NOX2 Deficiency Protects Against Streptozotocin-Induced β-Cell Destruction and Development of Diabetes in Mice

NOX2^−/− and C57BL/6 (wild-type [WT]) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Male NOX2^−/− and WT mice 8–12 weeks old were used in all experiments. Animals in this study were handled in accordance with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publ. no. 85-23, revised 1996).

Single-cell suspensions were prepared from spleens of NOX2^−/− and WT mice as previously described (19). Briefly, spleens were removed, minced, and filtered through sterile 100-μm filters. Erythrocytes were lysed, and the nucleated cells were washed resuspended in saline and injected via the lateral tail vein into irradiated (970 cGy) WT mice (10⁷ cell/mouse). Mice that received NOX2^−/− and WT splenocytes were termed WT^NOX2−/− and WT^WT, respectively. These mice were allowed to recover for 5 weeks before the induction of diabetes.

To induce diabetes, WT, NOX2^−/−, WT treated with apocynin (10 mg/day/kg body wt i.p.), WT^NOX2−/−, and WT^WT mice were treated with STZ (80 mg/kg body wt i.p. for 3 consecutive days). Additionally, some WT, NOX2^−/− mice were treated with saline as vehicle controls. Random blood glucose levels were monitored on day 2, 5, and 8 after the final injection of STZ with a glucose meter (OneTouch Ultra2; LifeScan Canada, Burnaby, BC). On day 9, an intraperitoneal glucose tolerance test (IPGTT) was performed using an intraperitoneal injection of d-glucose at 2 g/kg body wt after 16 h fasting. Blood glucose was analyzed at 0, 15, 30, 60, and 120 min after introducing glucose. Nonfasting serum was collected. Pancreata were weighed and collected. Diabetic hyperglycemia was defined as a nonfasting blood glucose concentration >11.1 mmol/l for two or more consecutive tests.

WT, NOX2^−/− mice were treated with alloxan (60 mg/kg body wt i.v.). Fasting blood glucose levels were measured, and IPGTT was performed as described above. Nonfasting serum was collected for measurement of insulin levels.

Pancreata from WT and NOX2^−/− mice killed on day 9 were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and serially cut into 5-μm sections at 50-μm intervals. Immunostaining was performed with an anti-mouse insulin antibody (1:2,000; Sigma, Oakville, ON) using a mouse-on-mouse immunodetection kit (Vector Laboratories, Burlingame, CA). The staining was visualized using diaminobenzidine. Sections were then counterstained with hematoxylin. The microscopic images were taken by a Zeiss microscope and analyzed using AxioVision software. Insulin-positive cells were considered functional β-cells. Relative β-cell and islet area to total pancreatic area ratios were calculated. The β-cell and islet mass were then evaluated by multiplying the area ratio with the pancreatic weight. The evaluation of insulitis score was assigned using the following scale: 0, intact islet; 1, peri-insulitis; 2, moderate insulitis (<50% of the islet infiltrated); and 3, severe insulitis (≥50% of the islet infiltrated) as previously described (19). For the analysis of pancreatic islet apoptosis, double staining of in situ cell death detection fluorescein and insulin fluorescent immunohistochemistry (20) was performed. Pancreata from WT and NOX2^−/− mice killed 8 h after the final injection of STZ were isolated, embedded, and sectioned as described above. Apoptosis and necrosis of the islet cells were assessed via hematoxylin staining by the chromatin morphology (21).

Pancreatic islets were isolated from NOX2^−/− and WT mice by liberase RI (Roche Diagnostics, Laval, QC) digestion as described previously (22). Isolated pancreatic islets were cultured free-floating in RPMI-1640 medium containing 11.1 mmol/l glucose, supplemented with 2 mmol/l l-glutamine, 10% FCS, 12 mmol/l HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 95% air and 5% CO₂ at 37°C overnight. Groups of 20 islets were then incubated in 200 μl Dulbecco's modified Eagle's medium containing 11.1 mmol/l glucose supplemented with 2 mmol/l l-glutamine, 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin with or without a cytokine mixture, which consisted of human recombinant IL-1β, IFN-γ, and TNF-α (2 ng/ml each; Sigma) for 48 h at 37°C in 5% CO₂.

The insulin secretion assay was performed as previously described (23). Briefly, islets from each experiment group were incubated in Krebs-Ringer bicarbonate buffer supplemented with 2 mg/ml BSA and 1.67 mmol/l glucose for 30 min, followed by a 90-min incubation in Krebs-Ringer bicarbonate HEPES buffer supplemented with 2 mg/ml BSA and in the presence of 1.67 or 16.7 mmol/l glucose. Insulin released during the 90 min was then measured and normalized by the DNA content of each 20-islet group. DNA content was determined by a CyQUANT-cell proliferation assay kit (Invitrogen Canada, Burlington, ON).

Total ROS production in isolated islets was determined by dichlorofluorescein diacetate assay. Islets were homogenized in 100 μl buffer solution (50 mmol/l Tris-HCl, pH 7.5, 50 mmol/l KCl, and 10 mmol/l MgCl₂) and incubated with 10 μmol/l dichlorofluorescein diacetate at 37°C for 3 h. Fluorescence was measured by excitation (485 nm) and emission (525 nm) spectra with a microplate reader (SpectraMax M5; Molecular Devices, Sunnyvale, CA). Superoxide production in serum and isolated islets was determined by dihydroethidium assay. Serum and islets were incubated with 50 μmol/l dihydroethidium at 37°C for 30 min. Fluorescence was measured by excitation (396 nm) and emission (579 nm) spectra, which detects 2-hydroxyethidium (a specific superoxide-derived dihydroethidium product) (24), using a microplate reader (SpectraMax M5). Superoxide production in splenocytes in response to concanavalin A (ConA) was measured by lucigenin assay (25). Briefly, 10⁶ splenocytes were treated with ConA (5 μg/ml) with or without 0.1 μmol/l diphenylene iodonium (NADPH inhibitor) for 5 min at 37°C followed by 50 μmol/l lucigenin. Luminescence was then measured using a microplate reader (SpectraMax M5).

Isolated splenocytes (5 × 10⁶) from WT and NOX2^−/− mice were cultured overnight and then incubated with ConA (5 μg/ml) for 48 h. TNF-α and IL-1β release in the media was determined by an ELISA kit (eBioscience, San Diego, CA).

Culture media of isolated islets in response to a 90-min stimulation of 1.67 and 16.7 mmol/l glucose were collected. Serum from nonfasting mice was obtained. Both culture media and serum were stored in −20°C, and their insulin concentrations were measured by a mouse insulin ELISA kit (ALPCO, Salem, NH). Serum insulin levels were expressed as micrograms per liter. Insulin concentrations in the culture media were adjusted by DNA content of the islets and expressed as nanograms per 100 μg islet DNA per 90 min. Serum total antioxidant levels were detected by a total antioxidant capacity assay kit (Abcam, Cambridge, MA) and were expressed as milliequivalents per liter of Trolox per liter (mEq Trolox/l).

Antioxidant enzymes manganese SOD (SOD-2) and Gpx1 protein levels were assessed by Western blotting. Briefly, cell lysates containing 80 μg protein were subjected to sodium dodecyl sulfate–PAGE using 12% gels, followed by electrotransfer to nitrocellulose membranes. Blots were probed with antibodies against SOD-2 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), Gpx1 (1:500; Abcam, MA), and GAPDH (1:8,000; Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase–conjugated secondary antibodies (1:2,000; Santa Cruz Biotechnology). Protein bands were detected using the enhanced chemiluminescence method.

Data are presented as means ± SEM. Statistical analysis was performed using two-way ANOVA for multiple group comparisons followed by Bonferroni's correction or Student t test for comparison between two groups. Difference in insulitis score between NOX2^−/− and WT mice was analyzed by the χ² test. A P value of <0.05 was considered statistically significant.

Under normal conditions, adult WT and NOX2^−/− mice showed equivalent levels of random blood glucose, serum superoxide, antioxidant, and insulin (Fig. 1). Two days after the final injection of STZ, five of seven WT mice developed hyperglycemia, whereas only one of seven NOX2^−/− mice had blood glucose level >11.1 mmol/l. On day 5, all WT mice became diabetic, but there were still two of seven NOX2^−/− mice that demonstrated normal blood glucose levels. Compared with WT mice, NOX2^−/− mice presented significantly lower random (Fig. 1,A and B) and fasting (Fig. 1,E) blood glucose levels after STZ treatment (P < 0.01). Consistently, the results of IPGTT performed on day 9 as an in vivo measure of β-cell function showed that STZ-induced glucose intolerance was significantly ameliorated in NOX2^−/− mice (P < 0.01, Fig. 1,C and D). A similar effect was also observed in WT mice treated with an NADPH oxidase inhibitor, apocynin (10 mg/kg body wt/day). In concordance to the blood glucose level results, the serum insulin level was restored in NOX2^−/− mice compared with WT mice (P < 0.05, Fig. 1,F). Serum superoxide production significantly increased after STZ treatment in both WT and NOX2^−/− mice (Fig. 1,G). However, compared with WT mice, NOX2^−/− mice presented significantly lower serum superoxide levels (P < 0.01, Fig. 1,G). No significant changes were observed in serum antioxidant activity levels or protein expression of antioxidant enzymes including SOD-2 and Gpx1 after STZ treatment (Fig. 1 H–J).

Apoptosis is an important mechanism in the development of diabetes. We examined β-cell apoptosis 9 days after STZ treatment by transferase-mediated dUTP nick-end labeling (TUNEL) staining and co-staining with insulin. Apoptotic β-cells were detected by immunofluorescence associated with fragmented DNA (Fig. 2,A). Compared with WT mice, there were significantly less TUNEL-positive β-cells in NOX2^−/− mouse islets 9 days after STZ treatment (P < 0.01, Fig. 2,B). Acute cell death in islets after STZ treatment was also investigated. Eight hours after the final STZ injection, a significant decrease in the number of apoptotic and necrotic cells in the islets was observed in NOX2^−/− mice compared with WT mice (P < 0.01, Fig. 2,C). Furthermore, necrosis was about 15-fold higher than apoptosis at this early stage of STZ-induced islet damage (Fig. 2 C).

STZ treatment caused destruction of pancreatic β-cells (representative histological images of pancreatic tissue are shown in Fig. 3,A). In WT mice, the number of functional (insulin immuno-positive) β-cells within islets was markedly decreased as a result of STZ treatment. However, in NOX2^−/− mice, β-cells appeared to be protected after the same dose regimen of STZ treatment (Fig. 3,A). Compared with controls, β-cell mass was significantly decreased in WT mice after STZ treatment (P < 0.01). However, NOX2 deficiency significantly preserved the β-cell mass (P < 0.05, Fig. 3 B).

Insulitis is an important process in β-cell destruction induced by STZ. A scoring system was used to evaluate the extent of insulitis after STZ treatment. An example of each score grade was shown in Fig. 4,A. Histological analysis showed the presence of insulitis in both groups of mice, with significant differences in the degree of leukocyte infiltration (P < 0.01). In NOX2^−/− mice, 43% of islets exhibited no insulitis, with 38% of islets showing only peri-insulitis. However, in WT mice, half of the islets showed grade 2–3 insulitis (Fig. 4 B).

Islets isolated from WT and NOX2^−/− mice showed a similar level of insulin release in response to glucose stimulation (Fig. 5,A). The cytokine treatment (2 ng/ml each of IL-1β, IFN-γ, and TNF-α) significantly decreased insulin release from the WT islets (P < 0.01). However, this impairment was abrogated in NOX2^−/− islets (P < 0.01, Fig. 5,A). Cytokine treatment significantly increased total ROS and superoxide production in both WT and NOX2^−/− islets (P < 0.01). When compared with WT, both total ROS (Fig. 5,B) and superoxide (Fig. 5 C) levels of NOX2^−/− islets were significantly decreased (P < 0.01).

Adoptive transfer was used to replace the WT immune environment with a NOX2-deficient one. Five weeks after adoptive transfer, basal blood glucose levels of WT^WT and WT^NOX2−/− mice remained normal. STZ treatment induced a severe hyperglycemia in WT^WT mice. However, the WT^NOX2−/− mice demonstrated a significantly lower level of random blood glucose (Fig. 6,A and B, P < 0.01) and fasting blood glucose (Fig. 6,E, P < 0.01), higher serum insulin level (Fig. 6,F, P < 0.01), and less intolerance to IPGTT compared with WT^WT (Fig. 6,C and D, P < 0.05). WT and NOX2^−/− splenocytes used to perform the adoptive transfer were stimulated with ConA. Superoxide production detected by lucigenin assay in response to ConA stimulation was significantly attenuated in NOX2^−/− splenocytes compared with WT (P < 0.01, Fig. 6,G). Moreover, 48 h after ConA stimulation, the proinflammatory cytokine release (TNF-α and IL-1β) was significantly impaired in NOX2^−/− splenocytes compared with WT (P < 0.05, Fig. 6 H and I).

After alloxan treatment (60 mg/kg i.v.), all WT mice developed hyperglycemia, whereas none of the NOX2^−/− mice had blood glucose levels >11.1 mmol/l on day 2 and 5. NOX2^−/− mice presented significantly lower random (Fig. 7,A and B) and fasting (Fig. 7,E) blood glucose levels compared with WT (P < 0.01). Consistently, IPGTT performed on day 9 showed that alloxan-induced glucose intolerance was abrogated in NOX2^−/− mice (P < 0.01, Fig. 7,C and D). Furthermore, serum insulin levels were significantly increased in NOX2^−/− compared with WT mice after alloxan treatment (P < 0.01, Fig. 7 F).

In the present study, we demonstrated for the first time that deficiency of NOX2 protects against the onset and development of STZ-induced diabetes in mice. STZ-induced hyperglycemia was significantly inhibited in NOX2^−/− compared with WT mice. In agreement with the blood glucose levels, the in vivo islet function measured by IPGTT and the serum insulin levels after STZ treatment were restored in NOX2^−/− mice. Histological investigation of pancreatic tissue revealed that the majority of β-cells in WT pancreatic islets was destroyed by the STZ, whereas the β-cell mass was well preserved in NOX2^−/− mice. These data clearly showed the protective effects of NOX2 deficiency against STZ-induced diabetes, suggesting a critical role of NOX2-containing NADPH oxidase in β-cell destruction in diabetes.

In the multiple-low-dose STZ model, diabetes is induced by two main mechanisms (3,26,27). One is the direct cytotoxic effect of STZ on β-cells by alkylation of nuclear and mitochondrial DNA. The other is the local infiltration in the pancreatic islets and inflammation by mononuclear cells including lymphocytes and macrophages, which simulate the autoimmune reaction in type 1 diabetes (28). Although ROS has been implicated in both mechanisms, the source of the ROS production during development of diabetes is still not clear. Recently, functional phagocyte-like NADPH oxidase has been found in T-cells (17) and β-cells (18). NOX has been shown to be quantitatively an important source of ROS in pancreatic islets (18,29). In the present study, cell death at 8 h after the third STZ treatment was significantly decreased in the NOX2^−/− mice. Although cytokine (IL-1β, IFN-γ, and TNF-α) treatment significantly increased total ROS and superoxide production of the WT islets, both total ROS and superoxide levels were significantly decreased in the NOX2^−/− islets. Furthermore, β-cell function in terms of insulin release was well preserved in NOX2^−/− islets. Additionally, serum insulin levels and IPGTT were restored in NOX2^−/− mice after STZ treatment in vivo. These data suggest that NOX2-containing NADPH oxidase plays an important role not only in the early islet cell death from STZ, but also cytokine-mediated ROS production in the islets leading to impairment of β-cell function.

Alloxan is another diabetogenic chemical used frequently because of its ability to selectively damage β-cells by inducing ROS formation (3). To further demonstrate that decreased ROS production due to NOX2 deficiency protects against diabetes, the alloxan diabetes model was used. Results showed that NOX2 deficiency prevented diabetes development after alloxan treatment. Our data provide convincing evidence for an important role of NOX2-mediated ROS production in diabetes development.

Type 1 diabetes is an autoimmune disease. Activation and recruitment of immune cells including β-cell–specific CD8⁺ T-cells have been implicated in the pathogenesis of diabetes through cytokine expression and ROS production leading to β-cell destruction (1). In fact, recent studies have shown that T-cells have functional NADPH oxidase enzyme, and deficiency in NOX2 decreases their ability to produce ROS according to antigen stimulation (17). In the present study, we isolated splenocytes and measured ConA-stimulated superoxide production and proinflammatory cytokine release, which reflects immune cell activation. Our data showed that superoxide production and proinflammatory cytokine release (TNF-α and IL-1β) were significantly decreased in NOX2-deficient splenocytes. To further investigate the contribution of NOX2 in immune cells to functional impairment and damage of β-cells, NOX2^−/− splenocytes were adoptively transferred into lethally irradiated WT mice to reproduce the altered immune system in the NOX2 animals. Consistent with our hypothesis, a significantly lower blood glucose level, higher serum insulin level, and better islet function were observed in the WT mice repopulated with NOX2 splenocytes compared with the control group after STZ treatment. Our results suggest that NOX2-containing NADPH oxidase in the activated immune cells including T-cells also plays a key role in the impairment of islet function after STZ treatment.

In type 1 diabetes and animal models of STZ-induced diabetes, leukocytic infiltration (insulitis) and the production of proinflammatory cytokines contribute significantly to β-cell destruction (30,–32). Infiltrated macrophages and T-cells generate cytotoxic mediators including cytokines and induce β-cell death through apoptosis (33,34). In our study, decreased insulitis and apoptosis were observed in the islets of NOX2^−/− mice, leading to the well-preserved β-cell mass after STZ treatment, which can be explained by a reduction of early islet cell death and the decreased ability of NOX2^−/− splenocytes to produce superoxide and release proinflammatory cytokines. Previous studies have also shown that NOX2 deficiency results in decreased superoxide production from polymorphonuclear neutrophils and macrophages (34) and decreased hydrogen peroxide generation from T-cells (17). Antigen-presenting cells deficient in NOX2 have an impaired ability to present antigens due to enhanced antigen degradation (35). Additionally, the peripheral blood memory B lymphocyte compartment is reduced in patients with chronic granulomatous disease, which mainly results from NOX2 deficiency (36). Taken together, NOX2 deficiency protects against β-cell destruction and preserves islet function in the STZ-induced diabetes by inhibiting insulitis. These beneficial effects are likely due to less early β-cell death from toxic effects of STZ and an inhibition of immune reaction, including decreased antigen presentation, impaired immune cell activation, and suppression of cytotoxic effects from T-cells, neutrophils, and macrophages during development of diabetes.

In conclusion, our study demonstrated that NOX2 deficiency attenuates the severity of hyperglycemia, β-cell mass loss, and insulitis in the multiple-low-dose STZ-induced diabetes in mice. The protective effects are a result of decreased β-cell apoptosis, decreased ROS production from the islets, and suppressed immune response. The ability of NOX2 in regulating the immune response in this model supports an important role of NOX2-containing NADPH oxidase in the development of diabetes induced by STZ. NOX2 may represent a potential therapeutic target for treatment of diabetes.

This study was supported by grants from the Canadian Institutes of Health Research (MOP-14653 to Q.F.) and the Heart and Stroke Foundation of Ontario (HSFO) (T-6040 to Q.F.). F.-L.X. was supported by a Doctoral Research Award from the Heart and Stroke Foundation of Canada. Q.F. is an HSFO Career Investigator.

No potential conflicts of interest relevant to this article were reported.

F.-L.X. researched data and wrote the manuscript. X.L. reviewed/edited the manuscript. B.S. researched data. D.J.H. reviewed/edited the manuscript. Q.F. wrote the manuscript.