Received for publication, July 10, 2002, and in revised form, December 12, 2002
Oxygen free radicals have been implicated in
-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have
thus far been defined indirectly by monitoring oxidative tissue damage
and the effects of antioxidants, free radical scavengers, and
overexpression of superoxide dismutase. We employed the
superoxide-mediated oxidation of hydroethidine to ethidium to
dynamically and directly assess the relative rates of mitochondrial
superoxide anion generation in isolated islets in response to glucose
stimulation. Superoxide content of isolated islets increased in
response to glucose stimulation. We next compared the oxyradical levels
in Zucker lean control and Zucker diabetic fatty rat islets by digital
imaging microfluorometry. The superoxide content of Zucker diabetic
fatty islets was significantly higher than Zucker lean control islets
under resting conditions, relatively insensitive to elevated glucose
concentrations, and correlated temporally with a decrease in
glucose-induced hyperpolarization of the mitochondrial membrane.
Importantly, superoxide levels were elevated in islets from young,
pre-diabetic Zucker diabetic fatty animals. Overproduction of
superoxide was associated with perturbed mitochondrial morphology and
may contribute to abnormal glucose signaling found in the Zucker
diabetic fatty model of type 2 diabetes mellitus.
 |
INTRODUCTION |
Oxidative stress is thought to contribute to the pathogenesis of
neurodegenerative diseases (1) and complications of diabetes mellitus
(2). Insulin-secreting
-cells may also be subject to injury from
oxidative stress. Formation of reactive oxygen species
(ROS)1 such as superoxide
anion (O2
), hydrogen peroxide, hydroxyl
radicals, and the concomitant generation of nitric oxide (NO) have been
implicated in
-cell death and dysfunction in type 1 (3) and type 2 (4, 5) diabetes mellitus. In actively respiring cells such as
hippocampal neurons, mitochondrial respiration is the main source of
O2
(6). Superoxide anions are generated by
single electron reduction of molecular oxygen in complex I and III of
the mitochondrial respiratory chain (7-10) and detoxified in cells by
superoxide dismutase (SOD), catalases, peroxidases, and other scavenger systems.
Although previous investigations implicating ROS in
-cell death or
damage have relied on the protective effects of antioxidants, scavengers, and overexpression of SOD to deduce the destructive presence of oxyradicals (11-14), no real-time assays of mitochondrial generation of ROS in insulin-secreting cells have been reported. The
oxidation of reduced, non-fluorescent probes to fluorescent products is
a well characterized optical technique to monitor the formation of ROS
and has proven useful in the study of the mechanisms underlying
apoptosis in a variety of cell types (e.g. 15, 16). To
investigate the role of free radical generation in
-cell function
and diabetes, we used hydroethidine (HEt) to measure the rate of
mitochondrial O2
production in islets
isolated from Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF)
rats, an animal model of type 2 diabetes mellitus. The oxidation of HEt
to the fluorescent cation ethidium (Et) is selective for
O2
(6, 17, 18), and the fluorescent
properties of Et are not affected by the presence of hydrogen peroxide,
perchlorate, NO, and hydroxyl radicals (6).
We report here that glucose stimulation rapidly increases the ROS
levels in ZLC islets. In contrast, ZDF islets exhibit an elevated basal
rate of O2
production but are insensitive to
glucose stimulation. These differences correlated with a marked
abnormality in the ZDF islet mitochondria morphology and function. Our
findings provide compelling evidence that
-cell production of ROS is
linked to mitochondrial metabolism and that ROS production is highly
aberrant in obesity-related diabetes. Together with previous studies,
these results further implicate oxidative stress in the pathophysiology
of
-cell dysfunction in type 2 diabetes mellitus.
 |
EXPERIMENTAL PROCEDURES |
Islet and Insulinoma Cell Culture--
Islets of Langerhans were
isolated from the pancreata of ZLC, ZDF, and Holtzmann rats and
C57BL/KsJ mouse islets by a collagenase digestion and discontinuous
Ficoll gradient separation method as described in Ref. 19. ZLC and ZDF
rats were between 6 and 13 weeks of age. Islets were cultured in RPMI
1640 medium supplemented with 11 mM glucose, 5% fetal calf
serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Islets were studied 2 to 7 days after isolation. MIN6 cells (20) were
grown in Dulbecco's modified Eagle's medium containing 25 mM glucose, 1 mM sodium pyruvate, 15% fetal
calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin in
humidified 5% CO2, 95% air at 37 °C. Cells were used
between passages 20 and 30.
Mitochondrial Membrane Potential (
m)
Measurement--
Rhodamine 123 (Rh123) was used as an indicator of

m as well as to visualize mitochondria using confocal
microscopy (21-24). Rh123 is a lipophilic cation that partitions
selectively into the negatively charged mitochondrial membrane.
Hyperpolarization of the mitochondrial membrane causes the uptake of
Rh123 into mitochondria and a decrease in fluorescence due to quenching
(21). Islets were incubated in Krebs-Ringer bicarbonate (KRB) buffer containing (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 25 NaHCO3, 2 glucose
supplemented with 10 µg/ml Rh123 for 20 min at 37 °C. Rh123
fluorescence was excited at 540 nm (to decrease phototoxicity) and
measured at 590 nm. Images were collected with an intensified video
camera as previously described (24). Data were normalized to the
average fluorescence intensity recorded during a 5-min period prior to
the application of glucose. All experiments were performed at
37 °C.
Measurements of O2
Content--
Cells
and islets were cultured on glass coverslips and loaded with HEt
(1 µg/ml; Molecular Probes, Eugene, OR). Production of
O2
was monitored at room temperature
(~24 °C) in KRB-HEPES buffer (KRB containing 10 mM
HEPES-NaOH, pH 7.4) by the appearance of red fluorescence using
rhodamine optics and fluorescence video microscopy with an intensified
CCD camera (6). HEt was present in all solutions. Minimum illumination
was used to limit photodamage to islets. The islet was focused using
maximum camera intensifier gain, the gain was then reduced to a
constant value, and the initial image was used as the background
correction for subsequent data collected in the presence of dye. Images
(8-frame average) were only captured every 30 s to further reduce
illumination effects. Most experiments were conducted in static bath
conditions. Solutions were replaced by adding 4-5× the bath volume
while simultaneously drawing off the old solution and were completed
within 30 s. A region of interest was drawn within the borders of
the islet (in the initial autofluorescence image) to monitor
fluorescence changes in real time. A region of interest located in
islet-free regions tracked changes in background fluorescence.
Observations were typically 20-30 min; after this period of time the
camera was saturated by the fluorescence signal. Although longer
observations might be attained by reducing camera gain, interpretation
of these experiments would be limited by increasing toxicity of
ethidium and its binding to cellular polynucleotides (25). We attempted to correct data for the thickness of islets obtained from the measured
optical cross-section area and estimates of islet volume; however,
islets in all cases were thicker than the illuminated volume of the
objective. Normalization of oxidation rates did not significantly
improve the variation within treatments (data not shown); thus no
correction is employed in data presented here. Slopes for fluorescence
intensity changes were fitted by linear regression for each experiment
as described previously (6). The data are expressed as the change in
fluorescence intensity (F) as a function of time (F/min).
Confocal Microscopy of Islets--
Islets from ZDF or ZLC rats
were incubated with HEt as above and visualized using an Ultraview
Nipkow disk, Zeiss 410, or Fluoview 200 laser scanning confocal
microscope. Some islets were double stained with HEt and Rh123 to
examine whether the Et was localized to mitochondria. In this series of
experiments Et fluorescence was selectively excited with a 547-nm laser
line, and Rh123 was excited at 488 nm. Following background
subtraction, images were corrected for cross-talk determined from
single-stained preparations under identical imaging conditions. Some
islets were co-incubated with mitotracker green FM (Molecular Probes)
in place of Rh123. In other experiments Rh123 was applied and laser
scanning confocal sections obtained with 0.3-µm steps for at least
one cell layer within the islet. Maximum intensity z-axis
projections for three sections (1 µm thick) were constructed with
MetaMorph. Image detail was enhanced by application of a 5 × 5 unsharp mask kernel. In parallel experiments, free radical generation
in islets was monitored with 2',7'-dichlorodihydrofluorescein diacetate
(H2DCFDA). H2DCFDA (33 µM, from
1000× stock in dimethylformamide) was acutely applied to live islets
on the stage of the confocal microscope. Images were collected every
few minutes or once after 5 min using minimum laser intensity.
H2DCFDA diffuses into cells where the free, non-fluorescent dichlorodihydrofluorescein is produced in the cytoplasm that is then
oxidized to the fluorescent moiety, dichlorofluorescein (DCF), by a
variety of ROS (26). Some islets were co-incubated with HEt and
H2DCFDA; images were collected into separate detector channels for subsequent colocalization analyses. Digitized images were
processed and pseudocolor overlaid using MetaMorph software.
 |
RESULTS |
HEt Oxidation in Islets--
Following exposure to a
HEt-containing solution, red fluorescence first appeared in the cytosol
of most cells in an islet, and the intensity increased over time (Fig.
1). Images shown are at 2-min intervals
for 16 min following dye addition. The fluorescence in the cytosol was
non-uniform and resembled the staining pattern of mitochondria. A
subset of elongated or spherical organelles was commonly observed to
stain quickly and intensely within minutes of HEt addition
(e.g. Fig. 1a and see below).

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Fig. 1.
Time-lapse imaging of HEt oxidation in a ZLC
islet. Confocal images shown are taken at 2-min intervals
following the application of HEt (1 µg/ml). Note that the increase in
ethidium fluorescence occurs throughout the cytosol of the islet cells.
Nuclei are not stained over the 16-min period. Scale bar, 20 µm.
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We examined living islets incubated with HEt and Rh123 by Nipkow disk
confocal microscopy at higher magnification. On this system Et was
selectively excited by the 547-nm laser line and Rh123 (and Et) by the
488-nm laser line; images were corrected for signal cross-talk.
Adequacy of the correction protocol was confirmed with single-stained
samples (data not shown). Et fluorescence was distributed throughout
the cytoplasm in a punctate and fusiform pattern, resembling a
mitochondrial distribution, superimposed upon a lower, diffuse
background (Fig. 2, upper
panel). The mitochondrial distribution was then revealed by
rescanning the optical plane for Rh123 staining and was very similar
(Fig. 2, middle panel). Organelles that stained rapidly and
more intensely by Et were co-labeled with Rh123 (Fig. 2, bottom
panel). It is evident that organelle movements occurred between
collection of the Et and Rh123 images, such as the obvious
displacements of otherwise identical structures like those in the
magnified inset (Fig. 2, merge panel, 2×). Similar results
were obtained with mitotracker green; however, this dye did not
penetrate islets beyond the first layer of cells and caused
mitochondrial swelling (data not shown).

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Fig. 2.
High magnification confocal imaging of a
mouse islet stained with HEt and Rh123. Images have been digitally
corrected to remove fluorescence contamination (cross-talk) between the
dyes (see "Experimental Procedures"). A,
(HEt) shows the distribution of Et fluorescence (547 nm
excitation) within cells of an islet of Langerhans. Et fluorescence
appears to be localized to a fusiform network of cytoplasmic organelles
in all islet cells. In some cells, the fluorescence is diffusely
distributed, suggesting a cytosolic localization. To determine
whether these were organelles mitochondria, the islet was double
stained with Rh123 (B, Rh123; 488 nm excitation).
C (merge) shows the co-localization
(red + green = yellow) of the
Rh123 fluorescence, indicating the location of mitochondria with the Et
pattern. The movement of some organelles in the live islet has produced
a slight offset of the two otherwise identical patterns. The
inset (2×) in the merge panel shows a magnified view of the
boxed region to the right. Nuclei of cells did not stain
with ethidium for at least 20-30 min under low illumination
conditions. Lack of nuclear staining under these conditions of
illumination is an important observation because the intensity of
ethidium fluorescence increases roughly 20-fold upon binding to DNA
(25) and can give rise to measurement artifacts (45). Similar data were
obtained in ZLC islets (data not shown). Scale bar, 10 µm.
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Effects of Temperature and Glucose on HEt Oxidation in
Islets--
The rate of HEt oxidation under basal conditions in 2 mM glucose (24 °C) required several min to attain a
steady linear rate (Fig. 3A),
then typically remained stable for 10 or more min. The exponential
increase observed during the first few minutes following HEt addition
may be because of dye penetration into the islet. Rates at 35 °C
were 4-5 times greater than at room temperature (data not shown). No
change in intrinsic fluorescence was observed in the absence of HEt.
Stabilized rates were well fit by a linear model (typically
p < 0.05; r2 > 0.95;
n > 75 islets).

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Fig. 3.
Effect of glucose on ethidium fluorescence in
ZLC islets. A, the graph shows the increase in Et
fluorescence as a function of time. After stabilization of the rate of
fluorescence intensity change, the glucose concentration was increased
to 10 mM (filled bar). The change in the rate of
ROS production was determined from the slope of the lines fit to the
data by linear regression (e.g. dashed line).
Note that the slope is increased following glucose stimulation
(asterisk). Data indicate the average Et intensity value
(expressed as arbitrary fluorescence units) measured in two adjacent
islets and are representative of 16 similar experiments. Data for the
final 10-min period of the incubation in 2 mM glucose
solution (between arrows) were well fit by linear
regression: adjusted r2 = 0.999, F = 46,609, p = 1.8 × 10 83. B, summary of HEt oxidation rates in ZLC
and ZDF rat islets. The glucose-induced increase in superoxide
production in ZLC islets is statistically significant as is the
elevated rate in ZDF islets in 2 mM glucose (see
"Results").
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Stimulation with 10 mM glucose increased the rate of HEt
oxidation in most islets after a delay of ~3-4 min (Fig.
3A). The rate of HEt oxidation remained relatively constant
for up to 10 min. After this period, the fluorescence intensity of Et
caused CCD camera saturation. Imaging the subcellular distribution of the fluorescence in islet cells by confocal microscopy revealed that
the increase in intensity was because of greater staining of the
cytosolic contents but not the nuclei. The rate increase was absent for
solution changes with identical glucose content and was smaller for 6 versus 10 mM glucose (data not shown). A parallel study showed the changes in Rh123 fluorescence
(hyperpolarization) after glucose exposure following a similar time
course to the visualization of Et fluorescence in mitochondria (data
not shown).
The average HEt oxidation rate in ZLC rat islets increased from 1.8 to
3.1 F/min (1.7-fold) following application of 10 mM glucose
(Fig. 3B). Similar values were observed in islets from Holtzmann rats, C57BL/KsJ mice, and MIN6 cells stimulated with glucose
(data not shown). The increase in 10 mM glucose was
effectively clamped by application of the complex III blocker
myxothiazol (200 nM; slope in myxothiazol was decreased to
39.4 ± 2.3% of that in 10 mM glucose;
n = 3), suggesting the mitochondria are the source of
HEt oxidation rather than a sink for the ethidium. The increased HEt
oxidation coincided with the hyperpolarization of the mitochondrial
membrane potential induced by the elevation of glucose concentration
(data not shown) and is thus not due to the potential (artifactual)
release of Et from within mitochondria.
These findings are in accord with parallel experiments that utilized
H2DCFDA to visualize the production of ROS.
H2DCFDA produces the fluorescent compound DCF when the
de-esterified parent compound is oxidized by a variety of intracellular
reactive species (26). Stimulation of ZLC rat islets with 10 mM glucose increased the rate of DCF production 1.21-fold
(2.23 ± 0.2 to 2.70 ± 0.15 F/min; p = 0.0008, paired t test; n = 12 islets). We
did not, however, pursue additional studies with H2DCFDA,
because of problems with photoconversion and photobleaching of the
indicator and phototoxicity associated with the blue excitation light
required for DCF excitation. Also, unlike HEt but similar to other
esterified fluorescent precursors, H2DCFDA did not
penetrate deeply into islets, staining only the outer layer of islet
cells. The pattern of DCF fluorescence was also primarily mitochondrial
(Fig. 4) as has been described in other
cell types (27). Taken together, these data strongly suggest that the
source of the oxyradicals in isolated islets is the mitochondria.

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Fig. 4.
Confocal imaging of Et and DCF fluorescence
in islets. A, shows the outer cell layer of a ZLC islet
exposed to HEt and (B) H2DCFDA. Both dyes
display a similar pattern of distribution and in most regions appear to
be restricted to mitochondria (white arrow). C,
an overlay of the Et and DCF images shows extensive colocalization
(merge, red + green = yellow). D, higher magnification view of the
DCF-stained mitochondria (e.g. arrow) in a
different ZLC islet. Intense illumination can promote DCF fluorescence
in non-mitochondrial structures; note the accumulation in the nuclear
space (e.g. pale disk-like areas, n). Scale
bars, 10 µm.
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HEt Oxidation Rates in ZDF Islets--
We next examined the rates
of Et production in islets isolated from ZDF rats. Under basal glucose
levels (2 mM), the rate of HEt oxidation in islets isolated
from hyperglycemic ZDF rats was significantly higher (1.8-fold) than in
ZLC rat islets. In ZDF islets the rate of Et formation was not
significantly affected by the presence of 10 mM glucose
(Fig. 3B; pooled animals >8 weeks: 1.2-fold increase
from 3.24 ± 0.29 to 3.96 ± 0.36 F/min; p = 0.12, t test; n = 34 and 32 ZDF islets
exposed to 2 and 10 mM glucose, respectively; 12-week old
ZDFs only: 3.11 ± 0.3 versus 2.72 ± 0.4 F/min;
p = 0.12, paired t test; n = 10). This indicates that O2
production was at
a near maximal level in the diabetic islets under low glucose.
We also examined the HEt oxidation rates in pre-diabetic ZDF rat
islets. The 6-week ZDF rats are not diabetic but are not normal in
secretory response even at this stage, and the male rats at this age
show impaired glucose tolerance. Hence they can be considered
"pre-diabetic." We found that the basal rate of O2
generation (in the presence of 2 mM glucose) was significantly elevated (2.58 ± 0.39 F/min; p = 0.05 versus ZLC (Fig.
3B), t test; n = 11 ZDF islets
isolated from 6-week-old animals). The increase in HEt oxidation
elicited by 10 mM glucose was modest but significant in
6-week ZDF islets (rate in 10 glucose = 3.11 ± 0.3;
p = 0.03, paired t test, n = 10). These findings suggest that the increase in
O2
levels and oxidative stress may be
important contributing factors to
-cell dysfunction and not
secondary epiphenomena associated with the diabetic state. Although
high levels of 02
were already present in
6-week-old pre-diabetic ZDF rat islets that already showed impaired
glucose tolerance, islets were still able to respond to glucose with
increased HEt oxidation similar to ZLC islets.
Altered Mitochondrial Morphology in ZDF Islets--
We examined
mitochondrial morphology in 1-µM-thick confocal
reconstructions (volume) from ZLC and ZDF islets stained with Rh123,
examples of which are shown in Fig. 5. In
ZLC islets the mitochondrial morphology was consistent with a network
of bright, filamentous structures (Fig. 5, left panels). In
ZDF islets, the mitochondria were generally short and swollen (Fig. 5,
right panels). Mitochondria were classified as either
elongated or short and swollen, and the percentage of cells containing
each type was determined. In a sample of 20 volumes from ZLC islets,
55% (11/20) of the volumes contained cells with elongated
mitochondria, and 40% (8/20) displayed a mixture of long and
short/swollen mitochondria (Fig. 5, left panels). Short,
swollen mitochondria were the predominant morphological type in a few
cells from only 5% of the volumes. In contrast, of 25 volumes from ZDF
islets, the proportion containing cells with each mitochondrial
morphology was: elongated 8%, combination of long and short/swollen,
44%, and short, swollen 48% (Fig. 5, right panels).

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Fig. 5.
Islet mitochondrial morphology.
Mitochondria were stained with Rh123. The three representative
images (confocal reconstructions) are maximum intensity projections,
1-µm thick, from individual isolated ZLC and ZDF rat islets. The
distribution of mitochondrial morphology from multiple confocal volumes
is described under "Results." Images have been digitally
sharpened and contrast balanced to reveal details of mitochondrial
morphology. Scale bar, 5 µm.
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 |
DISCUSSION |
Previous studies have implicated a role of ROS in islet damage
associated with diabetes using indirect approaches (e.g. 5, 13, 28). In this paper, we monitored the production of ROS in islets of
Langerhans using a fluorescent indicator of
O2
(Et) and microfluorometry. HEt oxidation
and consequent generation of Et fluorescence is stimulated specifically
by superoxide radicals. Previous investigations employing experimental
conditions similar to those used in our current studies demonstrated
that HEt oxidation and Et fluorescence were not affected by reactive
nitrogen species or H2O2 (6). Compared with
other free radical indicator dyes, HEt offers a selectivity for
superoxide under the conditions used and is largely insensitive to
photoconversion, photobleaching, and pH sensitivity that limits use of
indicators such as H2DCFDA or dihydrorhodamine. Utility of
HEt oxidation as a measure of ROS production in islets was validated by
several criteria, including concordance with DCF fluorescence, direct
visualization of the cytoplasmic localization of the Et signal, absence
of Et fluorescence in nuclei (indicative of binding of Et to DNA), and
an inhibition of oxyradical production in the presence of the oxidative
phosphorylation complex III inhibitor, myxothiazol. Nevertheless, Et
fluorescence will underestimate the rate of ROS production, because
O2
is rapidly converted to
H2O2 and peroxynitrite, lost to other competing
sinks, and removed by scavenging enzymes. Critical aspects of the
method are to use minimal dye concentration (to avoid toxic effects of
Et and quenching of Et fluorescence, to forestall nonlinear fluorescence increases that accompany Et binding to DNA, and to use
minimal light excitation (to minimize phototoxicity). The requirement
of minimal dye concentration implies detection of only a small fraction
of the total O2
generated. Notwithstanding
this limitation, HEt oxidation is sufficiently sensitive to detect
increased superoxide generation in islets following glucose stimulation.
We found two major differences in O2
regulation in ZLC and ZDF islets. First, under low glucose conditions,
the rate of O2
generation was significantly
higher in the ZDF islets. Second, stimulation with glucose caused a
large increase in O2
production in the ZLC
but not the ZDF islets. The findings suggest that development and
progression of diabetes is associated with a defect in one or more of
the mechanisms that regulate ROS content within islet cells. This
defect, which appears to be present prior to the onset of overt
diabetes, results in a sustained elevation of
O2
levels that likely contributes to
oxidative stress and
-cell dysfunction. It is noteworthy that the
fasting glucose levels of ZDF rats are already slightly elevated at
5-6 weeks of age but that the animals are not yet diabetic (29). Our
direct measurements of ROS content support the hypothesis that the
impairment of
-cell function associated with type 2 diabetes may
result in part from abnormal production of
O2
.
The reason for the significantly higher superoxide levels that occurred
in ZDF rat islets under normal glucose levels is uncertain. ZDF rats
have defective leptin receptors that result in altered expression of
fatty acid metabolism and synthetic pathways, leading to increased
islet triglyceride content (30). ZDF rats also have greatly
reduced UCP2 levels (31), and the consequent increase in mitochondrial
polarization could lead to an increase in superoxide production. This
concept is consistent with a role for UCP2 and oxidative stress in
diabetes. Overexpression of UCP2 has been shown to restore
glucose-induced insulin secretion in ZDF islets (30), and UCP2
overexpression has also been shown to protect INS-1 cells from
oxidative stress (32).
Elevated glucose concentrations are thought to alter metabolism and
create oxidative stress in many cell types in addition to
glucose-responsive
-cells (2). ROS generation in
-cells may be
more problematic than in other cells because of apparently low levels
of SOD, as reported in NOD mouse islets (33-36) and HIT and RIN
insulinoma cells (37). Levels of SOD may be higher in human islets than
in rodent islets, although the expression of SOD in purified human
-cells is unknown (38, 39). Any relative deficiency in SOD activity
may make
-cells more sensitive to the toxic effects of glucose,
because more superoxide produced in the mitochondrial generation of ATP
would be available to interact with any NO that is produced.
Interestingly, it has been reported that acute elevations of glucose
produce a closely correlated increase in SOD-1 activity in rat islets
(40). Our data might suggest this is a response to increased superoxide
leak from the electron transport chain observed with acute glucose elevation.
Oxyradical damage to islets as opposed to damage in other tissues has
been more closely examined in models of type 1 diabetes mellitus than
in type 2 diabetes. Inflammatory cytokines, while activating a variety
of enzymes, including proteases and phospholipases, may also activate
production of various damaging free radicals. In one model of the
pathogenesis of type 1 diabetes (41), immune activation perhaps induced
by viral damage was linked to antigen presentation, production of
cytokines, and poor
-cell defense against free radicals produced by
the
-cells themselves. Together these processes would result in
-cell destruction. The role of NO in this process is in some
dispute. Although the cytokine-induced destruction of human
-cells
involves oxygen free radical-mediated lipid peroxidation and aldehyde
production, NO does not seem to be cytotoxic (42). Our demonstration of
increased superoxide levels following acute glucose elevations implies
that NO will be more prone to form peroxynitrite, a much more reactive
and toxic species.
The most likely source of ROS in islets is the mitochondria. This is
supported by the staining pattern of Et and DCF that strongly resembles
that of the mitochondria as shown by Rh123 fluorescence. Similar
staining and conclusions were described for reoxygenated endothelial
cells (15). ROS damage could account for the altered mitochondrial
morphology we observed in ZDF islets, which exhibited a significantly
increased content of short swollen mitochondria in comparison with ZLC
islets. A previous report mentioned that abnormal mitochondria were
present in pre-diabetic and diabetic ZDF
-cells as observed in
transmission electron micrographs (43). Both the mitochondrial
alteration and onset of diabetes were ameliorated by troglitazone, an
insulin sensitizer (5). The change in morphology may be a consequence
of ROS damage. For example, H2O2 treatment and
depolarization of the mitochondrial membrane with chemical uncouplers
of oxidative phosphorylation can change filamentous mitochondria to
condensed/swollen morphologies (44). Our data show that the altered
morphology is predominant within ZDF islets. Taken together, these
findings suggest that the higher endogenous reactive oxygen species
present in diabetic islets may be responsible for the observed
alterations in mitochondrial morphology.
An alternative view of these data involves the possibility that ROS
serves as a critical signaling molecule, in this case for insulin
secretion. Thus, ROS might have a role in normal insulin secretion:
increased ROS initially causes the increased insulin secretion under
basal glucose conditions and may only indirectly be implicated in the
-cell mitochondrial damage that apparently occurs with time.
Supporting this hypothesis are the reportedly low levels of SOD in
-cells, which might actually be necessary for proper ROS signaling
to promote normal insulin secretion, rather than being the defect that
it is usually considered. On the other hand, overexpression of
SOD in
-cells did not impair insulin secretion (11, 13), and we
could find no evidence in the many reports using free radical
scavengers in diabetic models to show that free radical scavengers
impair insulin secretion. Indeed, the opposite is usually the case.
Given the recent interest in the possibility that uncoupling proteins
might function in part through a free radical mechanism (48) or not
(46), this remains an intriguing concept for further study in
insulin-secreting cells.
Our studies, using real-time, direct measurements of superoxide,
indicate that O2
generation is coupled to
metabolism, mitochondrial metabolism in particular. While this paper
was in review, a similar conclusion was reached by Sakai et
al. (47) without imaging mitochondria directly. We also found that
O2
generation is not only increased in
frankly diabetic ZDF rat islets but also in pre-diabetic ZDF rat islets
that already have mildly impaired insulin secretion and glucose
tolerance. This initial validation of an optical method for the
detection of ROS in islets and their localization in mitochondria with
parallel measures of membrane potential and calcium responses will
facilitate additional studies to identify and characterize the role of
ROS in insulin secretion and the onset of diabetes.
Published, JBC Papers in Press, January 4, 2003, DOI 10.1074/jbc.M206913200
1.
|
Kirkinezos, I. G.,
and Moraes, C. T.
(2001)
Semin. Cell Dev. Biol.
12,
449-457[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Nishikawa, T.,
Edelstein, D.,
Du, X. L.,
Yamagishi, S.-I.,
Matsumura, T.,
Kaneda, Y.,
Yorek, M. A.,
Beebe, D.,
Oates, P. J.,
Hammes, H.-P.,
Glardino, I.,
and Brownlee, M.
(2000)
Nature
404,
787-790[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Corbett, J. A.,
and McDaniel, M. L.
(1992)
Diabetes
41,
897-903[Abstract]
|
4.
|
Shimabukuro, M.,
Ohneda, M.,
Lee, Y.,
and Unger, R. H.
(1997)
J. Clin. Invest.
100,
290-295[Abstract/Free Full Text]
|
5.
|
Shimabukuro, M.,
Zhou, Y. T.,
Levi, M.,
and Unger, R. H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2498-2502[Abstract/Free Full Text]
|
6.
|
Bindokas, V. P.,
Jordan, J.,
Lee, C. C.,
and Miller, R. J.
(1996)
J. Neurosci.
16,
1324-1336[Abstract]
|
7.
|
Turrens, J. F.
(1997)
Biosci. Rep.
17,
3-8[Medline]
[Order article via Infotrieve]
|
8.
|
Korshunov, S. S.,
Skulachev, V. P.,
and Starkov, A. A.
(1997)
FEBS Lett.
416,
15-18[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Demin, O. V.,
Kholodenko, B. N.,
and Skulachev, V. P.
(1998)
Mol. Cell Biochem.
184,
21-33[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Nicholls, D. G.,
and Budd, S. L.
(2000)
Physiol. Rev.
80,
315-360[Abstract/Free Full Text]
|
11.
|
Kubisch, H. M.,
Wang, J.,
Luche, R.,
Carlson, E.,
Bray, T. M.,
Epstein, C. J.,
and Phillips, J. P.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9956-9959[Abstract/Free Full Text]
|
12.
|
Kaneto, H.,
Fujii, J.,
Myint, T.,
Miyazawa, N.,
Islam, K. N.,
Kawasaki, Y.,
Suzuki, K.,
Nakamura, M.,
Tatsumi, H.,
Yamasaki, Y.,
and Taniguchi, N.
(1996)
Biochem. J.
320,
855-863[Medline]
[Order article via Infotrieve]
|
13.
|
Kubisch, H. M.,
Wang, J.,
Bray, T. M.,
and Phillips, J. P.
(1997)
Diabetes
46,
1563-1566[Abstract]
|
14.
|
Laight, D. W.,
Desai, K. M.,
Gopaul, N. K.,
Anggard, E. E.,
and Carrier, M. J.
(1999)
Eur. J. Pharmacol.
377,
89-92[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Hempel, S. L.,
Buettner, G. R.,
O'Malley, Y. Q.,
Wessels, D. A.,
and Flaherty, D. M.
(1999)
Free Radic. Biol. Med.
27,
146-159[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Raha, S.,
and Robinson, B. H.
(2001)
Am. J. Med. Genet.
106,
62-70[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Macho, A.,
Castedo, M.,
Marchetti, P.,
Aguilar, J. J.,
Decaudin, D.,
Zamzami, N.,
Girard, P. M.,
Uriel, J.,
and Kroemer, G.
(1995)
Blood
86,
2481-2487[Abstract/Free Full Text]
|
18.
|
Prehn, J. H.,
Bindokas, V. P.,
Jordan, J.,
Galindo, M. F.,
Ghadge, G. D.,
Roos, R. P.,
Boise, L. H.,
Thompson, C. B.,
Krajewski, S.,
Reed, J. C.,
and Miller, R. J.
(1996)
Mol. Pharmacol.
49,
319-328[Abstract]
|
19.
|
Roe, M. W.,
Worley, J. F., III,
Tokuyama, Y.,
Philipson, L. H.,
Sturis, J.,
Tang, J.,
Dukes, I. D.,
Bell, G. I.,
and Polonsky, K. S.
(1996)
Am. J. Physiol.
270,
E133-140[Medline]
[Order article via Infotrieve]
|
20.
|
Miyazaki, J.,
Araki, K.,
Yamato, E.,
Ikegami, H.,
Asano, T.,
Shibasaki, Y.,
Oka, Y.,
and Yamamura, K.
(1990)
Endocrinology
127,
126-132[Abstract]
|
21.
|
Emaus, R. K.,
Grunwald, R.,
and Lemasters, J. J.
(1986)
Biochim. Biophys. Acta
850,
436-448[Medline]
[Order article via Infotrieve]
|
22.
|
Duchen, M. R.,
Smith, P. A.,
and Ashcroft, F. M.
(1993)
Biochem. J.
294,
35-42[Medline]
[Order article via Infotrieve]
|
23.
|
Rustenbeck, I.,
Herrmann, C.,
and Grimmsmann, T.
(1997)
Diabetes
46,
1305-1311[Abstract]
|
24.
|
Zhou, Y. P.,
Pena, J. C.,
Roe, M. W.,
Mittal, A.,
Levisetti, M.,
Baldwin, A. C.,
Pugh, W.,
Ostrega, D.,
Ahmed, N.,
Bindokas, V. P.,
Philipson, L. H.,
Hanahan, D.,
Thompson, C. B.,
and Polonsky, K. S.
(2000)
Am. J. Physiol.
278,
E340-351
|
25.
|
LePecq, J. B.,
and Paoletti, C.
(1967)
J. Mol. Biol.
27,
87-106[Medline]
[Order article via Infotrieve]
|
26.
|
Murrant, C. L.,
and Reid, M. B.
(2001)
Microsc. Res. Tech.
55,
236-248[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Lemasters, J. J.,
Qian, T.,
Trollinger, D. R.,
Cascio, W. E.,
Ohata, H.,
and Nieminen, A. L.
(2001)
Mitochondria in Pathogenesis
, pp. 21-49, Kluwer Academic/Plenum Publishers, New York
|
28.
|
Lortz, S.,
Tiedge, M.,
Nachtwey, T.,
Karlsen, A. E.,
Nerup, J.,
and Lenzen, S.
(2000)
Diabetes
49,
1123-1130[Abstract]
|
29.
|
Pick, A.,
Clark, J.,
Kubstrup, C.,
Levisetti, M.,
Pugh, W.,
Bonner-Weir, S.,
and Polonsky, K. S.
(1998)
Diabetes
47,
358-364[Abstract]
|
30.
|
Wang, M. Y.,
Shimabukuro, M.,
Lee, Y.,
Trinh, K. Y.,
Chen, J. L.,
Newgard, C. B.,
and Unger, R. H.
(1999)
Diabetes
48,
1020-1025[Abstract]
|
31.
|
Zhou, Y. T.,
Shimabukuro, M.,
Koyama, K.,
Lee, Y.,
Wang, M. Y.,
Trieu, F.,
Newgard, C. B.,
and Unger, R. H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6386-6390[Abstract/Free Full Text]
|
32.
|
Li, L. X.,
Skorpen, F.,
Egeberg, K.,
Jorgensen, I. H.,
and Grill, V.
(2001)
Biochem. Biophys. Res. Commun.
282,
273-277[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Gandy, S. E., III,
Galbraith, R. A.,
Crouch, R. K.,
Buse, M. G.,
and Galbraith, G. M.
(1981)
N. Engl. J. Med.
304,
1547-1548[Medline]
[Order article via Infotrieve]
|
34.
|
Malaisse, W. J.,
Malaisse-Lagae, F.,
Sener, A.,
and Pipeleers, D. G.
(1982)
Proc. Natl. Acad. Sci. U. S. A.
79,
927-930[Abstract]
|
35.
|
Papaccio, G.,
Frascatore, S.,
Pisanti, F. A.,
Latronico, M. V.,
and Linn, T.
(1995)
Life Sci.
56,
2223-2228[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Lenzen, S.,
Drinkgern, J.,
and Tiedge, M.
(1996)
Free Radic. Biol. Med.
20,
463-466[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Zhang, H.,
Ollinger, K.,
and Brunk, U.
(1995)
Diabetologia
38,
635-641[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Welsh, N.,
Margulis, B.,
Borg, L. A.,
Wiklund, H. J.,
Saldeen, J.,
Flodstrom, M.,
Mello, M. A.,
Andersson, A.,
Pipeleers, D. G.,
Hellerstrom, C.,
and Eizirik, D. L.
(1995)
Mol. Med.
1,
806-820[Medline]
[Order article via Infotrieve]
|
39.
|
Hausmann, D. H.,
Porstmann, T.,
Weber, I.,
Hausmann, S.,
Dummler, W.,
Liebe, S.,
and Emmrich, J.
(1997)
Int. J. Pancreatol.
22,
207-213[Medline]
[Order article via Infotrieve]
|
40.
|
Oliveira, H. R.,
Curi, R.,
and Carpinelli, A. R.
(1999)
Am. J. Physiol.
276,
C507-510[Medline]
[Order article via Infotrieve]
|
41.
|
Nerup, J.,
Mandrup-Poulsen, T.,
Helqvist, S.,
Andersen, H. U.,
Pociot, F.,
Reimers, J. I.,
Cuartero, B. G.,
Karlsen, A. E.,
Bjerre, U.,
and Lorenzen, T.
(1994)
Diabetologia
37,
S82-89[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Rabinovitch, A.,
Suarez-Pinzon, W. L.,
Strynadka, K.,
Lakey, J. R.,
and Rajotte, R. V.
(1996)
J. Clin. Endocrinol. Metab.
81,
3197-3202[Abstract]
|
43.
|
Higa, M.,
Zhou, Y. T.,
Ravazzola, M.,
Baetens, D.,
Orci, L.,
and Unger, R. H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11513-11518[Abstract/Free Full Text]
|
44.
|
Maechler, P.,
Jornot, L.,
and Wollheim, C. B.
(1999)
J. Biol. Chem.
274,
27905-27913[Abstract/Free Full Text]
|
45.
|
Budd, S. L.,
Castilho, R. F.,
and Nicholls, D. G.
(1997)
FEBS Lett.
415,
21-24[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Couplan, E.,
del Mar Gonzalez-Barroso, M.,
Alves-Guerra, M. C.,
Ricquier, D.,
Goubern, M.,
and Bouillaud, F.
(2002)
J. Biol. Chem.
277,
26268-26275[Abstract/Free Full Text]
|
47.
|
Sakai, K.,
Matsumotot, K.,
Nishikawa, T.,
Suefuji, M.,
Nakamura, K.,
Hirashmia, Y.,
Kawashima, J.,
Shirotani, T.,
Ichinose, K.,
Brownlee, M.,
and Araki, E.
(2003)
Biochem. Biophys. Res. Commun.
300,
216-222[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Echtay, K. S.,
Roussel, D.,
St-Pierre, J.,
Jekabsons, M. B.,
Cadenas, S.,
Stuart, J. A.,
Harper, J. A.,
Roebuck, S. J.,
Morrison, A.,
Pickering, S.,
Clapham, J. C.,
and Brand, M. D.
(2002)
Nature
415,
96-99[CrossRef][Medline]
[Order article via Infotrieve]
|