemoleculestobemarketed-calledinsulinlispro-is
engineeredsuchthatlysineandprolineresiduesontheC-terminalend
oftheBchainarereversed;thismodificationdoesnotalterreceptor
binding,butminimizesthetendencytoformdimersandhexamers.
•

14. Site; β-cells of Islets
1.Synthesis of Preproinsulin.
2.Conversion of preproinsulinto proinsulin.
3.Conversion of proinsulinto insulin.
Insulin is synthesized by ribosomesof the rough ER as a
larger precursor peptide that is then converted to the
mature hormone prior to secretion
Biosynthesis of Insulin –3 major steps
Biosynthesis of Insulin contd…..

15. Insulinissynthesizedintheformofahmwprecursor,preproinsulin(110
aa),processedviaanintermediateprecursor,proinsulin(86aa),tothe
matureinsulin(51aa)molecule
Biosynthesis of Insulin contd…..

16. Conversion of proinsulinto insulin
•Proinsulinundergoesmaturationintoactiveinsulinthroughactionofcellular
endopeptidasesknownasprohormoneconvertases(PC1andPC2),andthe
exoproteasecarboxypeptidaseE
•Theendopeptidasescleaveat2positions,releasingafragmentcalledtheC-
peptide,andleaving2peptidechains,theB-andA-chains,linkedby2disulfide
bonds
Intheβcells,insulincombineswithzinc
toformcomplexes
Inthisform,insulinisstoredingranules
ofthecytosolwhichisreleasedin
responsetovariousstimuli.

17. Collapsed cavityInsulin hexamer;Top view
TwoZn
2+
axialatomsperhexamericunit
Monomeristhebiologicallyactiveformofinsulin,hexamerservesas
thestorehouseofthehormone.
Stable cavity with water
Water molecules
Zn ²
+
Wefindthatthesewatermoleculesaredynamicallyslowerthanthebulkandweaveanintricate
hydrogenbondnetworkamongthemselvesandwithneighboringproteinresiduestogeneratea
robustbackboneatthecenterofthehexamerthatholdstheassociationstronglyfrominside
andmaintainsthebarrelshape.

18. Peptide Hormone Synthesis, Packaging, and Release
ECFCytoplasm Plasma
Capillary
endothelium
Messenger RNA on the
ribosomes binds amino
acids into a peptide chain
called a preprohormone.
The chain is directed into
the ER lumen by a signal
sequenceof amino acids.
mRNA
Ribosome
Endoplasmic reticulum (ER)
Preprohormone
1
1

19. ECFCytoplasm Plasma
Capillary
endothelium
Messenger RNA on the
ribosomes binds amino
acids into a peptide chain
called a preprohormone.
The chain is directed into
the ER lumen by a signal
sequenceof amino acids.
Enzymes in the
ER chop off the
signal sequence,
creating an
inactive
prohormone.
mRNA
Ribosome
Prohormone
Signal
sequence
Endoplasmic reticulum (ER)
Preprohormone
1 2
1
2
Peptide Hormone Synthesis, Packaging, and Release

20. Golgi complex
ECFCytoplasm Plasma
Capillary
endothelium
Messenger RNA on the
ribosomes binds amino
acids into a peptide chain
called a preprohormone.
The chain is directed into
the ER lumen by a signal
sequenceof amino acids.
Enzymes in the
ER chop off the
signal sequence,
creating an
inactive
prohormone.
The prohormone
passes from the
ER through the
Golgi complex.
mRNA
Ribosome
Prohormone
Signal
sequence
Transport
vesicle
Endoplasmic reticulum (ER)
Preprohormone
1 2 3
1
2
3
Peptide Hormone Synthesis, Packaging, and Release

21. 4
Active hormone
Golgi complex
Secretory
vesicle
ECFCytoplasm Plasma
Peptide
fragment
Capillary
endothelium
Messenger RNA on the
ribosomes binds amino
acids into a peptide chain
called a preprohormone.
The chain is directed into
the ER lumen by a signal
sequenceof amino acids.
Enzymes in the
ER chop off the
signal sequence,
creating an
inactive
prohormone.
The prohormone
passes from the
ER through the
Golgi complex.
Secretory vesicles containing
enzymes and prohormone
bud off the Golgi. The enzymes
chop the prohormone into one
or more active peptides plus
additional peptide fragments.
mRNA
Ribosome
Prohormone
Signal
sequence
Transport
vesicle
Endoplasmic reticulum (ER)
Preprohormone
1 2 3
1
2
3
4
Peptide Hormone Synthesis, Packaging, and Release

22. 4 5
Active hormone
Golgi complex
Secretory
vesicle
ECFCytoplasm Plasma
Peptide
fragment
Release
signal
Capillary
endothelium
Messenger RNA on the
ribosomes binds amino
acids into a peptide chain
called a preprohormone.
The chain is directed into
the ER lumen by a signal
sequenceof amino acids.
The secretory
vesicle releases
its contents by
exocytosis into
the extracellular
space.
Enzymes in the
ER chop off the
signal sequence,
creating an
inactive
prohormone.
The prohormone
passes from the
ER through the
Golgi complex.
Secretory vesicles containing
enzymes and prohormone
bud off the Golgi. The enzymes
chop the prohormone into one
or more active peptides plus
additional peptide fragments.
mRNA
Ribosome
Prohormone
Signal
sequence
Transport
vesicle
Endoplasmic reticulum (ER)
Preprohormone
1 2 3
1
2
3
4
5
Peptide Hormone Synthesis, Packaging, and Release

23. 4 5
To target
Active hormone
Golgi complex
Secretory
vesicle
ECFCytoplasm Plasma
Peptide
fragment
Release
signal
Capillary
endothelium
Messenger RNA on the
ribosomes binds amino
acids into a peptide chain
called a preprohormone.
The chain is directed into
the ER lumen by a signal
sequenceof amino acids.
The secretory
vesicle releases
its contents by
exocytosis into
the extracellular
space.
The hormone
moves into the
circulation for
transport to its
target.
Enzymes in the
ER chop off the
signal sequence,
creating an
inactive
prohormone.
The prohormone
passes from the
ER through the
Golgi complex.
Secretory vesicles containing
enzymes and prohormone
bud off the Golgi. The enzymes
chop the prohormone into one
or more active peptides plus
additional peptide fragments.
mRNA
Ribosome
Prohormone
Signal
sequence
Transport
vesicle
Endoplasmic reticulum (ER)
Preprohormone
1 2 3 6
1
2
3
4
5
6
Peptide Hormone Synthesis, Packaging, and Release

25. Leptin
Fasting

26. Regulation of insulin secretion by glucose in pancreatic βcells
.

27. •Similartootherneuroendocrinecells,insulin-secretingcellsareelectrically
excitable.
•Thismeansthatinsulinsecretioninresponsetoglucoseisdependenton
initiationofelectricalactivityfromabasalelectronegativerestingstate..Inthe
normalcourseofglucoseexcitationcouplingtoinsulinsecretion,glucoseenters
thecellviaGLUT2(andGLUT1)glucosetransportersandistrappedinthecell
byphosphorylationviaglucokinase,aspecializedhighKmhexokinase.
•ThehighKmofglucokinaseforglucoseunderliesitsroleasthe“glucose
sensor”oftheβcell,inthatitsenzymeactivityreflectsphysiologiccirculating
glucoseconcentrations.

28. Followingglucosephosphorylation,furthermetabolismviaglycolysis,
theKrebs(tricarboxycylicacid,TCA)cycle,andoxidativephosphorylation
inthemitochondrialeadstoanincreaseintheATP-to-ADPratiointhe
cytoplasm.
Interestingly,whileglucoseisthekeyphysiologicstimulatorofthe
insulin-secretingsystem,itcanbereplacedorenhancedbyotherenergy-
providingmetabolites(e.g.,leucine,andsuccinicacidmonomethylester)
thateitherfeedintoglycolysis,theTCAcycle,orrelatedoxidative
phosphorylationpathways.
TheATP/ADPratioincreaseisdrivenbymetabolismthatleadstoATP-
dependentpotassiumchannel(KATP)channelinhibition,inturnleading
toaccumulationofpositivecharge(K+andNa+)insidethecelland
causingthecellmembranetodepolarize.
Asthemembranepotentialreachesabout–20mVfromtherestinglevel
ofabout–70mV,voltage-dependentcalciumchannelsopen,allowing
entryofCa2+.

29. InteractionofnumerousK+,Ca2+,andNa+voltage-dependentchannels
contributestotheappearanceoftonicorperiodictransientsofboth
membranepotentialandCa2+.
OnceKATPclosesandCa2+entrybeginsviatheopeningofvoltage-
dependentcalciumchannels,avarietyofmechanismscontrolintracellular
Ca2+,whichisakeyregulatoryfactorforinsulinrelease.
Theseincluderegulationoftheplasmamembranepotentialbyother
voltage-andCa2+-dependentK+channelsthatservetohelprepolarizethe
plasmamembrane,andvoltagedependentNa+channelsthataccelerate
plasmamembranedepolarizationespeciallyinhuman,canine,andporcine
islets.

30. Insideofthecell,Ca2+canbesequesteredbytheactionofthe
sarcoplasmic/endoplasmicreticulumadenosinetriphosphatase
(SERCA)pump.
ReleaseofintracellularCa2+fromtheendoplasmicreticulum
throughactionofinositoltriphosphate(IP3)onIP3receptors,aswell
asviaryanodinereceptorsthatcanbeactivatedbyadditional
messengers,mayallplaykeyrolesinregulatingintracellularCa2+
transients.
SpatialandtemporalcontrolofCa2+signalsmaybehighly
regulatedinplasmamembraneCa2+microdomainswhereinsulin
granulesfuse.
MitochondriaarealsoimportantstoresofcalciumandtakeupCa2+
duringmetabolicactivityinparttoregulatethekeydehydrogenases
intheTCAcycle

31. •Otheranapleroticmoleculescomingfromthemitochondria,glutamateinparticular,
canbeimplicatedinregulationofsecretion.
•TheelevationinintracellularfreeCa2+activatesmultipleproteins(e.g.,smallG
proteinssuchasRabs,andsolubleN-ethylmaleimideattachmentproteinreceptor
[SNARE]pathways)regulatingtheCa2+-triggeredfusionofthe(predocked)insulin-
containinggranuleswiththecellmembrane,resultinginthefirstphaseofinsulin
secretion.

34. •Asinneurotransmitterrelease,βcellgranulefusiondependson
interactionsofsynaptosome-associatedproteinsv-SNAREs(VAMP2
andsynaptotagmin)withplasmamembranereceptorssuchasSNAP-
25,at-SNARE(targetlocalizedSNAPreceptor),andsyntaxins.
•Second-phaseinsulinsecretionreferstothecontinuedreleaseof
insulinfollowingtheinitialpeak.
•Inparallelwithfusionofthedockedinsulincontaininggranules,the
granuleslocatedfartherawayofplasmalemma(termedresting
newcomers)canberecruitedviamicrotubulesandassociated
kinases,chaperones,andsmallGTP-bindingproteins(syntaxin4,
Munc18)throughtheactinnetworkuntiltheytoocandockatt-
SNAREsitesandfusetotheplasmamembrane.158

36. Increased ATP closes the ATP-sensitive K
+
channels
K
+
efflux
Depolarizes the cell membrane
Open voltage sensitive calcium channels
Calcium enters the cell
Intracellular Ca
2+
Triggers insulin secretion by exocytosis
Glucose;keyregulator(aa,ketones,variousnutrients,GIPs,andNTs
alsoinfluenceinsulinsecretion.)
Glucose levels > 3.9 mmol/L (70 mg/dL) stimulate insulin synthesis,
primarily by enhancing protein translation and processing

37. Degradation of Insulin
Half-life;4-6minutes.
Liver-principalsiteofinsulindegradation
Hepaticglutathioneinsulintranshydrogenase–Reducesthedisulphide
bondsandthenindividualAandBchainsarerapidlydegraded
IDE–CleavesB-chainatTyr16-Leu17

38. Insulinpromotestheanabolicstatebychannelingmetabolismtowardsthe
storageofcarbohydratesandlipids,andtowardsproteinsynthesis.
Insulinpromotesenergystoragebystimulatingthesynthesisoffattyacids
andtriglyceridesintheliverandadiposetissue,glycogeninliverand
skeletalmuscle,andproteinsynthesisinmuscles.
Atthesametime,itopposesthecatabolismbyinhibitingthebreakdown
ofproteins,triglyceridesandglycogen,andbysuppressing
gluconeogenesisbytheliver.

39. Insulinactsonthreemaintargettissues:

40. Different organs and tissues handle fuels differently
Atrest,thebrainusesapproximately20%ofalloxygenconsumedbythebody.
Glucoseisnormallyitsonlyfuel:duringstarvation,however,thebrainadaptstothe
useofketonebodiesasanalternativeenergysource.
Thetwopathwaysthatprovideglucoseareglycogenolysisandgluconeogenesis.
Whenglucoseconcentrationintheextracellularfluiddecreases,itisfirst
replenishedbydegradingliverglycogen.However,whenthefastingperiodextends
gluconeogenesisisinitiated.Gluconeogenesistakesplacemostlyintheliver,and
thekidneysalsocontributeduringprolongedfast.
Itsmainsubstratesarelactate(fromanaerobicglycolysis),alanine(fromtheamino
acidsreleasedduringbreakdownofmuscleprotein)andglycerol(fromthe
breakdownoftriacylglycerolsintheadiposetissue.

41. Insulin Action on Carbohydrate Metabolism:
Liver:
Stimulatesglycolysis
Promotesglucosestorageasglycogen
Inhibitsglycogenolysis
Inhibitsgluconeogenesis
Muscle:
Stimulatesglucoseuptake(GLUT4)
Promotesglucosestorageasglycogen
AdiposeTissue:
Stimulatesglucosetransportintoadipocytes
Promotestheconversionofglucoseintotriglyceridesandfattyacids

42. Insulinpromotesglycolysis

44. Inliver,insulindecreasegluconeogenesisbysuppressingtheenzymespyruvate
carboxylase,phosphoenolpyruvatecarboxykinaseandglucose-6-phosphatase
InliverandmusclesinsulinincreasesGlycolysisandglycogensynthesis

45. Effect on lipogenesis:
Insulinfavoursthesynthesisoftriacylglycerolsfromglucosebyproviding
moreglycerol3-phosphate&NADPH.
InsulinincreasestheactivityofacetylCoAcarboxylase,akeyenzymein
fattyacidsynthesis.
Effect on lipolysis:
Insulindecreasestheactivityofenzyme-hormone-sensitivelipase&
reducesthereleaseoffattyacidsfromstoredfat.
Effect on ketogenesis:
InsulinreducesketogenesisbydecreasingtheactivityofHMGCoA
synthase.
Effectsonlipidmetabolism

46. Insulinregulatesglucoseuptakebyadipocytesandtriggersfattyacidtransport
proteintranslocationaswellasfattyaciduptakebyfatcells.
Bindingofinsulintoitsspecificcell-surfacereceptorproducestyrosine
phosphorylationandactivationoftheinsulinreceptor,whichleadstotheinteraction
withtheinsulinreceptorsubstrates(IRS-1andIRS-2),inturnactivatingthep
hosphatidylinositol3-kinase(PI3K)complex.
Insulinpowerfullyinhibitsbasalandcatecholamine-inducedlipolysisthrough
phosphorylation(viaaPKB/Akt-dependentaction)andactivationof
phosphodiesterase-3B(PDE-3B).
ThephosphodiesterasecatalysesthebreakdownofcAMPtoitsinactiveform,
therebydecreasingcAMPlevels,whichinturnreducesPKAactivationand,therefore,
alsotranslatesintopreventingHSLstimulation.
Insulinmayalsosuppresslipolysisthroughphosphorylationoftheregulatorysubunit
ofproteinphosphatase-1(PP-1),whichonceactivatedrapidlydephosphorylatesand
deactivatesHSL,thusdecreasingthelipolyticrate.

47. Effect on lipogenesis:
Effect on lipolysis:

48. Stimulatestheentryofaminoacidsintothecells,
EnhancesProteinSynthesisand
Reducesproteindegradation
Intheliver,insulindepressestherateofgluconeogenesisconservestheamino
acids
Effectsonproteinmetabolism
Insulin and Growth hormone interact
synergistically to promote growth

49. ImportantroleinPotassiumhomeostasis
StimulatesK+uptakebythecells
Inflammation and Vasodilation
Insulin’s actions within endothelial cells and macrophages have an anti-
inflammatory effect on the body.
Within endothelial cells, insulin stimulates the expression of endothelial
nitric oxide synthase(eNOS).
eNOSfunctions to releasenitric oxide (NO), which leads to vasodilation.

51. Insulinisreleasedfromtheisletintothebloodstreamandits
actionsaremediatedbytheinsulinreceptor(IR)onthe
surfaceoftargetcells.
Cell-surfacereceptor withtyrosine-kinaseactivity-
PhosphorylatesubstrateproteinsonTyrosineresidues
Heterotetramericglycoprotein-2extracellularαand
2transmembraneβsubunitslinkedtogetherbydisulfide
bonds–α2β2
Molecularweight300kDa
αsubunits-insulinbindingsite
βsubunits-tyrosinekinaseactivity,involvedinintracelular
signaling.
Expressedinmostmammaliantissues;adiposeandliver
havethehighestIRdensity(>300,000receptors/cell).
Insulin receptor
Tyrosine kinaseactivity
~

53. Activationofinsulinsignalingregulates
themetabolismsofglucoseandlipids,
andproteinsynthesis.
PhosphorylationatY972oftheβ-subunit
generatesanNPXpYmotif,leadingtothe
increaseintheaffinityoftheIRSbindingto
theIRandthetyrosinephosphorylationof
IRS.

54. Binding
of Insulin on α-subunit
Phosphorylation
of β-subunit
Phosphorylation
of IRS
Mechanism of action of insulin
Insulinregulatesbothmetabolic
enzymesandgeneexpression.
Doesnotentercells,butinitiatesa
signalthattravelsfromthecellsurface
receptorto-cytosolandtothenucleus.
Theinsulinreceptor(INS-R)isa
glycoproteinreceptorwithtyrosine
kinaseactivity.
GeneExpression Metabolism
Growth
CHANGES IN

55. SignalingthroughINS-Rbeginswhen-bindingofinsulinactivatesthe
Tyrkinaseactivity,andeachβsubunitphosphorylatesthreecriticalTyr
residuesnearthecarboxylterminusoftheotherβsubunit.
•Bindingofligandcausesaconformationalchangethatpromotes
dimerizationoftheextracellulardomainsofRTKs,whichbringstheir
transmembranesegments—andthereforetheircytosolicdomains—
closetogether.
Thisautophosphorylationopensuptheactivesitesothattheenzyme
canphosphorylateTyrresiduesofothertargetproteins.
aregionofthecytoplasmicdomain(anautoinhibitorysequence)that
normallyoccludestheactivesitemovesoutoftheactivesiteafterbeing
phosphorylated,openingupthesiteforthebindingoftargetproteins.
Activationoftheinsulinreceptor,whichleadstotheinteractionwiththe
insulinreceptorsubstrates(IRS-1andIRS-2),inturnactivatingthe
phosphatidylinositol3-kinase(PI3K)complex.

56. WhenINS-RisautophosphorylatedandbecomesanactiveTyr.
Kinase,itphosphorylatesInsulinreceptorsubstrate-1(IRS-1)
OncephosphorylatedonseveralofitsTyrresidues,IRS-1becomes
thepointofnucleationforacomplexofproteins,thatcarrythe
messagefromtheinsulinreceptortoendtargetsinthecytosoland
nucleus,throughalongseriesofintermediateproteins.

57. ActivationoftheTyrkinaseallowseachsubunittophosphorylatethreeTyr
residues(Tyr1158,Tyr1162,Tyr1163)ontheothersubunit.Theintroduction
ofthreehighlychargedP–Tyrresiduesforcesa30Åchangeintheposition
oftheactivationloop,awayfromthesubstrate-bindingsite,whichbecomes
availabletobindandphosphorylateatargetprotein.

58. Insulinsignallingisoneofthemostimportantsignallingnetwork,whichregulates
somefundamentalbiologicalfunctionssuchasglucoseandlipidmetabolism,
proteinsynthesis,cellproliferation,celldifferentiationandapoptosis.These
differentbiologicalresponsesareachievedbytheinsulinbindingtoitsreceptor
andbythesubsequentcombinedactivationofthreemajorpathways:
ThePI3K-AKTpathway,mostlyresponsibleforthemetabolicinsulinactionvia
thetranslocationoftheglucosetransportertype4(GLUT4)vesiclestothe
plasmamembrane,which,inturn,allowtheglucoseuptakeinmusclecellsand
adipocytes;
TheTSC1/2-mTORpathway,playingacriticalroleinproteinsynthesissince
mammaliantargetofrapamycin(mTOR)isacentralcontrollerforseveral
anabolicandcatabolicprocessesincludingRNAtranslation,ribosomebiogenesis
andautophagy,inresponsenotonlytogrowthfactorsandhormoneslikeinsulin,
butalsotonutrients,energyandstresssignals;
TheRAS-MAPKpathway,promotingcellsurvival,divisionandmotilityvia
extracellularsignal-regulatedkinase1/2(ERK1/2)complexthat,once
phosphorylated,translocatesintothenucleusactivatingmanytranscription
factors,thusconstitutinganimportantconnectionbetweenthecytoplasmicand
nuclearevents.
Insulin signalling

59. Insulinpowerfullyinhibitsbasalandcatecholamine-induced
lipolysisthroughphosphorylation(viaaPKB/Akt-dependent
action)andactivationofphosphodiesterase-3B(PDE-3B).
ThephosphodiesterasecatalysesthebreakdownofcAMPto
itsinactiveform,therebydecreasingcAMPlevels,whichin
turnreducesPKAactivationand,therefore,alsotranslates
intopreventingHSLstimulation.
Insulinmayalsosuppresslipolysisthroughphosphorylationof
theregulatorysubunitofproteinphosphatase-1(PP-1),which
onceactivatedrapidlydephosphorylatesanddeactivates
HSL,thusdecreasingthelipolyticrate.

60. STEPS
1.Ligandreception
2.ReceptorDimerizaton
3.Autophosphorylation–ActivationofTyrosinekinaseinINS-R
4.Phosphorylationoftargetproteins(eg;IRSproteins)
5.TyrosinephosphorylatedIRSproteinsactasabindingsitefor
signalingmoleculescontainingSH-2(Src-homology-2)domainssuch
asPI3’-kinase,andGrb2/sos,SHP2.
6.Insulinsignaling

61. Biological actions of insulin, initiated by its binding to INS-R and an have
short,intermediate, andlong-term effectsoncellularfunctions
Short term effects
Intermediate effects
Long term effects
Biological actions of insulin

62. Short term effects
Immediateeffects-occurwithinsecondsafterreceptoractivation
Activationofglucoseandion-transportsystemsand
Covalentmodifications(PhosphorylationandDephosphorylation)ofpre-
existingenzymes
Intermediate effects
Minutes to hours
Induction of genes and expression of certain proteins

63. Long term effects
Hourstoseveraldays
StimulateDNAsynthesis,cellproliferationcelldifferentiationandsome
geneexpressionevents.
These effects are the results not of a simple linear pathway, but
of the multiple diverging and converging pathways mediated
by INS-R.

65. Insulinsignallingisoneofthemostimportantsignaling
network,
It regulates most fundamental biological functions such
as;
Glucoseandlipidmetabolism,
Proteinsynthesis,
Cellproliferation,CelldifferentiationandApoptosis.
Insulin signaling

66. There are two major routes by which,
insulin signals are transmitted

67. PI3K-Akt pathway
•ThePI3K/AKTsignalingpathwayisakeyregulatorofnormalcellular
processesinvolvedincellgrowth,proliferation,metabolism,motility,
survival,andapoptosis.
•Responsibleforthemetaboliceffectsofinsulin
•Translocationoftheglucosetransportertype4(GLUT4)vesiclestothe
plasmamembrane;allowsglucoseuptakeinmusclecellsand
adipocytes
•AberrantactivationofthePI3K/AKTpathwaypromotesthesurvivaland
proliferationoftumorcellsinmanyhumancancers

70. TheactivatedINS-RphosphorylatesIRS.
PhosphorylatedIRSbindstotheSH2domainsofPI3K.
Binding of IRS, results in activation of the catalytic subunit of PI3K.
The PI3K-Akt Signaling Pathway
PIP3,whichremainsinthecytosolicleafletoftheplasmamembranerecruitstwo
proteinkinasestotheplasmamembraneviatheirPHdomains-
Akt(PKB-ProteinkinaseB)and
PDK1(Phosphoinositide-dependentproteinkinase1).
Bothare;Serine-threoninekinases

71. RecruitmentofPDK1totheplasmamembrane,incloseproximitytoPKB,
providesasettinginwhichPDK1canphosphorylateandactivatePKB.
PhosphorylationbyPDK1(atThr-308)isessential,butnotsufficientforactivation
ofPKB.
ActivationofPKBalsodependsonphosphorylation(atSer-473)byasecond
kinase,mTOR.
Remember Aktrequires two phosphorylationevents for
activation!!!
INS-R

72. AKTisactivatedbyphosphorylationofThr308initsactivationloopbythejuxtaposed
membrane-boundPDK1.
AKTisoformsregulatephosphorylationofproteinsthatcontrolcellsurvival,growth,
proliferation,angiogenesis,metabolism,andmigration.
Morethan100AKTsubstratesareknownandseveralareespeciallyrelevanttoinsulin
signaling—including
i.GSK3α/β(blocksinhibitionofglycogensynthesis);
ii.AS160(promotesGLUT4translocation);
iii.BAD•BCL2heterodimer(inhibitsapoptosis);
iv.TheFOXOtranscriptionfactors(regulatesgeneexpressioninliver,β-cells,
hypothalamus,andothertissues);
v.p21CIP1andp27KIP1(blockscell-cycleinhibition);
vi.eNOS(stimulatesNOsynthesisandvasodilatation);
vii.PDE3b(hydrolyzescAMP);and
viii.TSC2(tuberoussclerosis2tumorsuppressor)thatinhibitsmTORC1
(mechanistictargetofrapamycincomplex1)

73. ActivatedAktphosphorylatesvarioustargetproteinsattheplasmamembrane,as
wellasinthecytosolandnucleus.
Four of the critical downstream substrates of Aktare
mTOR,mammaliantargetofrapamycin,involvedintheregulationofprotein
synthesis
GSK3(glycogensynthasekinase3),involvedintheregulationofglycogen
synthesis
FoxO(forkheadbox-containingprotein,Osubfamily)transcriptionfactors,
especiallyFoxO1,involvedintheregulationofgluconeogenicandadipogenic
genesand
AS160(AKTsubstrateof160kDa),involvedinglucosetransport.

75. mTORCascade
mTOR(mechanistictargetofrapamycin)isaSer/Thrkinasethatisregulated
throughmultiplemechanisms.
ItbelongstothePI3K-relatedkinasefamilyandformstwolargefunctionally
distinctproteincomplexes;
mTORC1
mTORC2
Composedofcommonanduniquesubunits.
Bothcomplexesarecontrolledbygrowthfactorsandinsulinthroughthe
PI3K→AKTcascade,buttheyarerecruitedtodifferentcompartmentsandrespond
distinctlytonutrients,stress,hypoxia/energystatus,andotherstimulitocoordinate
adiversearrayofbiologicalprocesses—includingproteinandlipidsynthesis,
liposomebiogenesis,autophagy,andcellmigration,growth,andproliferation.

76. In addition to the common catalytic subunit, mTORC1 and mTORC2 share
I.mLST8(mammalianlethalwithsec-13protein8),
II.DEPTOR(Disheveled,Egl-10,andPleckstrindomaincontainingmTOR-
interactingprotein),and
III.Tti1(Telomeremaintenance2interactingprotein1).
mTORC1isdistinguishedbytwospecificcomponents,including
a.RAPTOR(RPTOR,regulatoryassociatedproteinofmTOR,complex1)and
b.AKT1S1(PRAS40,AKT1substrate1proline-rich).
mTORC2lacksthemTORC1-specificcomponents,butincludes
I.RICTOR(RAPTOR-independentcompanionofmTOR,complex
II.mSIN1(SAPKinteractingprotein1,orMEKK2interactingprotein1),and
III.PRR5(protor1/2,proteinobservedwithRictor1and2).
mTORC1isstronglyregulatedbynutrientconcentrationandinhibitedby
rapamycin,whereasmTORC2isinhibitedvariablybyrapamycinandis
insensitivetonutrientlevels.

77. ThecontrolofcellgrowthbythePI-3-kinase–Aktpathwaydependsinpart
onalargeproteinkinasecalledTOR(namedasthetargetofrapamycin,a
bacterialtoxinthatinactivatesthekinaseandisusedclinicallyasbothan
immunosuppressantandanticancerdrug).
TORwasoriginallyidentifiedinyeastsingeneticscreensforrapamycin
resistance;inmammaliancells,itiscalledmTOR,whichexistsincellsin
twofunctionallydistinctmultiproteincomplexes.
mTORcomplex1containstheproteinraptor;thiscomplexissensitiveto
rapamycin,anditstimulatescellgrowth—bothbypromotingribosome
productionandproteinsynthesisandbyinhibitingproteindegradation.
Complex1alsopromotesbothcellgrowthandcellsurvivalbystimulating
nutrientuptakeandmetabolism.
mTORcomplex2containstheproteinrictorandisinsensitiveto
rapamycin;ithelpstoactivateAkt,anditregulatestheactincytoskeleton
viaRhofamilyGTPases.

78. ThemTORincomplex1integratesinputsfromvarioussources,
includingextracellularsignalproteinsreferredtoasgrowthfactorsand
nutrientssuchasaminoacids,bothofwhichhelpactivatemTORand
promotecellgrowth.
ThegrowthfactorsactivatemTORmainlyviathePI-3-kinase–Akt
pathway.
AktactivatesmTORincomplex1indirectlybyphosphorylating,and
therebyinhibiting,aGAPcalledTsc2.
Tsc2actsonamonomericRas-relatedGTPasecalledRheb.
Rhebinitsactiveform(Rheb-GTP)activatesmTORincomplex1.
ThenetresultisthatAktactivatesmTORandtherebypromotescell
growth
ThemTORC1complexisactivatedbyAktwhereasmTORC2is
capableofactivatingAKT.
mTORC2,whichcanphosphorylateAktatS473invitroandinvivo,
therebyindicatingthatmTORcanactasbothasubstrateandeffector
oftheAktsignalingpathway.

80. AKTregulatesglucoseandlipidmetabolism.ActivatedAKT2,whichisprimarilyexpressed
ininsulin-responsivetissues,promotestranslationofglucosetransporter4(GLUT4).And
thedirecttargetofAKTisasubstrateof160kDa(AS160),alsoknownasTBC1D1
Inintracellularcompartments,AKTconvertsglucosetoglucose6-phosphatebystimulating
hexokinase.AKTregulatestwoprocessesbyglycolysisGlucose6-phosphateand
glycogensynthasekinase3(GSK3)toproducecellularenergyviaglycolysisand
promotesglycogenproductionbyinhibiting.
FoxOproteins,particularlyFoxO1,arethemaintargetofAKTandaffectenergy
homeostasisthroughoutbody.FoxO1andperoxisomeproliferator-activatedreceptor-
coactivator1α(PGC1α)coordinatelyregulategeneexpressiontoincrease
gluconeogenesisandfattyacidoxidatio.
Ontheotherhand,FoxO1inducestheexpressionofphosphoenolpyruvatecarboxykinase
(PEPCK)andglucose-6-phosphatasegene(G6PC),subsequentlyincreases
gluconeogenesis.
AKTdirectlyinhibitsFoxO1,reducingglucoselevels,FoxO1simultaneouslyactivatesAKT
toincreaseenergyproductionandinhibitsmTORcomplex1(mTORC1)toreducelipidand
proteinproduction.
Finally,GSK3inhibitsglycogensynthase(GS),whichpromotesglycogensynthesis.AKT
exertsaninhibitoryeffectonGSK3byphosphorylationofGSK3.
AKTregulateslipidmetabolismthroughsterolregulatoryelement-bindingproteins
(SREBP),whichincreasescholesterolandfattyacidaccumulation,includingSREBP-1c,
SREBP-1a,andSREBP-2.Therefore,PI3K/AKTregulatesglucosemetabolismthrough
FoxO1andGSK-3andlipidmetabolismthroughmTORC1andSREBP.

81. ForkheadboxO(FOXO);transcription
factors
Regulateexpressionoftargetgenesinvolved
inDNAdamagerepair,apoptosis,metabolism,
cellularproliferation,stresstolerance,and
longevity.
FOXO1 stimulatesthesynthesisof
gluconeogenicenzymesandsuppressesthe
synthesisoftheenzymesofglycolysis,the
HMPshunt,andTAGsynthesis.

82. RasMAP Kinasepathway
TheRas/MAPKpathwayregulates;celldifferentiation,celldivision,
cellproliferation,inflammation,cellstressresponse,metabolism,and
apoptosis.
Thepathwayrelaysextracellularsignalsfromtheplasmamembrane
throughthecytoplasmandintothenucleus.
TheMAPKpathwayisinvolvedinmediatinglongtermeffectsofinsulin-
Growthandmitogenesis
Players involved;
Grb2;Growthfactorreceptor-boundprotein2-Adaptor
Sos;SonofSevenless-GEF
Ras;GTPaseswitchproteins
Raf; a serine/threoninekinase; MAPKKK
MEK, serine/tyrosine/threoninekinase; MAPKK
ERK; serine/ threoninekinase; MAPK

83. Ras
Ras
RassuperfamilyofsmallGTPaseshelprelaysignalsfromRTKs
ThemonomericRasproteinbelongs
totheGTPasesuperfamilyof
intracellularswitchproteins.
Rasproteinsare“switch”proteins
thatalternatebetweenanactive“on”
statewithaboundGTPandan
inactive“off”statewithaboundGDP’
Rasactivationisacceleratedbya
guaninenucleotideexchangefactor
(GEF),whichbindstotheRas∙GDP
complex,causingdissociationofthe
boundGDP
Thiskinasecascadeculminatesin
activationofcertainmembersofthe
MAPkinasefamily,whichcan
translocateintothenucleusand
phosphorylate many different
proteins.

84. Recruitmentofthe
Grb2adaptorprotein
toPTresiduesofIRS
viaSH2domains.
(Grb2bringsIRSand
Sostogether)
Grb2adaptor,viaits
SH3domainbindsto
proline-richregionof
Sos.

85. STEPS
LigandinduceddimerizationandautophosphorylationofcytosolicdomainofINS-R
PhosphorylationofIRSbyactiveINS-R
RecruitmentoftheGrb2adaptorIRSviaSH2domains.
Grb2bindstoSosviaSH3domains.
WhenboundtoGrb2,SosactsasaGEF,replacesboundGDPwithGTPonRas.
ActiveRas-GTPrecruitstheproteinRaftothemembrane,whereitisactivated.
Raf-1 phosphorylatesMEK on two Ser residues, activating it.
MEK phosphorylatesERK on a Thrand a Tyr residue, activating it.
Once activated, MAPK undergoes nuclear translocation, where it phosphorylates
transcription factors.

86. Raspromotestheformationofasignaltransductioncomplex,containing
threesequentiallyactingproteinkinases,atthecytosolicsurfaceofthe
plasmamembrane.
•RascanexistineithertheGTP-bound(active)orGDP-bound(inactive)
conformation.
•WhenGTPbinds,Rascanactivateaproteinkinase,Raf-1thefirstof
threeproteinkinases—Raf-1,MEK,andERK—thatformacascadein
whicheachkinaseactivatesthenextbyphosphorylation.
•TheproteinkinasesMEKandERKareactivatedbyphosphorylationof
bothaThrandaTyrresidue.
•Whenactivated,ERKmediatessomeofthebiologicaleffectsofinsulinby
enteringthenucleusandphosphorylatingtranscriptionfactors,suchas
Elk1,thatmodulatethetranscriptionofabout100insulin-regulatedgene.

87. TheproteinsRaf-1,MEK,andERKaremembersofthreelarger
families,forwhichseveralnomenclaturesareemployed.
ERKisintheMAPKfamily(mitogenactivatedproteinkinases;
mitogensareextracellularsignalsthatinducemitosisandcell
division).
KinasesintheMAPKandMAPKKKfamiliesarespecificforSeror
Thrresidues,and
MAPKKs(here,MEK)phosphorylatebothaSerandaTyrresiduein
theirsubstrate,aMAPK(here,ERK).
Thetargetofphosphorylationisoftenanotherproteinkinase,which
thenphosphorylatesathirdproteinkinase,andsoon.
The result is a cascade of reactions that amplifies the initial signal
by many orders of magnitude

88. Regulation of Insulin signaling
IRsignalingisregulatedinavarietyofways.Twoproteintyrosine
phosphatases(PTPases)dephosphorylatetheIR,terminatinginsulin
actionwithoutdegradingtheIR.
Cell-surfaceIRdensityisregulatedbyadditionfromtheGolgiapparatus
andinsulin-stimulatedIRendocytosis.
IRserinephosphorylationalsocontributestothenegativeregulationof
IRsignaling.

89. Diabetes Mellitus
Theword‘diabetes’-derivedfromaGreekword,meaningtosiphonand
referstothemarkedlossofwaterbyurination,polyuria.
Theword‘mellitus’derivedfromtheLatinmeanssweetandthus
diabetesmellitusisknownassweeturinedisease.
Groupofmetabolicdiseasescharacterizedbyincreasedlevelsof
glucoseintheblood(hyperglycemia)resultingfromdefectsininsulin
secretion,insulinaction,orboth

90. Diabetes mellitus is classified into four broad categories:
Type1
Type2
Gestationaldiabetesand
Impairedglucosetoleranceandprediabetes
Person has high blood sugar.
Thishighbloodsugarproducestheclassicalsymptomsof
Glycosuria,
Polyuria(frequenturination),
Polydipsia(increasedthirst)and
Polyphagia(increasedhunger).

91. Diabetictissuedamageincludes
‘Microvascularcomplications’(e.g.Neuropathy,Retinopathyandnephropathy),
‘Macrovascularcomplications’(CHD,CVD,PVD,strokeandrenalarterystenosis)
and
Complications

92. Insulinoma
Adenomaofisletsoflangerhans
Increasedinsulinsecretion
Features:neuropsychiatricsymptoms,nervousness,confusionand
hypoglycemicattacks
Increased Insulin Level

93. Insulin shock
High level of insulin.
•Fall in blood glucose level.
•CNS depression.
•50-70 mg/dl CNS excitability
•20-50 mg/dl CONVULSION & COMA
•< 20 mg/dl COMA

94. What we have learnt…

96. References
AdultandPediatricEndocrinology,Volume2-LarryJameson,7
th
edition
Biochemistry-U. Satyanarayana
Human Endocrinology, Paul R. Gard
•Diabetes Mellitus: A Fundamental and Clinical Text-Derek LeRoith,
Simeon I. Taylor, Jerrold M. Olefsky
PrinciplesofBiochemistry–AlbertLehninger,7
th
edition
PrinciplesofMammalianBiochemistry-AbrahamWhite,EmilSmith,Philip
Handler.7
th
edition
Slideshare

97. Thank You