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  • 8/10/2019 Normal Cell Physiology, Cell Growth & Cell Metabolism

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    NORMAL CELL PHYSIOLOGY CELL DEVELOPMENT

    Surviving cells: the final stage of

    Cell growth (mass accumulation - size & maturity)

    Cell proliferation (cell division)

    Cell remodelingHomeostatic mechanism to maintain cell number and sizeTo preserve organ size & function

    A. Cell Growth & Cell Proliferation

    i. Cell growth vs cell proliferation

    Cell growth can be determined by its sizeCell size is the quality associate with cell function (multicellular) & fitness (unicellular)

    As cells grew larger, passive diffusion may limiting factor for cell growth

    Decrease surface are to volume; nutrient uptake as limiting factor

    Cell proliferationmultiplying the cells

    ii. Size homeostasis in proliferating cells coordination of cell growth & cell division

    Cell growth & cell division are tightly controlledCell will double its mass before enter cell divisionCell growth occur exponentially

    Bigger cells will add more mass over time compared to small cells

    Cell size checkpoint to limit the divergence of cell size

    Coordination relies on the stability & complex balancePositive & negative regulatory stimuliTo maintain tissues with different proliferative rate & metabolic activities (complex organism)

    Four coordination mechanisms

    Dependency of cell cycle to cell growth (Cell division will not be initiated until a cell reached certain size)

    Dependency of cell growth to cell cycle

    The coordinate control of growth & cell cycle progression

    Complete intertwining of growth & cell cycle progression

    Dependency of cell cycle to cell growth (Cell division will not be initiated until a cell reached certain size)Size requi rements for m ajor cel l cycle transi t ion

    Important to reach optimum size-maintain the cell size over generations (avoid cells become progressively small)

    Critical cell size thresholdDeprivation of nutrient & growth factor block cell growthcell arrest at G0 phase

    Cause lengthening time for G1 phase [cells require longer time to achieve standard size at G1 phase)Abundant nutrient, hyperactivation of growth regulation pathways drive cell cycle progression

    Short time of G1 phaseAsymmetric cytokinesis

    Daughter cells must grow until critical cell size threshold S-phase

    Variable G1 lengths while S2/G2/M phase is constantStill, cell growth & cell division can be regulated independently by distinct extracellular signals

    Occur either cells in or out of cell cycleConstant metabolism (making & degrading metabolism) to maintain biological functions

    Progression through cell cycle an ALL-OR-NOTHING, unidirectional process (no turning back)Triggered by threshold level of mitogenic signalingHigh mitogenic stimulationcells in cell cycleToo low mito enic stimulationcells ermanentl withdrawn / out of c cle

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    Both cell grow & cell division require instructive signals

    a) Regulation of cel l size in prol i ferating u nicel lular (yeast)

    Budding yeast (Saccharomyces cerevisiae) as eukaryote model to study coordination cell growth & cell divisionAsymmetric cytokinesis large mother cell & smaller daughter cellCoordination between cell growth & cell cycle occur at STARTSTART: short interval in late G1 phase yeast commit to divisionTo pass START, cell need to obtain critical cell size

    Large mother cells: short G1 phaseIf nutrition deprived, large mother cell will arrest

    Small daughter cells: long G1 phaseSTART ends in S phase entry

    G1/S specific cyclin Cln3; protein encoded by CLN3 geneBudding yeast G1 cyclinControl the timing of startMay be the key regulator; linking the cell growth & cell divisionBind & activate CDK (cdc28)Cln3/Cdc28Cln3 levels are different between the mother & daughter cellsCln3 as critical activator of STARTFunctions:

    a. Induce Cln1 & Cln2 expression by positive feedback loopCln1/2-Cdc28; primary complex that control cell cycle progression in budding yeast

    b. Activation of MBF & SBF transcription factor complexActivation require cells to attain critical sizeUltra-sensitive to Cln3-Cdc28Cln3-Cdc28 phosphorylate Whi-5 (inhibitor) & displace Whi-5 from repressing MBF & SBFTranscription of S-phase genes

    c. As cell size sensorProtein translation rate control STARTCell growth correspond with protein productionCell size sensor should bei. Unstable, levels depend on current translation rate not translation over timeii. Concentration remain constant with growth

    Cln3 as candidate of size sensor proteini. critical activator of STARTii. unstable as it localize in nucleusiii. unlike normal cyclins that will fluctuate, Cln3 expression is continuous throughout the cell cycle especially

    in G1; cell growth can occur continuously (whether in or out of cell cycle)Cln3 is not essential as cells lacking this gene (or low expression) do eventually pass START

    Nutrient modulation of critical size threshold is more significant

    nutrient effects the length of G1 phase (poor nutrient more longer yeast in G1 phase)Allow time for an important response to starvation (such as formation of spore)

    low nutrient quality decrease ribosome biogenesisrepress SBF/MPFLink nutrients to critical cell size threshold

    plasticity in size poor nutrient; smaller critical size

    Enable yeast to compete for limited & fluctuating nutrient resources

    b) Regulat ion of c el l s ize in pro l i ferating m ul t ice llu lar (mammals) & cel l d iv is ion checkpoints

    Cell cycle regulatory principlea. Cyclin & CDKb. Mechanisms of transcriptional controlc. Checkpoint signaling

    2 crucial aspects of cell cycle regulation to control proper cell divisiona. DNA structure checkpoints arrest cell cycle in response to DNA damage or incomplete replicationb. Restriction pointcommitment point; cell commit to enter cell cycle & progress through it

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    i) Commitment point in cell cycle progression: RESTRICTION POINT (R-POINT)

    Occur at G1/S checkpoint

    when the cell commit to enter cell division, G1/S phase transition

    the point of no return (unidirectional)all or nothing

    cell mass & cell signaling (growth, mitogenic & survival signals) affect the decisionMulticellular: Adult cells

    terminally differentiated cells (at G0, quiescent phases) specialized cells continue to divideDecision of cells to exit the cell cycle as G0 or to reenter the cell cycle in G1 phaseDecision made before S-phase; at restriction point. A point at G1

    Determined by extracellular signals (promotional & inhibitory signals)MAPK growth pathway

    Mechanism at R-point: activating cyclin-CDK dependent transcription (G1-S transcriptional activation)Depends on E2F transcription factorsG1 phase regulator: CyclinD-Cdk4/6G1-S phase regulator: CyclinE-Cdk2 (all-or-nothing switch)

    Early G1E2F repressed by pRB at promoter to repress activation

    Mid G1Cyclin D/Cdk4/6 expression increase (response to mitogen stimuli)

    (MAPK associate with growth pathways)Remove inhibition of Cyclin E/Cdk2 (G1-S phase regulator)Cyclin G1/Cdk phosphorylates pRB (inhibitors)pRB detached from E2F (tf)E2F activatedtranscription S-phase essential gene

    Late G1Expression of S-phase geneCell become committed to cell division

    Cells starved of serum before the restriction point enter a G0-like state,while cells starved after R are unaffected and continue through mitosis

    ii. Regulation of eukaryote cell division (DNA STRUCTURE CHECKPOINTS)

    Important to maintain genomic stabilityTo trigger processes to eliminate severely damaged or high risk cells from dividing poolsa) G1/S checkpoint Restrict damage cells from entering S-phase Hold cells at G1/S boundary until DNA damage & high risk factors are remove Severe damageapoptosis & senescence to eliminate the cells from cycling pool

    b) G2/M checkpointCheck for DNA damage (replication damage) before entry of M-phase Prevent premature cell to enter M-phase minimize chromosome segregation errors Cell ready for chromosome segregation at M-phase

    c) Mitotic spindle checkpoint Chromosome aligned correctly at spindle

    Segregationdaughter cells have correct equal chromosome numbers Minimize chromosome segregation errors

    (Regulation at cell cycle note)

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    B. Loss of Homeostasis Control

    i. Cell Death

    a) Progr ammed Cel l Death

    Damaged cellsCell cycle arrest & DNA repairCells that cannot be repaired (damage beyond repair) programmed cell deathGene encoded suicidal programInvolve biochemical events characteristic cell changes & death

    PCD & Cell PhysiologyMain role of PCDto balance cell death with survival of normal cellsAbnormal regulation wide variety of human diseases: immunological, developmental disorders, neurodegeneration,and cancer.

    a) PCD role in development- Complex & involve vast number of cells- PCD in human development from inner cell mass differentiation to maintenance of tissue homeostasis in adulthood- Apoptosis inhibition developmental abnormalities & pathologies (cancer & degenerative diseases)- PCD in neural development

    PCD is an adaptive mechanism to regulate the number of progenitor cells. Observed in post-mitotic cells

    PCD is identified in germinal areas of brain parts such as cerebral cortex & spinal cord. To optimize the connection between neurons and their afferent inputs and efferent targets To correct for errors in neurons that have migrated at abnormal, innervated incorrect targets, or have axons

    that have gone awry during path finding

    b) Sculpting structures- Organogenesis & tissue remodeling- Formation of digits (fingers) in higher vertebrates (human)PCD eliminates the interdigital web especially via apoptosis- Conversion of solid structures into hollow tube Lumina such as proamniotic cavity

    c) Deleting structures- Delete various structures that have transient function

    - EmbryogenesisDeletion of Mullerian duct in males & Wolffian duct in females

    d) Regulating cell numbers- In developing tissues & organs: Balance between cell division & PCD to get appropriate cell numbers- In nervous, immune & reproductive system, PCD removed overproduced cells

    PCD discards 80% oocytes before birth- Competition for limiting amounts of survival signals to match the numbers of different cell types in a tissue- Competition between cells that proliferate at different rate growth homeostasis (eliminate slower dividing cells)

    e) Elimination of unwanted & potentially dangerous cells- PCD as protective process (development & adult life)

    During B & T cell lymphocytes selection: PCD eliminates self-reactive cells to prevent autoimmunity Eliminate cells in response to viral infection, unrepaired DNA damage, cell cycle perturbation, fate &

    differentiation defects

    f) For survival- Cell degradation by PCD type II (Autophagy) during severe nutrient stress- To get the amino acids for metabolism homeostasis

    3 main forms of PCD1) Apoptosis2) Autophagy3) Programmed Necrosis

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    Type 1 ApoptosisInternally programmed cell death modeMajor mode of cell deathPhysiological response to specific suicide signals or lack of survival signalsOccurs normally during development & agingAs homeostatic mechanism to maintain cell populations in tissueAs defense mechanism (immune reactions, cell damage)

    Morphological Changes Biochemical ChangesCell shrinkageDense cytoplasm & organelles are more tightly packedNuclear condensation & fragmentationDynamic membrane blebbingLoss of adhesion to neighbours or to extracellular matrixFormation of small fragmented apoptotic bodies(have cytoplasmic contents enclosed in cell membrane-maintain organelle integrity)Apoptotic bodies phagocytosedNo inflammatory reaction

    Chromosomal DNA cleavage into internucleosomalfragmentsDNA breakdownPhosphatidylserine externalizationIntracellular substrate cleavages by specific proteolysisProtein crosslinkingActivation of caspases proteolytic activityExpression of cell surface markers early phagocyticrecognition

    Mechanism: Initiation MediationExecutionRegulation of apoptosisTwo major pathways:

    Extrinsic pathway (death receptor pathway) Caspase-dependence apoptosis Activated by apoptotic stimuli Induced by extrinsic signals such as binding of death inducing ligands to cell surface receptor

    (Transmembrane receptor-mediated interactions)

    Death Receptors Dependence Receptor Signaling

    Ligand binding to cell-surface membranereceptors initiate signaltransduction

    Type II cells: cells with smallamount of FASS & Caspase 8Hepatocytes & pancreatic cells

    Depend on ligand availabilityLigand binding to receptorCell survivalAbsence of molecule binding receptor ApoptosisLigand deprivation-induced dependence receptorsignaling

    Receptor:Patched & DCCUNC5B

    FasL/CD95LFAS/CD95 (receptor)

    TNFa & TNF ligand TNFa receptorTNF-related apoptosis inducing ligand (TRAIL) TRAIL receptor

    Death Receptor Signaling

    FasL bind to FAS/CD95 oligomerization

    Receptor interacting protein kinase 1 (RIP-1) phosphorylate FASreceptorActivate

    Activated FAS recruits

    Fas-associated protein with death domain (FADD)

    cellular inhibitor of apoptosis proteins (CIAP)

    c-FLIPS

    Procaspase-8

    Form Death Inducing Signaling Complex (DISC)

    activation of pro-caspase 8 Caspase 8Active Caspase-8

    Type I cells Type II cells Activate IntrinsicPathway

    Dependence Receptors:UNC5B

    Receptor: Patched& DCC

    Catalyze proteolyticmaturation of caspase-3

    Mediate protein cleavage of BH3-interacting domain death agonist

    (BID)

    Removal of netrin-1 fromUNC5B receptor

    Removal of ligand

    Active Caspase-3 Truncated BID form mitochondrialpermeabilizing fragmentform

    channel at mitochondria membrane

    Recruit Death associatedprotein kinase 1 (DAPK-1) &

    Protein phosphatase 2A(PP2A)

    RecruitCytoplasmic

    adaptor protein(DRAL)

    Executioner phase ofcaspase dependent

    Mitochondrial Outer Membrane Permeability (MOMP)

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    Release Cytochrome C

    Cytochrome C + Apoptotic protease activating factor 1 (APAF-1)

    Form ApoptosomeCaspase 9 activating multiprotein complex

    Active Caspase-9

    Catalyze proteolytic maturation of caspase-3

    Active Caspase-3

    Executioner phase of caspase dependent

    APOPTOSIS

    Intrinsic Pathway (mitochondrial-dependent) Caspase dependent pathway Caspase independent pathway Signals:Intracellular stress conditions: DNA damage, oxidative stress, cytosolic Ca2+ overload,

    accumulation of unfolded proteins in the endoplasmic reticulum Pro-apoptotic signals > Anti-apoptotic signals (released to cope with stress) cause apoptotic intrinsic pathway

    Intracellular stress: DNA damage

    Activate B cell lymphoma 2 (BCL-2)

    Activation BCL-2-associated X protein (BAX) and BCL-2 antagonist or killer (BAK)

    BAX and BAK and truncated BID (from extrinsic pathway-Type 2 cells) insert & permeabilize the outer mitochondrial

    membrane.Mitochondrial Outer Membrane Permeabilization (MOMP) become irreversible

    Opening of Permeability Transition Pore Complex (PTPC)

    Mitochondria release transmembrane space (IMS) toxic protein to cytosol

    Caspase dependent Caspase independent

    Cytochrome C DIABLO/SMAC(Second mitochondria

    derived activator ofcaspases)

    High temperaturerequirement protein

    A2 (HTRA2)

    Apoptosisinducing

    factor

    Endonucleas G(ENDOG)

    Cyt C bind to Apoptoticprotease activating factor 1

    (APAF-1)

    Promote Inhibit inhibitors of apoptosis protein(IAP)

    Relocating nucleusMediating large scale DNA

    fragmentation

    Form Apoptosome in the

    presence of dATP

    And cleave cellular

    subsrate:cytoskeletal protein,

    mitochondrialmembranes

    Apoptosome recruitsprocaspase-9

    Activation of caspase-9

    Caspase-9 catalyze proteolytic maturationof caspase-3

    Active Caspase-3

    Executioner phase of caspase dependent

    APOPTOSIS

    Type 2 Autophagy (Self-Canibalization) Catabolic mechanism involves cell degradation of unnecessary or dysfunctional cellular components

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    Actions of lysosome Crucial pro-survival role in cell homeostasis During periods of starvation or stress due to growth factor deprivation

    Autophagydegradation & recycling of cellular components

    MechanismsFormation of autophagosomes (double membrane-bound structures)Surround cytoplasmic membranes & organelles

    Massive cytoplasmic vacuolization with increased autophagy fluxFusion with lysosome autophagolysosomeDegradation of contents

    Type 3 Regulated Necrosis (Necroptosis) Necrosis: accidental death mechanism Absence of morphological trait of apoptosis

    or autophagy Can occur in regulated manner Triggers: alkylating DNA damage, excitotoxins &

    ligation of death receptors, under selectedconditions

    Necroptosis in neurodegeneration & cell deathIfnflicted with ischemia & infection

    b) Unprogrammed Cel l Death - Patholo gical Necrosis

    Accidental death mechanismUnordered & passive cellular explosionIn response to acute & overwhelming trauma(extreme variance from physiological conditions)

    Hypothermia, hypoxia, viral infection Damage to plasma membrane

    Not associate with activation of caspase

    MechanismsImpairment of cells ability to maintain homeostasis

    Water influx & extracellular ionsCell swelling - Swelling of organelles (mitochondria & ER)Plasma membrane rupturerelease cell contentsTotal cell lysisSwelling nucleus but intactRelease factors and ATP from damaged cellsTrigger inflammasomeInflammatory response

    ii. Cell Aging (+ Telomerase shortening)

    Telomeres repeating DNA sequenceCapping the end of every linear chromosomes

    Telomere & Cell DivisionAntiparallel DNA replication during S-phase

    DNA polymerase require 3end donorPolymerization initiated by RNA primerLagging strand: 53 require Okazaki fragments (RNA primers)(provide free 3end for polymerization)At the very end of chromosomeLagging strand: empty space at 5endTemplate strand: free 3 end

    Newly synthesized DNA strand is shorter than the originaltemplate at 5end

    Protect chromosomes from fusing with each other orrearrangingProtect genetic information from damage

    Telomeres shorten with each mitotic celldivisioncause cellular senescence (stop doubling)Hayflick limit: Cell stop divide after 40-60 division

    NORMAL CELL PHYSIOLOGY CELL METABOLISMTo regulate cell growth

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    Balance between rates of accumulation of macromolecules (by synthesis & uptake) and their loss (by degradation& secretion) determine the cells size & growth rate

    Vary depend on the respond of changing levels of growth factor signalingHomeostasis between macromolecule synthesis (anabolic) and degradation (catabolic)Require mechanism to sense nutrients & oxygen

    Four major signals that can affect cell growth: stress, energy status, oxygen and amino acid levels

    Energetic & stress signals regulate growth factor signaling

    Two key molecules for sensing

    o AMP-activated kinase (AMPK)o Mammalian target of rapamycin complex 1 (MTOR1)

    A. Growth pathways: IGF1/PI3K/AKT/mTORC1 pathway controlling growth rateMajor regulator of cell growth & determine cell sizeBinding IGF1 to its receptor activate PI3K/AKT/mTORC1 pathwaymTORC integrates inputs from 4 major signals

    Acts as signaling nodeenergetic & stress signals modulate growth factor signalingActivation of this pathwayProtein synthesis > DegradationStimulate fatty acid intakeGlucose import & synthesis of glycogenMacromolecular synthesis

    B. Mechanism of nutrient sensing

    i. Energy sensing by AMPKAMPK is a sensor of energy status to maintain homeostasisEnergy production: ATP *hydrolyse ADP / AMPThe ratio of ADP & AMP acts as barometer of cellular energy statusHowever, ADP concentration > AMP; ADP level will be main energy barometer (ADP > AMP)Under most situations ADP will bind to AMPK than AMPAt severe stress, AMP will be main barometer (AMP > ADP)High energy use, low ATP, high ADP@AMP activate AMPKUpregulate ATP production pathways (maintain homeostasis)

    AMPKAMP-activated kinaseA heterotrimers

    Catalytic subunit, : contain typical serine/kinase domainActivation loop conserved threonine residues: Thr-172

    2 regulatory subunits: and subunit has nucleotide phosphate binding sites (ATP,ADP & AMP binding site)

    Regulation of AMPKAt normal energy requirement: ATP bind to -subunit & inactivate AMPKAt stress (low energy): ADP/AMP replace ATP & bind to -subunitCause conformational change & activate AMPK

    [AMP bind conformational change AMPK activation (3 mechanisms)]

    There are 3 independent mechanisms to promote AMPK activation byADP/AMP binding

    1. promotion of Thr-172 phosphorylation2. inhibition of Thr-172 dephosphorylation by phosphatase3. allosteric activation of AMPK already phosphorylated on Thr-172

    Among the 3 mechanisms, mechanism 2 is most CRUCIAL for AMPK activationby AMP or ADPUnder severe energy stress, AMP become essential regulatorMechanism 3 AMP specific ability allosteric activation

    >200 fold of AMPK activation compared 10 fold activationat normal energy stress by AMP

    Increase fold activation of Thr-172 phosphorylation

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    3 mechanismsAMPK become ultrasensitiveSlight change of AMP/ADP will produce large relative change in the final output

    The major upstream kinase phosphorylating Thr-172: Serine-threonine liver kinase B1 (LKB1)Ca2+/calmodulin-dependent protein kinase (CaMKK) also phosphorylate Thr-172

    Ca2+ activated pathway swith on AMPK

    ii. Amino acid sensing by mammalian target of rapamycin (MTOR)Amino acid building blocks of protein, essential for synthesis of nucleic acid, glucose & ATPmTORConserved central cell-growth modulatorSerine/threonine kinaseTightly regulated by amino acid availabilityExist in 2 different complexes: mTORC1 & mTORC2

    mTORC1 sensitive to amino acids mTORC21 has accessory protein - Raptor use amino acids to synthesis nucleic acid & protein this anabolic pathways require large amount of energy

    mTORC1 is activated when the cells adequate energy (ATP) nutrient availability

    oxygen abundance proper growth factor

    Active mTORC1 upregulate protein synthesis pathwaysfor cell growth & proliferationActivation mTORC1 favour anabolism

    RegulationAmino acidactivate Rag proteinsto form heterodimers

    promoting GTP/GDP binding to the protein complexActive RagA/Cheterodimer physically interact with mTORC1 via Raptor

    Active Rag recruit mTORC1 to surface of lysosomeGrowth factor signalsfrom Igf/PI3K/AKT

    AKT phosphorylate TSC1/TSC2 (negative regulator of mTORC1)

    Alleviate the inhibitory effect by TSC on mTORC1TSC1/TSC2 regulate the GTPase RhebGTPase Rheb activate mTORC1 at lysosome

    activate anabolic pathwaysProper mTORC1 activation require amino acids & proper growth factor.Amino acid deficiency: activate General Control Nonderepressible-2 (GCN-2)kinase & regulate mTORC1 inhibition

    iii. Oxygen sensing by prolyl hydroxylase domain protein (PHD)

    Cells receive O2 and generate energy aerobicallyIn addition to nutrient & energy: O2 is also critical for cell growth & cellular activityThe challenge of fluctuating O2 levelsIschemic disease (imbalance in O2 demand & supply)

    In healthy conditions: not all cells exposed to same level of O2Significant differences in partial pressure of PO2 (distinct anatomical sites & physiological conditions) kidney medulla & bone marrow has low PO2 (1025 mmHg) towards inside of solid tumour (extreme hypoxia

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    PHD use molecular oxygen (OH) and a-ketoglutarate to hydroxylate the prolyly residues in HIF-1a & inhibit HIF-1a Prolyl is the activating subunit of the HIF heterodimeric

    Hydroxylated HIF-1a is recognized by von Hippel-Lindau tumour suppressor protein. Ubiquitination by VHL degrade hydroxylated HIF-1a

    High O2 level; HIF-1a is rapidyly hydroxylated by PHD & degraded by VHL+E3 ubiquitin

    In hypoxia: lack of O2 (cofactor) Inhibit PHD activityHIF-1a is not be hydroxylated & stabilizedNon-hydroxylated HIF-1a accumulationActivate transcriptional program to prepare cells to adapt the hypoxia conditionHIF1 (heterodimeric protein)two subunits

    HIF1, the expression of which is stabilized byhypoxia

    HIF1, which is constitutively expressedWhen HIF1 associates with HIF1, they form a transcriptionfactor that binds and activates the promoters of multipleglycolytic genes

    C. Regulation of Metabolism

    AMPK mTORC1 PHD

    Energy stress sensor Nutrient availability sensor Oxygen sensor

    Low ATP, high ADP/AMP (Low energy)Activate

    Nutrient (amino acid) sufficient &growth factorActivate

    High O2Activate

    Activate catabolic pathways Produce ATPInhibit anabolic pathways & mTORC1

    Activate anabolic pathways Protein synthesis utilizing ATP

    Hydroxylate & inhibit HIF-1a

    Normal energy level:ATP bind to Y-subunit of AMPK

    Nutrient & energy stress:Inhibited by AMPK

    Hypoxia:Inhibited by low O2Non-hydroxylate HIF-1aactivate

    Regulation by AMPK - Metabolism

    (i) Glucose homeostasisAMPK switch ON catabolic pathways that generate ATP AMPK switch OFF anabolic pathways that use ATP

    Upregulation of catabolism pathways

    Glucose uptakevia activation of both GLUT1 &

    GLUT4Up-regulate GLUT4 + activation of HIF-1Translocation of existing transporters to the plasmamembraneProbably due in part to phosphorylation of the Rab-GAP proteinTBC1D1 by AMPK

    A longer-term effect on transcription of the GLUT4 geneProbably due in part to phosphorylation of histone deacetylase-5

    Glycolys isvia phosphorylation and activation of two offour isoforms of 6-phosphofructo-2-kinase (PFK-2), whichsynthesizes the glycolytic activator fructose-2,6-bisphosphateEnhancing mi tochon dr ia l b iogenesis

    Energy factory

    increase energy through oxidativecatabolism; promote activation of mitochondrial genes

    Downregulation of anabolism pathways

    Inh ib i t g lycogen synthesisphosphorylation of

    glycogen synthase (GS)Inh ib i t g lucon eogenesisrepress glucose-6-phosphatase & phosphoenolpyruvate carboxykinasephosphorylation of CRTC2 & class IIA histonedeacetylases

    mTORC1 inactivated by AMPKLow ATP by mitochondrial inhibition also blocks mTORC1activation & lysosomal localization

    mTOR regulationHyperactivation mTORC1Phosphorylation of S6 kinase 1 & Grb10 (downstreamproteins) reduce insulin receptor substrate (IRS1) increase insulin sensitivity resistant of insulin signaling

    reduce glucose uptake & glycogen synthesisIncrease gluconeogenesis

    (ii) Lipid homeostasisAMPK switch ON catabolic pathways that generate ATP AMPK switch OFF anabolic pathways that use ATP

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    Activation of fatty acid oxidation

    Fatty acid uptakevia translocation of the fatty acidtransporter FAT/CD36Fatty acid -oxidationvia phosphorylation of theACC2 isoform of acetyl-CoA carboxylase, thus loweringmalonyl-CoA, an inhibitor of fatty acid uptake intomitochondria

    Inhibition of lipogenesis

    Inh ib i t fa t ty acid synthesisphosphorylation of acetyl coA carboxylase 1 (ACC1)phosphorylation of the transcription factor SREBP-1cinhibit proteolytic processing to the active, nuclear formInh ib i t isoprenoid synthesisphosphorylation ofHMG-CoA reductase,

    Inh ib i t tr ig lycer ide and p hospho l ip id synthesis

    inactivation of glycerol phosphate acyl transferase

    mTORC1 inactivated by AMPKSREBP1/2 low expression Decrease lipogenic geneexpression

    mTOR regulationLipid also a building material for membrane cellgrowth alongside with proteinPromote lipid synthesis for cell growthmTOR promote function SREBP transcription factorpromote peroxisome proliferator-activated receptor (PPAR): key regulator of lipid uptake & adipogenesis

    (iii) Protein homeostasisAMPK switch ON catabolic pathways that generate ATP AMPK switch OFF anabolic pathways that use ATP

    mTORC1 inactivated by AMPK

    Under severe energy / nutrient stresscells undergo autophagy (intracellular degradation)indirectly induced by AMPK by inhibiting mTORC1AMPK phosphorylate & activate autophagy essentialkinase ULK1

    Protein catabolismbreakdown of protein to amino acidsamino acids may be used in Krebs cycle to produceATP & recycled to make new amino acids

    Inh ib i t prote in synthesis

    Switch off mTORC1AMPK phosphorylate & activate TSC2 (mTORC1 negativeregulator)Phosphorylate Raptor & inhibit Raptor interaction withmTORC1

    mTORC1 regulationpromote protein synthesis (anabolism) by 4 process1. amino acid synthesis2. RNA synthesis3. transcription4. translationmTORC1 inhibit autophagy

    Metabolic adaptation to hypoxia [aerobic metabolism anaerobic metabolism]HIF-1a activation: switch on anaerobic glycolysis

    Induce expression of glycolytic genes: cell use oxygen independent glycolysis to produce energy to maintain essentialcellular activity

    glucose transporters (GLUT1, GLUT3 & GLUT4): increase glucose uptake hexokinases lactate dehydrogenases

    PyruvateLactate (produce ATP): to compensate loss of mitochondrial ATP productionOxygen conservation in mitochondria

    blocking of pyruvate from glycolysis to enter mitochondria (into TCA cycle) by pyruvate dehydrogenase kinase(PDK)

    reduce mitochondrial biogenesis / increase autophagy to lower the amount of mitochondria Fumarate as electron acceptor (substitute O2) in electron transport chain

    Stimulate expression of genes (VEGF) to promote blood vessel growth (angiogenesis)

    To increase O2 supply to hypoxic tissueInhibit mTORC1 to reduce ATP-consuming protein synthesis & increase autophagy

    D. Metabol ic regulat ion & cel l physio logy

    i. Factors determine the metabolic requirementsa) Cell functionb) Microenvironmentii. Cell physiology during complete nutrient deprivation - Ketosis

    E. Proliferating cells metabolism

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    i. Normal glucose metabolismControl system to prevent exceed nutrient availability for cell divisionGrowth factors regulate nutrient uptake of cells from extracellular environment\

    Under aerobic conditons + quiescent normal cellsGlucose[glycolysis]pyruvateacetyl-coA + CO2Acetyl-CoA[mitochondrial TCA cycle]ATPs & NADHNADH[mitochondrial oxidative phosphorylation]maximize ATP production (36 ATPs per one glucose oxidation)

    Anaerobic glycolyisGlucosePyruvateLactate (Lactic acid fermentation)(2 ATPs per one glucose oxidation)

    ii. Proliferative metabolismAerobic glycolysis

    A phenomenon known as Warburg EffectRegardless of the availability of O2 & nutrients, aerobic and anaerobicGlucose metabolismrely on AEROBIC GLYCOLYSIS

    High production of lactate [ GlycosePyruvateLactate ]Lactic acid fermentation

    a) Normal proliferative cells

    GlucosePyruvateLactate (Lactic acid fermentation)(2 ATPs per one glucose oxidation)For proliferative cells; aerobic glycolysis occurs (although with sufficient nutrients) proliferative metabolismGrowth signals (via PI3/AKT pathways) regulate the proliferative metabolism

    b) Cancer cellsWarburg effects: A cancer phenotype by proliferation-induced oncogeneAerobic glycolysis is common in cancer cellsWarburg effects occur

    1. Due to adaptation to hypoxia environment within tumourHypoxia response system: upregulate glucose transporter & multiple enzymes of glycolytic pathways

    2. Damage to mitochondria (evading apoptosis)impairing oxidative phosphorylation & low ATP productionStill, mitochondria can remain functional with some oxidative phosphorylation continues

    Genetic mutations altering the receptor-initiated signaling pathwaysClonal expansion of cells that acquire this phenotypeOncogenes & mutation on tumour suppressor genes in altered energy metabolism

    Associate with hallmark capabilities of cell proliferation, avoidance of anti-growth signals & evading apoptosis

    Self-sufficiency of growth signals (independence from growth factor signals + produce own signals)Uncontrolled uptake and metabolism of nutrients exceed demands for cell survival & fuel cell growthExceed glucose >> demand also drive oncogenic mutations

    Increased glycolysis produce intermediates that involve in various biosynthetic pathways nucleosides & amino acidssynthesis