Beneficial Effects of Antioxidants in Diabetes
Possible Protection of Pancreatic b-Cells Against Glucose
Toxicity
Hideaki Kaneto, Yoshitaka Kajimoto, Jun-ichiro Miyagawa, Taka-aki Matsuoka, Yoshio Fujitani,
Yutaka Umayahara, Toshiaki Hanafusa, Yuji Matsuzawa, Yoshimitsu Yamasaki, and Masatsugu Hori
Oxidative stress is produced under diabetic conditions
and possibly causes various forms of tissue damage in
patients with diabetes. The aim of this study was to
examine the involvement of oxidative stress in the pro-
gression of pancreatic b-cell dysfunction in type 2 dia-
betes and to evaluate the potential usefulness of
antioxidants in the treatment of type 2 diabetes. We
used diabetic C57BL/KsJ-db/db mice, in whom antioxi-
dant treatment (N-acetyl-L-cysteine [NAC], vitamins C
plus E, or both) was started at 6 weeks of age; its
effects were evaluated at 10 and 16 weeks of age.
According to an intraperitoneal glucose tolerance test,
the treatment with NAC retained glucose-stimulated
insulin secretion and moderately decreased blood glu-
cose levels. Vitamins C and E were not effective when
used alone but slightly effective when used in combi-
nation with NAC. No effect on insulin secretion was
observed when the same set of antioxidants was given
to nondiabetic control mice. Histologic analyses of the
pancreases revealed that the b-cell mass was signifi-
cantly larger in the diabetic mice treated with the
antioxidants than in the untreated mice. As a possible
cause, the antioxidant treatment suppressed apoptosis
in b-cells without changing the rate of b-cell prolifera-
tion, supporting the hypothesis that in chronic hyper-
glycemia, apoptosis induced by oxidative stress causes
reduction of b-cell mass. The antioxidant treatment
also preserved the amounts of insulin content and
insulin mRNA, making the extent of insulin degranula-
tion less evident. Furthermore, expression of pancreatic
and duodenal homeobox factor-1 (PDX-1), a b-cell
specific transcription factor, was more clearly visible in
the nuclei of islet cells after the antioxidant treatment.
In conclusion, our observations indicate that anti-
oxidant treatment can exert beneficial effects in dia-
betes, with preservation of in vivo b-cell function. This
finding suggests a potential usefulness of antioxidants
From the Department of Internal Medicine and Therapeutics (H.K., Y.K.,
T.M., Y.F., Y.U., Y.Y., M.H.) and the Department of Internal Medicine and
Molecular Science (J.M., T.H., Y.M.), Osaka University Graduate School of
Medicine, Suita, Japan.
Address correspondence and reprint requests to Dr. Y. Kajimoto,
Department of Internal Medicine and Therapeutics, A8, Osaka University
Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871,
Japan. E-mail: kajimoto@medone.med.osaka-u.ac.jp.
Received for publication 12 May 1999 and accepted in revised form
16 August 1999.
ABC, avidin-biotin complex; AEC, 3-amino-9-ethylcarbozol; BrdU, 5-bromo-
2-deoxyuridine; BSA, bovine serum albumin; DAB, 3,39-diaminobenzidine
tetrahydrochloride; NAC, N-acetyl- L-cysteine; PDX-1, pancreatic and duo-
denal homeobox gene-1; ROS, reactive oxygen species; TUNEL, TdT-
mediated dUTP-biotin nick end labeling.
2398
for treating diabetes and provides further support for
the implication of oxidative stress in b-cell dysfunc-
tion in diabetes. Diabetes 48:23982406, 1999
I n general, the development of type 2 diabetes is asso-
ciated with pancreatic b-cell dysfunction occurring
together with insulin resistance. Normal b-cells can
compensate for insulin resistance by increasing
insulin secretion, but insufficient compensation leads to the
onset of glucose intolerance. Once hyperglycemia becomes
apparent, b-cell function progressively deteriorates: glu-
cose-induced insulin secretion becomes further impaired
and degranulation of b-cells becomes evident, often accom-
panied by a decrease in the number of b-cells (14). The signi-
ficance of hyperglycemia as a direct cause of these phe-
nomena, i.e., b-cell glucose toxicity, has been demonstrated
by various studies in vivo (5) and in vitro (610). Chronic
hyperglycemia may impair b-cell function at the level of
insulin synthesis as well as insulin secretion; when b-cell
derived cell lines are exposed to a high glucose concentra-
tion for a long period of time, insulin gene transcription and
insulin content are dramatically reduced (610). These
changes are often accompanied by a decrease in expres-
sion of pancreatic and duodenal homeobox factor-1 (PDX-1)
(8,10). PDX-1 (also known as islet duodenum homeobox-1
[IDX-1], somatostatin transactivating factor-1 [STF-1], and
insulin promoter factor-1 [IPF1]) (1115), is a b-cellspe-
cific transcription factor that plays a major role in main-
taining normal b-cell function, probably by regulating mul-
tiple genes expressed in b-cells (14,1618).
Under diabetic conditions, reactive oxygen species (ROS)
are produced mainly through the glycation reaction (19,20),
which occurs in various tissues (21) and may play a role in the
development of complications in diabetes (22). Although the
induction of the glycation reaction in diabetes was originally
found in neural cells and the lens crystalline, which are also
known targets of diabetic complications, another target was
recently shown to be the b-cell (2325). Indeed, advanced gly-
cosylation end products (AGEs) were shown to be
detectable in b-cells kept under high glucose concentrations
(24), and the level of 8-hydroxy-29-deoxyguanosine (8-OHdG),
a marker for oxidative stress, is increased in b-cells of diabetic
Goto-Kakizaki (GK) rats (25). Also, the expression of antiox-
idant enzymes, such as superoxide dismutase, catalase, and
glutathione peroxidase, is known to be very low in islet cells
DIABETES, VOL. 48, DECEMBER 1999
 H. KANETO AND ASSOCIATES

compared with other tissues and cells (26). Therefore, once

b-cells face oxidative stress, they may be rather sensitive to
it, suggesting that glycation and subsequent oxidative stress
may in part mediate the toxic effect of hyperglycemia. As
direct support for this, we recently showed that glycation-
mediated ROS production reduces insulin gene transcrip-
tion (27) and also causes apoptosis of b-cells (23).
Although animals have their own antioxidant defense sys-
tems, the defense can be externally strengthened. This might
be especially true for the pancreas, since it has a relatively
weak intrinsic defense system against oxidative stress (26).
Antioxidants include N-acetyl-L-cysteine (NAC), which scav-
enges hydrogen peroxide, and vitamins C and E, which are
well-known dietary antioxidants. Vitamin E is lipophilic and
inhibits lipid peroxidation, scavenging lipid peroxyl radicals
to yield lipid hydroperoxides and the tocopheroxyl radical
(28). Vitamin C, a water-soluble vitamin, functions coopera-
tively with vitamin E by regenerating tocopherol from the
tocopheroxyl radical. To investigate the implication of
oxidative stress in b-cell glucose toxicity in vivo and also to
find a potential tool for protecting the b-cell function from
glucose toxicity, we examined the possible effects of antiox-
idant treatment (NAC, vitamins C plus E, or both) on the
preservation of b-cell function in diabetic C57BL/KsJ-db/db
mice. In this mouse, a well-known obese model for type 2 dia-
betes, hyperglycemia is induced because of increasing
insulin resistance and the subsequent insufficiency of the
b-cell compensation (29,30). Our results show that the
antioxidant treatment is beneficial for treating diabetes and
can provide protection to b-cells against glucose toxicity in
C57BL/KsJ-db/db mice.

RESEARCH DESIGN AND METHODS

Experimental protocol. We used C57BL/KsJ-db/db and nondiabetic C57BL/KsJ-
misty/misty female mice purchased from Clea Japan (Tokyo). The mice were
allowed free access to food and water in a specific pathogenfree environment.
At 6 weeks of age, C57BL/KsJ-db/db mice were divided into four groups. Mice in
the control group (group 1, n = 40) were kept on regular diet without antioxidants,
and mice in the other three groups were given high-antioxidant diets: group 2
(n = 20), 2.5% NAC; group 3 (n = 20), 0.5% vitamin C plus 0.5% vitamin E; group 4
(n = 40), both 2.5% NAC and 0.5% vitamin C plus 0.5% vitamin E. At 10 and 16 weeks
of age, certain numbers of mice in each group (see figure legends) were used for
physiologic or histologic analyses. The animal studies were conducted in accor-
dance with Principles of Laboratory Animal Care (National Institutes of Health
publication no. 8523).
Glucose tolerance test. After an overnight fast, mice were injected intraperi-
toneally with glucose (1.0 g/kg body weight). Blood samples were taken at vari-
ous time points (0120 min). Blood glucose concentrations were measured by the
glucose oxidase method using a glucose analyzer (MS-GR101; Terumo, Tokyo),
and serum insulin concentrations were determined using a Lebis radioim-
munoassay kit (Shibayagi, Gunma, Japan) with mouse insulin as the standard.
Insulin tolerance test. After an overnight fast, mice were injected intraperi-
toneally with 2 U/kg human regular insulin. Blood samples were taken at various
time points (090 min), and blood glucose concentrations were measured as
described above.
Preparation of pancreas sections and immunohistochemical analyses.
The mice were anesthetized using pentobarbital sodium. A midline abdominal inci-
sion was made, and pancreases were removed from the mice and fixed overnight
in a solution of 4% paraformaldehyde. Fixed tissues were processed routinely for
paraffin embedding, and ~5-m sections were prepared and mounted on slides.
Before each incubation with antibodies, the mounted sections were rinsed three
times with phosphate-buffered saline (PBS).
For detection of insulin, the avidin-biotin complex (ABC) method was per-
formed using Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). The
mounted sections were incubated for 30 min with guinea pig polyclonal anti-insulin
antibody (Dako, Glostrup, Denmark) diluted 1:3,000 in PBS containing 1% bovine
serum albumin (BSA). They were then incubated for 30 min with biotinylated anti-
guinea pig IgG (Vector Laboratories), diluted 1:200, as the secondary antibody. The
FIG. 1. Body weights (A) and nonfasting plasma glucose levels (B) in
C57BL/KsJ-db/db mice (10 and 16 weeks of age) maintained on a diet
with and without antioxidants. , group 1; , group 2; , group 3;
, group 4. Data are means SE (n = 10). *P < 0.05 vs. group 1.
sections were then incubated with ABC reagent for 30 min, and positive reactions
were visualized by incubation with the peroxidase substrate solution containing
3,39-diaminobenzidine tetrahydrochloride (DAB) (Zymed Laboratories, San Fran-
cisco, CA). Nuclei were counterstained with methyl green.
PDX-1 staining followed a similar procedure, except the mounted sections were
incubated with Target Retrieval Solution (Dako) at 90C for 5 min before ABC was
performed and antiPDX-1 antiserum (16) diluted 1:1,000 in PBS containing
1% BSA was used.
Northern blot analyses. Total RNA was isolated from mouse pancreases, and
Northern blot analyses were performed. Ten micrograms of total RNA was size-
fractionated and transferred onto a Hybond-N+ membrane. After hybridization with
a [32P]-labeled mouse insulin cDNA probe at 42C in the presence of 50% for-
mamide, the membrane was washed twice with 13 SSPE (180 mmol/l NaCl,
10 mmol/l sodium phosphate, 1 mmol/l EDTA, pH 7.4) and 0.1% SDS at 50C for
15 min. Kodak XAR film was exposed with an intensifying screen at 80C.
Measurement of insulin content. Four mice in each group (10 and 16 weeks
of age) were used to measure the insulin content. After a 6-h fast, the whole pan-
creas was excised, and insulin content in the pancreatic tissue was determined using
a Lebis radioimmunoassay kit (Shibayagi) with mouse insulin as the standard. The

DIABETES, VOL. 48, DECEMBER 1999 2399

 ANTIOXIDANT TREATMENT PROTECTS b-CELLS
data were normalized with respect to protein concentration in the extract, which
was measured using a protein assay (BioRad Laboratories, Richmond, CA).
Detection of apoptosis. Apoptotic cells were detected by the TdT-mediated
dUTP-biotin nick end labeling (TUNEL) method using an in situ apoptosis detec-
tion kit (Takara Biochemicals, Kyoto, Japan). Sections were treated with pro-
teinase K (20 g/ml) for 15 min at room temperature and incubated with TdT
enzyme for 60 min at 37C. After washing in PBS, the sections were further incu-
bated with antifluorescein isothiocyanate HRP conjugate for 30 min at 37C and
visualized with DAB. Using the same sections, insulin staining was also per-
formed as described above except 3-amino-9-ethylcarbozol (AEC) (Dako) was
used for visualization and nuclei were counterstained with hematoxylin.
Evaluation of cell proliferation. 5-Bromo-2-deoxyuridine (BrdU) (Sigma, St.
Louis, MO), freshly dissolved in saline, was injected intraperitoneally into
C57BL/KsJ-db/db mice at a dose of 100 mg/kg 6 h before excision of the pancreas.
Sections were incubated for 60 min at 37C with a mouse monoclonal anti-BrdU
antibody diluted 1:10. After washing in PBS, sections were incubated for 30 min
at 37C with sheep anti-mouse IgG antibody (diluted 1:10) conjugated with alka-
line phosphate (Cell Proliferation Kit; Amersham Japan, Tokyo) followed by
visualization with DAB. To detect insulin-producing cells, the same sections were
stained for insulin as described above using AEC for visualization, and nuclei were
counterstained with hematoxylin.
Morphometry. The total area of the pancreas was measured using a television
monitor connected to a light microscope and microvideoscope system with a tablet
measure unit (Krypton-40; Flovel, Tokyo). b-Cell numbers were calculated by
counting the nuclei surrounded by cytoplasm positive for insulin staining. The mea-
surement and calculation were done with a total of 12 sections of the pancreas
from 4 mice (3 sections per mouse) in each group. The total number of islets
included in the 12 sections from each group was approximately 500700.
Statistical analyses. Data are means SE. Statistical comparisons of means
among individual groups used analysis of variance (ANOVA) followed by post-hoc
testing with Fishers least significant difference test.
RESULTS
Glucose tolerance. No differences in food intake or body
weight were observed among the four groups (group 1,
untreated; group 2, NAC alone; group 3, vitamins C plus E;
2400
group 4, NAC and vitamins C plus E) at 10 or 16 weeks of
age (Fig. 1A). As shown in Fig. 1B, the nonfasting blood glu-
cose levels in mice in groups 2 and 4 were lower than those
in mice in group 1 at both 10 and 16 weeks of age. No signi-
ficant difference was observed between groups 1 and 3
(Fig. 1B). Intraperitoneal glucose tolerance tests performed
at 10 and 16 weeks of age revealed that glucose tolerance in
mice in groups 2 and 4 was ameliorated (Fig. 2A and B). Also,
insulin secretion during the glucose tolerance test was
improved in mice in groups 2 and 4 compared with those in
group 1 (Fig. 2C and D), indicating that NAC is beneficial for
preservation of glucose tolerance. In contrast, the effect of
antioxidative vitamins is not evident when used alone: there
was no difference in blood glucose or insulin levels
between groups 1 and 3 at either 10 or 16 weeks of age
(Fig. 2AD). When used in combination with NAC, how-
ever, the antioxidative vitamins exerted some beneficial
effect: there were significant differences in blood glucose
levels (at 30 and 60 min after the glucose injection) and
insulin levels (at 30 and 120 min) between groups 2 and 4 at
16 weeks of age (Fig. 2B and D). In contrast to the positive
effects of the antioxidants in diabetic C57BL/KsJ-db/db
mice, the same set of antioxidants (NAC and vitamins C plus
E) did not alter the glucose tolerance in nondiabetic
C57BL/KsJ-misty/misty mice (data not shown).
To investigate the possible effects of antioxidant adminis-
tration on insulin sensitivity/resistance, an intraperitoneal
insulin tolerance test was performed. As shown in Fig. 3,
reduction of blood glucose levels in response to the injected
insulin (2 U/kg) was similar in the four groups at both 10 and
16 weeks of age.
FIG. 2. Glucose tolerance in C57BL/KsJ-db/db
mice treated with antioxidants. Intraperitoneal
glucose tolerance tests were performed in
C57BL/KsJ-db/db mice in each group at 10
(A and C) and 16 (B and D) weeks of age. After
an overnight fast, glucose was injected intra-
peritoneally at a dose of 1 g/kg, and blood glu-
cose (A and B) and insulin (C and D) levels
were measured. s, Group 1 (untreated);
d, group 2 (diet with NAC supplementation);
h, group 3 (diet with vitamin C plus E supple-
mentation); j, group 4 (diet with NAC and vit-
amin C plus E supplementation). Data are
means SE (A, n = 10; B, n = 12; C and D, n = 6
each). *P < 0.05 vs. group 1; **P < 0.01 vs. group
1; #P < 0.05 vs. group 2.
DIABETES, VOL. 48, DECEMBER 1999

Islet morphology. To elucidate how glucose tolerance is pre-
served in antioxidant-treated mice, insulin immunostaining
was performed with the pancreases from C57BL/KsJ-db/db
mice in each group. The results revealed that islets of the
16-week-old mice in group 4 (Fig. 4B) were larger and more
numerous than those in group 1 mice (Fig. 4A and B). Mice
DIABETES, VOL. 48, DECEMBER 1999
H. KANETO AND ASSOCIATES
FIG. 3. Insulin resistance in C57BL/KsJ-db/db mice
treated with antioxidants. Intraperitoneal insulin tol-
erance tests were performed in C57BL/KsJ-db/db mice
(10 and 16 weeks of age) given diet with and without
antioxidants. After an overnight fast, insulin was
injected at a dose of 2 U/kg. s, Group 1; d, group 2; h,
group 3; j, group 4. Data are means SE (n = 8).
in group 2 showed immunostaining results similar to group 4,
while mice in group 3 were similar to group 1 (data not
shown), suggesting that the antioxidative vitamins did not
exert significant effects on islet morphology.
To further elucidate the potency of antioxidants in pre-
serving b-cell function in diabetes, we tried to examine the
FIG. 4. Effects of antioxidants on b-cell
mass. A representative result is shown
for the insulin immunostaining per-
formed with pancreatic tissue sections
derived from C57BL/KsJ-db/db mice (16
weeks of age) in group 1 (A) and group 4
(B). In group 1 mice, islets were irregu-
lar in shape and islet cells showed
marked insulin degranulation. In con-
trast, islets in group 4 mice were plump
and revealed intense insulin immuno-
staining. Bar, 200 m. C: b-Cell mass was
quantitatively evaluated by counting
b-cell numbers per square millimeter of
the pancreatic section. The counting was
performed in the mice (10 and 16 weeks
of age) in group 1 (j) and group 4 (h).
Data are means SE (n = 4). *P < 0.05.
2401
 ANTIOXIDANT TREATMENT PROTECTS b-CELLS

FIG. 5. Insulin content in C57BL/KsJ-db/db mice treated with antiox-

idants. Insulin content in pancreases was measured at 10 and
16 weeks of age in C57BL/KsJ-db/db mice in group 1 (j) and group 4
(h). Data are means SE (n = 4). *P < 0.05; **P < 0.01.

mechanism underlying the preservation of insulin secretion

and islet morphology. For this purpose, we focused on the
mice in group 4, in which we assumed the maximal potency
of antioxidants might be revealed.
b-Cell mass. Using pancreas sections from mice in groups
1 and 4, we quantitatively evaluated the b-cell mass. For
each group, sections containing ~500700 islets were pre-
pared, and the b-cell number per square millimeter was cal-
culated. As shown in Fig. 4C, the number of b-cells in the
10-week-old mice in group 4 was higher than that of the
mice in group 1, and this difference became more evident
at 16 weeks of age.
Insulin synthesis. The results of insulin staining also
revealed that insulin degranulation was less evident in the
islets of mice in group 4 (Fig. 4A and B). Data for the insulin
content (Fig. 5), which showed a significant difference FIG. 6. Insulin mRNA levels in C57BL/KsJ-db/db mice treated with
between the mice in groups 1 and 4, supported this observa- antioxidants. A representative result is shown for Northern blot
tion. Although the decrease in insulin content in the whole analyses performed using mouse insulin cDNA as the probe. Total
RNA was isolated from pancreases of C57BL/KsJ-db/db mice in group
pancreas was in part due to the decrease in b-cell mass 1 and group 4 at 10 (A) and 16 (B) weeks of age. C: Relative insulin
(Fig. 4C), the insulin content per cell, estimated by normal- mRNA levels were evaluated by densitometry, with those of 10-week-
izing the data (Fig. 5) with respect to b-cell number (Fig. 4C), old mice in group 1 arbitrarily set at 1. Data are means SE (n = 4).
was also higher in the mice in group 4 (data not shown). *P < 0.05; **P < 0.01.
Consistent with this observation, the results of Northern blot
analyses revealed that the insulin mRNA level in the mice in
group 4 is significantly higher than that in the mice in group 1 PDX-1 expression was markedly retained, with clear
(Fig. 6). Again, the preservation in the insulin mRNA in part immunostaining in the nuclei (Fig. 7).
depends on the preservation in b-cell mass (Fig. 4C), but the b-Cell apoptosis. To examine the background for the
insulin mRNA per cell, normalized with respect to b-cell num- preservation of b-cell mass in the antioxidant-treated mice,
ber, was also higher in the mice in group 4 (data not shown). b-cell apoptosis was evaluated. Using double-immunostain-
Immunostaining for PDX-1. To understand the back- ing for insulin and apoptotic cells, ~500700 islets were
ground for the preservation in insulin biosynthesis, we examined for each group of mice. In the islets of mice in
examined the possible effects of the antioxidants on the group 1, 4.8 TUNEL-positive b-cells per 100 islets were rec-
expression of a transcription factor, PDX-1 (1118). ognized. On the other hand, only a few TUNEL-positive b-cells
Immunostaining for PDX-1 was performed in the pancreases (0.6 per 100 islets) could be identified in the islets of the
of C57BL/KsJ-db/db mice in groups 1 and 4. Consistent with mice in group 4 (Fig. 8).
a previous observation obtained with b-cells after partial b-Cell proliferation rate. To further understand the back-
pancreatectomy (5), we found that PDX-1 expression was ground for the preservation of b-cell mass in antioxidant-
reduced in the mice in group 1. In group 4 mice, however, treated mice, we examined the possible effects of the antiox-
2402 DIABETES, VOL. 48, DECEMBER 1999
 H. KANETO AND ASSOCIATES

FIG. 7. Immunostaining for PDX-1 in C57BL/KsJ-db/db mice treated with antioxidants. Pancreases were immunostained for PDX-1 in mice (16
weeks of age) in group 1 (A) and group 4 (B). In group 1 mice, weak immunostaining for PDX-1 was observed in islet cells. This contrasts with
the clear immunostaining in nuclei of the islet cells in the mice in group 4. Bar, 50 m. Similar results were obtained with 12 pairs of sections
from 4 mice each of groups 1 and 4.

idants on the cell proliferation rate of the b-cells. For this pur-
pose, we measured the percentage of S-phase cells by mon-
itoring BrdU incorporation (31). In mice from groups 1 and 4,
~500700 islets were examined, and the ratio of the BrdU-pos-
itive b-cells was calculated. As shown in Fig. 9A and B, some
BrdU-positive cells were detectable in the islets of both
group 1 and group 4. Double-immunostaining for BrdU and
insulin revealed that most of the BrdU-positive cells in the
islets were insulin-containing b-cells. There was no difference
in the ratio of BrdU-positive b-cells between groups 1 and 4
at either 10 or 16 weeks of age (Fig. 9C).
DISCUSSION
In this study, we have shown that antioxidant treatment
using NAC can improve glycemic control with preservation
of pancreatic b-cell function in diabetic C57BL/KsJ-db/db
mice. Since the same set of antioxidants did not alter the glu-
cose tolerance in nondiabetic C57BL/KsJ-misty/misty mice,
the antioxidant treatment probably exerts its effect in asso-
ciation with the presence of hyperglycemia; i.e., by protect-
ing b-cells from the toxic effects of ROS produced under
hyperglycemic conditions. Indeed, the antioxidant treatment
increased the b-cell mass (Fig. 4) and preserved insulin con-
tent (Fig. 5) and mRNA amount (Fig. 6). Since decreases in
b-cell mass and insulin biosynthesis are considered to be
associated with the development of diabetes in various ani-
mal models with type 2 diabetes (32,33), it is likely that those
effects of the antioxidants contributed to partial preservation
of the glucose tolerance in the antioxidant-treated mice
(Figs. 1B and 2).

FIG. 8. Effects of antioxidants on b-cell apoptosis. A representative result for double-immunostaining for insulin (red) and apoptotic cells
(brown) (visualized by TUNEL method) performed on pancreases of C57BL/KsJ-db/db mice (16 weeks of age) in group 1 (A) and group 4 (B).
The arrow indicates the b-cell identified as being apoptotic. Bar, 50 m. In each group, 12 pairs of sections from 4 mice each of groups 1 and 4
were examined, and similar results were obtained.

DIABETES, VOL. 48, DECEMBER 1999 2403

 ANTIOXIDANT TREATMENT PROTECTS b-CELLS

FIG. 9. Effects of antioxidants on b-cell proliferation. A representative result for double-immunostaining for insulin (red) and BrdU (dark brown)
performed in pancreases of C57BL/KsJ-db/db mice (16 weeks of age) in group 1 (A) and group 4 (B). Several BrdU-positive b-cells per islet
(arrows) were recognized in both groups. Bar, 50 m. In each group, 12 pairs of sections from 4 mice each of groups 1 and 4 were examined,
and similar results were obtained. C: Percentage of BrdU-positive cells in b-cells calculated in mice (10 and 16 weeks of age) in group 1 (j)
or group 4 (h). Data are means SE (n = 4).

There was no difference in insulin sensitivity between
groups 1, 2, 3, and 4 in the insulin tolerance test (Fig. 3), sug-
gesting that improvement of glycemic control was mainly
initiated by a beneficial effect of antioxidant on b-cells. How-
ever, we cannot totally deny the possibility that the antioxi-
dant treatment could have exerted an influence on target tis-
sues other than the b-cells such as muscle and fat; it was
recently reported that oxidative stress impaired insulin-
induced GLUT4 translocation in 3T3-L1 adipocytes (34).
Thus, it would be safe to conclude that antioxidant treat-
ment has beneficial effects on preservation of b-cell function
in diabetes, although the effects may not be exerted totally
through its direct action on b-cells. Also, regardless of the
influence on insulin sensitivity, because the antioxidant treat-
ment indeed reduced blood glucose levels, it must have
2404
reduced glucose toxicity and contributed in part to the pre-
vention of a decrease of b-cell mass and insulin content.
Once the vicious circle of the glucose toxicity is prevented by
somehow achieving good glycemic control, the circle then
starts moving in the opposite direction: the toxic effects of
high glucose will be reduced, resulting in further improvement
of glycemic control. Nonetheless, we assume that the antiox-
idants used in this study probably kept providing protection
of b-cells from the toxic effects of glucose. While the average
nonfasting glucose levels in mice treated with antioxidants
(NAC and vitamins C plus E) were >20 mmol/l at 10 and
16 weeks of age (Fig. 1B), Robertson and colleagues (610)
have shown that chronic exposure of the b-cellderived HIT-
T15 or bTC6 cells to a glucose level of 11.1 mmol/l caused
dramatic reductions in insulin gene transcription and insulin
DIABETES, VOL. 48, DECEMBER 1999

content. Also, according to a result of fluorescence-activated
cell sorting (FACS) analysis, there was a significant increase
in ROS amount in the HIT-T15 cells kept under 20 mmol/l glu-
cose compared with cells kept under 5 mmol/l glucose (H.K.,
Y.K., unpublished observations). Thus, we assume that even
after the moderate decrease of blood glucose levels was
achieved in the antioxidant-treated mice, the blood glucose
levels in those mice remained high enough to cause glucose
toxicity, and that the antioxidants kept protecting the b-cells
against it by neutralizing the toxic effects of oxidative stress
provoked due to hyperglycemia.
As possible mechanisms underlying the preservation of
the b-cell mass, we found that apoptosis was suppressed in
the mice in group 4 (Fig. 8). b-Cell apoptosis has been sug-
gested as being involved in physiologic and pathologic
decrease of b-cell mass (33,35,36). According to a report by
Donath et al. (35), hyperglycemia induces b-cell apoptosis in
Psammomys obesus islets. Also, Pick et al. (33) showed that
apoptosis plays a role in the failure of b-cell mass compen-
sation in Zucker diabetic fatty rats, providing support for the
possible involvement of apoptosis in b-cell glucose toxicity.
Although not very many TUNEL-positive cells were identified
even in the untreated mice (group 1), it should be noted that
the frequency of apoptosis tends to be underestimated, since
apoptotic cells are usually eliminated rather quickly. While
glycation-mediated ROS production induces apoptosis in
b-cells in vitro (17), our present results support the hypoth-
esis that ROS, provoked by hyperglycemia in vivo, play a
significant role in decreasing the b-cell mass in type 2 diabetes
through induction of apoptosis. Failure to detect a differ-
ence in rate of b-cell proliferation between the groups with
and without antioxidant treatment also supports this possi-
bility (Fig. 9).
The homeodomain containing transcription factor PDX-1 is
involved in pancreas development and, possibly, also in
regeneration after birth (18,3740). It is expressed in mature
b-cells and functions as an important transcription factor
common to multiple genes essential for the b-cell function
(1418). When the b-cellderived cell line HIT-T15 was kept
under a high glucose concentration for a long period of time,
a reduction of PDX-1 activity was identified in association
with a reduction of insulin gene transcription (8,10). Also,
Zangen et al. (5) have shown that the expression level of
PDX-1 is reduced in islets from partially pancreatectomized
rats, which were exposed to chronic hyperglycemia in vivo.
Although the limitations of mouse islet cell preparation pre-
vented reliable quantification concerning PDX-1 expression
levels and DNA-binding activity, we observed clear immuno-
staining in nuclei of the islet cells in group 4, which contrasts
with weak immunostaining for PDX-1 in islet cells in group 1
(Fig. 7). These results provide further support for the vulner-
ability of PDX-1 to chronic high glucose conditions and also
suggest the involvement of oxidative stress in hyperglycemia-
dependent reduction of PDX-1 activity, agreeing with the
observation that PDX-1 activity in HIT cells was reduced by
induction of oxidative stress in vitro (27). Clinically, it was
shown that heterozygous mutations of PDX-1 cause diabetes,
typically maturity-onset diabetes of the young type 4
(MODY 4) (41,42). Also, a phenotype of the heterozygous
PDX-1 knockout mouse, which displays a decreased b-/a-cell
ratio and subsequent impaired glucose tolerance, indicates that
the PDX-1 gene has a dose-dependent effect (43). Whereas
DIABETES, VOL. 48, DECEMBER 1999
H. KANETO AND ASSOCIATES
these observations suggest that the amount of PDX-1 expres-
sion is important for maintaining normal b-cell function, it is
possible that the suppression of PDX-1 function by ROS in dia-
betes is implicated in further deterioration of b-cell function
and thus mediates glucose toxicity.
Although both NAC and vitamins C and E have antioxida-
tive activity, only NAC ameliorates glucose tolerance when
used alone (Figs. 1B and 2). The main function of vitamins C
and E is to suppress lipid peroxidation, which occurs in the
plasma membrane and damages membrane structure and
permeability. Therefore, the antioxidative vitamins C and E
may not have been able to exert an effect on intracellular
events, such as those involved in apoptosis and gene tran-
scription. In other words, inhibition of lipid peroxidation by
antioxidative vitamins (C and E) was not enough to improve
b-cell function. On the other hand, NAC scavenges hydrogen
peroxide, which is produced in the cytoplasm and probably
the nuclei of cells as a direct consequence of the glycation
reaction (19,20). Once produced, hydrogen peroxide has a rel-
atively long lifespan and can transfer anywhere by penetrat-
ing nuclear and plasma membranes. Indeed, hydrogen per-
oxide is known to mediate the glycation-dependent degra-
dation of several proteins and is widely involved in the
damage of various tissues in diabetes (20,44). Therefore, it
seems reasonable that NAC, which is known to decrease the
intracellular hydrogen peroxide level in b-cells (23), was
effective for preventing b-cell damage. Although the antiox-
idative vitamins did not reveal significant effects when used
alone, they exerted some beneficial effects when used in
combination with NAC (Fig. 2B and D). While many of the
molecules constituting the machinery for the glucose-respon-
sive insulin secretion are membrane proteins, it seems pos-
sible that vitamins C and E improved the outcome of NAC
treatment through their effects on such membrane proteins.
In conclusion, our present results show for the first time
that antioxidants can exert beneficial effects on pancreatic
b-cell function in diabetes. Thus a sufficient supply of antiox-
idants may prevent or delay b-cell dysfunction in diabetes by
providing protection against glucose toxicity.
ACKNOWLEDGMENTS
This work was supported in part by grants from Suzuken
Memorial Foundation (to Y.K.) and a grant-in-aid for Scien-
tific Research from the Ministry of Education of Japan (to
Y.K. and Y.Y.).
We thank Noriko Fujita and Katsumi Yamamori for the
excellent technical assistance. We also thank Dr. M. Alan
Permutt of the University of Washington School of Medicine
and Dr. John M. Chirgwin of the University of Texas Health
Science Center for kindly providing the mouse insulin II
cDNA plasmid.
REFERENCES
1. Porte D Jr: Banting lecture 1990: b-cells in type II diabetes mellitus. Diabetes
40:166180, 1991
2. DeFronzo RA, Bonadonna RC, Ferrannini E: Pathogenesis of NIDDM: a bal-
anced overview. Diabetes Care 15:318368, 1992
3. Yki-Jarvinen H: Glucose toxicity. Endocrine Rev 13:415431, 1992
4. Vinik A, Pittenger G, Rafaeloff R, Rosenberg L, Duguid W: Determinants of pan-
creatic islet cell mass: a balance between neogenesis and senescence/apo-
ptosis. Diabetes Rev 4:235263, 1996
5. Zangen DH, Bonner-Weir S, Lee CH, Latimer JB, Miller CP, Habener JF, Weir
GC: Reduced insulin, GLUT2, and IDX-1 in b-cells after partial pancreatectomy.
Diabetes 46:258264, 1997
2405
 ANTIOXIDANT TREATMENT PROTECTS b-CELLS
6. Robertson RP, Zhang H-J, Pyzdrowski KL, Walseth TF: Preservation of insulin
mRNA levels and insulin secretion in HIT cells by avoidance of chronic expo-
sure to high glucose concentrations. J Clin Invest 90:320325, 1992
7. Olson LK, Redmon JB, Towle HC, Robertson RP: Chronic exposure of HIT cells
to high glucose concentrations paradoxically decreases insulin gene tran-
scription and alters binding of insulin gene regulatory protein. J Clin Invest
92:514519, 1993
8. Sharma A, Olson LK, Robertson RP, Stein R: The reduction of insulin gene tran-
scription in HIT-T15b cells chronically exposed to high glucose concentration
is associated with loss of RIPE3b1 and STF-1 transcription factor expression.
Mol Endocrinol 9:11271134, 1995
9. Poitout V, Olson LK, Robertson RP: Chronic exposure of bTC-6 cells to sup-
raphysiologic concentrations of glucose decreases binding of the RIPE3b1
insulin gene transcription activator. J Clin Invest 97:10411046, 1996
10. Moran A, Zhang H-J, Olson LK, Harmon JS, Poitout V, Robertson RP: Differ-
entiation of glucose toxicity from beta cell exhaustion during the evolution
of defective insulin gene expression in the pancreatic islet cell line, HIT-T15.
J Clin Invest 99:534539, 1997
11. Ohlsson H, Karlsson K, Edlund T: IPF1, a homeodomain-containing-transac-
tivator of the insulin gene. EMBO J 12:42514259, 1993
12. Leonard J, Peers B, Johnson T, Ferreri K, Lee S, Montminy MR: Characteri-
zation of somatostatin transactivating factor-1, a novel homeobox factor that
stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol
7:12751283, 1993
13. Miller CP, McGehee RE, Habener JF: IDX-1: a new homeodomain transcrip-
tion factor expressed in rat pancreatic islets and duodenum that transactivates
the somatostatin gene. EMBO J 13:11451156, 1994
14. Stoffers DA, Thomas MK, Habener JF: Homeodomain protein IDX-1: a mas-
ter regulator of pancreas development and insulin gene expression. Trends
Endocrinol Metab 8:145151, 1997
15. Weir GC, Sharma A, Zangen DH, Bonner-Weir S: Transcription factor abnor-
malities as a cause of beta cell dysfunction in diabetes: a hypothesis. Acta Dia -
betol 34:177184, 1997
16. Watada H, Kajimoto Y, Umayahara Y, Matsuoka T, Kaneto H, Fujitani Y,
Kamada Y, Kawamori R, Yamasaki Y: The human glucokinase gene b-cell-type
promoter: an essential role of insulin promoter factor 1 (IPF1)/PDX-1 in its acti-
vation in HIT-T15 cells. Diabetes 45:14781488, 1996
17. Waeber G, Thompson N, Nicod P, Bonny C: Transcriptional activation of the
GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol
10:13271334, 1996
18. Kaneto H, Miyagawa J, Kajimoto Y, Yamamoto K, Watada H, Umayahara Y,
Hanafusa T, Matsuzawa Y, Yamasaki Y, Higashiyama S, Taniguchi N: Expres-
sion of heparin-binding epidermal growth factor-like growth factor during pan-
creas development: a potential role of PDX-1 in transcriptional activation.
J Biol Chem 272:2913729143, 1997
19. Sakurai T, Tsuchiya S: Superoxide production from nonenzymatically glyca-
tion protein. FEBS Lett 236:406410, 1988
20. Hunt JV, Smith CC, Wolff SP: Autoxidative glycosylation and possible involve-
ment of peroxides and free radicals in LDL modification by glucose. Diabetes
39:14201424, 1991
21. Myint T, Hoshi S, Ookawara T, Miyazawa N, Suzuki K, Taniguchi N: Immuno-
logical detection of glycated proteins in normal and streptozotocin-induced
diabetic rats using anti hexitol-lysine IgG. Biochem Biophys Acta 1272:7379,
1995
22. Baynes JW: Role of oxidative stress in development of complications in dia-
betes. Diabetes 40:405412, 1991
23. Kaneto H, Fujii J, Myint T, Miyazawa N, Islam KN, Kawasaki Y, Suzuki K, Naka-
mura M, Tatsumi H, Yamasaki Y, Taniguchi N: Reducing sugars trigger oxida-
tive modification and apoptosis in pancreatic b-cells by provoking oxidative
stress through the glycation reaction. Biochem J 320:855863, 1996
24. Tajiri Y, Moller C, Grill V: Long term effects of aminoguanidine on insulin
2406
release and biosynthesis: evidence that the formation of advanced glycosy-
lation end products inhibits B cell function. Endocrinology 138:273280, 1997
25. Ihara Y, Toyokuni S, Uchida K, Odaka H, Tanaka T, Ikeda H, Hiai H, Seino Y,
Yamada Y: Hyperglycemia causes oxidative stress in pancreatic b-cells of GK
rats, a model of type 2 diabetes. Diabetes 48:927932, 1999
26. Tiedge M, Lortz S, Drinkgern J, Lenzen S: Relation between antioxidant
enzyme gene expression and antioxidative defense status of insulin-produc-
ing cells. Diabetes 46:17331742, 1997
27. Matsuoka T, Kajimoto Y, Watada H, Kaneto H, Kishimoto M, Umayahara Y, Fuji-
tani Y, Kamada T, Kawamori R, Yamasaki Y: Glycation-dependent, reactive oxy-
gen species-mediated suppression of the insulin gene promoter activity in HIT
cells. J Clin Invest 99:144150, 1997
28. Stahl W, Sies H: Antioxidant defense: vitamins E and C and carotenoids. Dia -
betes 46 (Suppl. 2):1418, 1997
29. Hummel KP, Dick MM, Coleman DL: Diabetes, a new mutation in the mouse.
Science 153:11271128, 1966
30. Coleman DL, Hummel KP: Studies with the mutation, diabetes, in the mouse.
Diabetologia 3:238248, 1966
31. deFrazio A, Leary JA, Hedley DW, Tattersall NHN: Immunohistochemical
detection of proliferating cells in vivo. J Histochem Cytochem 35:571577, 1987
32. Tokuyama Y, Sturis J, DePaoli AM, Takeda J, Stoffel M, Tang J, Sun X,
Polonsky KS, Bell GI: Evolution of b-cell dysfunction in the male Zucker dia-
betic fatty rat. Diabetes 44:14471457, 1995
33. Pick A, Clark J, Kubstrup C, Levisetti M, Pugh W, Bonner-Weir S, Polonsky KS:
Role of apoptosis in failure of b-cell mass compensation for insulin resistance
and b-cell defects in the male Zucker diabetic fatty rat. Diabetes 47:358364,
1998
34. Rudich A, Tirosh A, Potashnik R, Hemi R, Kanety H, Bashan N: Prolonged
oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1
adipocytes. Diabetes 47:15621569, 1998
35. Donath MY, Gross DJ, Cerasi E, Kaiser N: Hyperglycemia-induced b-cell apo-
ptosis in pancreatic islets of Psammomys obesus during development of dia-
betes. Diabetes 48:783744, 1999
36. Scaglia L, Smith FE, Bonner-Weir S: Apoptosis contributes to the involution
of b cell mass in the post partum rat pancreas. Endocrinology 136:54615468,
1995
37. Jonsson J, Carlsson L, Edlund T, Edlund H: Insulin-promoter-factor 1 is
required for pancreas development in mice. Nature 37:606609, 1994
38. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan
BLM, Wright CVE: PDX-1 is required for pancreas outgrowth and differenti-
ation of the rostral duodenum. Development 122:983995, 1996
39. Waguri M, Yamamoto K, Miyagawa J, Tochino Y, Yamamori K, Kajimoto Y,
Nakajima H, Watada H, Yoshiuchi I, Itoh N, Imagawa A, Namba M, Kuwajima
M, Yamasaki Y, Hanafusa T, Matsuzawa Y: Demonstration of two different
processes of b-cell regeneration in a new diabetic mouse model induced by
a selective perfusion of alloxan. Diabetes 46:12811290, 1997
40. Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, Weir GC,
Habener JF, Bonner-Weir S: The homeodomain protein IDX-1 increases after
an early burst of proliferation during pancreatic regeneration. Diabetes
48:507513, 1999
41. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF: Pancreatic age-
nesis attributable to a single nucleotide deletion in the human IPF1 gene
coding sequence. Nat Genet 15:106110, 1997
42. Stoffers DA, Ferrer J, Clarke WL, Habener JF: Early-onset type-II diabetes mel-
litus (MODY4) linked to IPF1. Nat Genet 17:138139, 1997
43. Dutta S, Bonner-Weir S, Montminy M, Wright C: Regulatory factor linked to
late-onset diabetes? Nature 392:560, 1998
44. Sagara M, Satoh J, Wada R, Yagihashi S, Takahashi K, Fukuzawa M, Muto G,
Toyota T: Inhibition with N-acetylcysteine of development of peripheral neu-
ropathy in streptozotocin-induced diabetic rats. Diabetologia 39:263269,
1996
DIABETES, VOL. 48, DECEMBER 1999