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Protein oxidation in diabetes mellitus

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Protein Oxidation and Disease, 2006: ISBN: 81-308-0028-4

Editor: Jens Pietzsch

Protein oxidation in diabetes

mellitus
Ulrich Julius1, Irina G.Obrosova2 and Jens Pietzsch3
1
Medical Clinic and Outpatient Department III, University Hospital at the
University of Technology, Dresden, Germany; 2Pennington Biomedical
Research Center, Louisiana State University System, Baton Rouge, LA, USA
3
Department of Radiopharmaceutical Biology, Institute of Radiopharmacy
Research Center Rossendorf, Dresden, Germany

Abstract
Growing evidence indicates that diabetes mellitus
is accompanied by oxidative stress. In addition to
lipids and nucleic acids, proteins also undergo
oxidation. Diabetic patients have several groups of
oxidatively modified proteins, i.e., advanced glycation
end-products, carbonylated proteins, and advanced
oxidation protein products, in the circulation.
Oxidized proteins have also been detected in tissues of
animals with experimental diabetes. Oxidative
modifications of mitochondrial proteins are blunted in
diabetic conditions. Of special interest is the oxidation
Correspondence/Reprint request: Prof. Dr. Ulrich Julius, Medizinische Klinik und Poliklinik III
Universitätsklinikum Dresden, Fetscherstrasse 74, 01307 Dresden, Germany
2 Ulrich Julius et al.

of apolipoprotein B-100, the structural protein of low density lipoproteins.

Oxidatively modified apolipoprotein B-100 molecules had been detected in
atherosclerotic lesions as well as in the circulation. Oxidatively altered
proteins lead to vascular damage. Increased oxidative stress was also
observed in gestational diabetes. Protein oxidation plays a major role in the
development of diabetes complications and has been implicated in the
pathogenesis of diabetic nephropathy, neuropathy and retinopathy. In diabetic
nephropathy which develops as a result of complex interplay of hemodynamic
and metabolic factors, oxidative protein modifications contribute to
albuminuria, mesangial expansion, glomerulosclerosis and tubulo-interstitial
fibrosis. There is also strong experimental evidence that morphological
abnormalities of diabetic retinopathy, such as pericyte dropout, formation of
acellular capillaries, are associated with non-enzymatic glycosylation
(glycation) and accumulation of advanced glycation end-products. Protein
oxidation also takes place in the lens and plays a central role in the
development of cataract. Growing evidence implicates advanced protein
glycoxidation and lipoxidation in the pathogenesis of both somatic and
autonomic diabetic neuropathy, and, in particular, motor and sensory nerve
conduction deficits, neurovascular dysfunction, diabetic neuropathic pain and
loss of sensory function, and morphological abnormalities characteristic for
peripheral and autonomic neuropathy.

Oxidative stress in diabetes: An introduction

Increased production of reactive oxygen (ROS) and reactive nitrogen
(RNS) species and subsequent oxidative-nitrosative stress have been observed
in both Type 1 and Type 2 diabetes mellitus [1-4]. ROS and RNS directly
oxidize and damage nucleic acids, proteins, and lipids. In addition to their
ability to directly inflict damage on macromolecules, ROS and RNS indirectly
induce damage to tissues by activating a number of cellular stress-sensitive
pathways. This review deals with the influence of oxidative stress in diabetes
on proteins and on these pathways. Increasing evidence from both
experimental and clinical studies suggests that oxidative-nitrosative stress
plays a major role in the pathogenesis of both types of diabetes mellitus [3]. In
diabetic conditions, free radicals production via glucose oxidation, non-
enzymatic glycosylation (glycation) of proteins, and the subsequent oxidative
degradation of glycated proteins is increased [5]. Enhanced free radical
generation and simultaneous decline of antioxidant defense mechanisms lead
to damage of cellular organelles and enzymes, protein oxidation, and
development of insulin resistance. High blood glucose level leads to
overproduction of reactive oxygen species (ROS) by the mitochondria electron
transport chain. High reactivity of ROS determines chemical changes in
virtually all cellular components, leading to nucleic acid and protein
Protein oxidation in diabetes 3

modification and lipid peroxidation [6]. In high-fat diet-induced obese and

insulin-resistant mice (HF) adipocytes it has been shown that protein kinase
C-delta is responsible for elevated intracellular ROS production, and this is
mediated by high glucose and NADPH oxidase [7]. Glibenclamide also
stimulates ROS production in pancreatic beta-cells [8]

Advanced glycation endproducts (AGE)

Advanced glycation endproducts (AGE) are a class of compounds
resulting from glycation and oxidation of proteins, lipids or nucleic acids.
Glycation is a non-enzymatic addition or insertion of saccharide derivatives to
these molecules [9]. This process leads to the formation of intermediary Schiff
bases and Amadori products and finally to irreversible AGE. This classical
view has been modified in recent years with recognition of the importance of
oxidative and carbonyl stress in endogenous AGE formation [10]. These stable
compounds accumulate slowly throughout the life span and contribute to
structural and physiologic changes in the cardiovascular system such as
increased vascular and myocardial stiffness, endothelial dysfunction, altered
vascular injury responses, and atherosclerotic plaque formation. Mechanisms
underlying these alterations include AGE cross-linking of collagen and AGE
interactions with circulating proteins and AGE receptors. The clinical
manifestations of AGE accrual - isolated systolic hypertension, endothelial and
diastolic dysfunction, and atherosclerosis - underscore their role in increased
cardiovascular risk associated with aging as well as diabetes and hypertension
[11]. In the glycation reaction, alpha-dicarbonyl compounds such as
deoxyglucosone, methylglyoxal, and glyoxal are more reactive than the
parental sugars e.g., glucose, with respect to their ability to react with amino
groups of proteins, to form inter- and intramolecular cross-links of proteins,
stable end products called advanced Maillard products or AGE. It was
suggested that the formation of AGE not only modifies protein properties but
also induces biological damage in vivo. Evidently, glycation of protein
generates active centers for catalyzing one-electron oxidation-reduction
reactions. This active center, which exhibits enzyme-like character, is
suggested to be the cross-linked Schiff base radical cation of the protein. It
mimics the characteristics of the metal-catalyzed oxidation system. These
results together indicate that glycated proteins accumulated in vivo provide
stable active sites for catalyzing the formation of free radicals [12]. In one
study, AGE were found to be elevated only in Type 2 diabetic patients whereas
in Type 1 diabetic patients they were not significantly different from healthy
controls [13]. There was a tight correlation between AGE and advanced
oxidation protein products (AOPP) in both types of diabetes, and both AGE
and AOPP levels correlated with triglycerides. In Type 1 diabetes mellitus
only, AGE concentration correlated with HbA1c and blood glucose levels.
4 Ulrich Julius et al.

Protein carbonyl content

Protein carbonyl content is the most general and well-used biomarker of
severe oxidative protein damage. Direct reaction of proteins with ROS can lead
to formation of protein derivatives or peptide fragments possessing highly
reactive carbonyl groups (aldehydes, ketones) that, normally, are not present in
proteinogenic amino acids. Note that proteins containing reactive carbonyl
groups can also be generated by secondary reactions of primary amino groups
of lysine residues with reducing sugars or their oxidation products. Human
diseases associated with enhanced protein carbonylation include Alzheimer's
disease, rheumatoid arthritis, chronic lung disease, chronic renal failure,
diabetes and sepsis [14,15]. Assessment of protein carbonyl groups as
biomarkers of oxidative stress has some advantages in comparison with
measurements of other (non-protein) oxidation products because of relatively
early formation and relative stability of carbonylated proteins. Most of the
assays for detection of protein CO groups involve derivatisation of the
carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to
formation of a stable dinitrophenyl (DNP) hydrazone product. The latter can be
detected by various means, i.e., spectrophotometric methods, enzyme-linked
immunosorbent assay (ELISA), and one-dimensional or two-dimensional
electrophoreses followed by Western blot analysis. At present, assessment of
protein carbonyl groups after their derivatisation with DNPH is the most
widely employed test of protein oxidation [14]. Plasma protein carbonyl
groups have been found elevated in chronic diabetic Sprague-Dawley rats
compared with those in either acute diabetic rats or non-diabetic controls [16].
Such increase in plasma protein carbonyl groups in chronic but not in acute
diabetic rats suggests that persistent hyperglycemia is involved in the evolution
of oxidative protein damage [16]. Red blood cell plasma membrane protein
carbonyl content assessed by the DNPH method was found significantly higher
in Type 2 diabetic patients with either good or poor glycemic control compared
with non-diabetic subjects [17]. Furthermore, there was a significant difference
in protein carbonyl content between patients with poor and good glycemic
control.

Advanced oxidation protein products (AOPP)

AOPPs include several oxidation products potentially formed due to the
actions of chloride, hydroxylation, dimerisation, adduct formation, but also
fragmentation. The mechanisms of these reactions are not fully understood, but
the developed changes are irreversible. Oxidative stress plays an important role
in the formation of AOPP in diabetes mellitus [18]. Reaction of proteins with
hypochlorite (HOCl/OCl-) generated via myeloperoxidase (MPO) activity,
could represent one of the pathways for AOPP production in plasma proteins
Protein oxidation in diabetes 5

exposed to activated phagocytes [19]. AOPP can be measured by a

spectrophotometric assay [20]. In diabetic Sprague-Dawley rats, AOPP were
found elevated in plasma [16]. One group found AOPP increased in diabetic
subjects with Type 2, but not Type 1, diabetes [21]. Others reported increased
AOPP levels in Type 1 diabetic patients, too [22]. Furthermore, in Type 1
diabetes families, protein oxidation was confirmed by the presence of
increased AOPP [23]. Elevated AOPP concentrations have also been detected
in non-diabetic subjects with mild to moderate renal insufficiency
[24,20,25,26], also in critically ill patients (sepsis, reperfusion injury, heart
failure) [27]. It is interesting that these authors [24] found a decrease of AOPP
abundance in these patients after the administration of ramipril, probably due
to decreased oxidative stress.

Protein oxidation in diabetes

Mitochondrial oxidative damage
Protein carbonyl group, nitrotyrosine, AOPP, and lipid hydroperoxide
levels were found decreased in liver mitochondria of diabetic rats [28]. In the
same study, only AOPP and lipid hydroperoxide levels were decreased in
kidney mitochondria being not significantly different in muscle mitochondria.
Diabetes-associated decrease in mitochondrial oxidative protein damage may
be either due to upregulation of antioxidant defense mechanisms or a different
adaptive response of the cells to the increased extramitochondrial oxidative
stress. However, mechanism(s) preventing oxidative damage of mitochondrial
proteins in diabetes remain to be clarified.

Insulin oxidation
Taking into consideration the presence of enhanced oxidative stress in
diabetes mellitus, the effects of ROS on human insulin were studied in vitro
[29]. Human recombinant insulin was exposed to free hydroxyl radicals (HO•)
generated by the Fenton reaction. The following changes in insulin molecular
structure were recorded: a) a significant increase in absorbance (280 nm) due
to phenylalanine hydroxylation, b) peroxidation products formation on the
amino acid side branches (peroxyl and alcohoxyl group), c) increase in free
carbonyl groups with loss of secondary structure, and d) modification of
epitopes decreasing the insulin antigen-antibody reactivity measured as a
decrease in insulin concentration by radioimmunoassay.

Oxidation of ceruloplasmin and ferritin

Aminoacetone (AA), a putative endogenous source of cytotoxic
methylglyoxal, and ceruloplasmin (CP), an antioxidant plasma copper transporter,
are increased in diabetes. AA was recently shown in vitro to act as a pro-oxidant
6 Ulrich Julius et al.

toward ferritin and isolated mitochondria. AA-generated ROS have been

implicated in CP oxidation [30]. Incubation of human CP with AA resulted in
extensive protein aggregation. The role for ROS-driven thiol cross-linking in
CP aggregation is supported by demonstration of the inhibitory effects of
added superoxide dismutase, catalase, mannitol, and dithiothreitol. CP addition
to AA solutions accelerated oxygen consumption by AA and caused CP copper
ion release and loss of ferroxidase and aminoxidase activities. This reaction, if
operative in vivo, would impair the antioxidant role of CP and iron uptake by
ferritin and hence contribute to intracellular iron-induced oxidative stress
during AA accumulation in diabetes mellitus.

Protein oxidation in tissues

Peroxidized protein content (DNPH incorporation) was reported elevated
in the liver of diabetic rats, compared with controls, and this increase in DNPH
incorporation was inhibited by vitamin E, but not vitamin C [31]. In another
study, red blood cell glutathione (GSH) level and glutathione peroxidase
(G-Px), glutathione reductase (G-Red) and glutathione S-transferase (GST)
activities were measured in controls, as well as well controlled and poorly
controlled Type 2 diabetic subjects before and after in vitro incubation with
H2O2 [32]. Red blood cell GSH level, G-Px and G-Red activities decreased but
GST activity increased in red blood cells from all the groups after incubation
with H2O2. The percent decrease of G-Px and G-Red, but not GSH, was related
to glycemic control. The percent increase in GST was found independent from
the presence or absence of diabetes. It appeared that GSH and GSH-related
antioxidant enzymes in human red blood cells are susceptible to oxidation;
furthermore, G-Px and G-Red in Type 2 diabetic subjects with poorly
controlled diabetes were found particularly susceptible to oxidation. Another
study examined the effect of elevated glucose concentrations on protein
modification and functional changes in human red blood cells in vitro [33].
Erythrocytes were exposed to 5-45 mmol/L glucose concentrations, and
protein glycation and oxidation were evaluated in erythrocyte ghosts by
spectrophotometric methods. G-actin was measured by a DNase I inhibition
assay in cell lysates. In 24 h after exposure to different concentrations of
glucose, a significant protein oxidation was found in the samples incubated
with 25 and 45 mmol/L glucose whereas G-actin increase was observed for all
concentrations. In 48 h, a significant increase in glycation (for samples
incubated with 25 and 45 mmol/L glucose), protein oxidation, and G-actin was
observed in all samples. Significant positive correlations were observed
between glucose concentration and protein oxidation, glucose and G-actin
concentrations and protein oxidation and G-actin aconcentrations at the 24-h
and 48-h time points These findings indicate that oxidative effect of glucose on
red blood cells is determined by both glucose concentration and time of
Protein oxidation in diabetes 7

exposure. Another group detected enhanced oxidative stress in the small

intestine of streptozotocin-induced diabetes by measuring the extent of
oxidative damage as well as the status of the antioxidant defense system [34].
Significant increases in protein oxidation (38%) as measured by protein
carbonyl content were observed in diabetic rats with 6-week duration of
streptozotocin-induced diabetes.

Lipoprotein oxidation
Low density lipoproteins (LDL) incubated with pathophysiological
concentrations of glucose became selectively enriched in ortho- and meta-
tyrosine, implicating a hydroxyl radical-like species in protein damage. Model
system studies demonstrated that the reaction pathway requires both a reactive
carbonyl group and a polyunsaturated fatty acid, involves lipid peroxidation,
and is blocked by the carbonyl scavenger aminoguanidine [35]. Advanced
glycation is a major pathway of posttranslational modification of plasma and
tissue proteins. In this line, incubation of LDL with glucose or glucose-6-
phosphate produces AGE moieties on both the lipid and apolipoprotein
constituents [36]. In Type 2 diabetic patients, LDL were more glycated, more
susceptible to in vitro oxidation and had a higher percentage of electronegative
LDL. The glycation of apolipoprotein B-100 is proposed to be associated with
a significant increase of LDL oxidation in vivo and in vitro [37]. The oxidative
modification of LDL may be dependent or independent on lipid peroxidation.
This peroxidation may be initiated by metal ions, possibly in association with
phospholipase activity, or catalyzed by MPO independent of metal ions. It
results in generation of aldehydes, which substitute lysine residues in the
apolipoprotein B-100 moiety and thus in the generation of oxidized LDL.
These processes are associated with formation of reactive aldehyde which
causes oxidative modification of LDL in the absence of lipid peroxidation [38].
Formation of DNPH-reactive apolipoprotein modifications during in vitro
exposure of LDL to MPO has been described in the literature [39]. In contrast
to complex mixture of peptides formed during oxidation of LDL with the
reagent HOCl, MPO-induced in vitro oxidation produced a major tryptic
peptide with absorbance at 365 nm. These findings demonstrate that the
DNPH-reactive modifications others than aldehydes and ketones can be
formed in the course of protein oxidation. They also suggest a direct
interaction of the LDL particle with the MPO active site and indicate that
effects of protein microenvironment can greatly influence product formation
and stability. One study describes the oxidative modification of LDL by
hypochlorite (OCl-), a powerful oxidant produced from H2O2 and chloride in
MPO-catalyzed reaction, with the enzyme is released from activated
neutrophils and monocytes [40]. Exposure of LDL to enzymatically generated
OCl- at 4 or 37 degrees C resulted in immediate and preferential oxidation of
8 Ulrich Julius et al.

amino acid residues of apolipoprotein B-100. Lysine residues quantitatively

represented the major target and, like tryptophan, were oxidized to
approximately the same extent with enzymatically generated OCl-. In contrast,
LDL lipid oxidation was less favoured than protein oxidation. Treatment with
OCl- caused aggregation of LDL, as shown by an increased turbidity of the
oxidized LDL solution and elution from a size-exclusion HPLC column of
high-molecular-mass LDL complexes. Chemical modification of lysine
residues before oxidation with OCl- prevented aggregation, while it enhanced
the extent of lipid peroxidation. Treatment of LDL with OCl- also caused the
formation of carbonyl groups and release of ammonia; these two modifications
were inhibited by the lysine-residue modification before oxidation. These
results demonstrate that aggregation reactions are dependent on initial lysine
oxidation by OCl-, followed by deamination and carbonyl formation, but
do not involve lipid (per)oxidation. The authors proposed that the observed
OCl--mediated aggregation of LDL is caused, at least in part, by cross-linking
of apolipoproteins by Schiff base formation, independently of lipid
peroxidation. Some studies report the protein component of LDL to be the
major site for HOCl attack, whereas others describe extensive involvement of
lipid peroxidation. There is one study addressing this controversy [41]. The
obtained results are consistent with the hypothesis that radical-induced
oxidation of LDL lipids by HOCl is a secondary reaction, with most HOCl
consumed via rapid, non-radical reaction with apolipoprotein B-100. The
subsequent incubation of HOCl-treated LDL gives rise to lipid peroxidation
and antioxidant consumption in a time-dependent manner. Similarly, with
MPO/H2O2/Cl- (the source of HOCl in vivo), protein oxidation is rapid and is
followed by an extended period of lipid peroxidation during which further
protein oxidation does not occur. The initial reaction of low concentrations of
HOCl (400-fold or 800-fold molar excess) with LDL therefore seems to occur
primarily by two-electron reactions with side-chain sites on apolipoprotein
B-100. Some of the initial reaction products, identified as lysine-residue-
derived chloramines, subsequently undergo homolytic (one-electron) reactions
to form radicals that initiate antioxidant consumption and lipid oxidation via
tocopherol-mediated peroxidation [41]. In another study, the authors examined
time courses of lipid and protein oxidation during copper ion-mediated oxidation
of LDL [42]. They showed an early, lipid-mediated loss of 40-50% of the
tryptophan residues of the apolipoprotein B-100 protein. Concomitant with
tryptophan loss, the antioxidant alpha-tocopherol is consumed with subsequent
extensive lipid peroxidation. Further changes to the protein, including the copper
ion-dependent 3.5-fold increase in 3,4-dihydroxyphenylalanine and the copper ion-
independent 3-5-fold increase in o-tyrosine, i.e., the oxidation products of tyrosine
and phenylalanine, respectively, occur only after maximal lipid peroxidation.
Prolonged incubation periods result in 3,4-dihydroxyphenylalanine depletion,
Protein oxidation in diabetes 9

presumably reflecting further oxidative changes. Overall, copper ion-mediated

LDL oxidation appears to proceed initially by lipid radical-dependent
processes, even though some of the earliest detectable changes occur on the
apolipoprotein B-100 protein. This is followed by extensive lipid peroxidation
and subsequent additional oxidation of aromatic residues on apolipoprotein
B-100; though it is not yet clear whether this late protein oxidation is lipid-
dependent or occurs as a result of direct radical attack. Another study
evaluated carbonylation and fragmentation of apolipoprotein B during LDL
oxidation induced in vitro by copper ions and HO•/O2•- radicals generated by
radiolysis [43]. The authors used DNPH to detect apolipoprotein B
carbonylation and fragmentation. Apolipoprotein B carbonylation was found
present during the lag phase of LDL oxidation in the two oxidative processes
whereas apolipoprotein B fragmentation was not detected during the lag phase
of copper-oxidized LDL but was manifest during the propagation phase. By
contrast, apolipoprotein B fragmentation was detected in the lag phase of LDL
oxidized by HO•/O2•-. On the other hand, met-hemoglobin can transfer most of
its hemin to LDL, and the presence of H2O2 accelerated the process. In
contrast, met-myoglobin partially released hemin, but only in the presence of
H2O2. The minor amount of hemin transferred from met-myoglobin to LDL
appeared sufficient to trigger apolipoprotein B oxidation, due to formation of
covalent aggregates via inter-bityrosines [44]. This suggests that heme bound
to high affinity site(s) is responsible for oxidation. LDL components providing
the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra
revealed that the high affinity site of monomeric hemin is located on the LDL
apolipoprotein B. The complex heme-CO-apolipoprotein B underwent
instantaneous oxidation to hemin-apolipoprotein B, and the bound hemin then
slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented
LDL oxidation by trapping hemoprotein transferable heme. The authors
concluded that met-hemoglobin and met-myoglobin exert their oxidative
activity on LDL via transfer of heme, which serves as a vehicle for iron
insertion into the LDL protein. According to some authors, no evidence was
obtained for copper ion-induced stimulation of lipid peroxidation or direct
protein oxidation caused by glucose [45]. This study suggests that the earliest
significant event in this system is metal ion-independent glycation of the
protein component of LDL, whilst oxidative events (glycoxidation or direct
oxidation of lipid or proteins) occur to a significant extent only at a later time
point. In contrast, other authors describe that Cu2+-mediated LDL oxidation
was potentiated by glucose in a dose-dependent manner and increased the
chemotactic activity of oxidized LDL towards pro-inflammatory cells [46].
Incubation with glucose alone, under conditions in which very little oxidation
was observed, also increased the chemotactic property of LDL particles. As
has been discussed above, several groups have shown that hemin (Fe3+-
10 Ulrich Julius et al.

protoporphyrin IX) plays an active role in the oxidation of LDL apolipoprotein

B-100 [44, 47-50]. Oxidation of LDL apolipoprotein B-100 by hemin leads to
the formation of apolipoprotein B-100 crosslinks and loss of free amino
groups. Recently, it has been demonstrated that hemin may induce specific
oxidation of both proline and arginine residues of apolipoprotein B-100 in
vitro [51, 52]. This leads to the formation of γ-glutamyl-semialdehyde (γGSA)
which can be reduced to 5-hydroxy-2-aminovaleric acid (HAVA). γGSA arises
directly via oxidation of proline or via hydrogen abstraction and subsequent
loss of the guanidine group of arginine in the presence of reactive oxygen
species. The non-proteinogenic amino acid HAVA represents a highly specific
and sensitive marker of protein oxidation and can be sensitively quantified by
gas chromatography-mass spectrometry (GC-MS) [51]. In vitro experiments
provided the following results [53]. The hemin/H2O2 as well as the
Fe2+/EDTA/ascorbate systems induced a significant formation of HAVA.
The addition of the redox-inert iron chelator N,N-bis(2-hydroxybenzyl)
ethylenediamine-N,N-diacetic acid (HBED) to the system with hemin/H2O2
clearly inhibited the formation of HAVA. Oxidation systems containing Cu2+,
Cu2+/H2O2, and Fe2+ induced only a slight formation of HAVA. On the other
hand, oxidation systems containing MPO/H2O2 and HOCl, respectively, did
not produce significant amounts of HAVA. The addition of glucose alone to
native LDL did not induce any measurable increase of the relative
electrophoretic mobility (REM). In contrast, the oxidation of LDL by
hemin/H2O2 under the conditions employed doubled this parameter. Clearly
manifest REM increments have been seen with the increase of glucose
concentrations. In a similar experiment, the HAVA concentration has been
measured. Here comparable results have been obtained. Hemin/H2O2 led to an
increase of the HAVA concentration, which was further enforced by glucose
with a clear dependence on glucose concentration. The addition of manganese
superoxide dismutase completely inhibited the effect of glucose on the HAVA
formation. Furthermore, in groups with impaired glucose tolerance and with
diabetes mellitus the HAVA content in LDL apoB-100 was found to be
significantly increased [53]. 3-chlorotyrosine, a highly specific marker of
MPO-catalyzed protein oxidation, and 6-hydroxy-2-aminocaproic acid
(HACA), another highly specific marker of metal catalyzed protein oxidation,
an index of lysine side chain residue oxidation, can be assessed by sensitive
GC-MS methods [54,55]. Both HAVA and HACA determinations compare
well with the measurement of carbonyl groups that is generally accepted as a
nonspecific index of protein oxidation. Thus, both HAVA and HACA are
sensitive assays for studying specific apolipoprotein B-100 modification.
Assessment of oxidized LDL (oxLDL) in impaired glucose tolerance (IGT)
generated interesting results [56]. After correction for age and Body Mass
Index, serum levels of oxLDL were significantly increased in IGT versus
Protein oxidation in diabetes 11

subjects with normal glucose tolerance. OxLDL levels were not associated
with the following parameters of the oxidative/antioxidative balance in the
blood: total antioxidant capacity, urate-to-allantoin ratio, and circulating
phagocyte oxygenation activity. In stepwise multivariate analysis, LDL
cholesterol and triglycerides were the strongest predictors of circulating
oxLDL levels, followed by HDL cholesterol, 2-h postchallenge C-peptide,
fasting free fatty acids, and serum paraoxonase activity. The strong correlation
of oxLDL with LDL cholesterol and triglycerides indicates that LDL oxidation
in IGT is preferentially associated with dyslipidemia. The increase of oxLDL
may explain the high atherogenic potency of dyslipidemia in the prediabetic
state. Physico-chemical properties and secondary structure of very-low density
lipoprotein (VLDL) apolipoprotein B-100 are also modified in the course of
Cu2+ catalyzed oxidation [57]. Incubation of VLDL with copper ions resulted
in a decrease in tryptophan and lysine residues parallel to lipid peroxidation
products, conjugated dienes and TBARS. Fluorescence polarization showed an
increase in the molecular order at the lipoprotein surface of VLDL. The
secondary structure of apolipoprotein B-100 was investigated by infrared
spectroscopy. Increased order and structural changes, as observed after
oxidative stress on VLDL, could potentially be a determinant of the abnormal
interactions between lipoproteins and cell membranes.

Consequences of protein oxidation

Vascular damage
Enhanced oxidative stress in diabetic vascular tissues is mediated by
multiple mechanisms and, in particular, activation of protein kinase C (PKC)
[58]. High glucose-stimulated ROS production via a PKC-dependent activation
of NAD(P)H oxidase has been described for cultured aortic endothelial cells,
smooth muscle cells, and renal mesangial cells. Such changes can lead to
increased apoptosis [59]. In addition, expression of NAD(P)H oxidase
components was shown to be upregulated in vascular tissues and kidneys in
animal models of diabetes. Endothelial dysfunction and increased arterial
stiffness occur early in the pathogenesis of diabetic vasculopathy. They are
both powerful independent predictors of cardiovascular risk. Hyperglycaemia
also contributes to accelerated arterial stiffening by increasing formation of
AGE, which alter vessel wall structure and function [60]. Patients with
diabetes mellitus suffer from an increased incidence of complications
including cardiovascular disease. One characteristic of such complications is
an accumulation of AGE formed by the adduction of glucose or species
derived from glucose, such as low-molecular mass aldehydes, to proteins.
These reactions result in glycation or glycoxidation of LDL and its conversion
to a form that is recognized by the scavenger receptors of macrophages. This
12 Ulrich Julius et al.

results in the accumulation of cholesterol and cholesteryl esters within

macrophages and the formation of foam cells, a hallmark of atherosclerosis.
Several studies have therefore examined the nature, time course, and extent of
LDL modifications induced by glucose and two aldehydes, methylglyoxal and
glycolaldehyde. These agents have been found to modify arginine, lysine, and
tryptophan residues of the apolipoprotein B-100 protein of LDL, with the
extent of modification induced by the two aldehydes being more severe than
with glucose. These processes are rapid and are not affected by low
concentrations of copper ions. In contrast, lipid and protein oxidation are slow
processes that require copper ions. No evidence was obtained for the
stimulation of copper ion-induced lipid or protein oxidation by glucose or
methylglyoxal, whereas glycolaldehyde treatment resulted in a modest
stimulation of such reactions. These results suggest that the earliest significant
events in this system are metal ion-independent glycation (modification) of the
protein component of LDL, whilst oxidative events (glycoxidation or direct
oxidation of lipid or proteins) occur to at a later time point. This 'carbonyl
stress' is implicated the formation of foam cells in the pathogenesis of
atherosclerosis as well as vascular complications of diabetes [45]. AGE as well
as other protein ligands like S100/calgranulin and amphoterin mediate
receptor-independent and -dependent (via the interaction with the AGE
receptor, RAGE) effects. The ligand-RAGE-interaction results in activation of
NF-κB, increased expression of cytokines, chemokines, and adhesion
molecules, and induces oxidative stress. An important role of the ligand-
RAGE-interaction in accelerated atherosclerosis and increased neointima
formation in diabetes mellitus has been demonstrated in in vivo studies[61].
Increased oxidative stress in diabetes mellitus may underlie the development of
endothelial cell dysfunction by decreasing the availability of nitric oxide (NO•)
as well as by activating pro-inflammatory pathways. In the arterial wall, redox
imbalance and oxidation of tetrahydrobiopterin uncouples endothelial nitric
oxide synthase (eNOS). This results in decreased production and increased
consumption of NO• as well as in generation of other free radicals and oxidants
such as the superoxide anion radical and peroxynitrite [62]. Endothelial injury,
associated with increased production of free radicals, is associated with
increased prostaglandin synthesis and platelet adhesion/activation. These
processes are associated with the release of aldehydes, which induce LDL
oxidative modifications [38]. Oxidized LDL is a marker of coronary
atherosclerosis, whereas malondialdehyde (MDA)-modified LDL is a marker
of plaque instability. Free radicals damage both lipids and proteins, and both
oxidized lipids and proteins accumulate in tissues in aging as well as a number
of pathological conditions including atherosclerosis and diabetes. Oxidation of
the protein and lipid moieties of LDL is of particular interest due to its
potential role in the unregulated uptake of lipids and cholesterol by
Protein oxidation in diabetes 13

macrophages; this may contribute to the initial stage of foam cell formation in
atherosclerosis [42,63]. Several studies have demonstrated an association
between coronary heart disease (CHD) and increased plasma levels of oxidized
LDL. A higher prevalence of elevated oxidized LDL was demonstrated in
persons with high calculated CHD risk prior to events [64]. The odds of having
elevated oxidized LDL for subjects with high calculated CHD risk prior to
events were even higher than for those with diagnosed CHD. In the same
study, several metabolic syndrome components (high triglycerides, low high
density lipoprotein (HDL) cholesterol, glucose intolerance and diabetes)
predicted high levels of oxLDL, independently of LDL cholesterol. In
addition, elevated oxLDL predicted myocardial infarction in the Health ABC
cohort consisting of well-functioning elderly people, even after adjusting for
age, gender, race, smoking, and the metabolic syndrome. Endothelial
dysfunction is associated with pathological vascular conditions including
atherosclerosis, hypertension, and diabetes [65]. The oxidatively modified
form of LDL is recognized as a major cause of endothelial dysfunction in
atherogenesis. At least 11 different scavenger receptors which are collectively
categorized as the "scavenger receptor family" have been described [66]. The
lectin-like oxidized LDL receptor-1 (LOX-1) was identified as the receptor for
oxidized LDL in endothelial cells [65]. LOX-1 is up-regulated by products of
oxidative stress and molecules that induce oxidative stress. LOX-1 activation
induces ROS generation and decreases NO• released from endothelial cells.
LOX-1 activation further induces expression of endothelin-1, angiotensin II
type 1 (AT1) receptor, and cell adhesion molecules [65]. Together with these
properties, LOX-1 functions as an adhesion molecule for activated platelets
and neutrophils. The interrelation between oxidative stress and endothelial
dysfunction has been studied. Human aortic endothelial cells were exposed to
normal (5.5 mmol/L) and high (22.2 mmol/L) glucose. Glucose selectively
increased mRNA and protein expression of cyclooxygenase-2 (COX-2). Its
upregulation was associated with an increase of thromboxane A2 and a
reduction of prostacyclin (PGI2) release [67]. Glucose-induced activation of
protein kinase C resulted in the formation of peroxynitrite and tyrosine
nitration of PGI2 synthase. NO• release was reduced despite 2-fold increase of
endothelial nitric oxide synthase expression [67]. Superoxide (O2•-) is a key
risk factor for cardiovascular disease (CVD), including atherogenesis,
reperfusion injury, angina, restenosis following balloon angioplasty, and vein
graft failure. Axiomatically, O2•- reacts with NO• to form peroxynitrite
(ONOO-) resulting in a depletion of endogenous vascular NO•, which is now
firmly associated with cardiovascular disease [68]. Hyperglycemic LDL
receptor-deficient mice that were fed a cholesterol-free diet for 12 weeks did
not have elevated cholesterol levels compared with nondiabetic mice, and there
was no evidence of increased macrophage proliferation in atherosclerotic
14 Ulrich Julius et al.

lesions [69]. Glucose promoted lipid and protein oxidation of LDL in vitro.
Glucose-oxidized LDL resulted in phosphorylation of extracellular signal-
regulated kinase and protein kinase B/Akt and stimulated proliferation of
isolated macrophages. The mitogenic effect of glucose-oxidized LDL was
mediated by CD36, a class B scavenger receptor, and by extracellular signal-
regulated kinase activation induced by protein kinase C-dependent and
phosphatidylinositol 3-kinase-dependent pathways. Thus, hyperglycemia alone
is not sufficient to stimulate macrophage proliferation in lesions of
atherosclerosis or in isolated macrophages. A combination of hyperglycemia
and hyperlipidemia, however, stimulates macrophage proliferation by a
pathway that may involve the glucose-dependent oxidation of LDL. One study
demonstrated that serum concentrations of the antioxidant enzyme
paraoxonase are significantly lower in Type 1 (insulin-dependent) diabetic
patients compared with non-diabetic first-degree relatives, independent of
known gene polymorphisms. Concentrations are reduced to an extent that can
affect its anti-oxidant capacity. The results are consistent with the contention
that modifications to serum paraoxonase in Type 1 patients can increase risk of
lipoprotein oxidation and, consequently, risk of vascular disease [70].
Recently, evidence for the existence of HOCl-oxidized LDL in human
atherosclerotic lesions has been reported, and MPO, which is thought to act
through production of HOCl, has been identified in human atherosclerotic
lesions [39]. There is growing evidence, from experimental and clinical
studies, that oxidative stress may also be implicated in the pathogenesis of
atherosclerosis and other complications of end-stage renal disease, namely
dialysis-related amyloidosis, malnutrition, and anaemia [71]. Given that free
radicals have extremely short half-lives, e.g., 10-9 seconds for HO•, the clinical
assessment of oxidative stress is based on the measurement of different stable
oxidized compounds (such as lipid peroxidation products, advanced glycation
and oxidation lipid and protein products, nucleic acid oxidation derivatives) or
antibodies directed against oxidized epitopes (such as anti-oxidized low-
density lipoprotein antibodies). Various agonists, pathological conditions, and
therapeutic interventions lead to modulated expression and function of oxidant
and antioxidant enzymes, including NAD(P)H oxidase, endothelial nitric oxide
synthase, xanthine oxidase, MPO, superoxide dismutases, catalase, thioredoxin
reductase, and glutathione peroxidase. Data from numerous studies underline
the importance of dysregulated prooxidant and antioxidant enzymes for the
development and progression of atherosclerotic disease in animal models and
humans [72,73]. Diabetic patients with angiopathy had significantly higher
protein carbonyl content than the diabetic patients without angiopathy. These
data suggest that protein oxidation may be related to complications of diabetes
[17]. The carbonyl content of plasma proteins was measured as global index of
protein oxidation. Its levels were higher in diabetic patients than in controls.
Protein oxidation in diabetes 15

Likewise, both 11-dehydro-thromboxane B2 and prothrombin F1/2 levels were

higher in diabetics. By contrast, anticoagulant markers, such as activated
protein C, protein C activation peptide, and soluble thrombomodulin were
depressed in Type 2 diabetes mellitus. Thus, oxidative stress in Type 2
diabetes mellitus involves circulating proteins and is associated with an
unbalanced promotion of procoagulant reactions [74]. Growing evidence
suggests that RNS, such as peroxynitrite (ONOO-), a product of the reaction of
superoxide anion radicals (O2•-) with nitric oxide (NO•), plays an important
role in both diabetes and diabetes complications. The mechanisms by which
diabetes increases RNS, and those by which RNS modify vascular function,
are not fully understood. It was recently discovered that physiologically
relevant concentrations of ONOO- oxidize the zinc thiolate center in
endothelial nitric oxide synthase (eNOS). In active eNOS dimers, a
tetracoordinated zinc ion is held by four thiols, two from each 135-kDa
monomer. Because it remains partially positively charged, the zinc thiolate
center is subject to attack by the ONOO-. This oxidant disrupts the zinc thiolate
center, releasing zinc, and oxidizing the thiols. Upon thiol reduction, eNOS
dimers dissociate into monomers. This modification of eNOS results in
reduced NO• bioactivity and enhanced endothelial O2•- production, which
reacts with NO• with generation of ONOO- (eNOS uncoupling). In addition,
these studies also demonstrate that low concentrations of ONOO- selectively
nitrate and inactivate prostacyclin synthase (PGIS), which not only eliminates
the vasodilatory, growth-inhibiting, antithrombotic, and antiadhesive effects of
prostacyclin (PGI2), but also increases release of the potent vasoconstrictor,
prothrombotic, growth- and adhesion-promoting agents, prostaglandin H2
(PGH2) and thromboxane A2 (TxA2). eNOS is uncoupled in diabetic mice and
rats which results in increased PGIS tyrosine nitration. Thus, in diabetes, the
enzymes involved in biosynthesis of two major endogenous vasodilators
undergo oxidative inactivation by different mechanisms, which are, however,
tightly interdependent [75]. Hyperglycemia causes a decreased endothelial
reaction. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts
was determined using electrophoretic mobility-shift assays. An acute exposure
(4 h) of human aortic endothelial cells to 25 mmol/L glucose moderately
increased eNOS activity and eNOS mRNA and protein expression. In contrast,
chronic exposure to elevated glucose levels (25 mmol/L for 7 days) reduced
total nitrite levels (46% reduction), as well as eNOS mRNA (46% reduction)
and eNOS protein (65% reduction) expression. In addition, AP-1 DNA binding
activity was increased in chronic high glucose-cultured human aortic
endothelial cells, and this effect was reduced by inhibition of ROS production
in the mitochondrial electron transport chain. Mutation of AP-1 sites in the
human eNOS promoter reversed the effects of high glucose. Compared with
C57BL/6J control mice, eNOS mRNA expression in diabetic db/db mouse
16 Ulrich Julius et al.

aortic endothelial cells was reduced by 60%. This decrease was reversed by
overexpression of manganese superoxide dismutase using an adenoviral
construct [76]. In prediabetic and diabetic states an increased formation of
atherosclerotic lesions has been observed. Glucose-enhanced LDL
apolipoprotein B-100 oxidation may represent one mechanism of increased
atherosclerosis in the pre-diabetic and diabetic state. These data are supported
by recent findings demonstrating γGSA formation in LDL apolipoprotein
B-100 in atherosclerotic lesions [77]. The relationship between plasma levels
of C-reactive protein (CRP), fibrinogen, and AOPP, as selected markers of
inflammation and oxidative stress, and incident first occlusive atherosclerotic
cardiovascular (CV) events (ASCVEs) was examined in a single-center cohort
of uremic predialysis patients without diabetes [78]. Some patients developed
coronary, cerebral, or peripheral artery occlusive accidents. Plasma levels of
CRP, fibrinogen, and AOPP were significantly greater at baseline in these
patients than in those who did not develop atherosclerotic diseases, although
serum creatinine levels did not differ between the 2 groups. By multivariate
Cox regression analysis, age, CRP, fibrinogen, and AOPP levels were
identified as significant independent predictors of ASCVEs. Direct relation
between plasma AOPP level and CAD status was identified using multivariable
regression models in another study [79].

Heart damage
Oxidative stress plays an important role in diabetic heart disease.
Interesting results have been generated in a study aimed at elucidating role of
the potent oxidative and cellular stress-responsive system, heme oxygenase
(HO) [80], in the rat model. One month of streptozotocin-induced diabetes
caused increased oxidative stress manifested by increased number of
8-hydroxy-2'-deoxyguanosine (8-OhdG, a sensitive and specific marker of
DNA damage)-positive cardiomyocytes (to 80% compared with 11.25% in
controls), in association with increased HO isozyme mRNA (2.7-fold increase
compared with controls) and protein expression, and increased HO activity. In
addition, diabetic rats demonstrated increased number of cardiomyocytes with
stainable iron. Treatment with the potent HO inhibitor, tin protoporphyrin IX,
resulted in reduced number of 8-OHdG-positive cardiomyocytes (to 19.5%
compared with 80% in untreated diabetics), in parallel with reduced HO
activity. Note that non-diabetic rats treated with the HO agonist hemin (50
mg/kg/d) exhibited abnormalities similar to diabetic rats. These results provide
the first evidence indicating that diabetes-induced oxidative stress in the
heart is, in part, due to upregulated HO expression and activity. HO acts as
enzymatic pro-oxidant in the diabetic heart, and its effects may be mediated by
increased redox-active iron [80].
Protein oxidation in diabetes 17

Protein oxidation in gestational diabetes (GDM)

Normal pregnancy is accompanied by an increase in AOPP concentrations
[81]. Glutathione S-transferase (GST) activity was significantly increased in
insulin-dependent diabetic pregnancy (IDDP) when compared with non-
diabetic controls [82]. Maternal erythrocyte GST activity seems to be a
sensitive indicator of oxidative stress in IDDP before delivery. Selenium
dependent glutathione peroxidase (Se-GPx) was found to be significantly
increased in IDDP [82]. Thiobarbituric acid reactive-substances (TBARS)
were also significantly increased. Increased oxidative stress is observed in
gestational diabetes or in Type 1 diabetes with pregnancy [83]. NOS activity
is significantly increased in both groups of pregnant women compared with
non-pregnant ones, and in GDM women compared with pregnant controls.
Production of peroxynitrite was higher in GDM women than in pregnant
controls who also had significantly reduced production compared with
non-pregnant women. Basal levels of the platelet membrane peroxidation
evaluated either by hydroperoxide content and TBARS levels or the
susceptibility to peroxidation were increased in GDM patients in comparison
with both control groups [84]. Superoxide dismutase activity and protein
carbonyl content were elevated in placentae obtained from women with GDM
(P<0.04 and P<0.004 respectively), whereas there was no significant
difference in the activity of glutathione peroxidase [85]. In late gestation,
increased oxidative stress is observed in pregnancies complicated by diabetes.
This oxidative stress is manifest by increased lipid peroxide and isoprostane
concentrations and decreased expression and activity of enzymatic
antioxidants. Peroxynitrite-induced nitrosative stress, subsequent to oxidative
stress, is seen in the placenta in preeclampsia and diabetes in association with
altered placental function [86].

Oxidative stress and development of diabetic

complications
Hyperglycemia leads to diabetic complications via multiple mechanisms
from which increased aldose reductase (AR) activity, glycation/glycoxidation,
and activation of PKC are the best studied [87-89]. All three mechanisms
contribute to enhanced oxidative/nitrosative stress developing as a result of the
imbalance between production of free radicals and oxidants and their
neutralization by non-enzymatic antioxidants and antioxidative defense
enzymes. Enhanced oxidative/nitrosative stress is, in turn, responsible for
activation of important downstream pathways, i.e., mitogen-activated protein
kinases (MAPKs), cyclooxygenase-2, inflammatory response, and the nuclear
enzyme poly(ADP-ribose) polymerase (PARP). PARP activation in turn
depletes the intracellular concentration of its substrate NAD+, slowing the rate
18 Ulrich Julius et al.

of glycolysis, electron transport, and ATP formation, and produces an ADP-

ribosylation of the GAPDH [90,91]. Recently, it was demonstrated that the
control of cytosolic and mitochondrial redox balance and the cellular defense
against oxidative damage is one of the primary functions of NADP+-dependent
isocitrate dehydrogenase (ICDH), because it supplies NADPH for antioxidant
systems. When exposed to reducing sugars such as glucose, glucose 6-phosphate,
and fructose, ICDH was susceptible to oxidative modification and damage,
which was indicated by a loss of activity and fragmentation of the peptide as
well as by the formation of carbonyl groups [92]. The glycation-mediated
damage to ICDH may result in the perturbation of cellular antioxidant defense
mechanisms and subsequently lead to a pro-oxidant condition and may
contribute to various pathologies associated with the general aging process and
long-term complications of diabetes. Indicative parameters of protein oxidation
were measured in blood samples from young diabetic patients with recently
diagnosed (< 6 months) microangiopathy (+DC), diabetic patients without
complications (–DC), and healthy, age-matched controls [93]. Evidence of
oxidative damage to proteins was generated through quantification of plasma
protein carbonyl levels, which were significantly higher in –DC subjects,
compared with non-diabetic controls, and were further increased in the +DC
subjects. In addition, a marked increase in protein oxidation was demonstrated
in +DC subjects through assessment of AOPP considered to be an oxidized
albumin index; AOPP values were found significantly higher in +DC subjects
compared with –DC subjects and non-diabetic controls. These results suggest
that oxidatively modified proteins are involved in the pathogenesis of diabetes
complications. In two other studies [13,17], slight but not significant
differences in AGE and AOPP levels were observed in patients with or without
diabetic complications. Diabetic patients with angiopathy had significantly
higher protein carbonyl content than diabetic patients without angiopathy
(p<0.001). These observations suggest that impaired glycemic control is linked
to protein oxidation, and protein oxidation is implicated in diabetes
complications [17].

Nephropathy
The pathogenesis of diabetic nephropathy has been extensively studied in
animal models of diabetes, and involves complex interactions between
hemodynamic and metabolic factors [94]. The hemodynamic factors include
increased systemic and intraglomerular pressure and activation of various
vasoactive hormone pathways, e.g., the renin-angiotensin and endothelin (ET)
systems [94]. The metabolic mechanisms include increased sorbitol pathway
activity [87,95], non-enzymatic glycation/glycoxidation [96,97], activation of
protein kinase C [98,99], oxidative stress [100,101] and activation of
hexosamine pathway [102,103]. In diabetes, methylglyoxal (MGO)-induced
Protein oxidation in diabetes 19

hydroimidazolones are the predominant modification. In contrast to acute

hyperglycemia, mitochondrial respiration is depressed in chronic diabetes. To
determine if MGO-derived protein modifications result in abnormalities in
mitochondrial bioenergetics and superoxide formation, proteomics and
functional studies were performed in renal cortical mitochondria isolated from
rats with 2, 6, and 12 months of streptozotocin (STZ)-induced diabetes [104].
MGO-modified proteins belonged to two pathways: a) oxidative phosphorylation,
and b) fatty acid beta-oxidation. Mitochondria from rats with diabetes
exhibited a decreased rate of oxidative phosphorylation. A decrease in the
respiratory complex III activity significantly correlated with MGO-derived
mitochondrial protein hydroimidazolone content in both diabetic and control
animals. In diabetes, isolated renal mitochondria produced significantly
increased quantities of superoxide, and showed evidence of oxidative damage.
Post-translational modifications of mitochondrial proteins by MGO may
represent causative factor leading to renal mitochondrial oxidative stress
in chronic diabetes. Renal AGE accumulation, as determined by both
immunohistochemistry and serum and renal fluorescence, was increased in
diabetic animals. Nitrotyrosine, a marker of protein oxidation, also followed a
similar pattern [105]. The renin-angiotensin system and the accumulation of
AGE in experimental diabetic nephropathy may be linked through oxidative
stress [105]. Growing evidence obtained in diabetic animals (primarily,
streptozotocin(STZ)-diabetic rats and mice) [100,101,106,107] as well as cell
culture models [106,108,109] indicates an important role for oxidative stress in
both hemodynamic and metabolic mechanisms implicated in diabetic
nephropathy. Oxidative stress is linked to activation of mitogen-activated
protein kinases (MAPKs) [110-112], the nuclear transcription factor NF-κB
[113,114] and upregulation of growth factors such as cytokines [115,116], and
vascular endothelial growth factor (VEGF) [117,118] implicated in diabetic
renal disease [94, 119-122]. Diabetes-induced oxidative stress affects all three
compartments of the renal cortex, i.e., glomeruli [123], tubulo-interstitium
[124] and vasculature [125]. Renal lipid peroxide accumulation [126,100],
reduced glutathione (GSH) and ascorbate (AA) depletion, increased oxidized
glutathione (GSSG)/GSH and dehydroAA/AA ratios and upregulated NADH
oxidase, superoxide dismutase (SOD), GSH peroxidase, glutathion S-transferase
and GSSG reductase activities were demonstrated in early diabetes. Increased
lipid peroxidation and GSH depletion have also been documented in the model
of advanced diabetic nephropathy [101]. Oxidative stress in tissue-sites for
diabetic complications is caused primarily by hyperglycemia [127]; however,
other factors such as increased fatty acids [128,129], angiotensin II [94, 130]
and endothelin-1 [131] also contribute. Hyperglycemia induces ROS
formation via, at least, four mechanisms, i.e., glucose autoxidation [132],
glycation/glycoxidation [133,134], increase in aldose reductase (AR) [135,136]
20 Ulrich Julius et al.

and protein kinase C [137,138] activities. AGEs were found to produce free
radicals by interacting with their receptors [139,140]. ROS are also formed in
the Maillard reaction [141]. The AGE formation inhibitor aminoguanidine was
found to prevent hyperglycemia-induced lipid peroxidation in bovine
endothelial cells thus implicating the glycation mechanism in oxidative stress
in vascular endothelium [142]. In vitro glycation with formation of Amadori
product (prior to its transformation to AGE) causes downregulation of Cu,
Zn-SOD [143]. Amadori-product containing SOD has been found in tissues of
diabetic rats [144]. It was demonstrated [135,136,145-150] that increased AR
activity promotes a) superoxide and hydrogen peroxide formation; b) lipid
peroxidation; c) downregulation of antioxidative defense provided by both
non-enzymatic antioxidants, i.e. GSH, ascorbate, taurine, and antioxidative
enzymes; and d) nitrosative stress, in diabetic and hyperglycemic conditions.
Increased AR activity also contributes to oxidative stress indirectly, via other
metabolic pathways. Increased AR leads to formation of the AGE precursors,
i.e., fructose 3-phosphate, methylglyoxal, and 3-deoxyglucosone [151-153],
and the AGEs, i.e., pentosidine and carboxymethyllysine [154-156]. Fructose
produced by sorbitol pathway is 10-times more potent glycation agent than
glucose [157]. In several cell types [158-161] including glomerular mesangial
cells [159], increased AR activity leads to protein kinase C activation, an
important factor in hyperglycemia-associated oxidative stress. Protein kinase C
is essentially required for phosphorylation (activation) of NAD(P)H oxidase.
These studies support the role for superoxide in diabetic nephropathy;
however, the question about origin of superoxide in the diabetic kidney
requires further studies. In addition to mitochondrial mechanisms,
extramitochondrial sources such as NAD(P)H oxidase [162,163] and 12,15-
lipooxygenase [164] appear to be of great importance. It is unclear whether the
effects of superoxide are mediated via hydrogen peroxide, hydroxyl radicals,
or peroxynitrite, or the effects of these individual ROS in the diabetic kidney
are additive. One of the important downstream consequences of free radical
and oxidant-induced injury is PARP activation. An accumulation of
poly(ADP-ribosyl)ated proteins (an index of PARP activation) was found to
develop very early, i.e., within approximately 12 h of exposure of human aortic
endothelial and human mesangial cells to hyperglycemic conditions. This is
consistent with the recent study in bovine aortic endothelial cells [165] in
which PARP activity was assessed by a different method, i.e., 3H-NAD+
radiolabel incorporation into protein. On the other hand, PARP activation may
precede and contribute to free radical and oxidant-induced injury [166]. At the
end of an 8-week period nitric oxide release, lipid peroxidation and protein
oxidation were measured in kidney cortices, and urinary albumin excretion
(UAE) was determined in 24-h urine samples in rats [167]. Diabetic rats had
higher levels of lipid peroxidation and protein oxidation compared to healthy
Protein oxidation in diabetes 21

rats. NO• release was significantly lower in diabetic groups than healthy
controls. UAE levels showed a positive correlation with lipid peroxidation and
a negative correlation with NO• release. Consequently increased lipid
peroxidation as well as protein oxidation could be involved in the pathogenesis
of diabetic albuminuria [168]. It was also observed that protein carbonylation
was increased in non-diabetic subjects with chronic kidney disease [169-171].
There was a good correlation between variables of lipid (MDA) and protein
oxidation (protein carbonyls). Lipid and protein oxidation variables are higher
in subjects with chronic kidney disease compared with those with essential
hypertension despite similar blood pressure. This suggests that non-
hemodynamic factors such as inflammation and altered cellular redox state
may play an important role in the generation of oxidative stress in chronic
kidney disease.

Diabetic neuropathy
According to the vascular concept of diabetic neuropathy, diabetes-
induced endothelial dysfunction with resulting decrease in nerve blood flow
(NBF) and endoneurial hypoxia play a key role in functional and morphologic
changes in the diabetic nerve. Endothelial changes in vasa nervorum are
caused by multiple factors including increased AR activity [172], non-
enzymatic glycation and glycoxidation [173], activation of PKC [174],
oxidative stress [175-184], changes in arachidonic acid metabolism [185] and
others. Some of the afore mentioned mechanisms, i.e., increased AR and
protein kinase C activities, oxidative/nitrosative stress and impaired
neurotrophic support have also been implicated in the pathogenesis of diabetic
autonomic neuropathy [186-191]. The role for vascular vs. non-vascular
mechanisms in diabetic neuropathy remains a subject of debate. Evidence for
important role for oxidative/nitrosative stress and ROS and reactive nitrogen
species-initiated downstream mechanisms in both experimental [173,176-
180,192-194] and clinical [195,196] neuropathy is emerging. Diabetes-
associated oxidative stress in peripheral nervous system (PNS) is a result of
increased formation of ROS and downregulation of antioxidative defense
provided by both non-enzymatic antioxidants and antioxidative defense
enzymes. Enhanced oxidative stress has been documented in the circulation
(systemic oxidative stress [197]), endothelium [198] as well as the neural
tissues, i.e., peripheral nerve [175,177,179,199,181] and dorsal root and
sympathetic ganglia [175,179,194,200] of PNS in diabetic animal models.
Recently, considerable progress has been made in the detection of diabetes-
associated oxidative injury in PNS. New studies [201,202] have confirmed
previously established lipid peroxidation product accumulation, GSH depletion
and increase in GSSG/GSH ratio, and downregulation of SOD activity in the
diabetic peripheral nerve. In addition, new markers of ROS-induced injury
22 Ulrich Julius et al.

have been identified in peripheral nerve, vasa nervorum and dorsal root
ganglia (DRG) in experimental peripheral diabetic distal symmetric
sensorimotor polyneuropathy (PDN). Those include decreased catalase and
total quinone reductase activities, depletion of AA and taurine and increase in
dehydroAA/AA ratio in peripheral nerve [202], increased production of
superoxide in vasa nervorum [182-184], and accumulation of 8OHdG in DRG
of streptozotocin-induced (STZ)-diabetic rats [194]. Furthermore, accumulation
of nitrotyrosine (a footprint of peroxynitrite-induced protein nitration) has been
documented in peripheral nerve [203], vasa nervorum [182-184], and DRG
[200] of STZ-diabetic rats, peripheral nerve of STZ-diabetic mice [204], and in
ob/ob mice (Obrosova, I.G. and Szabó, C., unpublished results) indicating that
diabetes creates not just oxidative, but oxidative/nitrosative stress in PNS.
Numerous studies reveal the important role of oxidative/nitrosative stress in
nerve functional, metabolic, neurotrophic and morphological abnormalities
charateristic for PDN. Oxidative/nitrosative stress is implicated in a number of
diabetes-induced metabolic abnormalities including decreased Na+/K+-ATP-
ase activity [181,205], changes in NAD(P)+/NAD(P)H redox state and energy
state [181,204,206], increased formation of α-glycerophosphate [206,207],
methylglyoxal and diacylglycerol [198] due to diversion of the glycolytic flux
from glyceraldehyde 3-phosphate dehydrogenase, and activation of
hexosamine pathway [198]. Recently, it has been found that oxidative stress
affects downstream signal transduction mechanisms causing activation of
mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 and impairment
in Ca++ homeostasis and signaling in peripheral nerve and DRG neurons
[208-210]. These three mechanisms play important role in nerve conduction
deficit and another manifestations of PDN.

Diabetic retinopathy (DR)

Hyperglycemia in rats raised levels of o-tyrosine, m-tyrosine, and
oxygenated lipids in the retina, a tissue rich in polyunsaturated fatty acids [35].
Rats that received aminoguanidine did not show this increase in protein and
lipid oxidation. In contrast, hyperlipidemia in the absence of hyperglycemia
failed to increase protein and lipid oxidation products in the retina. These
findings suggest that generation of a hydroxyl radical-like species by a
carbonyl/polyunsaturated fatty acid pathway might promote localized
oxidative stress in tissues vulnerable to diabetic damage. An increased AR
activity has also been found to result in a variety of biochemical abnormalities
in the diabetic retina including pyruvate and α–ketoglutarate depletion [211],
mitochondrial and cytosolic NAD+/NADH redox imbalances [146,145],
upregulation of vascular endothelial growth factor, lipid peroxidation product
accumulation [211], dowregulation of antioxidative defense [211], glial
fibrillar acidic protein overexpression [212], activation of complement in the
Protein oxidation in diabetes 23

retinal vessel wall [213] as well as DNA fragmentation [214,215]. There is

also strong experimental evidence that morphological abnormalities of diabetic
retinopathy, such as pericyte dropout, formation of acellular capillaries,
endothelial proliferation and vascular occlusion, are associated with non-
enzymatic glycation and accumulation of advanced glycation end-products
[216,217]. Oxidative stress plays a role in the development of DR. Oxidative
stress, generated by increased AR activity [135], non-enzymatic glycation-
glycoxidation [133] and PKC activation [218], and, vice versa, contributing to
glycation and activation of diacylglycerol-PKC pathway [127], is a strong
candidate for the role of such a common mechanism. A number of studies
present evidence of increased lipid peroxidation and impaired antioxidative
defense in the retina both in early [219-221] and long-term [217,222] diabetes.
Evidence for important role of oxidative/nitrosative stress in DR is emerging
and a number of antioxidants (vitamin E, vitamin C, nicanartine, lipoic acid
[117,138, 222-225,]) as well as antioxidant mixture [224,225] have been
proven to effectively prevent biochemical, functional, and morphological
abnormalities in the diabetic retina. Findings from several laboratories
[117,118, 226-229] implicate oxidative/nitrosative stress in diabetes-associated
overexpression of retinal vascular endothelial growth factor (VEGF). VEGF is
a 45-kDa homodimeric glycoprotein existing as four isoforms in the human as
a result of alternative RNA splicing [230] and is produced by numerous retinal
cells including pigment epithelial cells, pericytes, endothelial cells, glial cells,
and ganglion cells [231-234]. The consequences of enhanced
oxidative/nitrosative stress such as MAPK and NF-κB activation, endothelin-1
and cyclooxygenase 2 overexpression, and inflammatory response are well
documented in diabetic retina and high glucose-exposed endothelial and
Muller cells [235-240]. One recent study [241] revealed that activation of
PARP, an important downstream effector of free radical and oxidant-induced
DNA damage, is present in inner nuclear and ganglion retinal cell layers of rats
with short-term (4 wks) STZ-diabetes.

Eye lens proteins

It is generally accepted that the sorbitol pathway-associated osmotic stress
is a leading factor in sugar cataractogenesis. However, other studies suggest
that factors like non-enzymatic glycation and oxidative stress also participate.
Biochemical analyses of eye lens proteins showed significant elevation of
carbonyl groups in diabetic animals in comparison to healthy controls [242].
The direct oxidative free radical damage of eye lens proteins does not seem to
be the key mechanism effective in the development of diabetic cataract [242].
Sugar cataractogenesis appears to be a complex process, in which multiple
mechanisms may be involved, including consequences of the overt oxidative
stress in diabetes (e.g., protein modifying potential of toxic aldehydes
24 Ulrich Julius et al.

generated as byproducts of carbohydrate autoxidation and lipid peroxidation).

One project studied the effect of high-glucose (HG) concentrations on the
protein oxidation in cultured rabbit lens cells and in crystalline protein solution
[243]. The authors found significantly higher levels of carbonyl protein in HG-
treated compared with normal glucose-treated lens cells and in crystalline
protein solution. This suggests that HG can cause the oxidation and
modification of proteins in the lens, and that vitamin B6 and n-acetylcysteine
supplementation may be helpful in slowing the oxidation of lens proteins.
Another study addressed the quantitative relationship between the degree of
protein oxidation and the rate of advanced cataract development in the widely
used model of streptozotocin-induced experimental diabetes in rats [244]. In
the course of lens opacification the authors observed a time-dependent increase
in the content of protein carbonyls and decrease in the concentration of protein
sulfhydryls in the lenses of diabetic animals.

Conclusion
Diabetes mellitus is associated with oxidative stress leading to protein,
lipid and DNA oxidation. Oxidized proteins may be involved in the
development of vascular damage, chronic diabetic complications (i.e.,
nephropathy, neuropathy and retinopathy) as well as cataract. Tight glycemic
control is the best measure against oxidative stress and – as it has been shown
in several studies – against diabetes complications. The preventive and
therapeutic effects of antioxidants still remain to be demonstrated.

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