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1
Biochemistry and molecular cell biology of diabetic
complications
A unifying mechanism
2
Pathophysiology of microvascular complication
Chronic hyperglycemia Initiating factor of microvascular diseases Magnitude & duration => positively correlates
to diabetic microvascular complication
3
Pathophysiology of microvascular complication
Early DM hyperglycemiablood flow, intracapillary pressure NO activity, ET-1, angiotensin II , VEGF permeability Retinal capillary damage and albumin excretio
n in glomerular capillary
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Pathophysiology of microvascular complication
Hyperglycemia Decrease production of trophic factor for end
othelial 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
5
Pathophysiology of macrovascular disease
Hyperglycemia/insulin resistance Insulin resistance correlates with
degree of atherosclerosis
IR adipocyte
FFA LDL, HDL
Atherosclerosis risk factorMacrovasucular complications
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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 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 perc
entage In hyperglycemia => increased emzymatic con
version 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 c
omplications : site, species, tissue specific
10
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
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Increased polyol pathway NO maintain AR in inactive
This suppression is relieved in diabetic tissue NO-derived adduct formation is cys298=> inhibition o
f 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
12
Mechanisms of hyperglycemia induced damage
Increased polyol pathway
Increased intracelllular Advanced Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
13
Increaed intracellular AGE formation
Advanced Glycation End product(AGE) Increased in diabetic retinal vessle, renal glo
meruli Hyperglycemia is primary initiating event in th
e formation of extra/intracellular AGEs AGE precursors(methylglyoxal) damage target
cells
14
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, VEGF
Macular edema and retinal neovascularization Early pahse of DM nephropathy
VEGF is stimulated Hyperfiltration, microalbuminuria
Treatment aminoguanidine to T1DM patients Lowered total urinary protein Slowed progression of nephropathy
15
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 express
ions
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Increaed intracellular AGE formation
Methylglyoxal(AGE precursor) Diabetic patient() 3~5times : 8uM Induction of apoptosis by DNA damage and oxidativ
e stress Changes matrix molecule functional properties
Tyep I collagen : decreased elasticity
18
AGE receptor
Blockade of RAGE Inhibits development of diabetic vasculopathy,
nephropathy and periodonatal disease Suppresses macrovasular disease in atheros
clerosis-prone T1DM mouse Reduce lesion size and structure, decreased
parameters of inflammation
19
Mechanisms of hyperglycemia induced damage
Increased polyol pathway
Increased intracelllular Advanced Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
20
Activation of PKC
hyperglycemiaDAG
PCK activation
Physiologicallly multiple effects
Phorbol esterROS
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Activation of PKC and physiological effects
PKC- overexpression Myocardium in diabetic mice
Connective tissue growth factor TGF Cardiomyophathy and cardiac fibrosis
isoform-specific PKC inhibitor Reduced PKC activity in retian, renal glomeruli of diab
etic mice Diabetic-induced retinal mean circultion time, glomer
ular filtration rate, urinary albumin excretion ameliorated db/db mice : glomerular mesangil expnsion inhibition
23
Mechanisms of hyperglycemia induced damage
Increased polyol pathway
Increased intracelllular Advanced Glycation End Product(AGE) formation
Activation of PKC isoforms
Increased hexosamine pathway
24
Increased hexosamine pathway flux
Excess intracellular glucose=> hexosamine pathway flux=>diabetic complication
Glucose=>g-6-P => f-6-P=> glycolysis
Inhibition of glutamine:fructose-6-P amidotransferase(GFAT) blocks PAI-1, TGF transcription
Meausred by UDP-GlcNAc accumlation
Hexosamine pathway
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Increased hexosamine pathway flux Sp1 site regulate hyperglycemia-induced activati
on 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 f
orm. Increased luciferase activity of PAI-1 promoterw/ s
p1 site Mutaitoin of sp1 site decreased activity
27
Glycosylation and phosphorylation of SP1
Sp1 O-GlcNacylation ->decrease of ser/Thr phosphorylation Competetion of O-GlcNacylation and phospho
rylation to sp1 Hypergycemiahexosamine activity in arotic
cellsincreased sp1 glycosylation/decreased phosphorylation
28
Nuclear and cytoplasmic protein and O-GlcNAc modification
Diabetic complications Inhibition of eNOS activity by hyperglycemia-in
duced 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
29
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
30
Increased hexosamine pathway flux
hyperglycemia
Hexosamine pathway activation
Diabetic-related gene expression andProtein function such as PAI-1
Diabetic complication
31
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
32
Inactivation of glucose-6-phosphate dehyrogenase
G6P-Dehydrogenase First rate-limiting enzyme in glycolysis Produce NADPH NADPH : critical intracellular reducint equivale
nt reduction of oxidized glutathione(against oxidative stress) Act as cofactor for eNOS activity
33
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
34
Decreased cAMP-response element-binding 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, end
othelin-1, IL-6
35
Decreased cAMP-response element-binding protein(CREB) activity and content
Hyperglycemia in VSCM CREB content, function increase of migr
ation and proliferation
CREB overexpression Completely restore hyperglycemia-induced pro
liferation and migration DM
CREB macrovascular complication
36
Decreased cAMP-response element-binding 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 piv
otal consequence of glycemia-mediated dysfunction in complications target tissue of diabetic complication
37
Mechanism of macrovascular damage induced by FFA
38
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 endot
helia cells metabolic linkage between IR and macrovascular disease
39
Mechanism of hyperglycemia-induced 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
40
Hyperglycemia-mitochondria superoxide
ETS through complexes I, III, IV generation proton gradient that drive ATP synthase
gradinet superoxide production By Hyperglycemia By FFA
41
Mitochondrial superoxide production
42
Overexpression of UCP-1 Decrease Proton gradient Prevent hyperglycemia induced ROS
Overexpression of MnSOD MnSOD(manganase superoxide dismutase)
Abolish ROS signal by hyperglycemia
43
UCP-1 / MnSOD and polyol pathway
Inhibition of hyperglycemia induced superoxide production by UCP1 and MnSOD Prevent incresed polyol pathway flux in endot
helia cells Sorbitol accumulation increased
Cultured cell, 530mM glucose media Mt superoxide production inhibition no change o
f sorbitol in 30mM glucose media
44
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
45
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 incr
eased GAPDH inhibition by hyperglycemiatriose increase
dmethylglyoxal formationAGE
46
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
47
UCP-1 / MnSOD and hexosamine pathway acitivity
Hyperglycemia-induced superoxide by inhibition of UCP1 and MnSOD Prevent hexosamine pathway acitivity in endothelial cell
s 5mM30mM glucose medium : UDP-GlcNAc Mt superoxide prevented30mM: UDP-GlcNAc was not increa
sed Hyperlgycemia
more F6P ROSinhibition of GAPDHF6P GFAT hexosamine pathway
GAPDH antisense : increase hexosamine pathway flux in the absence of hyperglycemia
48
49
hyperglycemia and NFkB
Hyperglycemia-induced activation of redoxsensitive transcription factor NFkB was prevented by inhibition of Mt superoxide overproduction
50
Overexpression of UCP-1 and MnSOD
Prevent hyperglycemia-induced inactivation of GAPDH
SOD mimetic Loss of CREB, PDGF recector- reversed in N
OD mice CREB and Bcl-2 expression restored
51
Overexpression of UCP-1, MnSODand 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 monocy
te adhesion to endothelial cells Anti-atherogenic enzyme
Hyperglycemiainhibits prostacyclin synthetaseprevented by overexpression of UCP-1/MnSOD
52
Overexpression of UCP-1 and MnSOD
Prevent Hyperglycemia-induced eNOS inhibition STZ animal
STZ-wild STZ-human Cu++/Zn++ superoxide dismutase overex
pressed transgenic mice Albumiuria, glomerular hypertrophy, TGF in glomerular was at
tenuated
db/db mice SOD transgene mice
Attenuation Glomerular mesngial matrix expansion
53
Norglycemia and FFA
ExcessFFA
Inhibitor of CPT-1
Superoxide
MnSODUCP-1
PhysiologicallyAdverse effect
Hyperglycemia
Mt ETS
IR adipose tissue
Macrovascular damage by IRMicrovascular damage by Hyperglycemia