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Biochemistry and molecular cell biology of diabetic

complications

A unifying mechanism

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Pathophysiology of microvascular complication

Chronic hyperglycemia Initiating factor of microvascular diseases Magnitude & duration => positively correlates

to diabetic microvascular complication

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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

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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

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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

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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

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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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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

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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

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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 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

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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

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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

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

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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

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

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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

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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

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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 andProtein 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 equivale

nt 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 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

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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

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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

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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 endot

helia cells metabolic linkage between IR and macrovascular disease

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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

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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

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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

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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

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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

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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 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

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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- reversed in N

OD mice CREB and Bcl-2 expression restored

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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

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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 overex

pressed transgenic mice Albumiuria, glomerular hypertrophy, TGF in glomerular was at

tenuated

db/db mice SOD transgene mice

Attenuation Glomerular mesngial matrix expansion

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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