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 hyperglycemiablood
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
6
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
7
8
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
9
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
14
How AGE precursors damage target
cell?

Intracellular protein
modification(glycation)function altered

Extracellular matrix components modification by
AGE precursorsabnormally interact with matrix
component and with matrix receptor(integrin)

Plasma protein modification by AGE precursors
 Endothelial, mesengial cells, macrophage
 ROS productionNFkBpathologic change of gene
expressions
15
16
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
17
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
18
Mechanisms of hyperglycemia
induced damage

Increased polyol pathway

Increased intracelllular Advanced
Glycation End Product(AGE) formation

Activation of PKC isoforms

Increased hexosamine pathway
19
Activation of PKC
DAG
hyperglycemia
Phorbol ester
ROS
PCK activation
Physiologicallly
multiple effects
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21
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
22
Mechanisms of hyperglycemia
induced damage

Increased polyol pathway

Increased intracelllular Advanced
Glycation End Product(AGE) formation

Activation of PKC isoforms

Increased hexosamine pathway
23
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
24
25
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
promoterw/ sp1 site
 Mutaitoin of sp1 site decreased activity
26
Glycosylation and phosphorylation of
SP1

Sp1 O-GlcNacylation ->decrease of
ser/Thr phosphorylation
 Competetion of O-GlcNacylation and
phosphorylation to sp1
 Hypergycemiahexosamine activity in
arotic cellsincreased sp1
glycosylation/decreased phosphorylation
27
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,



Hyperglycemiahexosamine pathway
activiationMMP-2,-9
HyeprglycemiaIncreased carotid plaque
 O-GlcNAc modified protein
28
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
29
Increased hexosamine pathway flux
hyperglycemia
Hexosamine pathway
activation
Diabetic-related gene expression and
Protein function such as PAI-1
Diabetic complication
30
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
31
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
PKAinhibit by phosphorylation of
G6PDH
 These inhibition increase oxidative stress

Decreased G6PDH activity  decrease
endothelium derived bioavailable NO
33
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
34
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
36
Mechanism of macrovascular
damage induced by FFA
37
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
38
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
40
Mitochondrial superoxide production
41

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, 530mM glucose media
 Mt superoxide production inhibition no
change of sorbitol in 30mM glucose media

43
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
 HyperglycemiaMethylglyoxal-derived AGE
 5mM30mM glucose medium : AGE
 Mt superoxide prevented30mM: AGE was not
increased
 GAPDH inhibition by hyperglycemiatriose
increasedmethylglyoxal formationAGE 
45
UCP-1 / MnSOD and PKC activation

Hyperglycemia-induced superoxide by
inhibition of UCP1 and MnSOD
 Decrease PKC activation in endothelial cells
 HyperglycemiaPKC activation


5mM30mM glucose medium : PKC
Mt superoxide prevented30mM: PKC was not
increased
 HyperglycemiaGAPDH inhibition de novo
synthesis of DAGPKC activation
 GAPDH antisense : activation of PKC in
physiologic glucose conc.
 PKCNADPH oxidase activationsuperoxide
production
46
UCP-1 / MnSOD and hexosamine
pathway acitivity

Hyperglycemia-induced superoxide by
inhibition of UCP1 and MnSOD
 Prevent hexosamine pathway acitivity in endothelial
cells



5mM30mM glucose medium : UDP-GlcNAc 
Mt superoxide prevented30mM: UDP-GlcNAc was not
increased
Hyperlgycemia
 more F6P
 ROSinhibition of GAPDHF6P   GFAT
hexosamine pathway

GAPDH antisense : increase hexosamine
pathway flux in the absence of hyperglycemia
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48
hyperglycemia and NFkB

Hyperglycemia-induced activation of
redoxsensitive transcription factor NFkB
was prevented by inhibition of Mt
superoxide overproduction
49
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
50
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

Hyperglycemiainhibits prostacyclin
synthetaseprevented by overexpression of UCP1/MnSOD
51
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
52
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
53