Circulation research

Hyperglycemia causes tissue damage through 5 major mechanisms: (1) increased flux of glucose and other sugars through the polyol pathway; (2) increased intracellular formation of AGEs (advanced glycation end products); (3) increased expression of the receptor for AGEs and its activating ligands; (4) activation of protein kinase (PK)C isoforms; and (5) overactivity of the hexosamine pathway.

Several lines of evidence indicate that all 5 mechanisms are activated by a single upstream event. all 5 mechanisms are activated by a single upstream event: mitochondrial overproduction of the ROS. In many of these tissues, glucose uptake is mediated by insulin-independent GLUTs; intracellular glucose concentrations, therefore, rise in parallel with hyperglycemia. Several mechanisms have been proposed to explain how hyperglycemia-induced increases in polyol pathway flux could damage the tissues involved. The most cited is an increase in redox stress caused by the consumption of NADPH. Because NADPH is a cofactor required to regenerate reduced glutathione (GSH), and GSH is an important scavenger of ROS, this could induce or exacerbate intracellular oxidative stress.

Intracellular production of AGE precursors can damage cells by 3 general mechanisms. Firstly, intracellular proteins modified by AGEs have altered function. Secondly, extracellular matrix components modified by AGE precursors interact abnormally with other matrix components and with matrix receptors (integrins) that are expressed on the surface of cells. Finally, plasma proteins modified by AGE precursors bind to AGE receptors on cells such as macrophages, vascular endothelial cells, and vascular smooth muscle cells. Receptor for AGE (RAGE) binding induces the production of ROS, which in turn activates the pleiotropic transcription factor nuclear factor (NF)-_B, causing multiple pathological changes in gene expression.28 Persistent and excessive activation of several PKC isoforms operates as a third common pathway mediating tissue injury induced by diabetes-induced ROS. Hyperglycemia and insulin resistanceinduced excess fatty acid oxidation also appear to contribute to the pathogenesis of diabetic complications by increasing the flux of fructose

6-phophate into the hexosamine pathway.7174 In this pathway, fructose 6-phosphate is diverted from glycolysis to provide substrate for the rate-limiting enzyme of this pathway, glutamine:fructose 6-phosphate amidotransferase (GFAT). GFAT converts fructose 6-phosphate to glucosamine 6-phosphate, which is then converted to UDP-NAcetylglucosamine. Specific O-GlcNAc transferases use this for posttranslational modification of specific serine and threonine residues on cytoplasmic and nuclear proteins by O-GlcNAc. Inhibition of GFAT blocks hyperglycemia-induced increases in the transcription of both TGF-_71 and TGF-_1.72 One new class of potential therapeutic agents is transketolase activators. When increased superoxide inhibits GAPDH activity, the concentration of glycolytic intermediates above the enzyme accumulates which increases the flux into the 5 pathways of hyperglycemic damage. Two of these glycolytic intermediates, fructose 6-phosphate and glyceraldehyde 3-phosphate, are also the final products of the transketolase reaction, which is the rate-limiting enzyme in the nonoxidative part of the pentose phosphate pathway.

A second new class of mechanism-based potential therapeutic agents are PARP inhibitors. In cultured arterial endothelial cells, a specific PARP inhibitor prevents hyperglycemia-induced activation of PKC, NF-_B, intracellular AGE formation, and the hexosamine pathway. In animal models of diabetes, PARP inhibition prevents arterial endothelial cell injury and podocyte apoptosis, ameliorates nephropathy, and alleviates sensory neuropathy.141143 A third class of mechanism-based therapeutics are SOD/ catalase mimetics. Excess superoxide itself directly inhibits critical antiatherosclerosis endothelial enzymes independent of activating the 5 damaging pathways implicated in metabolite-induced diabetic complications. Both of these enzymes (eNOS and prostacyclin synthase) are inhibited in diabetic patients and diabetic animals. To prevent oxidative inactivation of these key enzymes, in addition to preventing activation of the pathways discussed above, it is necessary to directly reduce the amount of superoxide. Conventional antioxidants are unlikely to do this effectively because conventional antioxidants neutralize reactive oxygen molecules on a one-for-one basis, whereas hyperglycemia-induced overproduction of superoxide is a continuous process. Based on observations of the beneficial effects of overexpression of antioxidant enzymes in mouse models, what is needed is a new type of antioxidant, a catalytic antioxidant, such as an SOD/catalase mimetic.144 Hyperglycemia-induced reactive

oxygen overproduction directly reduces eNOS activity in diabetic aortas by 65%. Similarly, but more dramatically, hyperglycemia-induced reactive oxygen overproduction directly reduces prostacyclin synthase activity in diabetic aortas by 95%. Treatment of these diabetic animals with an SOD/catalase mimetic completely prevents diabetes-induced oxidative inactivation of aortic prostacyclin synthase, and also normalizes all 5 of the pathways implicated in hyperglycemic damage.4 Inhibition of hyperglycemia-induced ROS production in diabetic mice using either transgenic antioxidant enzyme expression or combinations of antioxidant compounds prevents the development of experimental diabetic retinopathy, nephropathy, neuropathy and cardiomyopathy. 91,145149 Together, these data strongly suggest that therapeutic correction of diabetes-induced superoxide overproduction may be a powerful approach for preventing diabetic complications. Masukkan summary

Cardiovascular research
A number of mechanisms have been proposed to mediate hyperglycemia-induced toxicity. They include protein kinase C (PKC) activation, increased flux through the hexosamine pathway, increased advanced glycation end-product formation, and increased production of reactive oxygen species (ROS)10. One source of ROS is the one-electron reduction of O2 to superoxide anion by NADPH oxidase. This enzyme was first described in macrophages and has later been identified in several other cell types, including cardiomyocytes. NADPH oxidases are multimeric enzymes, which contain a heterodimeric membrane-bound cytochrome b558 made of either NOX2 or NOX4 and p22phox subunits. Moreover, high glucose-dependent NADPH oxidase activation and ROS production lead to an increase in apoptosis13. Superoxide generation by NADPH oxidase also depends on the availability of reducing equivalents. NADPH generated by the oxidative part of the pentose phosphate pathway (PPP) has been suggested to fuel NADPH oxidase and sustain ROS production in the heart14,15. Therefore, we investigated the connection between glucose transport, metabolism, NADPH oxidase activation and subsequent ROS production under hyperglycemia. In this paper, we describe for the first time that NADPH oxidase activation and ROS production in response to high glucose concentrations do not require glucose metabolism but rather its transport through a novel cardiac glucose transport system, the sodium-dependent glucose cotransporter, SGLT119. Results Chronic exposure to high glucose induces cell death by increasing ROS production

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An imbalance between reactive oxygen species (ROS) generation and antioxidant capacity favoring the former leads to oxidative stress and oxidative damage (30). Oxidative damage in various tissues may be controlled or prevented by enzymic and nonenzymic antioxidant defense systems, which include reduced glutathione (GSH), superoxide dismutase, catalase, glutathione peroxidase (1), and heme oxygenase (10). Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress. HO-1 metabolizes the prooxidant heme to the antioxidant biliverdin, carbon monoxide, and free iron. Biliverdin is reduced to another antioxidant, bilirubin, by biliverdin reductase (10, 31, 33). SOD is another key antioxidant enzyme that catalyzes the conversion of superoxide to hydrogen peroxide and molecular oxygen (9, 22). Heart failure, diabetes, and obesity are recognized as states of chronic inflammation. Inflammatory cytokines may play a role in all three of these conditions (52).

DISCUSSION

It has become evident that ROS play a crucial role in the development of diabetic complications. NADPH oxidase has emerged as the main source of ROS in the cardiovascular tissues with contributions from other sources, such as xanthine oxidase and mitochondrial respiration (24, 29, 30, 32, 46). Recent studies have provided evidence for increased levels of NADPH oxidase subunits in blood vessels (17, 47) and kidney from diabetic rats (3, 5, 14). Bhatti et al. (5) also reported that the antioxidant -lipoic acid attenuated the increased expression of NADPH oxidase subunits p22phox and p47phox in the kidney of streptozotocin-induced diabetic rats. The main new findings of this study are that NAC treatment prevents the membrane translocation of p67phox and the increased expression of p22phox and reduced myocardial superoxide formation in the diabetic rat hearts. We showed by Western blot analysis that the cardiac protein expression of p67phox and p22phox was enhanced in diabetic rats. This is consistent with the recent report that the NADPH oxidase activity in membrane fractions of diabetic hearts is significantly increased (47). Furthermore, since apocynin inhibition blocked NADPH-stimulated increases of superoxide production (Fig. 6), these data would suggest that the increase in NADPH oxidase activity is a major cause of diabetic myocardial superoxide production. In the present study, NAC reduced the expression of p67 phox in the membrane but not in the cytosolic fraction, indicating that it inhibited translocation of the p67phox subunit to the membrane fraction. The increased expression of p22phox, one membranebound subunit, was also prevented by NAC. Another critical subunit, p47phox, did not change significantly with diabetes and was not altered by NAC. Thus a mechanism of NADPH oxidase inhibition by NAC may be the suppression of translocation of the cytosolic p67phox component to the membrane fraction, with associated suppression of its anchor, p22 phox, where it forms a molecular cluster to activate NADPH oxidase. The enhanced expression of the NADPH oxidase subunits

results in the increase of ROS in diabetic rat hearts. In response to the increased ROS, antioxidant enzymes such as HO-1 and SOD, which act as a defense system, are induced to protect against cellular and tissue injury (7, 10, 25). Our study showed that the total SOD activity and the protein expression of Cu-Zn-SOD and HO-1 were upregulated in diabetic rat hearts, which may be a compensatory response in the face of elevated NADPH oxidase-derived ROS. The reason why the increase in HO-1 was accompanied by an increase in SOD may be that an increase in HO-1 in diabetic rats brought about a robust increase in extracellular SOD, one of three kinds of SOD, and the cytoprotective mechanism of HO-1 against oxidative stress required an increase in extracellular SOD (45). Oxidative stress is determined by the balance between the generation of ROS and the antioxidant defense system (24). Hyperglycemia increases ROS production. Fig.9 bagus mba