Visualizing Superoxide Production in Normal and Diabetic Rat Islets of Langerhans*

Vytautas P. BindokasDagger , Andrey Kuznetsov§, Seamus Sreenan, Kenneth S. Polonsky||, Michael W. Roe§, and Louis H. Philipson§**

From the Dagger  Department of Neurobiology, Pharmacology, Physiology and the § Department of Medicine, The University of Chicago, Chicago, Illinois 60637, the  Department of Endocrinology, James Connolly Memorial Hospital, Dublin 15, Ireland, and the || Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, July 10, 2002, and in revised form, December 12, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxygen free radicals have been implicated in beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidative stress is thought to contribute to the pathogenesis of neurodegenerative diseases (1) and complications of diabetes mellitus (2). Insulin-secreting beta -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 beta -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 beta -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 beta -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 beta -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 beta -cell dysfunction in type 2 diabetes mellitus.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta Psi m) Measurement-- Rhodamine 123 (Rh123) was used as an indicator of Delta Psi 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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").

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.

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 beta -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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -cells (2). ROS generation in beta -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 beta -cells is unknown (38, 39). Any relative deficiency in SOD activity may make beta -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 beta -cell defense against free radicals produced by the beta -cells themselves. Together these processes would result in beta -cell destruction. The role of NO in this process is in some dispute. Although the cytokine-induced destruction of human beta -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 beta -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 beta -cell mitochondrial damage that apparently occurs with time. Supporting this hypothesis are the reportedly low levels of SOD in beta -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 beta -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.

    ACKNOWLEDGEMENT

We thank Anshu Mittal for excellent technical assistance.

    FOOTNOTES

* This work has been supported by National Institutes of Health Grants DK44840, DK48494, and DK20595 (to The Diabetes Research and Training Center at the University of Chicago) and the Blum-Kovler Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Medicine, MC-1027, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 773-702-9180; Fax: 773-702-9194; E-mail: l-philipson@uchicago.edu.

Published, JBC Papers in Press, January 4, 2003, DOI 10.1074/jbc.M206913200

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; O2-, superoxide; NO, nitric oxide; SOD, superoxide dismutase; HEt, hydroethidine; Et, ethidium; ZLC, Zucker lean control; ZDF, Zucker diabetic fatty; Rh123, rhodamine 123; H2DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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