1. 1 Biochemistry and molecular cell biology of diabetic complications A unifying mechanism

2. 2 Pathophysiology of microvascular complication  Chronic hyperglycemia  Initiating factor of microvascular diseases  Magnitude & duration => positively correlates to diabetic microvascular complication

3. 3 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

4. 4 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

5. 5 Pathophysiology of macrovascular disease  Hyperglycemia/insulin resistance  Insulin resistance correlates with degree of atherosclerosis IR adipocyte FFA  LDL , HDL  Atherosclerosis risk factor Macrovasucular complications

6. 6 Mechanisms of hyperglycemia induced damage  Increased polyol pathway  Increased intracelllular Advanced Glycation End Product(AGE) formation  Activation of PKC isoforms  Increased hexosamine pathway

7. 7 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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9. 9 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

10. 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

11. 11 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

12. 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. 13 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

14. 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. 15 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

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17. 17 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

18. 18 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

19. 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. 20 Activation of PKC hyperglycemia DAG  PCK activation Physiologicallly multiple effects Phorbol ester ROS

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22. 22 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 diabetic mice  Diabetic-induced retinal mean circultion time, glomerular filtration rate, urinary albumin excretion  ameliorated  db/db mice : glomerular mesangil expnsion inhibition

23. 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. 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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26. 26 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

27. 27 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

28. 28 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 

29. 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. 30 Increased hexosamine pathway flux hyperglycemia Hexosamine pathway activation Diabetic-related gene expression and Protein function such as PAI-1 Diabetic complication

31. 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. 32 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

33. 33 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

34. 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, endothelin-1, IL-6

35. 35 Decreased cAMP-response element- binding 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

36. 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 pivotal consequence of glycemia-mediated dysfunction in complications target tissue of diabetic complication

37. 37 Mechanism of macrovascular damage induced by FFA

38. 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 endothelia cells  metabolic linkage between IR and macrovascular disease

39. 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. 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. 41 Mitochondrial superoxide production

42. 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. 43 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

44. 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. 45 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 

46. 46 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

47. 47 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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49. 49 hyperglycemia and NFkB  Hyperglycemia-induced activation of redoxsensitive transcription factor NFkB was prevented by inhibition of Mt superoxide overproduction

50. 50 Overexpression of UCP-1 and MnSOD  Prevent hyperglycemia-induced inactivation of GAPDH  SOD mimetic  Loss of CREB, PDGF recector-  reversed in NOD mice  CREB and Bcl-2 expression restored

51. 51 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 UCP- 1/MnSOD

52. 52 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

53. 53 Norglycemia and FFA Excess FFA Inhibitor of CPT-1 Superoxide  MnSOD UCP-1 Physiologically Adverse effect Hyperglycemia Mt ETS IR adipose tissue Macrovascular damage by IR Microvascular damage by Hyperglycemia