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Paradoxical Effects of Green Tea (Camellia Sinensis)
and Antioxidant Vitamins in Diabetic Rats
Improved Retinopathy and Renal Mitochondrial Defects
but Deterioration of Collagen Matrix Glycoxidation and
Cross-Linking
Georgian T. Mustata,
1
Mariana Rosca,
2
Klaus M. Biemel,
3
Oliver Reihl,
3
Mark A. Smith,
1
Ashwini Viswanathan,
1
Christopher Strauch,
1
Yunpeng Du,
2
Jie Tang,
2
Timothy S. Kern,
2
Markus O. Lederer,
3
† Michael Brownlee,
4
Miriam F. Weiss,
2
and Vincent M. Monnier
1,5
We tested the hypothesis that green tea prevents diabe-
tes-related tissue dysfunctions attributable to oxida-
tion. Diabetic rats were treated daily with tap water,
vitamins C and E, or fresh Japanese green tea extract.
After 12 months, body weights were decreased, whereas
glycated lysine in aorta, tendon, and plasma were in-
creased by diabetes (P < 0.001) but unaffected by
treatment. Erythrocyte glutathione and plasma hy-
droperoxides were improved by the vitamins (P < 0.05)
and green tea (P < 0.001). Retinal superoxide produc-
tion, acellular capillaries, and pericyte ghosts were
increased by diabetes (P < 0.001) and improved by
green tea and the vitamins (P variable). Lens crystallin
fluorescence at 370/440 nm was ameliorated by green
tea (P < 0.05) but not the vitamins. Marginal effects on
nephropathy parameters were noted. However, sup-
pressed renal mitochondrial NADH-linked ADP-depen-
dent and dinitrophenol-dependent respiration and
complex III activity were improved by green tea (P
variable). Green tea also suppressed the methylglyoxal
hydroimidazolone immunostaining of a 28-kDa mito-
chondrial protein. Surprising, glycoxidation in tendon,
aorta, and plasma was either worsened or not signifi-
cantly improved by the vitamins and green tea. Glu-
cosepane cross-links were increased by diabetes (P <
0.001), and green tea worsened total cross-linking. In
conclusion, green tea and antioxidant vitamins im-
proved several diabetes-related cellular dysfunctions
but worsened matrix glycoxidation in selected tissues,
suggesting that antioxidant treatment tilts the balance
from oxidative to carbonyl stress in the extracellular
compartment. Diabetes 54:517–526, 2005
G
reen tea is being widely studied for its alleged
beneficial properties in the treatment or pre-
vention of human diseases. To date, ⬎1,500
articles referencing “green tea” are listed in
Medline. Green tea is reported to delay or prevent certain
forms of cancer, arthritis, and cardiovascular and other
disorders (rev. in 1). To our knowledge, no systematic
study of its efficacy in the prevention of the long-term
complications of diabetes exists.
This study was motivated by three considerations. First,
diabetes complications have been linked to oxidant stress,
in particular the formation of superoxide (2). Second, the
major biological mechanisms of action of green tea are
being attributed to its antioxidant properties. Third, in a
senior high school science research project carried out in
part in our laboratory, a commercial green tea extract that
was fed to aging C57BL/6 mice delayed collagen cross-
linking and fluorescent advanced glycation end product
(AGE) accumulation by a mechanism that was duplicated
by the combination of vitamins C and E (3). Therefore, this
project was designed to examine the ability of green tea to
prevent some of the complications and biochemical dys-
functions of diabetes in the rat.
Green tea is rich in catechins, i.e., polyphenolic com-
pounds whose antioxidant oxidant activity is severalfold
higher than that of vitamins C and E. According to one
study, the total equivalent antioxidant capacity of cat-
echins increases from 0.99 mmol/l for vitamins C and E to
2.40, 2.50, 3.01, 3.82, 4.75, and 4.93 mmol/l for catechin,
epicatechin, gallic acid, epigallocatechin, epigallocatechin
gallate, and epicatechin gallate, respectively (4). Overall,
the catechins represent up to one-third of green tea dry
weight (5). Evidence suggests that catechins can prevent
lipid hydroperoxide formation and toxicity (6) and scav-
enge superoxide and other free radicals (7) and peroxyni-
From the
1
Department of Pathology, School of Medicine, Case Western
Reserve University, Cleveland, Ohio; the
2
Department of Medicine, School of
Medicine, Case Western Reserve University, Cleveland, Ohio; the
3
Institut fu¨r
Lebensmittelchemie, Universita¨ t Hohenheim, Stuttgart, Germany; the
4
Albert
Einstein College of Medicine, Bronx, New York; and the
5
Department of
Biochemistry, School of Medicine, Case Western Reserve University, Cleve-
land, Ohio.
Address correspondence and reprint requests to Vincent M. Monnier, Case
Western Reserve University, Department of Pathology, School of Medicine,
2085 Adelbert Rd., Cleveland, OH 44106. E-mail: vmm3@po.cwru.edu.
Received for publication 11 May 2004 and accepted in revised form 21
September 2004.
†Deceased.
AGE, advanced glycation end product; CML, carboxymethyl-lysine; DNP,
dinitrophenol; ERC, electron respiratory chain; HPLC, high-performance
liquid chromatography; TCC, tricarboxylic acid cycle.
© 2005 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
DIABETES, VOL. 54, FEBRUARY 2005 517
trite (8), all of which have been implicated in some aspect
of diabetes complications. Catechins were also shown to
alter the catalytic activity of oxidative enzymes (7) and to
chelate iron and copper, thus preventing metal-catalyzed
free-radical formation (9). The latter has been associated
with neuropathy in diabetic rats (10).
We tested the hypothesis that green tea displays antidi-
abetic and antiglycoxidation properties. To better inter-
pret any biological effect, we included a control group that
consisted of the classical combination of vitamins C and E.
Impact of these treatments on food intake; body weight;
indexes of glycemia; lipemia; plasma hydroperoxides;
erythrocyte glutathione; retinopathy; nephropathy; renal
mitochondrial functions; lens AGE fluorescence; and sev-
eral markers of glycoxidation, advanced glycation, and
cross-linking have been determined in plasma, skin, ten-
don, and aorta.
RESEARCH DESIGN AND METHODS
Most chemicals were from Sigma-Aldrich (St. Louis, MO). D
8
-lysine and
D
4
-carboxymethyl-lysine were a gift of Dr. Susan Thorpe. Upper-grade (Sen-
cha, Kawanecha) Japanese green tea (East/West Cultural, Kingston, NY) was
prepared daily as described by the manufacturer: 10 g of dry tea was added to
750 ml of deionized boiled water cooled to 90°C, brewed for 3 min, decanted,
vacuum filtered, placed on ice, and protected from light with aluminum foil.
Diabetic rats drank ⬃100 ml/day, i.e., ⬃10 times more than expected daily
human consumption. Vitamin C dose was 1 g/l drinking water, and vitamin E
10 IU/kg body wt solubilized in Tween 80 (1:3 parts) was administered daily by
gavage.
Thirty-eight male Lewis rats (⬃150 g body wt; Charles River, Wilmington,
MA) were divided into four groups: control (n ⫽ 6), diabetic (n ⫽ 10), diabetic
with vitamins C and E (n ⫽ 10), and diabetic with green tea (n ⫽ 10) and
housed under specific pathogen-free conditions. Diabetes was induced intra-
venously with 45 mg/kg streptozotocin (Sigma) in 0.1 mol/l citrate buffer. After
12 months, there were six, nine, eight, and nine survivors in each group,
respectively. Animals received 2 IU of ultralente insulin (Humulin; Eli Lilly,
Indianapolis, IN) three times per week.
Glycemia was monitored monthly, and glycated hemoglobin was moni-
tored at 3, 6, 9, and 12 months. Glycation of plasma, skin, tendon, and aorta
proteins was determined at 12 months by furosine method (see below),
glycohemoglobin by affinity chromatography (Sigma kit #442-B, now discon-
tinued), plasma triglycerides with Sigma kit #37 (GPO Trinder), plasma
hydroperoxides using the FOX assay (Pierce kit #23285), and erythrocyte
glutathione using a Calbiochem kit #354102.
Rats were anesthetized with pentobarbital, and blood was drawn by heart
puncture. Eyes, aorta, and kidneys were removed. Kidneys were fixed in
methacarn (60% methanol, 30% chloroform, 10% acetic acid). Aorta, shaved
dorsal skin (1–2 cm
2
), plasma, and erythrocytes (red blood cells) were stored
at ⫺80°C. One eye was placed in buffered formalin for isolation of the retinal
vasculature by the trypsin digest technique (11). The other retina was frozen
for biochemical analysis. Lenses were removed and stored at ⫺80°C.
Lenses were prepared as water-soluble and -insoluble fractions as previ-
ously described (12). The water-insoluble pellet was solubilized with twice 2%
(wt/wt) pronase E in phosphate buffer that contained 0.05% sodium azide at
37°C in 30 h. Digested fractions were centrifuged and filtered sterile. Protein
concentration in water-soluble fractions was estimated by BCA Protein Assay
(Pierce) and in water-insoluble enzyme digest by the ninhydrin method (13).
AGE-like fluorescence was determined at 370/440 nm.
Histopathology was performed in retinal vasculature isolated by the trypsin
digest technique as previously described (11,14). Acellular capillaries and
pericyte ghosts were quantified in a masked manner.
Detection of superoxide ion in retina. Superoxide anion production was
determined by the lucigenin method (15). Freshly isolated retina was equili-
brated in Krebs-HEPES buffer in the dark at 37°C in 95% O
2
/5% CO
2
for 30 min.
After equilibration, 0.5 mmol/l lucigenin was added, and photon emission was
measured over 10 s. Repeated measurements were made over a 10-min period
in a luminometer (Analytical Luminescence Laboratory, San Diego, CA).
Blanks that contained all components except retinas were counted and
subtracted from all other readings.
Assessment of nephropathy. Urinary albumin and creatinine were deter-
mined by automated instrumentation at University Hospitals of Cleveland.
Collagen type IV immunostaining was determined in methacarn fixed renal
tissue and sections (5 m) stained with rabbit antimouse collagen ␣1
antibodies (Chemicon, Temecula, CA) or nonimmune rabbit IgG for assess-
ment of nonspecific staining. Staining was quantitatively assessed with a Zeiss
Image Analysis System (Carl Zeiss Optical, Chester, VA). Twenty-five to 30
glomeruli per area were assessed in blinded manner.
Histochemical detection of redox active iron in kidney sections was
performed according to Smith et al. (16) using deparaffinized kidney sections
that were fixed in methacarn. The extent of iron deposition was analyzed in a
semiquantitative manner by a blinded investigator.
Mitochondrial functions. Mitochondria were isolated from the renal cortex
of 12-month Lewis rats according to a previously published standard tech-
nique (17). Oxidative phosphorylation was determined using a polarographic
Clark electrode (Instech Laboratories, Philadelphia, PA) (18). Coupled respi-
ration was measured by adding 300 nmol of ADP. Uncoupled mitochondrial
respiration was measured in the presence of 60 mol/l dinitrophenol (DNP).
DNP increases O
2
consumption independent of ADP transport or ATP
synthesis, but its effect is dependent on the supply of reducing equivalents and
electron respiratory chain components.
Electron respiratory chain complex activity. For analysis of complex I, 50
g/ml of mitochondrial proteins were suspended in a hypotonic buffer (25
mmol/l KH
2
PO
4
, 0.5 mmol/l EDTA). On addition of 5 mol/l antimycin A, 2
mmol/l potassium cyanide, 60 mol/l ubiquinone-1, and 100 mol/l NADH,
consumption of NADH was monitored spectrophotometrically at 340 nm
(molar absorptivity ε ⫽ 6,200 mol 䡠 l
⫺1
䡠 cm
⫺1
). For complex III activity, 2.5
g/ml proteins in hypotonic buffer were used. Upon addition of 40 mol/l
reduced decylubiquinone, 50 mol/l cytochrome c, and 2 mmol/l potassium
cyanide, the reduction of cytochrome c was measured at 550 nm (ε ⫽ 18,500
mol 䡠 l
⫺1
䡠 cm
⫺1
). The coenzyme Q analog decylubiquinone was reduced with
NaBH
4
to decylubiquinol and used freshly from a stock concentration of 5
mmol/l (pH 7.0). Complex IV activity was assayed polarographically after
addition of 5 mmol/l ascorbate, 250 mol/l TMPD (N,N,N⬘,N⬘-tetramethyl-P-
phenylenediamine), and 10 mol/l cytochrome c to 100 g of mitochondrial
proteins.
For Western blot detection of methylglyoxal hydroimidazolone–positive
bands, mitochondrial proteins (14 g/lane) were loaded on duplicate 4 –12%
(7 ⫻ 7 cm) Novex NuPAGE gels (Invitrogen, Carlsbad, CA). Monoclonal
antibody to methylglyoxal-derived imidazole AGE (1H7G5) was used at
1:6,000 dilution (2). Antimouse IgG conjugated to horseradish peroxidase
(1:40,000 dilution) and chemiluminescent substrate (SuperSignal West Femto,
Rockford, IL) were from Pierce.
Isolation and solubilization of collagen. Samples of skin, aorta, and
tendon were frozen in dry ice with methanol. Fat and hair were removed from
skin with a razor blade. Tissues were minced and delipidated. Insoluble
collagen was prepared as described (19). Aliquots (5 mg) of insoluble collagen
in buffer H (0.02 M HEPES, 0.1 M CaCl
2
[pH 7.5], and 1 l of toluene and
chloroform per ml) were solubilized with 140 units of collagenase (type VII;
Sigma C-0773) at 37°C for 24 h with continuous shaking. Tubes were
centrifuged, and the pellet was washed and redigested as before. The
supernatants were combined, dried, and stored at ⫺80°C for AGE
determination.
Determination of glucosepane and other glycation-related cross-
links. To the solubilized collagen samples was added 15 l of internal
standard solution that contained 155.5 pmol of
13
C
6
-glucosepane, 78.2 pmol of
13
C
6
-DOGDIC, 99.5 pmol of
13
C
6
-MODIC, and 103.0 pmol of
13
C
6
-GODIC
cross-links (see below). Digestion at 37°C was carried out by sequential
addition every 24 h of 0.120 units of peptidase (Peptidase P-7500; Sigma) in 25
l of HEPES buffer (pH 7.1), followed by 1 unit of pronase from Streptomyces
griseum (Roche Diagnostics, Indianapolis, IN) in 50 l of 0.02 M HEPES (pH
7.1) and 0.2 units of aminopeptidase M (Roche). The freeze-dried product was
diluted to 1.5 ml with water and subjected to preparative high-performance
liquid chromatography (HPLC) as previously described (20), except for a
change in the gradient ammonium formate buffer (10 mmol/l [pH 4.0])/MeOH:
0(0)-2.5(3)-35(12)-95(13–15)-0(18 –23). The residue of the lyophilized collected
fraction was suspended in 40 l of water and subjected to liquid chromatog-
raphy (electrospray ionization) mass spectrometry analysis for glucosepane
and other AGEs. The HPLC and LC/MS/MS system was as previously de-
scribed (21). DOGDIC-ox isomers were monitored mass at m/z 445.
Pentosidine, furosine, and carboxymethyl-lysine. Pentosidine was deter-
mined by HPLC with fluorescence detector in 5 mg of acid hydrolyzed tissue
samples as previously described (22). Carboxymethyl-lysine (CML) and fu-
rosine were determined as trifluoroacetic acid methyl ester derivatives in acid
hydrolyzed delipidated protein samples by gas chromatography/MS using an
isotope dilution method as described by Miyata et al. (23) using a Hewlett-
Packard 5890 Series II Gas Chromatograph with a 5971 Series Mass Selective
Detector and a 6890 Series Automatic Injector, a 25-m ⫻ 0.2-mm ⫻ 0.33-mol/l
Ultra2 column, and the temperature program of Dunn et al. (24). Internal
GREEN TEA AND DIABETES COMPLICATIONS
518 DIABETES, VOL. 54, FEBRUARY 2005
standards were 121.2 nmol of D
8
-lysine and 195 pmol of D
4
-CML. Lysine,
D
8
-lysine, CML, D
4
-CML, and furosine were monitored at ions m/z ⫽ 320, 328,
392, 396, and 110, which eluted at 23 (lysine, D
8
-lysine), 28 (CML, D4-CML),
and 35 (furosine) min.
Tendon breaking time assay. Tendon collagen cross-linking was assayed by
the tendon breaking time method as previously described (3,25).
Statistical analysis. The data are expressed as means ⫾ SD. Univariate
ANOVA was performed between diabetes and the individual intervention
group. Tukey multicomparison tests were also computed using SSPS software
for Windows (SPSS, Chicago, IL). Superscripts that share identical letters
indicate no significant difference among groups. Nonidentical superscripts
indicate statistical difference among groups at P ⬍ 0.05 level or better.
RESULTS
Body weights and food intake. The body weight of
diabetic animals at the end of the study was lower than
that of controls (P ⬍ 0.0001; Table 1). No significant
differences between diabetic groups were observed, i.e.,
neither vitamins E and C nor green tea ameliorated the
weight loss. More food was consumed by diabetic than
normal rats (P ⬍ 0.001). Green tea tended to decrease food
intake in diabetic rats (P ⫽ 0.07).
Green tea and antioxidant vitamins have no effect on
mean glycemia. The rats developed mild to moderate
diabetes with plasma glucose approximately three times
higher than in controls (Table 1, Fig. 1A). Neither vitamins
E and C nor green tea exerted a significant effect on
glycemia. Glycated hemoglobin increased with time, prob-
ably as a result of worsening glucose tolerance. Furosine
(i.e., acid hydrolyzed glycated lysine residues) was ele-
vated in diabetic rat plasma proteins, aorta, and tendon
and unaffected by treatment, except in aorta (Fig. 2), in
which green tea decreased it to ⬃30% of diabetic controls
(P ⬍ 0.01). This tissue-specific decrease in glycation by
FIG. 1. The effect of diabetes, vitamins E and C, and green
tea on glycated hemoglobin (A) and plasma triglyceride (B)
(P < 0.05). Bars that do not share identical letter super-
scripts are different at P < 0.05.
TABLE 1
Body weight, food intake, and glycemia in studied groups of rats
n Final weight (g)
Food intake
(g/rat/day)
Plasma glucose
(mmol/l)
Nondiabetic control 8 637.75 ⫾ 38.39 17.58 ⫾ 3.56 5.33 ⫾ 1.45
Diabetic control 10 347.55 ⫾ 64.74* 32.13 ⫾ 4.28* 18.61 ⫾ 2.31*
EC-treated diabetic rats 10 316.88 ⫾ 41.82* 33.69 ⫾ 5.17* 19.34 ⫾ 2.38*
GT-treated diabetic rats 10 328 ⫾ 80.95* 27.17 ⫾ 4.00* 19.32 ⫾ 3.75*
Data are means ⫾ SD. Measurements are shown for 1 year and are representative for the results obtained throughout the study. EC, vitamin
E ⫹ vitamin C; GT, green tea. *P ⬍ 0.0001 vs. nondiabetic control. Food intake was lower in GT vs. diabetic control (P ⫽ 0.07).
G.T. MUSTATA AND ASSOCIATES
DIABETES, VOL. 54, FEBRUARY 2005 519
green tea is unexpected and suggests either green tea–
induced improvement in collagen turnover or selective
blockage of glycation sites by oxidized green tea
catechins.
Green tea and antioxidant vitamins tend to suppress
hyperlipidemia. Plasma triglycerides were significantly
increased in diabetic compared with nondiabetic rats (P ⬍
0.0001; Fig. 1B). At 6, 9, and 12 months, values were
consistently lowered by the vitamins and green tea, but the
P value was ⬍0.05 only at 6 months in the green tea group.
Antioxidant vitamins and green tea suppress plasma
hydroperoxides and normalizes erythrocyte glutathi-
one. Plasma hydroperoxides were mildly increased in the
diabetic rats at 12 months (P ⬍ 0.05; Fig. 3A). Both
vitamins E and C and green tea decreased hydroperoxides
by ⬃40% (each, P ⬍ 0.001). It is interesting that vitamins E
and C and green tea decreased the hydroperoxides below
control levels by 20% (P ⬍ 0.001 and P ⬍ 0.05, respec-
tively), suggesting intense peroxidative activity even in
absence of diabetes. The 28% decrease in erythrocyte
glutathione (P ⬍ 0.01) was completely restored by both
vitamins E and C and green tea (P ⬍ 0.05; Fig. 3B).
Antioxidant vitamins and green tea improve but do
not normalize retinal superoxide formation and mor-
phologic abnormalities. Superoxide production in the
retina of diabetic rats was increased by 316% compared
with controls (P ⬍ 0.001; Fig. 4A). Vitamins E and C and
green tea induced a 49.4% (P ⬍ 0.05) and 34% (P ⬍ 0.05)
decrease, respectively. As expected, there was a signifi-
cant loss of pericytes in diabetic retinas (Fig. 4B). Thus,
2.9 times more pericyte ghosts were counted in diabetic
than control retinas (P ⬍ 0.05). This was decreased by 14
and 19% (NS) by vitamins E and C and green tea, respec-
tively. Acellular capillaries were almost doubled in the
diabetic state (P ⬍ 0.001; Fig. 4C), and green tea decreased
them by 23% (P ⬍ 0.05). The decrease achieved by
vitamins E and C was nonsignificant.
Antioxidant vitamins and green tea decrease lens
crystallin-bound fluorescence. Previous data revealed
that diabetes increased crystallin-bound fluorescence at-
tributable to glycoxidation (12) or dihydropyridinium
cross-links (26) and that green tea ameliorated selenite
cataract in the rat (27). Here, crystallin-bound fluores-
cence at 370/440 nm was higher in water-soluble fraction
(Fig. 5) in diabetic animals but unchanged in water-
FIG. 2. The effect of diabetes, vitamins E and C, and green tea on
furosine accumulation in tail tendon and aorta collagen and plasma
proteins. Bars that do not share identical letter superscripts are
different at P < 0.05.
FIG. 3. The effect of diabetes, vitamins E and C, and green tea on
plasma hydroperoxide levels (A) and erythrocyte glutathione (B)at
the end of the experiment. Bars that do not share identical letter
superscripts are different at P < 0.05.
GREEN TEA AND DIABETES COMPLICATIONS
520 DIABETES, VOL. 54, FEBRUARY 2005
insoluble fraction (data not shown). Diabetes induced a
2.9-times increase in relative fluorescence compared with
controls (P ⬍ 0.001). Vitamins E and C and green tea
decreased the relative fluorescence by 26 and 32%, respec-
tively. However, only green tea was significant (P ⬍ 0.05).
Nephropathy. Urinary albumin at 12 months was six
times higher in the diabetic compared with control rats
(P ⬍ 0.01; data not shown) but was mild in absolute terms.
Neither vitamins E and C nor green tea improved this
parameter. Glomerular staining for redox active iron in-
creased 1.7-fold (P ⬍ 0.05; data not shown) and glomerular
collagen IV by 23% (P ⬍ 0.05; data not shown), but neither
the vitamins nor green tea induced a significant decrease.
No significant difference in tubular iron staining among the
groups was noticed. The failure to develop dramatic
alterations in renal parameters may relate to the moderate
hyperglycemia of these rats (average HbA
1c
was 10 –11%,
whereas it is often 14 –15% in other studies [28,29]).
Protective effect of green tea on renal mitochondrial
respiration. The growing interest in the role of mitochon-
drial function in diabetes complications led us to examine
this relationship. Renal cortical mitochondria from 12-
month normal rats had a NADH-linked state 3 (ADP
dependent) and state 4 respiratory rate of 201.2 ⫾ 16.4 and
48.4 ⫾ 4.8 nmol O
2
䡠 mg mitochondrial protein
⫺1
䡠 min
⫺1
,
respectively (Fig. 6A). Diabetes induced an 18% decline in
state 3 respiration (P ⬍ 0.002) that was significantly
improved by green tea (P ⬍ 0.05). State 4 respiration was
unchanged under these experimental conditions.
Uncoupling agents collapse the proton gradient, thereby
promoting maximum rates of mitochondrial respiration
dependent on tricarboxylic acid cycle (TCC) and electron
respiratory chain (ERC) activity. The renal mitochondria
demonstrated decrease of uncoupled respiration (Fig. 6B),
supporting the idea that diabetes exerts its effect on either
TCC or ERC. Green tea induced a significant improvement
of uncoupled respiration, suggesting a protective effect on
TCC and/or ERC.
For further defining the activity of NADH-linked ERC
complexes, the activities of complexes I and IV of the ERC
were determined and found not to be affected by chronic
diabetes. However, complex III activity was decreased by
20% (P ⬍ 0.05), and both green tea and the vitamins
improved this parameter but significance was reached
only in the vitamin group (P ⬍ 0.05; Fig. 6C).
Green tea was found to protect against carbonyl-in-
duced modifications of mitochondrial proteins. Western
blot analysis of renal mitochondrial proteins revealed the
presence of three methylglyoxal hydroimidzolone– con-
taining proteins between 28 –38 kDa and ⬃14 kDa (Fig. 6D,
inset). The signal intensity of the ⬃28-kDa band was
increased in mitochondria from all diabetic compared with
control animals (P ⬍ 0.01), and green tea but not vitamins
E and C suppressed the immunoreactivity (P ⬍ 0.05).
Antioxidant vitamins worsen collagen glycoxidation
in selected tissues. Pentosidine and CML were quantified
in plasma and in insoluble collagen from tendon, skin, and
aorta. Diabetes increased pentosidine (Fig. 7A)inall
tissues and, surprisingly, the vitamins markedly worsened
its levels in tendon, aorta, and skin, whereas green tea
worsened levels in tendon and aorta. Thus, the 41%
diabetes-related increase in tail tendons (P ⬍ 0.0001) was
further increased by 55% in the vitamins E and C group
(P ⬍ 0.0001). The 66% increase in skin (P ⬍ 0.0001) was
further elevated by 60% (P ⬍ 0.0001) in the vitamin group.
Similar results were obtained in aorta with an additional
increase of 63% (P ⬍ 0.05) over diabetes control. The effect
of green tea was approaching significance (P ⫽ 0.06).
Green tea had the most enhancing effect in tendons (218%
over diabetes control; P ⬍ 0.001) and a 36% additional
increase approaching significance in aorta (P ⫽ 0.06).
The vitamins and green tea worsened CML in tendon
(P ⬍ 0.05) and plasma (NS; Fig. 7B). Similar to glycated
lysine (Fig. 2), there was a tendency for green tea to
decrease CML in aorta, although not significantly.
FIG. 4. The effect of diabetes, vitamins E and C, and green tea on
superoxide anion production in rat retina (A), frequency of pericyte
ghosts (B), and acellular capillaries (C) in retinas of diabetic rats. Bars
that do not share identical letter superscripts are different at P < 0.05.
G.T. MUSTATA AND ASSOCIATES
DIABETES, VOL. 54, FEBRUARY 2005 521
Effect of antioxidant treatment on glucosepane and
other cross-links of the Maillard reaction. The recently
described AGEs and lysine-arginine cross-links glu-
cosepane and imidazoline cross-links MODIC, GODIC, and
DOGDIC were quantified by LC/MS in enzymatic tendon
digests. Glucosepane is derived from Amadori products,
whereas the imidazolins are derived from methylglyoxal,
glyoxal, and 3-deoxyglucosone, respectively (20).
DOGDIC-ox, an oxidized form of DOGDIC (30), was also
determined.
Glucosepane was 195% increased compared with con-
trols (P ⬍ 0.0001; Fig. 8). Vitamins E and C slightly
decreased glucosepane formation by 17.6% (P ⬍ 0.05),
whereas the effect of green tea was not significant. Among
other cross-links, only DOGDIC was increased by diabetes
(NS; Fig. 8D). In contrast, MODIC and GODIC behaved like
FIG. 5. The effect of diabetes, vitamins E and C, and green
tea on relative fluorescence in water-soluble lens fraction.
Bars that do not share identical letter superscripts are
different at P < 0.05.
FIG. 6. The effect of diabetes, vitamins E and C, and green tea on NADH-dependent (A) and uncoupled (B) mitochondrial respiration,
mitochondrial complex III activity (C), and methylglyoxal modification of a 28-kDa protein probed with an anti-imidazolone antibody (D). Bars
that do not share identical letter superscripts are different at P < 0.05.
GREEN TEA AND DIABETES COMPLICATIONS
522 DIABETES, VOL. 54, FEBRUARY 2005
a mirror image of glucosepane, i.e., they were depressed
(NS) rather than increased in all diabetic animals regard-
less of the treatment modality.
Effect of antioxidant vitamins and green tea on total
tendon cross-linking. Tendon breaking time in urea, a
parameter of total cross-linking, increased in diabetic
animals from 45 ⫾ 12 min in controls to 1,010 ⫾ 680 min
and 1,002 ⫾ 352 min in the diabetes control and vitamins
E and C groups, respectively (each P ⬍ 0.001). Levels
further increased to 1,640 ⫾ 450 min in the green tea group
(P ⬍ 0.001). Large SDs were observed because the extent
of cross-linking far exceeded the linearity of the method.
DISCUSSION
This study reveals a number of important findings on the
relationship between diabetes complications, glycemic
and oxidant stress, and the chronic consumption of green
tea, i.e., a beverage widely promoted for its beneficial
properties that are attributed to its potent antioxidants,
the catechins (5). This study is timely in that oxidative
stress and the formation of mitochondria-derived super-
oxide have been recently proposed to be at the center of
the pathogenesis of diabetes complications (2).
Overall, the data indicate 1) that green tea was success-
ful at ameliorating oxidation end points such as plasma
hydroperoxides, erythrocyte glutathione, retinal superox-
ide formation, and renal mitochondrial respiratory chain
defects and 2) that it was in essence acting similarly to the
combination of vitamins E and C. The effects on nephrop-
athy were inconclusive, possibly because only mild ne-
phropathy developed. Thus, additional studies will be
needed to clarify its effects on this complication, as well as
neuropathy and atherosclerosis.
Despite significant effects of the antioxidant vitamins
and green tea on the development of biochemical and
functional tissue damage, it is surprising that the effects
were not more robust in comparison with drugs such as
FIG. 7. The effect of diabetes, vitamins
E and C, and green tea on pentosidine in
tendon, skin, aorta, and plasma (A) and
on CML in aorta and tendon collagen
and plasma proteins (B). Bars that do
not share identical letter superscripts
are different at P < 0.05.
G.T. MUSTATA AND ASSOCIATES
DIABETES, VOL. 54, FEBRUARY 2005 523
aminoguanidine (31), pyridoxamine (32), or benfotiamine
(33). This suggests that mere suppression of oxidant stress
without acting at a “higher” (34) or different level might
not be sufficient. This notion is particularly apparent in the
pyridoxamine trial with diabetic rats in which pyridoxam-
ine almost completely suppressed albuminuria and reti-
nopathy, whereas ␣-lipoic acid, enalapril, and vitamin E
had milder or no effect (35). One pronounced effect of
pyridoxamine was its ability to markedly lower hyperlip-
idemia to levels not observed in other studies, thereby
providing support for the proposed role of lipids in the
pathogenesis of diabetic nephropathy (36). In our study,
however, only a minor hypolipidemic affect was observed
in both treatment groups. Thus, it seems likely that the
ability of pyridoxamine to trap both advanced glycation
and lipoxidation products may contribute to its powerful
activity against diabetes complications.
The marked suppressive effects of diabetes on renal
mitochondrial respiratory function and their partial ame-
lioration by the combination of antioxidant vitamins and
green tea point to a key role of oxidative stress in
mediating damage to the respiratory chain. However,
although direct oxidative damage to components of the
respiratory chain would be a logical outcome, the finding
of a methylglyoxal-modified 28-kDa protein suggests an
indirect mechanism involving AGE formation and damage
to critical arginine residues. Such a pathway would be
fully compatible with a previous demonstration that mito-
chondrial proteins are subcellular targets of dicarbonyl-
induced modifications (17). The capacity of green tea to
diminish this modification reinforces the connection be-
tween oxidative and carbonyl stress.
The most unexpected and counterintuitive result of this
study was the observation that both green tea and antiox-
idant vitamins worsened a number of glycoxidation pa-
rameters in the extracellular matrix. Why would the
antioxidant treatment result in increased pentosidine,
CML, and cross-linking in selected tissues? A possible
explanation was recently offered by Culbertson et al. (36),
who reported increased pentosidine, CML, and generic
cross-link formation during in vitro glycation reactions in
the presence of antioxidants and other inhibitors. In short,
both the complexity of the pathways involved in glycoxi-
dation reactions and the multiple pharmacological prop-
erties of the various “inhibitors” are such that these may
favor alternative nonoxidative pathways of “glycoxida-
tion.” Thus, certain inhibitors may actually tilt oxidant
stress toward carbonyl stress. In their study, the vitamin E
analogue Trolox dramatically increased pentosidine yield
during protein ribosylation. Similar effects were noted on
CML formation from both ribose and glucose when metals
were absent from the incubation buffer.
Except for aorta, CML also increased beyond diabetic
control values in tendon and plasma in both the antioxi-
dant vitamin and green tea groups. Because the antioxi-
dant treatment tended to suppress rather than increase
lipidemia, it seems less likely that CML in these tissues
would originate from lipid peroxidation. The latter mech-
anism, however, might explain CML formation in aorta,
whereby either myeloperoxidase-mediated serine oxida-
tion or peroxynitrite-mediated Amadori product oxidation
could be involved (37).
FIG. 8. The effect of diabetes, vitamins E and C, and green tea on
glucosepane, MODIC, GODIC, DOGDIC, and DODIC-ox cross-link ac-
cumulation in tail tendon collagen. Bars that do not share identical
letter superscripts are different at P < 0.05.
GREEN TEA AND DIABETES COMPLICATIONS
524 DIABETES, VOL. 54, FEBRUARY 2005
Evidence in support of the importance of anaerobic
pathways of AGE formation in diabetes is reflected in the
high tendon levels of the collagen cross-link glucosepane
(Fig. 8A). As expected, levels were not affected by treat-
ment, because no oxidation is required for its formation.
The nonsignificant decrease in the vitamin group reflects
potential competition for identical reactive sites by ascor-
bylation products from vitamin C, such as pentosidine and
CML. A similar competition for identical modification sites
by the minor methylglyoxal and glyoxal-derived cross-
links MODIC and GODIC is apparent. In fact, these are
present in 10- to 20-fold lower quantities than glucosepane,
thereby explaining why levels are depressed and not
increased by diabetes.
Finally, the current results differ from our previous
study in nondiabetic mice in which decreased cross-
linking and AGE formation were observed in several
tissues. One possible explanation is that the increased
vascular permeability in diabetic tissues led to higher
tissue concentrations of oxidized catechins, which are
themselves potent tendon cross-linking agents (data not
shown). However, the similarity of the green tea data with
the vitamin group may also point to the tilting of oxidant to
carbonyl stress and anaerobic pathways of cross-link
formation. Future studies with various doses of green tea
and antioxidant vitamins will be needed to understand
precisely the discrepancy between the mice and the dia-
betic rat study.
In summary, the data support the notion that oxidative
stress plays a role in the pathogenesis of diabetes compli-
cations in the rat. However, the study also reveals that
interventions based on antioxidants alone, in contrast to
carbonyl trapping and other agents such as pyridoxamine,
aminoguanidine, and thiamine, only incompletely prevent
diabetes-like complications. Finally, the study also pro-
vides strong evidence for a dissociation between cellular
and extracellular processes, as reflected by a paradoxical
increase in tissue glycation, glycoxidation, and cross-
linking that might be attributed to the antioxidant treat-
ment itself. Although the primary focus of this study was
on green tea, further studies will be needed to clarify
which vitamin has the most deleterious effect on collagen
glycoxidation and cross-linking.
ACKNOWLEDGMENTS
This work was supported by grants from the National
Institutes of Health (NIDDK P031, DK-57733, and DK-
45619 to T.S.K., V.M.M., and M.F.W.; NIA 18619 and EY
097099 to V.M.M.); the Leonard B. Rosenberg Renal Re-
search Foundation of the Center for Dialysis Care, Cleve-
land, Ohio (to M.F.W.); a mentorship grant from the
American Diabetes Association (to V.M.M.); and a fellow-
ship grant from the Juvenile Diabetes Association (to
M.R.).
We thank Emi Satake for technical assistance and Drs.
Susan R. Thorpe and John W. Baynes for generous gifts of
D
4
-labeled CML.
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