NORMAL CELL PHYSIOLOGY CELL DEVELOPMENT

Surviving cells: the final stage of

Cell growth (mass accumulation - size & maturity)
Cell proliferation (cell division)
Cell remodeling
Homeostatic mechanism to maintain cell number and size
To preserve organ size & function
A. Cell Growth & Cell Proliferation
i. Cell growth vs cell proliferation
Cell growth can be determined by its size
Cell 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
Occur either cells in or out of cell cycle
Constant metabolism (making & degrading metabolism) to maintain biological functions

Cell proliferation multiplying the cells

Progression through cell cycle an ALL-OR-NOTHING, unidirectional process (no turning back)
Triggered by threshold level of mitogenic signaling
High mitogenic stimulation cells in cell cycle
Too low mitogenic stimulation cells permanently withdrawn / out of cycle
ii. Size homeostasis in proliferating cells coordination of cell growth & cell division
Cell growth & cell division are tightly controlled
Cell will double its mass before enter cell division
Cell 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 balance

Positive & negative regulatory stimuli
To 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 requirements for major cell cycle transition
Important to reach optimum size-maintain the cell size over generations (avoid cells become progressively small)
Critical cell size threshold
Deprivation of nutrient & growth factor block cell growth cell 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 phase
Asymmetric cytokinesis
Daughter cells must grow until critical cell size threshold S-phase
Variable G1 lengths while S2/G2/M phase is constant
Still, cell growth & cell division can be regulated independently by distinct extracellular signals

Both cell grow & cell division require instructive signals

a) Regulation of cell size in proliferating unicellular (yeast)
Budding yeast (Saccharomyces cerevisiae) as eukaryote model to study coordination cell growth & cell division
Asymmetric cytokinesis large mother cell & smaller daughter cell
Coordination between cell growth & cell cycle occur at START
START: short interval in late G1 phase yeast commit to division
To pass START, cell need to obtain critical cell size
Large mother cells: short G1 phase
If nutrition deprived, large mother cell will arrest
Small daughter cells: long G1 phase
START ends in S phase entry
G1/S specific cyclin Cln3; protein encoded by CLN3 gene
Budding yeast G1 cyclin
Control the timing of start
May be the key regulator; linking the cell growth & cell division
Bind & activate CDK (cdc28) Cln3/Cdc28
Cln3 levels are different between the mother & daughter cells
Cln3 as critical activator of START
Functions:
a. Induce Cln1 & Cln2 expression by positive feedback loop
Cln1/2-Cdc28; primary complex that control cell cycle progression in budding yeast
b. Activation of MBF & SBF transcription factor complex
Activation require cells to attain critical size
Ultra-sensitive to Cln3-Cdc28
Cln3-Cdc28 phosphorylate Whi-5 (inhibitor) & displace Whi-5 from repressing MBF & SBF
Transcription of S-phase genes
c. As cell size sensor
Protein translation rate control START
Cell growth correspond with protein production
Cell size sensor should be
i.
Unstable, levels depend on current translation rate not translation over time
ii.
Concentration remain constant with growth
Cln3 as candidate of size sensor protein
i.
critical activator of START
ii.
unstable as it localize in nucleus
iii.
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 biogenesis repress SBF/MPF
Link nutrients to critical cell size threshold
plasticity in size poor nutrient; smaller critical size
Enable yeast to compete for limited & fluctuating nutrient resources
b) Regulation of cell size in proliferating multicellular (mammals) & cell division checkpoints
Cell cycle regulatory principle
a. Cyclin & CDK
b. Mechanisms of transcriptional control
c. Checkpoint signaling
2 crucial aspects of cell cycle regulation to control proper cell division
a. DNA structure checkpoints arrest cell cycle in response to DNA damage or incomplete replication
b. Restriction point commitment point; cell commit to enter cell cycle & progress through it

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 decision
Multicellular: Adult cells
terminally differentiated cells (at G0, quiescent phases)
specialized cells continue to divide
Decision of cells to exit the cell cycle as G0 or to reenter the cell cycle in G1 phase
Decision 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 factors
G1 phase regulator: CyclinD-Cdk4/6
G1-S phase regulator: CyclinE-Cdk2 (all-or-nothing switch)
Early G1
E2F repressed by pRB at promoter to repress activation
Mid G1
Cyclin 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 activated transcription S-phase essential gene
Late G1
Expression of S-phase gene
Cell 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 stability
To trigger processes to eliminate severely damaged or high risk cells from dividing pools
a) 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 damage apoptosis & senescence to eliminate the cells from cycling pool
b) G2/M checkpoint
Check 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
Segregation daughter cells have correct equal chromosome numbers
Minimize chromosome segregation errors

(Regulation at cell cycle note)

B. Loss of Homeostasis Control

i. Cell Death
a) Programmed Cell Death
Damaged cells Cell cycle arrest & DNA repair
Cells that cannot be repaired (damage beyond repair) programmed cell death
Gene encoded suicidal program
Involve biochemical events characteristic cell changes & death
PCD & Cell Physiology
Main role of PCD to balance cell death with survival of normal cells
Abnormal 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
- Embryogenesis Deletion 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 PCD
1) Apoptosis
2) Autophagy
3) Programmed Necrosis

Type 1 Apoptosis
Internally programmed cell death mode
Major mode of cell death
Physiological response to specific suicide signals or lack of survival signals
Occurs normally during development & aging
As homeostatic mechanism to maintain cell populations in tissue
As defense mechanism (immune reactions, cell damage)
Morphological Changes
Cell shrinkage
Dense cytoplasm & organelles are more tightly packed
Nuclear condensation & fragmentation
Dynamic membrane blebbing
Loss of adhesion to neighbours or to extracellular matrix
Formation of small fragmented apoptotic bodies
(have cytoplasmic contents enclosed in cell membranemaintain organelle integrity)
Apoptotic bodies phagocytosed
No inflammatory reaction

Biochemical Changes
Chromosomal DNA cleavage into internucleosomal
fragments
DNA breakdown
Phosphatidylserine externalization
Intracellular substrate cleavages by specific proteolysis
Protein crosslinking
Activation of caspases proteolytic activity
Expression of cell surface markers early phagocytic
recognition

Mechanism: Initiation Mediation Execution Regulation of apoptosis

Two 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
Type II cells: cells with small
Ligand binding to cellamount of FASS & Caspase 8
surface membrane
Hepatocytes & pancreatic cells
receptors initiate signal
transduction
FasL/CD95L FAS/CD95 (receptor)
TNFa & TNF ligand TNFa receptor
TNF-related apoptosis inducing ligand (TRAIL) TRAIL receptor
Death Receptor Signaling
FasL bind to FAS/CD95 oligomerization
Receptor interacting protein kinase 1 (RIP-1) phosphorylate FAS
receptor Activate
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 8
Active Caspase-8
Type II cells Activate Intrinsic
Type I cells
Pathway
Catalyze proteolytic
Mediate protein cleavage of BH3maturation of caspase-3
interacting domain death agonist
(BID)
Active Caspase-3
Truncated BID form mitochondrial
permeabilizing fragment form
channel at mitochondria membrane
Executioner phase of
caspase dependent

Dependence Receptor Signaling

Depend on ligand availability
Ligand binding to receptor Cell survival
Absence of molecule binding receptor Apoptosis
Ligand deprivation-induced dependence receptor
signaling
Receptor:
Patched & DCC
UNC5B

Dependence Receptors:
UNC5B
Removal of netrin-1 from
UNC5B receptor

Recruit Death associated

protein kinase 1 (DAPK-1) &
Protein phosphatase 2A
(PP2A)
Mitochondrial Outer Membrane Permeability (MOMP)

Receptor: Patched
& DCC
Removal of ligand

Recruit
Cytoplasmic
adaptor protein
(DRAL)

Release Cytochrome C
Cytochrome C + Apoptotic protease activating factor 1 (APAF-1)
Form Apoptosome Caspase 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
High temperature
Apoptosis
Endonucleas G
(Second mitochondria
requirement protein
inducing
(ENDOG)
derived activator of
A2 (HTRA2)
factor
caspases)
Cyt C bind to Apoptotic
Promote Inhibit inhibitors of apoptosis protein
Relocating nucleus
protease activating factor 1
(IAP)
Mediating large scale DNA
(APAF-1)
fragmentation
Form Apoptosome in the
And cleave cellular
presence of dATP
subsrate:
cytoskeletal protein,
mitochondrial
membranes
Apoptosome recruits
procaspase-9
Activation of caspase-9
Caspase-9 catalyze proteolytic maturation
of 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

Actions of lysosome
Crucial pro-survival role in cell homeostasis
During periods of starvation or stress due to growth factor deprivation
Autophagy degradation & recycling of cellular components

Mechanisms
Formation of autophagosomes (double membrane-bound structures)
Surround cytoplasmic membranes & organelles
Massive cytoplasmic vacuolization with increased autophagy flux
Fusion with lysosome autophagolysosome
Degradation 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 selected
conditions
Necroptosis in neurodegeneration & cell death
Ifnflicted with ischemia & infection
b) Unprogrammed Cell Death - Pathological Necrosis
Accidental death mechanism
Unordered & passive cellular explosion
In response to acute & overwhelming trauma
(extreme variance from physiological conditions)
Hypothermia, hypoxia, viral infection
Damage to plasma membrane
Not associate with activation of caspase
Mechanisms
Impairment of cells ability to maintain homeostasis
Water influx & extracellular ions
Cell swelling - Swelling of organelles (mitochondria & ER)
Plasma membrane rupture release cell contents
Total cell lysis
Swelling nucleus but intact
Release factors and ATP from damaged cells
Trigger inflammasome
Inflammatory response
ii. Cell Aging (+ Telomerase shortening)
Telomeres repeating DNA sequence
Capping the end of every linear chromosomes
Protect chromosomes from fusing with each other or
rearranging
Protect genetic information from damage
Telomeres shorten with each mitotic cell
division
cause cellular senescence (stop doubling)
Hayflick limit: Cell stop divide after 40-60 division

Telomere & Cell Division

Antiparallel DNA replication during S-phase
DNA polymerase require 3end donor
Polymerization initiated by RNA primer
Lagging strand: 53 require Okazaki fragments (RNA primers)
(provide free 3end for polymerization)
At the very end of chromosome
Lagging strand: empty space at 5end
Template strand: free 3 end
Newly synthesized DNA strand is shorter than the original
template at 5end

NORMAL CELL PHYSIOLOGY CELL METABOLISM

To regulate cell growth

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 signaling
Homeostasis 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 rate
Major regulator of cell growth & determine cell size
Binding IGF1 to its receptor activate PI3K/AKT/mTORC1 pathway
mTORC integrates inputs from 4 major signals
Acts as signaling node energetic & stress signals modulate growth factor signaling
Activation of this pathway
Protein synthesis > Degradation
Stimulate fatty acid intake
Glucose import & synthesis of glycogen
Macromolecular synthesis

B. Mechanism of nutrient sensing

i. Energy sensing by AMPK
AMPK is a sensor of energy status to maintain homeostasis
Energy production: ATP *hydrolyse ADP / AMP
The ratio of ADP & AMP acts as barometer of cellular energy status
However, ADP concentration > AMP; ADP level will be main energy barometer (ADP > AMP)
Under most situations ADP will bind to AMPK than AMP
At severe stress, AMP will be main barometer (AMP > ADP)
High energy use, low ATP, high ADP@AMP activate AMPK
Upregulate ATP production pathways (maintain homeostasis)
AMPK AMP-activated kinase
A heterotrimers
Catalytic subunit, : contain typical serine/kinase domain
Activation loop conserved threonine residues: Thr-172
2 regulatory subunits: and
subunit has nucleotide phosphate binding sites (ATP,ADP & AMP binding site)
Regulation of AMPK
At normal energy requirement: ATP bind to -subunit & inactivate AMPK
At stress (low energy): ADP/AMP replace ATP & bind to -subunit
Cause conformational change & activate AMPK
[AMP bind conformational change AMPK activation (3 mechanisms)]
There are 3 independent mechanisms to promote AMPK activation by
ADP/AMP binding
1. promotion of Thr-172 phosphorylation
2. inhibition of Thr-172 dephosphorylation by phosphatase
3. allosteric activation of AMPK already phosphorylated on Thr-172
Among the 3 mechanisms, mechanism 2 is most CRUCIAL for AMPK activation
by AMP or ADP
Under severe energy stress, AMP become essential regulator
Mechanism 3 AMP specific ability allosteric activation
>200 fold of AMPK activation compared 10 fold activation
at normal energy stress by AMP
Increase fold activation of Thr-172 phosphorylation

3 mechanisms AMPK become ultrasensitive

Slight 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 & ATP
mTOR
Conserved central cell-growth modulator
Serine/threonine kinase
Tightly regulated by amino acid availability
Exist 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 pathways
for cell growth & proliferation
Activation mTORC1 favour anabolism
Regulation
Amino acid activate Rag proteins to form heterodimers
promoting GTP/GDP binding to the protein complex
Active RagA/C heterodimer physically interact with mTORC1 via Raptor
Active Rag recruit mTORC1 to surface of lysosome
Growth factor signals from Igf/PI3K/AKT
AKT phosphorylate TSC1/TSC2 (negative regulator of mTORC1)
Alleviate the inhibitory effect by TSC on mTORC1
TSC1/TSC2 regulate the GTPase Rheb
GTPase Rheb activate mTORC1 at lysosome
activate anabolic pathways
Proper 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 aerobically
In addition to nutrient & energy: O2 is also critical for cell growth & cellular activity
The challenge of fluctuating O2 levels
Ischemic disease (imbalance in O2 demand & supply)
In healthy conditions: not all cells exposed to same level of O2
Significant differences in partial pressure of PO2 (distinct anatomical sites & physiological conditions)
kidney medulla & bone marrow has low PO2 (10 25 mmHg)
towards inside of solid tumour (extreme hypoxia <0.1 mmHg)
O2 levels are not constant & fluctuate dynamically
Require mechanism to sense sensitive changes in O2 tension to adjust metabolism to O2 supply
a) Oxygen sensor: Prolyl hydroxylase domain protein (PHD)
b) Key mediators of cellular transcriptional response to hypoxia: Hypoxia-inducible transcription factor (HIF-1)
Under hypoxia: mechanism to restore oxygenation

Mechanisms
In normoxia: High PHD level + cofactor: O2 & a-ketoglutarate

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 activity
HIF-1a is not be hydroxylated & stabilized
Non-hydroxylated HIF-1a accumulation
Activate transcriptional program to prepare cells to adapt the hypoxia condition
HIF1 (heterodimeric protein) two subunits
HIF1, the expression of which is stabilized by hypoxia
HIF 1, which is constitutively expressed
When HIF1 associates with HIF1, they form a transcription factor that binds and activates the promoters of multiple
glycolytic genes

C. Regulation of Metabolism
AMPK
Energy stress sensor
Low ATP, high ADP/AMP (Low energy)
Activate
Activate catabolic pathways Produce ATP
Inhibit anabolic pathways & mTORC1
Normal energy level:
ATP bind to Y-subunit of AMPK

mTORC1
Nutrient availability sensor
Nutrient (amino acid) sufficient &
growth factor Activate
Activate anabolic pathways
Protein synthesis utilizing ATP
Nutrient & energy stress:
Inhibited by AMPK

PHD
Oxygen sensor
High O2 Activate
Hydroxylate & inhibit HIF-1a
Hypoxia:
Inhibited by low O2
Non-hydroxylate HIF-1a activate

Regulation by AMPK - Metabolism

(i) Glucose homeostasis
AMPK switch ON catabolic pathways that generate ATP

AMPK switch OFF anabolic pathways that use ATP

Upregulation of catabolism pathways

Downregulation of anabolism pathways

Glucose uptake via activation of both GLUT1 &

GLUT4
Up-regulate GLUT4 + activation of HIF-1
Translocation of existing transporters to the plasma
membrane

Inhibit glycogen synthesis phosphorylation of

glycogen synthase (GS)
Inhibit gluconeogenesis repress glucose-6phosphatase & phosphoenolpyruvate carboxykinase
phosphorylation of CRTC2 & class IIA histone
deacetylases

Probably due in part to phosphorylation of the Rab-GAP protein

TBC1D1 by AMPK

A longer-term effect on transcription of the GLUT4 gene

Probably due in part to phosphorylation of histone deacetylase-5

Glycolysis via phosphorylation and activation of two of

four isoforms of 6-phosphofructo-2-kinase (PFK-2), which
synthesizes the glycolytic activator fructose-2,6bisphosphate
Enhancing mitochondrial biogenesis
Energy factory increase energy through oxidative
catabolism; promote activation of mitochondrial genes
mTORC1 inactivated by AMPK
Low ATP by mitochondrial inhibition also blocks mTORC1
activation & lysosomal localization

mTOR regulation
Hyperactivation mTORC1
Phosphorylation of S6 kinase 1 & Grb10 (downstream
proteins)

reduce insulin receptor substrate (IRS1)

increase insulin sensitivity

resistant of insulin signaling

reduce glucose uptake & glycogen synthesis
Increase gluconeogenesis
(ii) Lipid homeostasis

AMPK switch ON catabolic pathways that generate ATP

AMPK switch OFF anabolic pathways that use ATP

Activation of fatty acid oxidation

Inhibition of lipogenesis

Fatty acid uptake via translocation of the fatty acid

transporter FAT/CD36
Fatty acid -oxidation via phosphorylation of the
ACC2 isoform of acetyl-CoA carboxylase, thus lowering
malonyl-CoA, an inhibitor of fatty acid uptake into
mitochondria

Inhibit fatty acid synthesis

phosphorylation of acetyl coA carboxylase 1 (ACC1)
phosphorylation of the transcription factor SREBP-1c
inhibit proteolytic processing to the active, nuclear form
Inhibit isoprenoid synthesis phosphorylation of
HMG-CoA reductase,
Inhibit triglyceride and phospholipid synthesis
inactivation of glycerol phosphate acyl transferase
mTORC1 inactivated by AMPK
mTOR regulation
SREBP1/2 low expression Decrease lipogenic gene
Lipid also a building material for membrane cell
expression
growth alongside with protein
Promote lipid synthesis for cell growth
mTOR promote function SREBP transcription factor
promote peroxisome proliferator-activated receptor
(PPAR): key regulator of lipid uptake & adipogenesis
(iii) Protein homeostasis
AMPK switch ON catabolic pathways that generate ATP

mTORC1 inactivated by AMPK

Under severe energy / nutrient stress
cells undergo autophagy (intracellular degradation)
indirectly induced by AMPK by inhibiting mTORC1
AMPK phosphorylate & activate autophagy essential
kinase ULK1
Protein catabolism
breakdown of protein to amino acids
amino acids may be used in Krebs cycle to produce
ATP & recycled to make new amino acids

AMPK switch OFF anabolic pathways that use ATP

Inhibit protein synthesis

Switch off mTORC1
AMPK phosphorylate & activate TSC2 (mTORC1 negative
regulator)
Phosphorylate Raptor & inhibit Raptor interaction with
mTORC1
mTORC1 regulation
promote protein synthesis (anabolism) by 4 process
1. amino acid synthesis
2. RNA synthesis
3. transcription
4. translation
mTORC1 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 essential
cellular activity
glucose transporters (GLUT1, GLUT3 & GLUT4): increase glucose uptake
hexokinases
lactate dehydrogenases
Pyruvate Lactate (produce ATP): to compensate loss of mitochondrial ATP production
Oxygen 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 tissue
Inhibit mTORC1 to reduce ATP-consuming protein synthesis & increase autophagy
D. Metabolic regulation & cell physiology
i. Factors determine the metabolic requirements
a) Cell function
b) Microenvironment
ii. Cell physiology during complete nutrient deprivation - Ketosis

E. Proliferating cells metabolism

i. Normal glucose metabolism

Control system to prevent exceed nutrient availability for cell division
Growth factors regulate nutrient uptake of cells from extracellular environment\
Under aerobic conditons + quiescent normal cells
Glucose [glycolysis] pyruvate acetyl-coA + CO2
Acetyl-CoA [mitochondrial TCA cycle] ATPs & NADH
NADH [mitochondrial oxidative phosphorylation] maximize ATP production (36 ATPs per one glucose oxidation)
Anaerobic glycolyis
Glucose Pyruvate Lactate (Lactic acid fermentation)
(2 ATPs per one glucose oxidation)
ii. Proliferative metabolism Aerobic glycolysis
A phenomenon known as Warburg Effect
Regardless of the availability of O2 & nutrients, aerobic and anaerobic
Glucose metabolism rely on AEROBIC GLYCOLYSIS
High production of lactate [ Glycose Pyruvate Lactate ] Lactic acid fermentation
a) Normal proliferative cells
Glucose Pyruvate Lactate (Lactic acid fermentation)
(2 ATPs per one glucose oxidation)
For proliferative cells; aerobic glycolysis occurs (although with sufficient nutrients) proliferative metabolism
Growth signals (via PI3/AKT pathways) regulate the proliferative metabolism
b) Cancer cells
Warburg effects: A cancer phenotype by proliferation-induced oncogene
Aerobic glycolysis is common in cancer cells
Warburg effects occur
1. Due to adaptation to hypoxia environment within tumour
Hypoxia response system: upregulate glucose transporter & multiple enzymes of glycolytic pathways
2. Damage to mitochondria (evading apoptosis) impairing oxidative phosphorylation & low ATP production
Still, mitochondria can remain functional with some oxidative phosphorylation continues
Genetic mutations altering the receptor-initiated signaling pathways
Clonal expansion of cells that acquire this phenotype
Oncogenes & 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 growth
Exceed glucose >> demand also drive oncogenic mutations
Increased glycolysis produce intermediates that involve in various biosynthetic pathways nucleosides & amino acids
synthesis