Ouabain Suppresses Glucose-Induced Mitochondrial
ATP Production and Insulin Release by Generating
Reactive Oxygen Species in Pancreatic Islets
Mariko Kajikawa, Shimpei Fujimoto, Yoshiyuki Tsuura, Eri Mukai, Tomomi Takeda,
Yoshiyuki Hamamoto, Mihoko Takehiro, Jun Fujita, Yuichiro Yamada, and Yutaka Seino
We examined the effects of reduced Naⴙ/Kⴙ-ATPase
activity on mitochondrial ATP production and insulin
release from rat islets. Ouabain, an inhibitor of Naⴙ/KⴙATPase, augmented 16.7 mmol/l glucose–induced insulin
release in the early period but suppressed it after a
delay of 20 –30 min. Unexpectedly, the ATP content in
an islet decreases in the presence of 16.7 mmol/l glucose
when Naⴙ/Kⴙ-ATPase activity is diminished by ouabain,
despite the reduced consumption of ATP by the enzyme.
Ouabain also suppressed the increment of ATP content
produced by glucose even in Ca2ⴙ-depleted or Naⴙdepleted conditions. That mitochondrial membrane hyperpolarization and O2 consumption in islets exposed to
16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced
mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of
site III of the mitochondrial respiratory chain. An antioxidant, ␣-tocopherol, also blocked ouabain-induced
ROS production as well as the suppressive effect of
ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial
ATP production originating from succinate and ADP.
These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic
alternation that may account in part for the suppressive
effect on insulin secretion. Diabetes 51:2522–2529, 2002
G
lucose stimulates insulin secretion by both
triggering and amplifying signals in pancreatic
␤-cells (1). The triggering pathway includes
entry of glucose into ␤-cells, acceleration of
glycolysis in cytosol and glucose oxidation in mitochondria, increase in ATP content and ATP/ADP ratio, closure
From the Department of Metabolism and Clinical Nutrition, Graduate School
of Medicine, Kyoto University, Kyoto, Japan.
Address correspondence and reprint requests to Mariko Kajikawa, 54
Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail: kajikawa@
metab.kuhp.kyoto-u.ac.jp.
Received for publication 2 July 2001 and accepted in revised form 29 April
2002.
[Ca2⫹]i, intracellular Ca2⫹ concentration; CM-DCF, 5-chloromethyl-2⬘,7⬘dichlorofluorescein; ⌬⌿m, mitochondrial membrane potential; DAPP,
diadenosine pentaphosphate; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; IC50, half-maximal inhibitory concentration; JC-1,
5,5⬘,6,6⬘-tetrachloro-1,1⬘,3,3⬘-tetraethylbenzimidazolcarbocyanine iodide;
KATP channel, ATP-sensitive K⫹ channel; KRBB, Krebs-Ringer bicarbonate
buffer; [Na⫹]i, intracellular Na⫹ concentration; ROS, reactive oxygen
species; VDCC, voltage-dependent Ca2⫹ channel.
2522
of ATP-sensitive K⫹ channels (KATP channels), membrane
depolarization, opening of voltage-dependent Ca2⫹ channels (VDCCs), increase in Ca2⫹ influx through VDCCs,
raised intracellular Ca2⫹ concentration ([Ca2⫹]i), and exocytosis of insulin granules. The KATP channel–independent
amplifying action of glucose consists of increased Ca2⫹
efficacy in stimulation-secretion coupling, which also is
dependent on the accelerated glucose metabolism that
correlates with increments in the ATP/ADP ratio. Regulation of the ATP level in pancreatic ␤-cells, therefore, plays
a crucial role in insulin secretion.
Na⫹/K⫹-ATPase is involved in maintaining Na⫹ and K⫹
gradients across the ␤-cell plasma membrane and is
thought to consume a large amount of ATP in the maintenance of homeostasis (2,3). Accordingly, the role of Na⫹/
K⫹-ATPase in the regulation of the intracellular ATP levels
in ␤-cells is of interest. We have reported that the ATP
content of an islet unexpectedly decreases in the presence
of glucose when Na⫹/K⫹-ATPase activity is diminished by
ouabain, despite reduced consumption of ATP by the
enzyme, whereas decrease of ATP content is not observed
using thapsigargin, an inhibitor of another ATP consumer,
the Ca2⫹-ATPase in endoplasmic reticulum (4,5).
Inhibition by ouabain of glucose oxidation (6) and
glucose utilization (7) has been reported previously in rat
islets. However, the effect of ouabain on glucose-induced
insulin release is complex. Ouabain has a stimulatory
effect on glucose-induced insulin secretion in the early
phase (8 –10), probably owing to increased Ca2⫹ influx
(10 –12). Such increased Ca2⫹ influx by ouabain should be
due to depolarization, since the electrogenic effect of
Na⫹/K⫹-ATPase is to hyperpolarize the membrane potential (2). On the other hand, ouabain decreases glucoseinduced insulin release in the late phase, which is
explained by the fall in intracellular K⫹ concentration
following blockade of the sodium pump by the glycoside
(13).
Ouabain has been shown in recent studies to enhance
reactive oxygen species (ROS) production in cardiac myocytes (14 –16). Because ROS inhibits mitochondrial ATP
production directly and suppresses insulin release in
␤-cells (17,18), we investigated its role in the suppression
of ATP content and insulin release by ouabain in rat
pancreatic islets.
RESEARCH DESIGN AND METHODS
Measurement of insulin release from isolated rat pancreatic islets. Male
Wistar rats weighing 180 –230 g were fed standard lab food ad libitum with free
DIABETES, VOL. 51, AUGUST 2002
M. KAJIKAWA AND ASSOCIATES
access to water in an air-conditioned room with a 12-h light, 12-h dark cycle.
Islets of Langerhans were isolated by collagenase digestion. Insulin release
from intact islets was monitored using either batch incubation or a perifusion
system as previously described (19). For batch incubation experiments, islets
were preincubated at 37°C for 30 min in Krebs-Ringer bicarbonate buffer
(KRBB) supplemented with 2.8 mmol/l glucose and 0.2% BSA (fraction V).
Groups of five islets were then batch-incubated for 60 min in 0.7 ml KRBB with
test materials. In some experiments, NaCl and NaHCO3 were replaced by
choline chloride and choline bicarbonate, respectively, and 5 ␮mol/l atropine
sulfate was added to prevent cholinergic effects. At the end of the incubation
period, islets were pelleted by centrifugation, and aliquots of the buffer were
sampled. For perifusion experiments, groups of 20 islets were placed in
parallel chambers (400 ␮l each) of a perifusion apparatus and perifused with
the same medium at a rate of 0.7 ml/min at 37°C. The medium was
continuously gassed with 95% O2 and 5% CO2. Islets were perifused for 30 min
to establish a stable insulin secretory rate at the basal level of glucose.
Ouabain and the stimulating level of glucose were added to the medium
according to each experimental protocol. The samples were collected at the
times indicated in the figures. The amount of immunoreactive insulin was
determined by radioimmunoassay, using rat insulin as a standard. Experiments using the same protocol were repeated three times to ensure reproducibility.
Measurement of O2 consumption in islets. The oxygen consumption in
isolated islets was measured by the method of Hutton and Malaisse (20) with
slight modification. O2 consumption was determined by the fluctuating levels
of pO2, which was monitored using a dual-channel oxygen monitoring system
with a Clark-type micro-electrode (YSI model 5300; Instech Laboratories,
Horsham, PA). The perifusate, KRBB medium equilibrated with 95% O2/5%
CO2 (vol/vol) atmosphere, flowed through the chamber (70 ␮l volume)
containing 1,000 islets, separated by a circular spacer of cellulose acetate, at
a rate of 0.25 ml/min at 37°C. Islets were perifused for at least 30 min to attain
a steady state in the presence of 2.8 mmol/l glucose, and the experiment was
then performed. Ouabain (1 mmol/l) was introduced into the medium 15 min
before 16.7 mmol/l glucose. The relationship between pO2 and O2 content of
the medium was determined from the O2 solubility data, with appropriate
corrections for atmospheric pressure and H2O vapor pressure.
Measurement of ATP content in islets. Isolated islets were cultured for
14 –18 h with RPMI 1640 medium (containing 10% FCS, 100 IU/ml penicillin,
100 ␮g/ml streptomycin, and 11.1 mmol/l glucose) at 37°C in humidified air
containing 5% CO2. The ATP content in islets was determined by luminometric
method as previously described (21). In brief, after groups of 10 islets were
preincubated at 2.8 mmol/l glucose for 30 min, they were batch-incubated for
60 min in 0.8 ml KRBB with 16.7 or 2.8 mmol/l glucose in the presence or
absence of 1 mmol/l ouabain. Ouabain was applied to the medium 15 min
before 16.7 mmol/l glucose. Ca2⫹-free media were prepared with Ca2⫹-free
KRBB plus 1 mmol/l EGTA. In some experiments, NaCl and NaHCO3 were
replaced by choline chloride and choline bicarbonate, respectively, and 5
␮mol/l atropine sulfate was added. The reaction was stopped by immediate
addition and mixing of trichloroacetic acid (TCA), to a final concentration of
5%. The islets were sonicated at 4°C and centrifuged (2,000g, 3 min), and a
fraction (0.7 ml) of the supernatant was mixed with 1 ml water-saturated
diethylether. The ether phase containing TCA was discarded. The step was
repeated four times, and a fraction of the extracts (0.1 ml) was diluted with 0.1
ml of 20 mmol/l HEPES (pH 7.4 with NaOH). The ATP concentration in the
solutions was measured by adding 0.1 ml luciferin-luciferase solution (Turner
Designs, Sunnyvale, CA) in a bioluminometer (model 20e; Turner Designs). To
draw a standard curve, blanks and ATP standards were run through the entire
procedure, including the extraction steps.
Because the 16.7 mmol/l glucose-induced ATP increment is small because
of the large stable ATP pool in fresh islets, as previously reported (22), we
have presented data from cultured islets, in which the increment of ATP
content by glucose is easier to observe because of the small stable ATP pool.
Experiments using the same protocol were repeated three times to ensure
reproducibility.
Fluorescence measurement of mitochondrial membrane potential. Mitochondrial membrane potential (⌬⌿m) was measured by 5,5⬘,6,6⬘-tetrachloro1,1⬘,3,3⬘-tetraethylbenzimidazolcarbocyanine iodide (JC-1) fluorescence as
previously reported (23,24). JC-1 exhibits potential-dependent accumulation
in mitochondria by a fluorescence emission shift from green (⬃525 nm) to red
(⬃595 nm). Isolated islets were incubated for 15 min in KRBB containing 2.8
mmol/l glucose and 0.2% BSA at room temperature in the dark with 10 ␮g/ml
JC-1. The islets were washed in PBS and incubated with 0.25% trypsin and 1
mmol/l EDTA solution (Life Technologies, Grand Island, NY) for 3 min at
37°C, diluted by 20 ml cold PBS, and dispersed by pipetting. The dispersed
islet cells were resuspended in 400 ␮l Ca2⫹-free medium and applied to glass
cuvettes. After preincubation for 20 min at 37°C, the fluorescence was
DIABETES, VOL. 51, AUGUST 2002
determined using a spectrofluorophotometer (RF 5000; Shimadzu, Kyoto,
Japan) with excitation wavelength at 490 nm and emission wavelength at 590
nm and with stirring medium containing dispersed cells in cuvettes at 37°C. At
time zero, basal fluorescence was determined, when glucose and ouabain, at
final concentrations of 16.7 mmol/l and 1 mmol/l, respectively, were added.
Cuvettes were incubated in humidified air containing 5% CO2 at 37°C, and
determinations were performed at the times indicated in the figures. Fluorescence was corrected by subtracting parallel blanks in which islet cells were
not loaded with JC-1 and by DNA content, which was measured as previously
described (21). Experiments using the same protocol were repeated three
times to ensure reproducibility.
Fluorescence measurement of ROS. ROS production in islet cells under
Ca2⫹-free conditions was measured by 5- (and 6-) chloromethyl-2⬘,7⬘-dichlorofluorescein (CM-DCF) fluorescence as previously reported (15,16,24). The
dispersed islet cells prepared by cold PBS and trypsin EDTA were incubated
in KRBB containing 10 ␮mol/l CM-DCFH diacetate, a reduced CM-DCF
conjugated with an acetate group, and 2.8 mmol/l glucose for 20 min at 37°C,
and then washed in Ca2⫹-free medium three times. During loading, the acetate
groups on CM-DCFH diacetate are removed by intracellular esterase, trapping
the probe inside the cells. Production of ROS could be measured by changes
in fluorescence, since oxidation of CM-DCFH produced fluorescent product
CM-DCF in the cells. At time zero, basal fluorescence was determined using a
spectrofluorophotometer (RF 5000) with excitation wavelength at 505 nm and
emission wavelength at 540 nm and with stirring medium containing dispersed
cells in cuvettes at 37°C. After 60-min incubation in 400 ␮l Ca2⫹-free KRBB
containing test materials and 16.7 mmol/l glucose, ROS production was
determined. Fluorescence was corrected by subtracting parallel blanks in
which islet cells were not loaded with CM-DCFH diacetate and by DNA
content. Experiments using the same protocol were repeated three times to
ensure reproducibility.
Measurement of mitochondrial ATP production. The mitochondrial suspension from freshly isolated islets was prepared by repeated centrifugation,
as previously reported (25,26), with slight modification. First, isolated islets
were homogenized in medium A consisting of (mmol/l) 50 HEPES, 100 KCl, 1.8
ATP, 1 EGTA, and 2 MgCl2 and 0.5 mg/ml BSA (electrophoretically homogeneous) (pH 7.00 at 37°C with KOH). After precipitation of cell debris and
nuclei by centrifugation, the supernatant was more rapidly centrifuged
(10,000g) to obtain a pellet containing the mitochondrial fraction. Afterward,
the precipitation diluted by 200 ␮l of solution A was centrifuged again, and
finally rinsed three times in medium B consisting of (mmol/l) 20 HEPES, 3
KH2PO4, 1 EGTA, 12 NaCl, 0.3 MgCl2, 148 KCl, and 4 carnitine and 0.5 mg/ml
BSA (electrophoretically homogeneous) (pH 7.10 with KOH). In some experiments, the ratio of NaCl and KCl was changed as indicated in the figures. The
mitochondrial fraction in 500 ␮l medium B was kept on ice until use. The
reaction was started by adding 5 ␮l mitochondrial suspension to 495 ␮l
prewarmed medium B (37°C) supplemented with 0.5 mmol/l succinate, 50
␮mol/l ADP, and 1 ␮mol/l diadenosine pentaphosphate (DAPP). The reaction
was stopped by the addition of 0.5 ␮mol/l antimycin A. The samples were
cooled to room temperature, and the ATP concentration in the solutions was
measured by adding luciferin-luciferase solution to each sample with a
bioluminometer.
To draw a standard curve, blank and ATP standards were added to parallel
samples containing the complete incubation mixture except the mitochondrial
suspension.
Materials. Thapsigargin, myxothiazol, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), ADP, DAPP, antimycin A, and ␣-tocopherol were
purchased from Sigma (St. Louis, MO); succinate was purchased from Aldrich
(Steimheim, Germany); ATP was purchased from Kohjin (Tokyo, Japan); and
JC-1 and CM-DCFH diacetates were purchased from Molecular Probes (Eugene, OR). All other agents, including ouabain, were obtained from Nacalai
Tesque (Kyoto, Japan).
Statistical analysis. Results are expressed as means ⫾ SE. Statistical
significance was evaluated by unpaired Student’s t test or paired t test. P ⬍
0.05 was considered significant.
RESULTS
Effect of ouabain on insulin release from pancreatic
islets. Ouabain inhibited 16.7 mmol/l glucose–stimulated
insulin release from 60-min–incubated islets in a concentration-dependent manner (Table 1).
When the glucose concentration was raised from 2.8
to 16.7 mmol/l, 1 mmol/l ouabain had a dual effect on
glucose-induced insulin release (Fig. 1A): 1 mmol/l ouabain
significantly increased insulin release in the presence of
2523
EFFECT OF OUABAIN ON ATP PRODUCTION
TABLE 1
Concentration dependence of the effect of ouabain on glucoseinduced insulin release
Ouabain (mol/l)
Insulin release (ng 䡠 islet⫺1 䡠 60 min⫺1)
0
10⫺6
10⫺5
10⫺4
10⫺3
3.70 ⫾ 0.19
2.77 ⫾ 0.34
2.94 ⫾ 0.16*
2.08 ⫾ 0.14†
1.81 ⫾ 0.11†
Data are means ⫾ SE of five determinations per experiment. Groups
of five islets were incubated for 60 min at 16.7 mmol/l glucose with
(10⫺6 to 10⫺3 mol/l) or without ouabain. *P ⬍ 0.05, †P ⬍ 0.01 vs.
control.
2.8 mmol/l glucose and enhanced the secretion more
intensively after application of 16.7 mmol/l glucose (Fig.
1B); however, the compound inhibited insulin secretion 22
min after administration of 16.7 mmol/l glucose (Fig. 1A).
The inhibition was reversed within 35 min after withdrawal of ouabain from the medium, whereas continuous
exposure to ouabain sustained the inhibition of insulin
secretion (Fig. 1A). The suppressive effect of 1 mmol/l
FIG. 2. Time course of O2 consumption in 16.7 mmol/l glucose– exposed
islets with or without 1 mmol/l ouabain. Groups of 1,000 islets were
perifused for 30 min at 2.8 mmol/l glucose (G) and for 60 min at 16.7
mmol/l glucose with (F; n ⴝ 4) or without (E; n ⴝ 3) ouabain. The
withdrawal effect of ouabain for 60 min is also shown. Values are
means ⴞ SE from several experiments. *P < 0.05, ouabain vs. control.
ouabain on 16.7 mmol/l glucose–induced insulin secretion
was greater than the stimulatory effect during 60-min
incubation. Total release during 60-min incubation was
calculated from the data shown in Fig. 2A (control 129.6 ⫾
7.9 vs. ouabain 86.9 ⫾ 2.5 ng 䡠 20 islets–1 䡠 60 min–1; P ⬍
0.01).
To determine the influence of the cationic changes
produced by ouabain, we examined insulin release under
conditions of replacement of extracellular Na⫹ by choline,
in which ouabain fails to raise intracellular Na⫹ concentration (27). In the absence of Na⫹, insulin release from
both 10-min– and 60-min–incubated islets in the presence
of 16.7 mmol/l glucose was inhibited significantly compared with that in the presence of Na⫹. However, ouabain
still produced an inhibitory effect on insulin release from
60-min–incubated islets, although it had no effect on
insulin release from 10-min–incubated islets in the absence of ambient Na⫹ (Table 2).
TABLE 2
Effect of Na⫹ deprivation on ouabain-induced alterations of
insulin release
FIG. 1. Effect of ouabain on insulin release. A: Time course of the effect
of 1 mmol/l ouabain on 16.7 mmol/l glucose–induced insulin release for
120 min. Two groups of islets were perifused with 2.8 mmol/l glucose
(G) for 30 min and with 16.7 mmol/l glucose for 120 min in the presence
(F; n ⴝ 5) or absence (E; n ⴝ 5) of ouabain. To observe the withdrawal
effect of ouabain, one group of islets perifused with ouabain was
perifused without ouabain for the last 60 min (Œ; n ⴝ 5). B: Insulin
release during the first 10 min after exposure to 16.7 mmol/l glucose
with (F; n ⴝ 6) or without (E; n ⴝ 6) 1 mmol/l ouabain. In A and B,
ouabain was introduced 15 min before 16.7 mmol/l glucose administration. (Experiments in A and B were performed separately.) Values are
means ⴞ SE of five or six determinations in the same experiment. *P <
0.05, ouabain vs. control (16.7 mmol/l glucose alone); †P < 0.05,
ouabain (withdrawal) vs. control.
2524
Glucose
(mmol/l)
Na⫹
(mmol/l)
Ouabain
(mmol/l)
Incubation
time (min)
Insulin release
(ng/islet)
16.7
16.7
16.7
16.7
16.7
16.7
16.7
16.7
154
154
0
0
154
154
0
0
0
1
0
1
0
1
0
1
10
10
10
10
60
60
60
60
0.40 ⫾ 0.02
0.55 ⫾ 0.04*
0.21 ⫾ 0.01*
0.20 ⫾ 0.01†
2.74 ⫾ 0.07
2.02 ⫾ 0.11*
1.23 ⫾ 0.14*
0.84 ⫾ 0.06†§
Data are means ⫾ SE of six determinations per experiment. *P ⬍
0.01 vs. 16.7 mmol/l glucose and 154 mmol/l Na⫹ without ouabain;
†P ⬍ 0.01 vs. 16.7 mmol/l glucose, 154 mmol/l Na⫹ with 1 mmol/l
ouabain; §P ⬍ 0.01 vs. 16.7 mmol/l glucose and 0 mmol/l Na⫹ without
ouabain. The 10- and 60-min incubation experiments were done
independently.
DIABETES, VOL. 51, AUGUST 2002
M. KAJIKAWA AND ASSOCIATES
TABLE 3
Effect of Ca2⫹ and Na⫹ deprivation on ouabain-induced suppression of ATP content increased by glucose
Glucose
(mmol/l)
Ca2⫹
(mmol/l)
Na⫹
(mmol/l)
Ouabain
(mmol/l)
ATP
content
(pmol/islet)
2.8
16.7
2.8
16.7
2.8
16.7
2.8
16.7
2.8
16.7
2.8
16.7
2.8
2.8
2.8
2.8
0
0
0
0
0
0
0
0
154
154
154
154
154
154
154
154
0
0
0
0
0
0
1
1
0
0
1
1
0
0
1
1
5.7 ⫾ 0.8
11.5 ⫾ 1.2*
6.8 ⫾ 0.6
6.6 ⫾ 0.7†
8.1 ⫾ 0.5
10.1 ⫾ 0.7‡
8.1 ⫾ 0.3
8.0 ⫾ 0.4†
12.6 ⫾ 0.9
16.2 ⫾ 1.1‡
10.0 ⫾ 1.1
9.9 ⫾ 0.6§
Data are means ⫾ SE of six determinations per experiment. *P ⬍
0.01, ‡P ⬍ 0.05 vs. control (2.8 mmol/l glucose without ouabain);
†P ⬍ 0.05, §P ⬍ 0.01 vs. 16.7 mmol/l glucose without ouabain. Zero
mmol/l Ca2⫹ indicates free Ca2⫹ with 1 mmol/l EGTA. Each set of
experiments (with or without Ca2⫹ and Na⫹) was done independently.
Effect of ouabain on O2 consumption in islets. The
elevation of O2 consumption remained for 120 min with
16.7 mmol/l glucose present. ⌬O2 consumption at 60 min
was 2.82 ⫾ 0.61 pmol 䡠 islet–1 䡠 min–1 in control samples. On
the other hand, O2 consumption in the presence of 1
mmol/l ouabain remained at a higher level until 30 min and
was subsequently reduced (⌬O2 consumption at 60 min
–1.65 ⫾ 0.73 pmol 䡠 islet–1 䡠 min–1; P ⬍ 0.05 vs. control).
However, the decrease was restored with a time delay of
about 30 min after withdrawal of the compound (Fig. 2).
Effect of ouabain on ATP content. In medium containing physiological concentrations of Ca2⫹ and Na⫹, ATP
content in islets incubated with 16.7 mmol/l glucose was
significantly greater than in islets with 2.8 mmol/l glucose.
However, in the presence of 1 mmol/l ouabain, 16.7 mmol/l
glucose failed to increase ATP content. In the presence of
the basal level of glucose, ATP content was not affected by
ouabain (Table 3). In islets, increasing [Ca2⫹]i increases
ATP consumption (28), which leads to a decrease of
intra-islet ATP content. Ouabain depolarizes the plasma
membrane and increases Ca2⫹ influx via voltage-dependent Ca2⫹ channels. To eliminate the effect of increased
[Ca2⫹]i induced by ouabain, the ATP content under ambient Ca2⫹-free conditions was examined. Although 16.7
mmol/l glucose increased the ATP content in the absence
of Ca2⫹, the increase also was abolished by ouabain (Table
3). This inhibitory effect of ouabain occurred in a concentration-dependent manner. Ouabain at 10⫺5 mol/l decreased ATP content significantly and at 10⫺3 mol/l
maximally (Table 4). When extracellular Na⫹ was replaced
by choline, ouabain still inhibited the ATP increase induced by 16.7 mmol/l glucose (Table 3). Ouabain also
decreased ATP content in fresh (noncultured) islets in the
presence of 16.7 mmol/l glucose (data not shown).
Effect of ouabain on ⌬⌿m. To evaluate the effect of
ouabain on ⌬⌿m, JC-1 fluorescence was measured in the
presence of 16.7 mmol/l glucose without Ca2⫹. After
addition of 16.7 mmol/l glucose to the medium, the fluorescence increased gradually, indicating hyperpolarization
DIABETES, VOL. 51, AUGUST 2002
TABLE 4
Concentration dependence of the effects of ouabain on ATP
content in the presence of 16.7 mmol/l glucose under Ca2⫹deprived conditions
Ouabain (mmol/l)
ATP content (pmol/islet)
0
10⫺6
10⫺5
10⫺4
10⫺3
3 ⫻ 10⫺3
11.5 ⫾ 0.2
10.6 ⫾ 0.6
9.3 ⫾ 0.7*
8.3 ⫾ 0.6†
7.7 ⫾ 0.3†
7.9 ⫾ 0.2†
Data are means ⫾ SE of five determinations per experiment. *P ⬍
0.05, †P ⬍ 0.01 vs. control.
of ⌬⌿m, whereas the basal level of fluorescence continued
in the presence of 2.8 mmol/l glucose during the measurement. Ouabain significantly inhibited glucose-induced hyperpolarization of ⌬⌿m 30 min after administration. JC-1
fluorescence decreased to less than basal value after the
addition of 1 ␮mol/l FCCP (Fig. 3).
Effect of ouabain on ROS production. The generation
of ROS was examined in the presence of 16.7 mmol/l
glucose in the absence of Ca2⫹. The fluorescence showed
concentration-dependent increments when islet cells were
incubated for 60 min with various concentrations of
hydrogen peroxide (0 to ⬃2 mmol/l) (Fig. 4A).
Myxothiazol, which inhibits the respiratory chain at site
III (29), inhibited ROS production significantly (71.3 ⫾ 2%;
P ⬍ 0.01 vs. control). FCCP, which increases proton leak
of the mitochondrial membrane, also suppressed it (85 ⫾
3%; P ⬍ 0.01 vs. control). Thapsigargin, an inhibitor of
Ca2⫹-ATPase in endoplasmic reticulum, did not enhance
FIG. 3. Time course of effect of 1 mmol/l ouabain on ⌬⌿m. After 1 ␮g/ml
JC-1 was loaded, islets were dispersed to cells by cold PBS and trypsin
EDTA as described in RESEARCH DESIGN AND METHODS. They were preincubated for 30 min at 2.8 mmol/l glucose. At time zero, basal fluorescence was determined, and cells were incubated in Ca2ⴙ-free KRBB in
the presence of 16.7 mmol/l glucose with (F; n ⴝ 3) or without (E; n ⴝ
3) 1 mmol/l ouabain. 䡺, incubation with 2.8 mmol/l glucose throughout,
n ⴝ 3. At 60 min, FCCP (final concentration 1 ␮mol/l) was added to the
medium. Results are means ⴞ SE of the change in fluorescence
(arbitrary units) versus time zero from the same experiment. *P <
0.01, 16.7 mmol/l glucose vs. 16.7 mmol/l glucose plus ouabain; †P <
0.01, 16.7 mmol/l glucose vs. 2.8 mmol/l glucose.
2525
EFFECT OF OUABAIN ON ATP PRODUCTION
FIG. 4. Effect of ouabain on ROS production. A: Concentration-dependent effect of hydrogen peroxide on CM-DCF fluorescence. After islets
were dispersed to cells by cold PBS and trypsin EDTA as described in
RESEARCH DESIGN AND METHODS, 10 ␮mol/l CM-DCFH diacetate was loaded
for 30 min. The cells then were washed in Ca2ⴙ-free KRBB three times
and incubated in Ca2ⴙ-free KRBB in the presence of 16.7 mmol/l
glucose with various concentrations of H2O2 for 60 min. Values are
expressed as % of control (0 mmol/l H2O2) in the presence of 16.7
mmol/l glucose. B: Effect of 1 mmol/l ouabain on ROS production in the
presence of 16.7 mmol/l glucose. Dispersed islet cells were incubated
for 30 min with CM-DCFH diacetate in the presence of 2.8 mmol/l
glucose, then washed in Ca2ⴙ-free KRBB three times. After CM-DCF
fluorescence was determined at time zero, islets cells were incubated
for 60 min in Ca2ⴙ-free medium containing 16.7 mmol/l glucose with 5
␮mol/l myxothiazol (Mx), 1 mmol/l ouabain (Ou), 5 ␮mol/l myxothiazol
plus 1 mmol/l ouabain (OuⴙMx), 1 ␮mol/l thapsigargin (Th), or 1
␮mol/l FCCP. Fluorescence was measured again. Values are means ⴞ
SE (n ⴝ 3–5) as a percentage of control (cont) (16.7 mmol/l glucose
alone) from the same experiment, which was calculated by the change
in fluorescence (arbitrary units) from time zero. *P < 0.05, **P < 0.01
vs. control.
ROS production (99.9 ⫾ 1.4%). However, ROS production
was significantly higher in islet cells exposed to 1 mmol/l
ouabain (114 ⫾ 3.7%; P ⬍ 0.05); the increment is approximately equivalent to that by 50 ␮mol/l hydrogen peroxide.
In the presence of myxothiazol, there was no ouabaininduced increase in ROS production (Fig. 4B).
Protective effect of ␣-tocopherol on ouabain-induced
inhibition of ATP production and of insulin secretion. To evaluate the effect of ROS on ouabain-induced
inhibition of ATP production, 100 ␮mol/l ␣-tocopherol, an
antioxidant, was added to the medium 30 min before
ouabain administration. ␣-Tocopherol significantly inhibited the ouabain-induced increase in ROS generation in the
2526
FIG. 5. Effect of ␣-tocopherol (vitamin E) on increased ROS production, decreased ATP content, and decreased insulin release by ouabain
in the presence of 16.7 mmol/l glucose. A: Effect of 100 ␮mol/l ␣-tocopherol on ROS production induced by 1 mmol/l ouabain in the
absence of Ca2ⴙ. Values are means ⴞ SE (n ⴝ 3–5) as a percentage of
control (16.7 mmol/l glucose alone) from the same experiment, which
was calculated by the change in fluorescence (arbitrary units) from
time zero. B: Effect of 100 ␮mol/l ␣-tocopherol on ATP content
suppressed by 1 mmol/l ouabain in the absence of Ca2ⴙ. Values are
means ⴞ SE of five or six determinations in the same experiment. C:
Effect of 100 ␮mol/l ␣-tocopherol on insulin release suppressed by 1
mmol/l ouabain in the presence of 2.7 mmol/l Ca2ⴙ. Insulin release was
determined by static incubation. ␣-Tocopherol was added to the medium 30 min before ouabain administration. Values are means ⴞ SE of
five determinations in the same experiment. A, B, and C: §P < 0.05,
†P < 0.01 vs. 16.7 mmol/l glucose alone. Cont, control; Ou, ouabain; Vit
E, ␣-tocopherol; AA, antimycin A. D: Time course of effect of 100 ␮mol/l
␣-tocopherol on insulin release suppressed by 1 mmol/l ouabain for 60
min. Two groups of islets were perifused with 2.8 mmol/l glucose (G)
and 100 ␮mol/l ␣-tocopherol for 45 min, and with 16.7 mmol/l glucose
and 100 ␮mol/l ␣-tocopherol (Vit. E) for 60 min in the presence (F; n ⴝ
5) or absence (E; n ⴝ 5) of ouabain. Ouabain was introduced 15 min
before 16.7 mmol/l glucose administration. Values are means ⴞ SE of
five determinations in the same experiment. *P < 0.05 vs. control (16.7
mmol/l glucose and 100 ␮mol/l ␣-tocopherol).
DIABETES, VOL. 51, AUGUST 2002
M. KAJIKAWA AND ASSOCIATES
FIG. 6. Effect of 1 mmol/l ouabain on mitochondrial ATP production.
The mitochondrial suspension from freshly isolated islets was prepared by repeated centrifugation as described in RESEARCH DESIGN AND
METHODS and was incubated for 60 min in medium B containing 50
␮mol/l ADP and 1 ␮mol/l DAPP with test materials indicated below the
graph. Ou, 1 mmol/l ouabain; H2O2, 50 ␮mol/l hydrogen peroxide; AA,
0.5 ␮mol/l antimycin A. Values from three determinations in several
experiments. *P < 0.05, **P < 0.01 vs. control (0.5 mmol/l succinate
with 12 mmol/l Naⴙ without ouabain).
presence of 16.7 mmol/l glucose without Ca2⫹ (ouabain
122.8 ⫾ 3% vs. ouabain ⫹ ␣-tocopherol 98.8 ⫾ 4%; P ⬍
0.05) (Fig. 5A). Although ouabain decreased ATP content
significantly (control 9.3 ⫾ 0.8 vs. ouabain 5.8 ⫾ 0.9
pmol/islet; P ⬍ 0.05), ouabain did not affect ATP content in
the presence of ␣-tocopherol (␣-tocopherol 8.9 ⫾ 0.9 vs.
ouabain ⫹ ␣-tocopherol 8.2 ⫾ 0.8 pmol/islet; NS) (Fig. 5B).
Early enhancement of insulin release by ouabain was still
observed in the presence of ␣-tocopherol. However, the
antioxidant prevented the delayed inhibitory effect of
ouabain (Fig. 5D). The total insulin release during 60-min
incubation calculated from the data shown in Fig. 5D was
not significantly different in control and ouabain-treated
islets (␣-tocopherol 146.2 ⫾ 10.0 vs. ␣-tocopherol ⫹
ouabain 128.5 ⫾ 4.8 ng 䡠 20 islets–1 䡠 60 min–1; NS).
Effect of ouabain on mitochondrial ATP production.
We examined the direct effect of ouabain and the Na⫹
concentration on ATP production from isolated mitochondria of islets. ATP production in the presence of succinate
and ADP was linear within 60 min (data not shown).
However, ATP production from mitochondria in the absence of succinate and in the presence of succinate and
antimycin A did not show any increase within 60 min.
Ouabain had no significant effect on ATP production from
mitochondria compared with control values, but exposure
to 50 ␮mol/l hydrogen peroxide inhibited it. In addition,
the increasing Na⫹ concentration in medium B enhanced
ATP production from mitochondria (Fig. 6).
DISCUSSION
In the present study, we demonstrate that ouabain suppresses the ATP content increase by glucose in pancreatic
islets regardless of intracellular cationic changes by reducing mitochondrial ATP production rather than by increasing ATP consumption. We also show that the inhibition by
ouabain of ATP production in islets is caused by increased
ROS production in the mitochondria, as was found recently in cardiac myocytes (14 –16).
Because 1 mmol/l ouabain, at which concentration
Na⫹/K⫹-ATPase activity is maximally suppressed, is commonly used to investigate the effect of the compound on
DIABETES, VOL. 51, AUGUST 2002
insulin secretion (7,10,12,30), we used that concentration
in all experiments in the present study. In addition, 1
mmol/l ouabain does not have irreversible, cytotoxic effects, as shown by the complete recovery of insulin
secretion after withdrawal of the compound.
Inhibition of Na⫹/K⫹-ATPase activity is known to depolarize the plasma membrane abruptly (2), resulting in Ca2⫹
influx and increased [Ca2⫹]i (10 –12). The augmentation of
insulin secretion in the basal state and in the early phase
during glucose stimulation in the presence of ouabain,
accordingly, should be attributable to the rise in [Ca2⫹]i,
consistent with previous reports (8 –10,13). Under Na⫹deprived conditions, early enhancement by ouabain was
not observed. Ouabain is reported not to alter intracellular
Na⫹ concentration ([Na⫹]i) under Na⫹-deprived conditions (27) in which Na⫹ influx and Na⫹/K⫹ exchange do
not occur. It should not cause membrane depolarization
under those conditions because the depolarizing effect
depends on blockade of the electrogenic effect of Na⫹/K⫹ATPase due to Na⫹/K⫹ exchange. Ca2⫹ influx through
voltage-dependent Ca2⫹ channels, therefore, should not be
triggered. These results support attribution of the early
enhancement of insulin release to the [Ca2⫹]i increase by
ouabain.
Because ouabain generates a higher level of [Ca2⫹]i and
enhances insulin release, it might raise ATP consumption
by increasing exocytosis of insulin granules (31–33) and by
enhancing Ca2⫹ uptake into endoplasmic reticulum
through Ca2⫹-ATPase (34,35), thus reducing the ATP content in the islets. Furthermore, a preceding high [Ca2⫹]i
level has been reported to desensitize to increased intramitochondrial Ca2⫹ concentration, which impairs the insulin secretory response (36). Therefore, we performed
experiments under Ca2⫹-depleted conditions to eliminate
ATP consumption and desensitization due to the depolarizing effect of the compound. The intracellular ATP content was reduced even in such conditions. The effect of
ouabain was maximal at ⬃10⫺3 mol/l and half-maximal
(IC50) at ⬃10⫺5 mol/l, which is higher than the concentration-dependent effects of the compound on Na⫹/K⫹ATPase inhibition, for which its IC50 is ⬃10⫺7 mol/l (37).
Mitochondrial ATP production is driven by the protonmotive force that includes the mitochondrial membrane
potential generated by the electron transport chain, and
the rate of ATP synthesis in mitochondria is closely
correlated with ⌬⌿m (38). Because ouabain reduced the
hyperpolarizing effect of glucose on ⌬⌿m in a Ca2⫹depeleted condition, the compound decreases mitochondrial ATP production independently of changes in [Ca2⫹]i.
Moreover, we have reported previously that ouabain also
inhibited the ATP increment induced by ␣-ketoisocaproate, which is metabolized in mitochondria (4). Accordingly, the ouabain-induced inhibition of ATP content is a
reflection of the reduction of ATP production, especially
that in mitochondria.
Ouabain reduced not only the increment in ATP content
and the hyperpolarization of ⌬⌿m by glucose, but also the
increment in O2 consumption by glucose. Because increased O2 consumption occurs in uncoupling (20),
ouabain-induced suppression of mitochondrial ATP production is not mediated by uncoupling, and the suppression should derive from direct or indirect effects on the
2527
EFFECT OF OUABAIN ON ATP PRODUCTION
respiratory chain. Because ouabain did not affect ATP
production from isolated mitochondria directly, the effect
should be indirect via intracellular signal transduction.
Ouabain induces an increment in [Na⫹]i and a reduction
in intracellular K⫹ concentration in ␤-cells in medium
containing physiological concentrations of monovalent
cations (27,30). Extracellular manipulation of monovalent
cation concentrations without using ouabain has been
reported to affect O2 consumption, glucose utilization, and
glucose oxidation, suggesting that intracellular concentrations of monovalent cations may affect mitochondrial
metabolism (39). However, these results were not necessarily due to intracellular cationic alternation without
change in Na⫹/K⫹-ATPase activity, because the manipulations themselves can affect Na⫹/K⫹-ATPase activity (2,37).
Ouabain suppressed the insulin release and the increment
in ATP content induced by glucose in islets even under the
Na⫹-deprived condition in which ouabain failed to raise
[Na⫹]i (27). This suggests that ouabain does not necessarily require altered [Na⫹]i to suppress mitochondrial metabolism, a theory supported by the observation that ATP
production from isolated mitochondria is not suppressed
by increasing the Na⫹ concentration in medium but rather
is enhanced, as previously reported (25).
We found that hydrogen peroxide, the most abundant
ROS inhibited mitochondrial ATP production from isolated mitochondria. Meachler et al. (17) previously demonstrated that transient exposure to hydrogen peroxide
suppresses hyperpolarization of ⌬⌿m, the increment in
insulin secretion, and the increase in ATP content induced
by glucose in pancreatic ␤-cells. Moreover, Xie and colleagues (14 –16) have shown recently that ouabain initiates
signal cascades independently of changes in [Ca2⫹]i and
[Na⫹]i and that it enhances Ras-dependent ROS generation
in cardiac myocytes. Ouabain-induced ROS production
also was observed in A7r5 cells and HeLa cells (16).
Recently, ROS have been regarded as intracellular second
messengers that act to regulate activation of downstream
effectors of Ras and Rac (40,41). The interaction of Na⫹/
K⫹-ATPase with ouabain initiates the activation of multiple interrelated signal pathways beginning with activation
of Src kinase through protein kinase C, followed by
Src-induced transactivation of epidermal growth factor,
recruitment and activation of Ras, and activation of Rasdependent pathways that communicate with mitochondria
to increase ROS generation in cardiac myocytes (42,43).
In pancreatic islets, as reported in other tissues (29,44),
metabolic inhibitors such as myxothiazol (an inhibitor of
the site III complex of mitochondrial electron transport)
and FCCP (an uncoupler) decrease ROS production. Moreover, ouabain induces ROS production in islets, which also
occurs independently of [Ca2⫹]i. Ouabain-induced ROS
production was found to be completely abolished in the
presence of myxothiazol, suggesting that its generation
takes place mainly in mitochondria, due to increased
synthesis or increased release. We noticed in this study
that inhibition of Ca2⫹-ATPase at the endoplasmic reticulum, another ATP consumer, by thapsigargin (35) increased ATP content in the presence of glucose (5) but did
not enhance ROS generation. These data indicate that
ouabain-induced ROS generation decreases mitochondrial
ATP production.
2528
We examined the effect of ouabain on ATP content in
the presence of ␣-tocopherol, a ROS scavenger. Because
␣-tocopherol restored the ouabain-induced suppression of
ATP content and the late-phase inhibition of glucoseinduced insulin release while early enhancement of insulin
release was not affected, ROS clearly plays a more important role in the late suppression of insulin release by the
compound.
Ouabain is known to be present endogenously in rat and
human plasma as a kind of adrenal cortex hormone (45)
and as a paracrine hormone secreted from the central
nervous system (46). In type 2 diabetic patients, the
plasma endogenous ouabain concentration is reported to
be higher (47), while the Na⫹/K⫹-ATPase activity in erythrocytes is significantly lower (48), than in nondiabetic
subjects. Whereas even at 10⫺5 mol/l, the effect of the
compound on ATP consumption and insulin release was
pronounced in the present study, that concentration of
ouabain is higher than in human plasma (10⫺10 to 10⫺9
mol/l) (47), so the physiological and pathophysiological
role of ouabain in Na⫹/K⫹-ATPase inhibition in insulin
secretion remains to be determined.
In conclusion, we show that ouabain, a Na⫹/K⫹-ATPase
inhibitor, suppresses mitochondrial ATP production by
generating ROS via signal transduction, not involving
cationic change. This mechanism may underlie the inhibitory effect of the compound on glucose-induced insulin
release. To determine if the increment in ROS generation
is Ras-dependent, as it is in cardiac myocytes, further
investigation is required.
ACKNOWLEDGMENTS
This study was supported in part by Grants-in-Aids for
Creative Basic Research (10NP0201) and for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, a grant from “Research
for the Future” Program of the Japan Society for the
Promotion of Science (JSPS-RFTF 97 I 00201), and Health
Sciences Research Grants for Research on Human Genome, Tissue Engineering and Food Biotechnology from
the Ministry of Health, Labor and Welfare. E.M. is a
research fellow of the Japan Society for Promotion of
Science.
The authors thank S. Nawata for technical assistance.
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