Biochemistry and molecular cell biology of diabetic complications

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

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

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22. 22 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

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

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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

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-a reversed in NOD mice CREB and Bcl-2 expression restored

51. 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 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