Biochemistry and molecular cell biology of diabetic
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Transcript Biochemistry and molecular cell biology of diabetic
Biochemistry and molecular cell
biology of diabetic
complications
A unifying mechanism
1
Pathophysiology of microvascular
complication
Chronic hyperglycemia
Initiating factor of microvascular diseases
Magnitude & duration => positively
correlates to diabetic microvascular
complication
2
Pathophysiology of microvascular
complication
Early DM hyperglycemiablood
flow, intracapillary pressure
NO activity,
ET-1, angiotensin II ,
VEGF permeability
Retinal capillary damage and albumin
excretion in glomerular capillary
3
Pathophysiology of microvascular
complication
Hyperglycemia
Decrease production of trophic factor for
endothelial and neuronal cells
Connective tissue growth factor(CTGF)
Key intermediate molecule involved in the
pathogenesis of fibrosing chronic disease in
diabetic animal(kidney, myocardium, aorta)
Micro, macrovascular disease caused by DM
4
Pathophysiology of macrovascular disease
Hyperglycemia/insulin resistance
Insulin resistance correlates with degree
of atherosclerosis
IR adipocyte
FFA
LDL, HDL
Atherosclerosis risk factor
Macrovasucular complications
5
Mechanisms of hyperglycemia
induced damage
Increased polyol pathway
Increased intracelllular Advanced
Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
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Increased polyol pathway
Aldose reductase(AR)
First enzyme in Polyol pathway
Monomeric oxidoreducatese
Catalyze reduction of carbonyl
compound(e.g glucose)
Low affinity for glucose
Contribute to glucose utilization in small
percentage
In hyperglycemia => increased emzymatic
conversion to the polyalcohol sorbitol
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Increased polyol pathway
Sorbitol is oxidized to fructose by
sorvitol dehydrogenase(SDH) with
NAD+ reduce to NADH
Flux through polyol pathway during
hyperglycemia varied form 33% in
rabbit lens to 11% in human erythrocyte
The contribution of this pathway to
diabetic complications : site, species,
tissue specific
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Increased polyol pathway
AR deplete reduced glutathione(GSH)
Consume NADPH
Intracellular oxidative stress
Transgenic mice(AR overexpression)
Decreased GSH in lens
Homozygous KO mice mice : diabetic
10
Increased polyol pathway
NO maintain AR in inactive
This suppression is relieved in diabetic tissue
NO-derived adduct formation is cys298=>
inhibition of AR
Diabetic => decreased NO => polyol flux
AR inhibition in dogs
prevent diabetic nephropathy
but failed to prevent retinopathy, capillary
basement membrane thickening in the retina,
kidney, muscle
AR inhibition in human
Zenarestat(AR inhibitor) =>positive effect on
neuropathy
11
Mechanisms of hyperglycemia
induced damage
Increased polyol pathway
Increased intracelllular Advanced
Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
12
Increaed intracellular AGE formation
Advanced Glycation End product(AGE)
Increased in diabetic retinal vessle, renal
glomeruli
Hyperglycemia is primary initiating event in
the formation of extra/intracellular AGEs
AGE precursors(methylglyoxal) damage
target cells
13
Increaed intracellular AGE formation
AGEs and DM complications
AGE inhibitors prevent(animals)
Diabetic microvascular disease in retina,
kidney, nerve
AGE formation in human diabetic retina,
Early pahse of DM nephropathy
VEGF
Macular edema and retinal neovascularization
VEGF is stimulated
Hyperfiltration, microalbuminuria
Treatment aminoguanidine to T1DM patients
Lowered total urinary protein
Slowed progression of nephropathy
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How AGE precursors damage target
cell?
Intracellular protein
modification(glycation)function altered
Extracellular matrix components modification by
AGE precursorsabnormally interact with matrix
component and with matrix receptor(integrin)
Plasma protein modification by AGE precursors
Endothelial, mesengial cells, macrophage
ROS productionNFkBpathologic change of gene
expressions
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Increaed intracellular AGE formation
Methylglyoxal(AGE precursor)
Diabetic patient() 3~5times : 8uM
Induction of apoptosis by DNA damage and
oxidative stress
Changes matrix molecule functional properties
Tyep I collagen : decreased elasticity
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AGE receptor
Blockade of RAGE
Inhibits development of diabetic
vasculopathy,nephropathy and
periodonatal disease
Suppresses macrovasular disease in
atherosclerosis-prone T1DM mouse
Reduce lesion size and structure,
decreased parameters of inflammation
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Mechanisms of hyperglycemia
induced damage
Increased polyol pathway
Increased intracelllular Advanced
Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
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Activation of PKC
DAG
hyperglycemia
Phorbol ester
ROS
PCK activation
Physiologicallly
multiple effects
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Activation of PKC and physiological
effects
PKC-b overexpression
Myocardium in diabetic mice
Connective tissue growth factor
TGFb
Cardiomyophathy and cardiac fibrosis
b isoform-specific PKC inhibitor
Reduced PKC activity in retian, renal glomeruli of
diabetic mice
Diabetic-induced retinal mean circultion time,
glomerular filtration rate, urinary albumin
excretion ameliorated
db/db mice : glomerular mesangil expnsion inhibition
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Mechanisms of hyperglycemia
induced damage
Increased polyol pathway
Increased intracelllular Advanced
Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
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Increased hexosamine pathway flux
Excess intracellular glucose=>
hexosamine pathway flux=>diabetic
complication
Glucose=>g-6-P => f-6-P=> glycolysis
Hexosamine pathway
Inhibition of glutamine:fructose-6-P
amidotransferase(GFAT) blocks PAI-1,
TGF transcription
Meausred by UDP-GlcNAc accumlation
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Increased hexosamine pathway flux
Sp1 site regulate hyperglycemia-induced
activation of the PAI-1 promoter
Covalent modification of sp1 by N-
acetylglucosamine
Hexosamine pathway activiation and hyperglycemia
induced PAI-1 expression
Glucosamine activate the PAI-1 promoter
through Sp1 site.
Glycosylated sp1 is more active than
deglycosylated form.
Increased luciferase activity of PAI-1
promoterw/ sp1 site
Mutaitoin of sp1 site decreased activity
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Glycosylation and phosphorylation of
SP1
Sp1 O-GlcNacylation ->decrease of
ser/Thr phosphorylation
Competetion of O-GlcNacylation and
phosphorylation to sp1
Hypergycemiahexosamine activity in
arotic cellsincreased sp1
glycosylation/decreased phosphorylation
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Nuclear and cytoplasmic protein and
O-GlcNAc modification
Diabetic complications
Inhibition of eNOS activity by
hyperglycemia-induced O-GlcNAc at the
Akt site of the eNOS protein
T2DM coronary artery endothelial cells,
Hyperglycemiahexosamine pathway
activiationMMP-2,-9
HyeprglycemiaIncreased carotid plaque
O-GlcNAc modified protein
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Increased hexosamine pathway flux
hyperglycemia increase GFAT activity in
arotic SMC
Hyperglycemia qulitatively and
quantitatively alters the glycosylation of
expression of many O-GlcNAc
modified protein in the nucleus
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Increased hexosamine pathway flux
hyperglycemia
Hexosamine pathway
activation
Diabetic-related gene expression and
Protein function such as PAI-1
Diabetic complication
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Other possible mechanisms of
hyperglycemia-induced damage
Inactivation of glucose-6-phosphate
dehyrogenase
Decreased cAMP-response element-binding
protein(CREB) activity and content
Mechanism of macrovascular damage
induced by FFA
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Inactivation of glucose-6-phosphate
dehyrogenase
G6P-Dehydrogenase
First rate-limiting enzyme in glycolysis
Produce NADPH
NADPH : critical intracellular reducint
equivalent reduction of oxidized
glutathione(against oxidative stress)
Act as cofactor for eNOS activity
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Inactivation of glucose-6-phosphate
dehyrogenase
Hyperglycemia inhibits G6PDH in
bovine aortic endothelial cell by
PKAinhibit by phosphorylation of
G6PDH
These inhibition increase oxidative stress
Decreased G6PDH activity decrease
endothelium derived bioavailable NO
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Decreased cAMP-response elementbinding protein(CREB) activity and
content
CREB
Located in cAMP signal downstream
Important roles in VSMC
Inhibition of proliferation and migration
Decrease expression of GF-receptor for PDGF,
endothelin-1, IL-6
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Decreased cAMP-response elementbinding protein(CREB) activity and
content
Hyperglycemia in VSCM
CREB content, function increase of
migration and proliferation
CREB overexpression
Completely restore hyperglycemia-
induced proliferation and migration
DM
CREB macrovascular complication
35
Decreased cAMP-response elementbinding protein(CREB) activity and
content
Decreased level of CREB
Insulin resistant/deficient mice
Nervous system in DM
STZ animal’s hippocampus and nerve
Thus,
Change and function of CREB represent a
pivotal consequence of glycemiamediated dysfunction in complications
target tissue of diabetic complication
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Mechanism of macrovascular
damage induced by FFA
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Mechanism of macrovascular
damage induced by FFA
In vitro
Low glucose cultured arotic endothelial cell and
elevated FFA
AGE, PKC activation, hexosamine pw , NFkB
The same extent as hyperglycemia
In vivo
Fatty Zuker rat(insulin resistant but no DM)
Above pathway blocked by inhibition of lipolysis with
nicotinic acid
Thus,
Increased of FFA from visceral adipocyte to arterial
endothelia cells metabolic linkage between IR and
macrovascular disease
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Mechanism of hyperglycemiainduced mitochondrial superoxide
overproduction
Polyol pathway flux from glucose
Hexosamine pathway flux from F6P
PKC activation from Glyceraldehyde-3-P
AGE formation from Glyceraldehyde-3-P
39
Hyperglycemia-mitochondria
superoxide
ETS through complexes I, III, IV
generation proton gradient that drive
ATP synthase
gradinet superoxide production
By Hyperglycemia
By FFA
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Mitochondrial superoxide production
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Overexpression of UCP-1
Decrease Proton gradient
Prevent hyperglycemia induced ROS
Overexpression of MnSOD
MnSOD(manganase superoxide dismutase)
Abolish ROS signal by hyperglycemia
42
UCP-1 / MnSOD and polyol pathway
Inhibition of hyperglycemia induced
superoxide production by UCP1 and
MnSOD
Prevent incresed polyol pathway flux in
endothelia cells
Sorbitol accumulation increased
Cultured cell, 530mM glucose media
Mt superoxide production inhibition no
change of sorbitol in 30mM glucose media
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UCP-1 / MnSOD and GAPDH activity
Hyperglycemia-induced superoxide by
inhibition of UCP1 and MnSOD
66% decrease of GAPDH activity
GAPDH inhibition ROS induced DNA
strand break
Polyol flux increased
44
UCP-1 / MnSOD and AGE formation
Hyperglycemia-induced superoxide by
inhibition of UCP1 and MnSOD
Decrease AGE formation in endothelial cell
HyperglycemiaMethylglyoxal-derived AGE
5mM30mM glucose medium : AGE
Mt superoxide prevented30mM: AGE was not
increased
GAPDH inhibition by hyperglycemiatriose
increasedmethylglyoxal formationAGE
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UCP-1 / MnSOD and PKC activation
Hyperglycemia-induced superoxide by
inhibition of UCP1 and MnSOD
Decrease PKC activation in endothelial cells
HyperglycemiaPKC activation
5mM30mM glucose medium : PKC
Mt superoxide prevented30mM: PKC was not
increased
HyperglycemiaGAPDH inhibition de novo
synthesis of DAGPKC activation
GAPDH antisense : activation of PKC in
physiologic glucose conc.
PKCNADPH oxidase activationsuperoxide
production
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UCP-1 / MnSOD and hexosamine
pathway acitivity
Hyperglycemia-induced superoxide by
inhibition of UCP1 and MnSOD
Prevent hexosamine pathway acitivity in endothelial
cells
5mM30mM glucose medium : UDP-GlcNAc
Mt superoxide prevented30mM: UDP-GlcNAc was not
increased
Hyperlgycemia
more F6P
ROSinhibition of GAPDHF6P GFAT
hexosamine pathway
GAPDH antisense : increase hexosamine
pathway flux in the absence of hyperglycemia
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hyperglycemia and NFkB
Hyperglycemia-induced activation of
redoxsensitive transcription factor NFkB
was prevented by inhibition of Mt
superoxide overproduction
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Overexpression of UCP-1 and MnSOD
Prevent hyperglycemia-induced
inactivation of GAPDH
SOD mimetic
Loss of CREB, PDGF recector-a reversed
in NOD mice
CREB and Bcl-2 expression restored
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Overexpression of UCP-1, MnSOD
and diabetic complications
MnSOD : suppress the increase cllagen synthesis
caused by hyperglycemia in glomerular cell
MnSOD overexpressed mice: decrease
programmed cell death caused by hyperglycemia
in DRG neuron
UCP-1 overexpression in embryonic DRG
Caspase inhibition
In aortic cells
UCP-1/MnSOD blocking of hyperglycemid-induced
monocyte adhesion to endothelial cells
Anti-atherogenic enzyme
Hyperglycemiainhibits prostacyclin
synthetaseprevented by overexpression of UCP1/MnSOD
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Overexpression of UCP-1 and MnSOD
Prevent Hyperglycemia-induced eNOS
inhibition
STZ animal
STZ-wild
STZ-human Cu++/Zn++ superoxide dismutase
overexpressed transgenic mice
Albumiuria, glomerular hypertrophy, TGF in glomerular
was attenuated
db/db mice
SOD transgene mice
Attenuation Glomerular mesngial matrix expansion
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Norglycemia and FFA
IR adipose tissue
Inhibitor of CPT-1
Hyperglycemia
Excess
FFA
Mt ETS
Superoxide
MnSOD
UCP-1
Physiologically
Adverse effect
Macrovascular damage by IR
Microvascular damage by Hyperglycemia
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