A Protective Role for Heme Oxygenase1 in INS1 Cells and Rat Islets that are Exposed to High Glucose Conditions

Abstract

Heme oxygenase-1 (HO-1) has been described as an inducible protein that is capable of cytoprotection via radical scavenging and the prevention of apoptosis. Chronic exposure to hyperglycemia can lead to cellular dysfunction that may become irreversible over time, and this process has been termed glucose toxicity. Yet little is known about the relation between glucose toxicity and HO-1 in the islets. The purposes of the present study were to determine whether prolonged exposure of pancreatic islets to a supraphysiologic glucose concentration disrupts the intracellular balance between reactive oxygen species (ROS) and HO-1, and so this causes defective insulin secretion; we also wanted to evaluate a protective role for HO-1 in pancreatic islets against high glucose levels. The intracellular peroxide levels of the pancreatic islets (INS-1 cell, rat islet) were increased in the high glucose media (30 mM glucose or 50 mM ribose). The HO-1 expression was induced in the INS-1 cells by the high glucose levels. Both the HO-1 expression and glucose stimulated insulin secretion (GSIS) was decreased simultaneously in the islets by treatment of the HO-1 antisense. The HO-1 was upregulated in the INS-1 cells by hemin, an inducer of HO-1. And, HO-1 upregulation induced by hemin reversed the GSIS in the islets at a high glucose condition. These results suggest HO-1 seems to mediate the protective response of pancreatic islets against the oxidative stress that is due to high glucose conditions.

Full text

INTRODUCTION
Antioxidant enzymes such as superoxide dismutase, cata-
lase, glutathione peroxidase, heat shock protein and heme
oxygenase-1 (HO-1) are induced by various stimuli, and they
represent a powerful endogenous protective mechanism that
act within the pancreatic islets against free radicals (1, 2).
HO-1 catalyzes the rate-limiting step in heme catabolism and
it generates CO and bilirubin, and bilirubin has been demon-
strated as a potent antioxidant (3). In addition, HO-1 has
been described as an inducible protein that is capable of cyto-
protection via radical scavenging and preventing apopotosis.
Glucose toxicity is defined as nonphysiological and poten-
tially irreversible cellular damage that results in defective
insulin gene expression, and this is caused by chronic expo-
sure to supraphysiologic glucose concentrations (4-9). The
beta cell in type 2 diabetes is also adversely affected by chronic
hyperglycemia and, in this sense, is also a target for secondary
complications. As hyperglycemia worsens, the beta cell steadi-
ly undergoes deterioration, secretes less and less insulin, and
becomes a participant in a downward spiral of loss of func-
tion (9). Recently, the overexpression of antioxidant gene
products has been induced in the islets, and along with using
antioxidant drugs, this helps to protect against oxidative stress
(10-14). However, the relation between glucose toxicity and
HO-1 in the islets is still not known completely.
Thus, we would like to determine whether prolonged expo-
sure of the pancreatic islets to a supraphysiologic glucose
con-
centration disrupts the intracellular balance between reactive
oxygen species (RO
S) and HO-1, and if this causes defective
insulin secretion; we also wanted to evaluate a protective role
for HO-1 against high glucose concentrations in the pancre-
atic islets.
MATERIALS AND METHODS
INS-1 cell culture
The INS-1 cells (15) were grown in 5% CO2-95% air at
37
℃
in RPMI-1640 medium containing 11.1 mM pyru-
vate, 10 mM HEPES, 50 M 2-mercaptoethanol, 100 U
penicillin/mL and 100 g streptomycin/mL. The RPMI-1640
medium used in all the experiments contained the supple-
Kyu Chang Won, Jun Sung Moon,
Mi Jung Eun, Ji Sung Yoon,
Kyung Ah Chun
*
, Ihn Ho Cho
*
,
Yong Woon Kim
�
, Hyoung Woo Lee
Department of Internal Medicine, Department of
Nuclear Medicine
*
, Department of Physiology
�
,
College of Medicine, Yeungnam University, Daegu,
Korea
Address for correspondence
Kyu Chang Won, M.D.
Department of Internal Medicine, College of Medicine,
Yeungnam University, 317-1 Daemyung-dong,
Nam-gu, Daegu 705-717, Korea
Tel : +82.53-620-3846, Fax : +82.53-654-8386
E-mail : kcwon@med.yu.ac.kr
*This study was supported by a Grants of Yeungnam
University Medical Center.
418
J Korean Med Sci 2006; 21: 418-24
ISSN 1011-8934 Copyright
�
The Korean Academy
of Medical Sciences
A Protective Role for Heme Oxygenase-1 in INS-1 Cells and Rat
Islets that are Exposed to High Glucose Conditions
Heme oxygenase-1 (HO-1) has been described as an inducible protein that is capa-
ble of cytoprotection via radical scavenging and the prevention of apoptosis. Chronic
exposure to hyperglycemia can lead to cellular dysfunction that may become irre-
versible over time, and this process has been termed glucose toxicity. Yet little is
known about the relation between glucose toxicity and HO-1 in the islets. The pur-
poses of the present study were to determine whether prolonged exposure of pan-
creatic islets to a supraphysiologic glucose concentration disrupts the intracellular
balance between reactive oxygen species (ROS) and HO-1, and so this causes
defective insulin secretion; we also wanted to evaluate a protective role for HO-1
in pancreatic islets against high glucose levels. The intracellular peroxide levels of
the pancreatic islets (INS-1 cell, rat islet) were increased in the high glucose media
(30 mM glucose or 50 mM ribose). The HO-1 expression was induced in the INS-1
cells by the high glucose levels. Both the HO-1 expression and glucose stimulated
insulin secretion (GSIS) was decreased simultaneously in the islets by treatment
of the HO-1 antisense. The HO-1 was upregulated in the INS-1 cells by hemin, an
inducer of HO-1. And, HO-1 upregulation induced by hemin reversed the GSIS in
the islets at a high glucose condition. These results suggest HO-1 seems to medi-
ate the protective response of pancreatic islets against the oxidative stress that is
due to high glucose conditions.
Key Words : HMOX1 protein, human; HO-1; Glucose Toxicity; Oxidative Stress; Islets of Langerhans
Received : 5 August 2005
Accepted : 4 November 2005

Heme Oxygenase-1 Protects Glucose Toxicity in INS-1 Cells 419
ments described above. The cells were passaged weekly after
they has been detached with trypsin-EDTA. All the studies
were performed on INS-1 cells that were between passages
21 and 29.
Pancreatic islet isolation
The pancreata from male Wistar rats were infused with
10 mL of 1.5 mg/mL collagenase type XI (Sigma, St. Louis,
MO, U.S.A.)/1% fetal bovine serum/2 units/mL RQ1 DNase
(Promega, U.S.A.) solution in Medium 199 (Sigma). After
it was surgically excised, the pancreas was incubated in the
collagenase solution at 37
℃
. The undigested tissue was re-
moved by using a 500 m screen,
and the recovered tissue
was washed twice with ice-cold Hanks
’ balanced salt solution
containing 0.1% bovine serum albumin; this was followed
by centrifugation at 250×g for 4 min. The islets in the pellet
were separated
by using a Histopaque-1077 gradient (Sigma),
and then islet
s were hand-picked and cultured in RPMI medi-
um 1640 containing 10% fetal bovine serum, 11.1 mM glu-
cose and penicillin/streptomycin/amphotericin B before the
experimentation.
In vitro induction of HO-1
Hemin (Sigma) was dissolved in 0.1N NaOH and it was
diluted 1:1 in phophate-buffered saline (PBS); the pH was
adjusted to 7.4 and the solution was sterilized by filtration.
The INS-1 cell or islets were incubated at 37
℃
for 24 hr with
the selected concentration of hemin.
Evaluation of ROS with flow cytometry
The intracellular peroxide levels (16) were detected by flow
cytometric analysis with using an oxidation-sensitive fluo-
rescein-labeled dye, carboxylated dichlorodi-hydrofluores-
cein diacetate (carboxy-H2DCFDA, Molecular Probes, Carls-
bad, CA, U.S.A.). Upon oxidation by intracellular ROS, the
non-fluorescent dye is
converted into its fluorescent form.
The islets and INS-1 cells
were labeled with 100 M car-
boxy-H2DCFDA for 1 hr at 37
℃
. Following the cell load-
ing of the dye, the islets were washed twice with PBS and
then put back into culture conditions for 2 hr. The islets and
INS-1 cells were then harvested, washed twice with PBS
and resuspended in trypsin-EDTA (0.25% trypsin, 2 mM
Na4-EDTA, Invitrogen) for 5 min at 37
℃
. To disperse the
islets into a single cell suspension, the islets and INS-1 cells
were gently passed 20 times in and out of a 200-1,000- L
tip. The cells were then washed twice with ice-cold PBS. The
pellet was then resuspended in ice-cold PBS, and 2 g/mL
propidium iodide was added. The cells were analyzed using
a 488 nm argon laser EPICS XL-MCL flow cytometer that
was controlled by EXPO 32-ADC software (Beckman Coul-
ter, Fullerton, CA, U.S.A.). The ROS values were analyzed
using a histogram plot of carboxy-H2DCFDA (the log of the
fluorescence). The results were calculated as the fold differ-
ence from the untreated control cells.
Glucose stimulated insulin secretion (GSIS)
Static incubation of the islets in Krebs-Ringer buffer that
contained either non-stimulatory or stimulatory concentrations
of glucose was performed for 1 hr (4). The insulin levels in the
Krebs-Ringer buffer samples collected from the static incuba-
tions (5.6 mM and 30 mM glucose conditions) from the islets
and INS-1 cells by using a 95.5 ethanol:hydrochloric
acid solu-
tion were measured by using a sensitive rat insul
in radioimmu-
noassay kit (Linco Research Immunoassay, St. Charles, MO,
U.S.A.).
HO activity assay
The HO activity was assayed by preparing a cell homogenate
from the INS-1 cells that were treated with hemin. The cell
homogenate was incubated with 50 M heme, 2 mg/mL rat
liver cytosol (the source of the biliverdin reductase), 1 mM
MgCl2, 3 U of glucose 6-phosphate dehydrogenase, 1 mM
glucose 6-phosphate, and 2 mM NADP+in 0.5 mL of 0.1
M potassium phosphate buffer, (pH 7.4) for 30 min at 37
℃
.
Placing the tubes on ice terminated the reaction, and the
bilirubin was extracted with chloroform as described by Wa-
gener et al. (2). The amount of bilirubin generated was deter-
mined by using a scanning spectrophotometer (Lambda 17
UV/VIS, Perkin-Elmer Cetus Instruments, Norwalk, CT,
U.S.A.) and this was defined as the difference between the
wavelengths 464 and 530 nm (the extinction coefficient for
bilirubin was 40/mM/cm). The HO activity was expressed
as the picomoles of bilirubin formed per milligram of INS-1
cell protein per hour.
HO-1 sense and antisense oligodeoxynucleotide (ODN)
treatment
For inhibition of HO-1, pretreated HO-1 antisense ODNs
(2.5 mg/mL) were transfected using a lipofection reagent to
the INS-1 cells (35). The INS-1 cells at 50% confluence were
washed three times with PBS and then they were cultured
in medium containing 10% Nu-serum, which lacks nuclease
activity (Collaborative Research Inc., Waltham, MA, U.S.A.),
for 5 hr before the addition of the ODNs. N-[-(2,3-dioleoy-
loxy)propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP; Boehringer Manngeim Diagnostics, Indianapolis,
IN, U.S.A.) was used as a vehicle for transfecting the cells
with the ODNs. The proportions used were 2 g ODN/1 g
DOTAP/mL of medium and the protocol used was described
by the manufacturer. The phosphorothioated oligonucleotides
derived from the rat HO-1 sequence were synthesized at the
Life technologies, U.S.A.. The sense/antisense ODNs for the

420 K.C. Won, J.S. Moon, M.J. Eun, et al.
HO-1 were directed against the flanking translation initia-
tion codon in the human HO-1 cDNA. The antisense se-
quence for the HO-1 was 5
′
-GCGCTCCATCGCGGG-3
′
,
whereas the sense sequence for the HO-1 was 5
′
-CCCGC-
GATGGAGCGC-3
′
and for HO-1 scramble, it was 5
′
-GG-
CCCTCTACGGGCG-3
′
. Each ODN was phospho-rothioat-
ed on the first three bases on the 3
′
end and then it was puri-
fied by HPLC. The cells were incubated for 24 hr with the
ODNs, and then the medium was replaced with fresh medi-
um containing 10% fetal bovine serum; the cells were then
incubated for 24 hr in the absence or presence of heme.
Western blot analyses
The pancreatic islets and the INS-1 cells are washed once
in ice-cold PBS buffer. The cells were lysed in buffer (140
mM NaCL, 10 mM Tris pH 7.4, 1 mM CaCl2, 1 mM MgCl2,
10% glycerol, 1% Nonidet P-40 and 1×complete protease
inhibitors [Roche, Indianapolis, ID, U.S.A.]) and then homo-
g
enized by sonication for 2 to 20 sec on ice. The cell homog
e-
nates are centrifuged at 12,000 rpm at 4
℃
for 10 min to
pellet the insoluble material. The supernatant is used for
Western
blot analyses. The protein was determined by the
bicinchonic
acid (BCA) assay (Pierce, Rockford, IL, U.S.A.).
The protein was fractionated by SDS-polyacrylamide gel elec-
trophoresis and then it was electroblotted to a nitrocellulose
membrane. Nonspecific binding sites are blocked by nonfat
dry milk for 1 hr at room temperature. The blots are then
incubated with the specific primary antibodies against HO-1
at a dilution of 1:2,000-1:5,000 for 2 hr at room temperature;
this was followed by a 1 hr incubation period with the peroxi-
dase-labeled secondary antibody at a dilution of 1:10,000.
The protein bands are visualized by chemiluminescence with
using the NEN detection system.
Data analysis
All the values are expressed as means
±
SE. Analysis of
variance and Student’s t-test were used for the statistical anal-
ysis, and the differences between groups were considered to
be significant at pvalues <0.05.
RESULTS
Intracellular peroxide levels in the islets at the high glucose
condition
The INS-1 cells cultured for 3 days in glucose concentra-
tions ranging from 5.6 to 30 mM had progressively greater
peroxide levels with the higher glucose concentrations (Fig.
1A, p<0.05). The rat islets cultured for 3 days in 30 mM or
50 mM ribose had greater peroxide levels than that in the
rat islets cultured in 11.1 mM glucose (Fig. 2B, p<0.05). In
addition, the cells at higher glucose or ribose concentrations
displayed a decreased GSIS (p<0.05).
HO-1 was induced in the INS-1 cells by the high glucose
levels
The INS-1 cells were cultured for 3 days in 5.6 mM or 30
mM glucose concentrations. Compared with 5.6 mM glu-
cose, 30 mM glucose caused an increase of the HO-1 expres-
sion and activity in the INS-1 cells (Fig. 2, p<0.05).
HO-1 downregulation in the INS-1 cells by the HO-1 anti-
sense
After 3 days culture (5 hrs exposure of the ODN) of the
INS-1 cells at 5.6 mM or 30 mM glucose concentrations,
the intracellular peroxide level, the HO-1 expression and
the GSIS were measured. HO-1 and GSIS were decreased
90
10
0
10
1
10
2
10
3
INS-1 cell
5.6 mM
22.2 mM
30 mM
A
Events
36
010
0
10
1
10
2
10
3
10
4
Fluorescence Intensity
Rat islet cell
11.1 mM Glucose
30 mM Ribose
50 mM Ribose
B
Events
Fluorescence intensity
6
5
4
3
2
1
05.6 mM G 30 mM G
Intracellular peroxide level
*
p<0.05 vs. 5.6 mM G
Fig. 1. The effects of high glucose on the intracellular peroxide
level and Glucose stimulating insulin secretion (GSIS) in the INS-
1 cells and rat islets. (A) INS-1 cells were incubated at 5.6, 22.2
or 30 mM glucose for 3 days. INS-1 cells incubated at 30 mM glu-
cose increased levels of intracellular peroxides compared with
the 5.6 mM concentration of glucose. (B) Isolated rat islets were
incubated with 11.1 mM glucose or 30 mM ribose for 3 days. 30
mM ribose caused an increase of intracellular peroxide levels
compared with the 11.1 mM glucose. Each cell at the high glucose
or ribose concentrations showed decreased GSIS (p<0.05). Data
are means
±
SD from 3 separate experiments.
Insulin secretion ( U/mL)
1,200
1,000
800
600
400
200
0
5.6 mM G 30 mM G
GSIS
4 mM G
Fluorescence intensity
25
20
15
10
5
011.1 mM G 30 mM R
Intracellular peroxide level
*
p<0.05 vs. 11.1 mM G
16.7 mM G
Insulin secretion ( U/Islet)
1,000
800
600
400
200
0
11.1 mM G 30 mM G
GSIS
5.6 mM G
16.7 mM G
*
*
*

Heme Oxygenase-1 Protects Glucose Toxicity in INS-1 Cells 421
simultaneously by treatment of the HO-1 antisense (Fig. 3,
p<0.05), suggesting GSIS is associated with HO-1.
HO-1 upregulation in the islets by hemin
The INS-1 cells cultured for 3 days (with 1day pre-expo-
sure of the hemin) in hemin concentrations ranging from
0.1 mM to 10 mM had progressively greater HO-1 levels
with the higher hemin concentrations, and the cells had pro-
gressively smaller peroxide levels with the higher hemin con-
centrations (Fig. 4A, p<0.05). Similar results were also ob-
tained in the rat islets (Fig. 4B, p<0.05).
HO-1 upregulation induced by hemin increased GSIS in
the INS-1 cells at high glucose conditions
The GSIS in both the INS-1 cells and rat islets after 3 days
subculture with high glucose concentration was increased
in a dose-dependent manner by additional treatment of hemin
for 1 day, which was associated with HO-1 upregulation
induced by hemin (Fig. 5, p<0.05).
DISCUSSION
Glucose toxicity is defined as the nonphysiological and
potentially irreversible cellular damage that results in defec-
tive insulin gene expression, and this is caused by chronic
exposure to supraphysiologic glucose concentrations (4-9).
With using HIT-T15 cells, Robertson et al. (4) observed that
cells chronically cultured for 6 months in media containing
11.1 mM glucose, a concentration exceeding what is neces-
sary to elicit maximal insulin responses, caused a marked
loss of insulin mRNA, greatly diminished levels of the insulin
content and almost a complete disappearance of insulin secre-
tion. In contrast, the HIT-T15 cells of the same passage that
were serially cultured in media containing 0.8 mM glucose
for 6 months retained their insulin mRNA, insulin content
and their glucose-induced insulin secretion (4). The concept
of glucose autoxidation along with the consequent excess
generation of ROS in relation to diabetes mellitus has been
proposed as early as 1987 by Wolff and Dean (17). Hunt et
al. (18) demonstrated that glucose autoxidation produces
hydroxyl radicals and that the hydroxyl radical scavengers
protected against the glucose-induced fragmentation of pro-
tein. Earlier work by Grankvist et al. (19) demonstrated that
the pancreatic islets contained relatively small amounts of
Fig. 2. The HO-1 expression and activity after 3 days subculture
of the INS-1 cells. Compared with the 5.6 mM glucose concen-
tration, 30 mM glucose caused an increase in the HO-1 expres-
sion and activity in the INS-1 cells (p<0.05). Data are means
±
SD
from 3 separate experiments.
Glucose concentrations
5.6 mM 5.6 mM 5.6 mM 30 mM 30 mM 30 mM
6
5
4
3
2
1
05.6 mM G 30 mM G
*
HO-1 expression
6
5
4
3
2
1
05.6 mM G 30 mM G
*
HO-1 activity
p<0.05 vs. 5.6 mM G
nmol bilirubin/mg protein/hr Intensity
Fluorescence intensity
6
5
4
3
2
1
05.6 mM G 30 mM G Scramble Sense Antisense
HO-1 expresion
*
p<0.05 vs. 30 mM G
*
p<0.05 vs. 30 mM G
Fig. 3. The intracellular peroxide level, HO-1 expression and GSIS
after 3 days culture (5 hrs exposure to the ODNs) in the INS-1 cells.
HO-1 was downregulated in the INS-1 cells by the HO-1 antisense
ODNs (p<0.05). Data are means
±
SD from 3 separate experi-
ments.
*
6
5
4
3
2
1
05.6 mM G 30 mM G Scramble Sense Antisense
30 mM G
Intracellular peroxide level
*
30 mM G
Insulin secretion (mU/mL)
1,200
1,000
800
600
400
200
05.6 mM G 30 mM G Scramble Sense Antisense
4 mM G
16.7 mM G
*

422 K.C. Won, J.S. Moon, M.J. Eun, et al.
antioxidant enzymes such as Cu, Zn-superoxide dismutase,
Mn-superoxide dismutase, catalase and glutathione peroxi-
dase. Oliveira et al. (20) reported that superoxide dismutase
activity increases with the increasing glucose concentration.
These observations set the stage for the increased risk for ROS-
induced damage. We also found that the intracellular perox-
Fluorescense intensity
7
6
5
4
3
2
1
05.6 mM G 30 mM G 0.1 M 1 M 10 M Hemin
INS-1 cells
HO-1 expression
Fig. 4. The intracellular peroxide level and the HO-1 expression and activity after 3 days subculture (1 day pre-exposure of Hemin) in the
INS-1 cells (A) and rat islets (B). HO-1 was upregulated in the INS-1 cells and rat islets by Hemin (p<0.05). Data are means
±
SD from 3
separate experiments.
A
Fluorescense intensity
12
10
8
6
4
2
011.1 mM G 50 mM R 0.1 M 1 M 10 M Hemin
Rat Islets
B
*
nmol bilirubin/mg protein/hr
9
8
7
6
5
4
3
2
1
05.6 mM G 30 mM G 0.1 M 1 M 10 M Hemin
HO-1 activity
*
Fluorescense intensity
6
5
4
3
2
1
05.6 mM G 30 mM G 0.1 M 1 M 10 M Hemin
HO-1 activity
*
*
Fluorescense intensity
25
20
15
10
5
011.1 mM G 50 mM R 0.1 M 1 M 10 M Hemin
*
*
p<0.05 vs. 30 mM G Glucose
*
p<0.05 vs. 50 mM G Ribose
30 mM G 50 mM R
Insulin secretion (mU/mL)
1,000
800
600
400
200
05.6 mM G 30 mM G 0.1 M 1 M 10 M Hemin
30 mM G
A
*
4 mM G
16.7 mM G
*
p<0.05
INS-1 Cell
Insulin secretion (mU/mL)
1,000
800
600
400
200
011.1 mM G 50 mM R 0.1 M 1 M 10 M Hemin
50 mM R
B
*
5.6 mM G
16.7 mM G
*
p<0.05
Rat Islets
Fig. 5. GSIS after 3 days subculture (1 day pre-exposure of Hemin) in the INS-1 cells (A) and rat islets (B). Hemin induced HO-1 upregula-
tion and it reserved the GSIS in the islets at the high glucose condition (p<0.05). Data are means
±
SD from 3 separate experiments.

Heme Oxygenase-1 Protects Glucose Toxicity in INS-1 Cells 423
ide level was increased by a chronic exposure of high glucose
conditions in both the INS-1 cells as well as rat islets, which
was accompanying with a decreased GSIS (Fig. 1).
The heme oxygenases play critical roles in physiological
iron homeostasis, antioxidant defense, and, as has been shown
from the accumulating evidences, in the signaling pathways
that employ CO as a messenger (21). Three mammalian iso-
forms of HO have been identified: HO-1, an inducible en-
zyme that is most highly concentrated in the tissues that are
heavily involved in the catabolism of heme proteins (22);
HO-2, a non-inducible isoform that is present at the high-
est concentrations in the brain and testes, and it is thought
to be particularly involved in signaling pathways (23); and
HO-3, an isoform with low catalytic activity and an uncer-
tain physiological role (24). HO uses dioxygen and nicoti-
namide-adenine dinucleotide phosphate as cofactors, and
the resulting products of the reaction are carbon monoxide,
iron and biliverdin (21). Biliverdin is converted to bilirubin
by a ubiquitous cytosolic enzyme biliverdin reductase (22).
Both HO-2 and an inducible HO-1 have been identified in
rat pancreatic islets (23-25), as well as in other tissues (26).
Pancreatic islets respond to stress through the induction and
activation of several stress-activated proteins. Interleukin-1
(IL-1 ) induces an inflammatory response in pancreatic islets
that is characterized by increased levels of inducible nitric
oxide synthase (iNOS) and increased nitric oxide (NO)/nitrite
levels (27-30). IL-1 and heat shock protein increase the
expression of hsp70 (31, 32), as well as HO-1 (23, 33). The
protective effect of heat shock protein (HSP) on the islet cells
may be associated with the reduced lysis from NO, reactive
oxygen intermediates and streptozotocin (32); but the response
is nonspecific because many HSPs respond to these stimuli.
On the other hand, liposomal delivery of hsp70 into islet
cells protected the cells from the IL-1 effects on insulin
secretion (34), suggesting that heightened levels of specific
HSP can protect cells from the inhibitory effects of the
cytokine. Hemin induces the synthesis of HO-1 and so it
partly counteracts the IL-1 induced inhibition of aconitase
activity and glucose oxidation (33), and perhaps this occurs
through antioxidant mechanisms. However, hemin has also
been reported to increase insulin and glucagon secretion from
normal rat islets (24). Ye and Laychock (1) reported that HO,
which is also known as a heat shock protein (hsp 32), is an
enzyme that may protect cells by reducing the heme levels
that catalyze the oxygen radical reactions, and it appears to
be a protective agent for pancreatic islets against interleukin-
1 . Similarly, our study has shown that compared with 5.6
mM glucose, 30 mM glucose caused an induction of HO-1
expression and activity in the INS-1 cells (Fig. 2, p<0.05).
The HO-1 expression increases in response to heme and to
such stressors as UV radiation and oxidative stress, as well as
to endotoxin, hormones and heavy metals (26). HO-1 induc-
tion may protect cells by reducing the heme levels that cat-
alyze oxygen radical reactions and by elevating bilirubin,
which has antioxidant properties (21). Bilirubin inhibits the
autoxidation or peroxyl-radical-induced oxidation of the
unsaturated fatty acids, and it apparently does so through
its peroxyl radical-trapping antioxidant abilities (35-37). In
the present study, we hypothesized that HO-1 can protect
the suppression of GSIS resulted from glucose toxicity.
To evaluate our hypothesis, we measured GSIS after cul-
tured with high glucose conditions in both HO-1 downreg-
ulated state and upregulated state. We observed that the
GSIS was decreased by treatment of HO-1 antisense ODNs,
which was accompanying with a downregulation of HO-1
expression (Fig. 3, p<0.05). Moreover, the GSIS was increased
incompletely by hemin administration associating with the
upregulation of HO-1 expression and activity (Fig. 4, 5).
These results in this study supported our hypothesis. Thus,
our results suggest that HO-1 seems to mediate the protec-
tive responses of the pancreatic islets against the oxidative
stress that is due to high glucose conditions. Also, we sug-
gested that HO-1 is one of the targets for preserving islets
from glucose toxicity in diabetic state.
REFERENCES
1. Ye J, Laychock SG. A protective role for heme oxygenase expres-
sion in pancreatic islets exposed to interleukin-1 . Endocrinology
1998; 139: 4155-63.
2. Wagener FA, da Silva JL, Farley T, de Witte T, Kappas A, Abraham
NG. Differential effects of heme oxygenase isoforms on heme medi-
ation of endothelial intracellular adhesion molecule 1 expression. J
Pharmacol Exp Ther 1999; 291: 416-23.
3. Pileggi A, Molano RD, Berney T, Cattan P, Vizzardelli C, Oliver R,
Fraker C, Ricordi C, Pastori RL, Bach FH, Inverardi L. Heme oxy-
genase-1 induction in islet cells results in protection from apoptosis
and improved in vivo function after transplantation. Diabetes 2001;
50: 1983-91.
4. Robertson RP, Zhang HJ, Pyzdrowski KL, Walseth TF. Preservation
of insulin mRNA levels and insulin secretion in HIT cells by avoid-
ance
of chronic exposure to high glucose concentration. J Clin Inves
t
1992; 90: 320-5.
5. Won KC. Oxidative stress in pancreatic islet beta-cells exposed to
high glucose concentration. J Korean Diabetes Assoc 2004; 28: 250
-
54.
6. Harmon JS, Stein R, Robertson RP. Oxidative stress-mediated, post-
translational loss of MafA protein as a contributing mechanism to
loss of insulin gene expression in glucotoxic beta cells. J Biol Chem
2005; 280: 11107-13.
7. Sharma A, Olson LK, Robertson RP, Stein R. The reduction of insulin
gene transcription in HIT-T15 beta cells chronically exposed to high
glucose concentration is associated with the loss of RIPE3b1 and
STF-1 transcription factor expression. Mol Endocrinol 1995; 9: 1127
-
34.
8. Harmon JS, Tanaka Y, Olson LK, Robertson RP. Reconstitution of
glucotoxic HIT-T15 cells with somatostatin transcription factor-1

424 K.C. Won, J.S. Moon, M.J. Eun, et al.
partially restores insulin promoter activity. Diabetes 1998; 47: 900-4.
9. Robertson RP. Chronic oxidative stress as a central mechanism for
glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem
2004; 279: 42351-4.
10. Tiedge M, Lortz S, Munday R, Lenzen S. Complementary action of
antioxidant enzymes in the protection of bioengineered insulin-pro-
ducing RINm5F cells against the toxicity of reactive oxygen species.
Diabetes 1998; 47: 1578-85.
11. Kralik PM, Xu B, Epstein PN. Catalase transfection decreases hydro-
gen
peroxide toxicity in a pancreatic beta cell line. Endocr Res 1998
;
24: 79-87.
12. Tiedge M, Lortz S, Munday R, Lenzen S. Protection against the co-
operative toxicity of nitric oxide and oxygen free radicals by overex-
pression of antioxidant enzymes in bioengineered insulin-producing
RINm5F cells. Diabetologia 1999; 42: 849-55.
13. Hohmeier HE, Thigpen A, Tran VV, Davis R, Newgard CB. Stable
expression of manganese superoxide dismutase (MnSOD) in insuli-
noma cells prevents IL-1 -induced cytotoxicity and reduces nitric
oxide production. J Clin Invest 1998; 101: 1811-20.
14. Moriscot C, Pattou F, Kerr-Conte J, Richard MJ, Lemarchand P,
Benhamou PY. Contribution of adenoviral-mediated superoxide
dismutase gene transfer to the reduction in nitric oxide-induced cy-
totoxicity on human islets and INS-1 insulin-secreting cells. Diabe-
tologia 2000; 43: 625-31.
15. Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB. Estab-
lishment
of 2-mercaptoethanol-dependent differentiated insulin-secret-
in
g cell lines. Endocrinology 1992; 130: 167-78.
16. Tanaka Y, Tran PO, Harmon J, Robertson RP. A role for glutathione
peroxidase in protecting pancreatic beta cells against oxidative stress
in a model of glucose toxicity. Proc Natl Acad Sci USA 2002; 99:
12363-8.
17. Wolff SP, Dean RT. Glucose autoxidation and protein modification.
The potential role of autoxidative glycosylation in diabetes. Biochem
J 1987; 245: 243-50.
18. Hunt JV, Dean RT, Wolff SP. Hydroxyl radical production and autox-
idative glycosylation. Glucose autoxidation as the cause of protein
damage in the experimental glycation model of diabetes mellitus and
ageing. Biochem J 1988; 256: 205-12.
19. Grankvist K, Marklund SL, Taljedal IB. CuZn-superoxide dismutase,
Mn-superoxide dismutase, catalase and glutathione peroxidase in
pancreatic islets and other tissues in the mouse. Biochem J 1981;
199: 393-8.
20. Oliveira HR, Curi R, Carpinelli AR. Glucose induces an acute increase
of superoxide dismutase activity in incubated rat pancreatic islets.
Am J Physiol Cell Physiol 1999; 276: 507-10.
21. McDonagh AF. Is bilirubin good for you? Clin Perinatol 1990; 17:
359-69.
22. George JW, Nulk K, Weiss A, Bruss ML, Cornelius CE. Biliverdin
reductase activity in cattle, sheep, rabbits and rats. Int J Biochem
1989; 21: 477-81.
23. Helqvist S, Polla BS, Johannesen J, Nerup J. Heat shock protein induc-
tion in rat pancreatic islets by recombinant human interleukin 1 .
Diabetologia 1991; 34: 150-6.
24. Henningsson R, Alm P, Lundquist I. Occurrence and putative hor-
mone regulatory function of a constitutive heme oxygenase in rat
pancreatic islets. Am J Physiol Cell Physiol 1997; 273: 703-9.
25. Welsh N, Margulis B, Borg LA, Wiklund HJ, Saldeen J, Flodstrom
M, Mello M, Andersson A, Pipeleers DG, Hellerstrom C, Eizirid DL.
Differences in expression of heat-shock proteins and antioxidant
emzymes between human and rodent pancreatic islets: implications
for the pathogenesis of insulin-dependent diabetes mellitus. Mol Med
1995; 1: 806-20.
26. Abraham NG, Lin JH, Schwartzman ML, Levere RD, Shibahara S.
The physiological significance of heme oxygenase. Int J Biochem
1998; 20: 543-58.
27. Southern C, Schulster D, Green IC. Inhibition of insulin secretion
by interleukin-1 and tumor necrosis factor necrosis factor- via
an L-arginine-dependent nitric oxide generating mechanism. FEBS
Lett 1990; 276: 42-4.
28. Welsh N, Eizirik DL, Bendtzen K, Sandler S. Interleukin-1 -induced
nitric oxide production in isolated rat pancreatic islets requires gene
transcription and may lead to inhibition in isolated of the Krebs cycle
enzyme aconitase. Endocrinology 1991; 129: 3167-73.
29. Corbett JA, Wang JL, Hughes JH, Wolf BA, Sweetland MA, Lan-
caster JR Jr, McDaniel ML. Nitric oxide and cyclic GMP formation
induced by interleukin 1 in islets of Langerhans: evidence for a role
of nitric oxide in islet dysfunction. Biochem J 1992; 287: 229-35.
30. Corbett JA, Sweetland MA, Wang JL, Lancaster JR, McDaniel ML.
Nitric oxide mediates cytokine-induced inhibition of insulin secretion
by human islets of Langerhans. Proc Natl Acad Sci USA 1993; 90:
1731-5.
31. Eizirik DL, Welsh M, Strandell E, Welsh N, Sandler S. Interleukin-1
beta depletes insulin messenger ribonucleic acid and increases the
heat shock protein hsp 70 in mouse pancreatic islets without
impair-
ing the glucose metabolism. Endocrinology 1990; 127: 2290-7.
32. Bellmann K, Wenz A, Radons J, Burkhart V, Kleemann R, Kolb H.
Heat shock induces resistance in rat pancreatic islet cells against
nitric oxide, oxygen radicals and streptozotocin toxicity in vitro. J
Clin Invest 1995; 95: 2840-5.
33. Welsh N, Sandler S. Protective action by hemin against interleukin-
1 induced inhibition of rat pancreatic islet function. Mol Cell En-
docrinol 1994; 103: 109-14.
34. Margulis BA, Sandler S, Eizirik DL, Welsh N, Welsh N, Welsh M.
Liposomal delivery of purified heat shock protein hsp 70 into rat
pancreatic islets as protection against interleukin-1 -impaired -
cell function. Diabetes 1991; 40: 1418-22.
35. Polte T, Abate A, Dennery PA, Achroder H. Heme oxygenase-1 is a
cGMP-inducible endothelial protein and mediates the cytoprotective
action of nitric oxide. Arterioscler Thromb Vasc Biol 2000; 20: 1209-
15.
36. Farrera JA, Jauma A, Ribo JM, Peire MA, Parallada PP, Roques-
Choua S, Bienvenue E, Seta P. The antioxidant role of bile pigments
evaluated by chemical tests. Bioorg Med Chem 1994; 2: 181-5.
37. Choi MK, Alam J. Heme oxygenase-1: function, regulation and
implication of a novel stress-inducible protein in oxidant-induced
lung injury. Am J Respir Cell Mol Biol 1996; 15: 9-19.