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.
Hyperglycemia menyebabkan kerusakan jaringan melalui 5 mekanisme utama: ( 1 ) meningkat fluksi
dari glukosa dan gula lainnya melalui jalur polyol; ( 2 ) intraselular yang meningkat pembentukan
usia ( maju glycation produk mengakhiri ); ( 3 ) meningkat ekspresi dari reseptor tersebut untuk abad
ke abad dan mengaktifkan nya ligands; ( 4 ) aktivasi protein kinase ( pk ) c isoforms; dan ( 5 )
overactivity dari jalur hexosamine.

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.
Beberapa jalur bukti menunjukkan bahwa semua mekanisme 5 diaktifkan oleh satu peristiwa hulu.
Semua 5 mekanisme diaktifkan oleh satu peristiwa hulu: mitokondria berlebih 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.
Karena NADPH merupakan suatu kofaktor yang diperlukan untuk meregenerasi berkurang
glutathione (GSH), dan GSH pemulung penting dari ROS, ini dapat menyebabkan atau memperburuk
stres oksidatif intraseluler.

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.
Expression of antioxidant
enzymes or treatment with antioxidant compounds can
ameliorate both triglyceride-associated oxidative stress and
diabetic cardiomyopathy.126,127 In vitro studies of cultured
myocytes and endothelial cells have shown that excess
saturated long chain fatty acids promote generation of
ROS.128,129 Several mechanisms have been implicated including
lipid activation of NADPH oxidase activity through the
downstream production of ceramides, and signaling through
pathways that converge on NF-_B.130,131 Oxidative stress
may also be intimately linked to lipid-induced endoplasmic
reticulum stress.132

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


H1278
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 p67phox 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, p22phox, 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

S227 full
Various pathophysiological and
biochemical mechanisms have been proposed to explain the
adverse effects of hyperglycemia on vascular cells (3 6). Among various possible
mechanisms, it is widely accepted that
high glucose level and a diabetic state induce the persistent
activation of the diacylglycerol (DAG)-protein kinase C (PKC)
pathway in micro- and macrovascular tissues of diabetic animals
and of patients with diabetes (712). However, accumulating evidence has shown that
oxidative
stress also may play a role in the development of diabetic
vascular complications.
It has been postulated
that ROS production in diabetes may be enhanced by hyperglycemia
through various mechanisms such as enhanced formation
of glycation products (17), altered polyol pathway
activity (18), and increased superoxide release from mitochondria
(19). In contrast, attention is increasingly focused on
NAD(P)H oxidase as the most important source of ROS production
in blood vessels (20 23). Recent reports have implicated
that this oxidase may be involved in the pathophysiology
of various vascular diseases, including hypercholesterolemia
(24), atherosclerosis (2527), and hypertension (28). In this
review, we show that a PKC-dependent activation of
NAD(P)H oxidase may be an essential mechanism responsible
for increased ROS production in diabetic vascular tissues. This
may provide novel insights into antioxidative therapy for preventing
diabetic vascular complications. In animals with chemically or genetically induced diabetes,
the PKC activities in the membrane pool, which is the active
fraction, have been shown to be elevated in many vascular
tissues such as the aorta, heart, retina, and renal glomeruli by
us and other investigators (711). The glucose-
induced activation of the DAG-PKC pathway in the vascular
cells probably could be linked to the dysfunction of
vascular walls in diabetes.
The role of oxidative stress in atherogenesis has received
increasing attention in recent years. Oxidative modification of
lipoproteins is critical for atheromatous lesion formation. In
addition, ROS reacts with nitric oxide, resulting in loss of nitric
oxides antiatherogenic properties. ROS affect a large number
of various signaling pathways and proteins and cause DNA
damage in vascular tissues. Recent reports have indicated that
vascular NAD(P)H oxidase driven ROS production may play
a role in the pathophysiology of various vascular diseases,
including hypercholesterolemia, atherosclerosis, and hypertension. Therefore, we examined
the role of NAD(P)H oxidase in
high glucose levelinduced ROS production in cultured aortic
endothelial cells, smooth muscle cells, and renal mesangial
cells using electron spin resonance (ESR) spectroscopy (34).
Antioxidative Agents Targeting the Mechanism
of PKC-Dependent Activation of NAD(P)H
Oxidase
Inhibition of oxidative stress using various antioxidants has
shown some success at preventing the diabetic vascular complications. One possible reason
for
its ineffectiveness is that radical scavengers such as vitamin E
may serve not only as an antioxidant but also as a pro-oxidant. For example, vitamin E reacts
with radicals and subsequently
generates tocopheroxyl radicals. Another problem in antioxidative
therapy is the lack of a direct and sensitive method to
evaluate oxidative stress in vivo. This in vivo ESR method
has been recently developed for a noninvasive, sensitive in vivo
measurement of free radical generation in living animals. Free
radicals such as ROS have unpaired electrons, which can be
detected selectively and sensitively by ESR spectroscopy.
The effect of the 3-hydroxy-3-methylglutaryl CoA reductase
inhibitors (statins) on cardiovascular diseases is mainly attributed
to their cholesterol-lowering properties, but accumulating
evidence has shown that some beneficial effects of these agents
may be independent of plasma cholesterol levels. Notably,
recent reports have revealed that statins may inhibit ROS
production in vascular cells probably via inhibition of angiotensin
IIinduced NAD(P)H oxidase activation (48,49). Therefore,
we expected that statins might also inhibit the high
glucoseinduced NAD(P)H oxidase activation and finally prevent
the increase in ROS production in diabetes.
The inhibition of increased oxidative stress in diabetes by
these agents strongly supports the notion that the PKC-dependent
activation of NAD(P)H oxidase may be a main source of
ROS production in diabetes. The mechanism of PKC-dependent
activation of vascular NAD(P)H oxidase may be a new
target of antioxidative therapy for preventing the diabetic vascular
complications (Figure 2). Figure2nya bagus mba
Conclusion
We showed here that a PKC-dependent activation of vascular
NAD(P)H oxidase may play a major role in the increase in
ROS production in diabetes and finally may contribute to the
onset or development of diabetic micro- or macrovascular
complications. Such a mechanism may be a new target of
antioxidative therapy for preventing diabetic vascular
complications.