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(The FASEB Journal. 1999;13:1845-1854.)
© 1999 FASEB

Supplementation of N-acetylcysteine inhibits NF{kappa}B activation and protects against alloxan-induced diabetes in CD-1 mice

EMILY HO, GOUMAN CHEN* and TAMMY M. BRAY1

Department of Human Nutrition, The Ohio State University, Columbus, Ohio 43210-1295, USA; and
* Department of Clinical Studies, University of Guelph, Guelph, Ontario, Canada

1Correspondence: Department of Human Nutrition, The Ohio State University, 347 Campbell Hall, 1787 Neil Ave., Columbus, OH 43210-1295, USA. E-mail: bray.21{at}osu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS) are involved in the destruction of pancreatic ß cells and the development of insulin-dependent diabetes mellitus (IDDM). However, the cellular mechanism responsible for ß cell death is still unclear. We hypothesize that activation of NF{kappa}B by ROS is the key cellular signal in initiating a cascade of events leading to ß cell death. Thus, enhancement of pancreatic GSH, a known antioxidant and key regulator of NF-{kappa}B, should protect against IDDM. Weanling CD1 mice (n=5) were injected with alloxan (50 mg/kg i.v.) to induce IDDM. Using EPR spin trapping techniques, we demonstrated that alloxan generated ROS in the pancreas 15 min after administration. Activation of NF{kappa}B in pancreatic nuclear extracts was observed 30 min after alloxan injection, as assessed by an electrophoretic mobility shift assay. Fasting blood glucose levels were monitored for 14 days. Supplementation with N-acetylcysteine (NAC, 500 mg/kg), a GSH precursor, inhibited alloxan-induced NF{kappa}B activation and reduced hyperglycemia. Thus, NF{kappa}B activation by ROS may initiate a sequence of events leading to IDDM. Inhibition of NF-{kappa}B activation by NAC attenuated the severity of IDDM. This research will contribute to the understanding of the etiology of IDDM and may lead to the development of better strategies for disease prevention.—Ho, E., Chen, G., Bray, T. M. Supplementation of N-acetylcysteine inhibits NF{kappa}B activation and protects against alloxan-induced diabetes in CD-1 mice.


Key Words: free radicals • antioxidants • GSH • transcription factor • IDDM


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TYPE 1 DIABETES or insulin-dependent diabetes mellitus (IDDM)2 is known to be a multifactorial disease (1 2 3) . Despite different triggering factors, the final outcome is characterized by profound destruction of the insulin-producing ß cells. The precise cellular mechanisms leading to pancreatic ß cell death have not yet been fully elucidated. However, there is growing evidence implicating reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the destruction of ß cells in the pathogenesis of IDDM (4 5 6 7) . Recently, ROS/RNS have gained increasing attention as physiologically and pathologically important cellular messengers (8) . Evidence suggests that ROS/RNS act as signal molecules in the regulation of gene expression, cell proliferation, and cell death (9) . Moreover, ROS/RNS are now known to play a critical role in up-regulation of the expression of genes involved in the inflammatory and autoimmune responses (10) .

Nuclear transcription factor {kappa}B (NF{kappa}B) is a transcription factor that can be activated by a variety of stresses such as oxidants, viruses, metals, xenobiotics, and proinflammatory cytokines (11 , 12) . NF{kappa}B is usually stored in the cytosol in its inactive form bound to the inhibitory unit i{kappa}B. Activation of NF{kappa}B in response to extracellular stimuli involves the release of ikB, resulting in a rapid translocation of NF{kappa}B to the nucleus (13 , 14) . ROS/RNS appear to be a key factor in initiating NF{kappa}B activation (15 16 17) . Once activated, NF{kappa}B binds to nuclear DNA and modulates the expression of several genes for adhesion molecules, such as selectins, intercellular adhesion molecule 1, and vascular adhesion molecule 1 (18) , and up-regulates the production of various proinflammatory cytokines such as IL-2 (19) , IL-6 (20) , tumor necrosis factor {alpha} (21) , and inducible nitric oxide synthase (iNOS) (22) .

IDDM is generally considered an autoimmune disease of the pancreas (23) . The inherent lack of antioxidant protection in the pancreas (24 , 25) may increase its sensitivity to diabetogenic agents that trigger ROS/RNS production. We propose that the critical determinant in ß cell destruction in IDDM may be the inappropriate activation of NF{kappa}B, which starts a cascade of events that results in the up-regulation of genes involved in the inflammatory and autoimmune response. Once an autoimmune/inflammatory response is launched, the invading immune cells amplify ROS/RNS production, which ultimately destroys the ß cells. Thus, ROS are not simply cytotoxic agents that damage ß cells, but are key modulators of the cellular response pathways that initiate ß cell death and the development of IDDM.

Glutathion (GSH), a cysteine-containing tripeptide, is the most abundant nonprotein thiol in mammalian cells (26) . It is a substrate of the ROS defense enzyme GSH peroxidase and the GSH transferase family of detoxification enzymes. GSH also protects sulfhydryl groups from oxidation (26 , 27) . GSH is not only an effective antioxidant, but also modulates cellular metabolism and gene expression by affecting cellular thiol redox status (28 29 30) . Intracellular redox status appears to be a critical determinant of NF{kappa}B activation (28 , 29 , 31) Hence, if ROS-induced NF{kappa}B activation modulates and amplifies the processes leading to ß cell death, inhibition of NF{kappa}B activation by GSH should prevent this damage and the development of IDDM.

The objectives of this study were to determine whether free radical-mediated NF{kappa}B activation in the pancreas is a key event leading to the onset of IDDM in an in vivo model and to assess the efficacy of supplementation of the GSH precursor N-acetylcysteine (NAC) on NF{kappa}B activation and the development of alloxan-induced diabetes in CD-1 mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Weanling male CD-1 mice (Harlan, Indianapolis, Ind.) weighing 20–25 g were used in all experiments. All animals were housed in individual cages in a temperature-controlled environment (22±2°C) with a light period between 0600 to 1800 h. Animals were fed their respective experimental diets and allowed free access to the diet with the exception of fasting periods for blood glucose determination. Animal protocol was approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.

Hyperglycemic effect of alloxan
To determine the dose-response of alloxan-induced diabetes, fasting blood glucose levels were monitored in CD1 mice after administration of varying doses of alloxan. After an overnight fast, weanling CD-1 mice were injected with 50, 100, 150, 200, or 300 mg/kg alloxan (i.p). Control animals received a sham saline injection. Fasting blood glucose levels were monitored for 9 days. Blood was obtained from the intraorbital sinus using a 10 µl capillary tube. Glucose concentrations were measured using the ONE-TOUCH II complete blood glucose monitoring kit. To minimize the effects of diurnal fluctuations, blood samples were collected at the same time every day.

In vitro and in vivo EPR spin trapping of free radicals
In vitro studies
Pancreatic tissue (0.5g) was homogenized in 150 mM phosphate buffer. Homogenates were then incubated with 0–80 mg/ml alloxan and 14 mg of spin trap, {alpha}-phenyl-t-butyl-nitrone (PBN; Sigma, St. Louis, Mo.) at 37°C for 90 min. Control samples contained PBN only. After the incubation period, PBN spin adducts were extracted with 6 ml of benzene, concentrated to a 0.2 ml sample, and degassed with nitrogen gas before EPR analysis. Samples were placed in a quartz round cell and analyzed at room temperature with a Bruker X band EPR spectrometer operating at 9.78 GHz. To study the effect of GSH on free radical formation in pancreatic homogenate, reduced GSH (0, 2.5, 5, 10, or 20 mM) was added to pancreatic homogenates. All samples were then incubated with 10 mg alloxan and 14 mg PBN at 37°C for 90 min. Extractions and concentration was performed as described previously and PBN adducts were analyzed by a Bruker X band spectrometer.

In vivo studies
Weanling CD-1 mice were injected with 150 mg/kg PBN dissolved in saline. Fifteen minutes later, animals were injected (i.v.) with 50 mg/kg alloxan or saline control. Fifteen minutes after alloxan injection, animals were killed; pancreatic tissue was removed and immediately frozen in liquid N2. Five pancreas samples were pooled and homogenized in phosphate buffer. PBN adducts were extracted and concentrated as described previously and analyzed by EPR spectrometer.

Determination of NF{kappa}B activation in vivo
For NF{kappa}B activation analysis, mice were injected with 50 mg/kg alloxan (i.v.) and killed 0, 15, 30, and 60 min after injection (n=5 in each time interval). A lower dose of alloxan is required to produce a similar hyperglycemic effect when it is injected i.v. It has been established in our laboratory that an i.v. injection of 50 mg/kg body weight (BW) of alloxan produces a similar hyperglycemic profile as an intraperitoneal (i.p.) injection of 200 mg/kg BW (data not shown). Pancreas and liver tissues were immediately removed. NF{kappa}B activation was determined using an electrophoretic mobility shift assay (EMSA). To determine the effect of NAC supplementation on alloxan-induced NF{kappa}B activation, animals were injected with NAC (500 mg/kg i.p.) 90 min before alloxan administration.

Crude nuclear extracts were prepared from pancreatic and liver tissues as described by Deryckere and Gannon (32) . Double-stranded synthetic oligonucleotides probes for NF{kappa}B (5'-AGTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, Wis.) were end-labeled using [{gamma}-32P] (Amersham, Arlington Heights, IL) and T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, Ind.). Binding reactions containing equal amounts of protein (~7 µg) and labeled oligonucleotide probes were conducted for 20 min at room temperature in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, pH 8.0, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris, 50 µg/ml poly [dI-dC]. Specific binding was confirmed using 100- to 400-fold excess unlabeled NF{kappa}B oligonucleotide as a specific competitor. Protein–DNA complexes were separated using 6% nondenaturing polyacrylamide gel electrophoresis, followed by radiography to detect the level of retardation produced by binding to NF{kappa}B probe.

Effect of dietary supplementation of NAC on alloxan-induced hyperglycemia
Mice were randomly allocated to one of three treatment groups (n=6): control, alloxan, or alloxan + NAC. Control and alloxan grouped animals were fed a purified 76AIN rodent diet. NAC-supplemented animals were fed a modified 76AIN purified rodent diet containing 0.4% NAC (w/w). All animals were fed their respective diets for 1 wk before alloxan administration. To induce diabetes, alloxan monohydrate (200 mg/kg BW) dissolved in saline was injected i.p. after an overnight fast. Control animals received a sham saline injection. Animals were allowed free access to diets, with the exception of fasting periods for blood glucose determination. Severity of hyperglycemia was measured by monitoring fasting blood glucose levels for 14 days using the ONE-TOUCH II system, as described previously. On day 14, animals were anesthetized and killed via cervical dislocation. Pancreas and liver were removed, immediately frozen in liquid nitrogen, and stored at -80°C for further analysis.

Effect of dietary supplementation of NAC on pancreatic GSH concentration
The effect of NAC supplementation on pancreatic GSH concentration was assessed by comparing total GSH content in NAC-supplemented and unsupplemented controls using the method of Tietze (33) . Pancreatic tissue was removed from mice after the 2 wk feeding period. Due to the small size of the tissue, four pancreases were pooled and homogenized in 5% trichloroacetic acid (TCA). Samples were centrifuged at 10,000 x g and supernatant was used for GSH analysis.

Expression of iNOS protein
Expression of iNOS protein in the pancreas in control, alloxan, and alloxan + NAC (ALX+NAC) groups was determined by Western blot analysis. Pancreatic homogenates taken 30 min after alloxan injection were mixed with an equal volume of sample buffer (125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% ß-mercaptoethanol, bromphenol blue) and boiled for 5 min. SDS electrophoresis was carried out under standard condition (34) . Protein was transferred from the SDS gel to nitrocellulose membranes (Bio-Rad, Hercules, Calif.) at 50 mA overnight. iNOS blots were blocked with in 5% non-fat dry milk for 1 h at 37°C. Blots were incubated for 3 h at 37°C with rabbit anti-mouse iNOS (Alexis Biochemical, San Diego, Calif.) at a dilution of 1:2000. The blots were washed five times with phosphate-buffered saline + 0.2% Tween 20, then incubated with horseradish peroxidase-conjugated donkey anti-rabbit antisera (Sigma) at a dilution of 1:5000, for 1 h at 37°C. iNOS was detected by enhanced chemiluminescence using Hyperfilm and ECL reagents (Amersham, Piscataway, N.J.).

Nitrite analysis
Pancreatic tissue was homogenized in 100 mM HEPES buffer and centrifuged at 100 000 x g for 60 min. Nitrite formation was used as an indirect measure of nitric oxide (NO) production. Nitrite concentration was determined using the Griess reaction as described by Hevel and Marletta (35) . Protein concentration was determined using the Lowry method (36) .

Statistics
All data were analyzed using SAS analytical software. Two-way analysis of variance was performed to assess differences between groups and time. Differences in means between and within treatments was tested by using Tukey's test ({alpha}=0.05). For nitrite analysis, one-way analysis of variance was used to detect differences between groups. Differences in means between groups was tested using Tukey's test ({alpha}=0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mice injected i.p. with alloxan concentrations of 150 mg/kg BW or higher display symptoms characteristic of diabetes including decreased plasma insulin levels (data not shown), elevated fasting blood glucose levels (hyperglycemia), polyuria, and weight loss. Figure 1 demonstrates the dose of alloxan required to induce hyperglycemia is 150 mg/kg BW or above when it is i.p. injected. Dose-dependent increase in hyperglycemic response after injection (i.p.) of increasing doses of alloxan is observed. EPR spin trapping studies were performed to assess free radical concentration in the pancreas after exposure to alloxan. Figure 2 shows the EPR spectra of PBN spin adducts of alloxan-induced free radicals in pancreatic homogenate (in vitro) and in the pancreas (in vivo) of CD1 mice. We were able to trap identical alloxan-induced free radicals in both in vitro and in vivo, as shown by the identical hyperfine splitting constants AN = 14.61 and AH = 3.2. These splitting constants suggest that the radical detected is a carbon-centered radical. However, further chemical analysis is needed to specifically identify the chemical structure of free radical species.



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  		Figure 1. Dose-dependent increase in hyperglycemic response with alloxan administration. On day 0, CD1 mice were injected with 50, 100, 150, 200, or 300 mg/kg alloxan i.p. after an overnight fast. Control animals received saline injection. Fasting blood glucose levels were monitored for 9 days. Values are expressed as mean ± SE (n=4).



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  		Figure 2. EPR spectra of PBN spin adducts of alloxan-induced free radicals in pancreas trapped in vitro and in vivo. A) In vitro study. Alloxan monohydrate (10 mg/ml) and PBN (14 mg/ml) were incubated in 0.5 g pancreatic tissue for 90 min at 37°C. PBN spin adducts were extracted and analyzed by EPR. B) In vivo study: mice were injected i.p. with PBN (150 mg/kg body weight) 15 min before injection i.v. of alloxan (50 mg/kg body weight). Pancreases were removed 15 min after alloxan injection. PBN spin adducts were extracted and analyzed by EPR. Instrument settings: microwave power, 20 mW; modulation amplitude, 1 G; receiver gain, 5 x 105; time constant 100 ms, scan range, 100 G, and sweep time, 50 s.

We were also able to establish a dose-dependent increase in free radical generation with alloxan in the pancreas by using in vitro EPR spin trapping techniques. Signal peak height is a relative measure of free radical concentration. Figure 3 shows that signal peak height increases when increasing concentrations of alloxan are added to pancreatic homogenates. Thus, these experiments demonstrate that alloxan can generate free radicals in a dose-dependent manner.



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  		Figure 3. Relative concentrations of alloxan-induced free radicals trapped by PBN in pancreatic tissue homogenate. The relative concentration of free radicals was assessed according to relative peak height of the EPR spectra. Alloxan monohydrate (0–80 mg/ml) and PBN (14 mg/ml) were incubated in 0.5 g pancreatic tissue homogenate for 90 min at 37°C. PBN spin adducts were extracted and analyzed by EPR. Instrument settings: microwave power, 20 mW; modulation amplitude, 1 G; receiver gain, 5 x 105; time constant 100 ms, scan range, 100 G and sweep time, 50 s.

Figure 4 A is a representative EMSA radiograph that depicts the 32P-DNA/NF{kappa}B complex present in the nuclear extracts of the pancreas after injection of alloxan (50 mg/kg i.v.) at various time points in CD-1 mice. Lane 1 is a positive control, using HeLa cells that contain activated NF{kappa}B. The presence of binding is indicative of the translocation and activation of NF{kappa}B (lane 5 and 7) in the pancreas. The specificity of NF{kappa}B binding was confirmed by using an excess of unlabeled DNA oligo, which contains NF{kappa}B binding sites as a specific competitor (lane 6 and 8). Significant binding of NF{kappa}B was seen 30 min after alloxan injection in pancreatic nuclear extracts. The presence of NF{kappa}B in nuclear extracts disappeared by 60 min. The result demonstrates that administration of alloxan activates NF{kappa}B rapidly in the pancreas of CD1 mice. Identical EMSA experiments were performed with liver extracts; however, no evidence of NF{kappa}B activation could be seen with alloxan administration in the liver (Fig. 4B ). These experiments illustrate both the time course of alloxan-induced NF{kappa}B activation and the specificity of NF{kappa}B activation to the pancreas in the in vivo system.



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  		Figure 4. Time course of NF-{kappa}B activation in the pancreas and liver of mice injected with alloxan. Mice were injected with 50 mg/kg alloxan i.v. A) Five pancreas samples were pooled together for nuclear extract preparation at each time point. Lane 1 is a positive control using HeLa cells (Promega) that contain activated NF{kappa}B. Lanes 2, 4, 6, 8, and 10 represent pancreatic extracts incubated with specific competitor (unlabeled NF{kappa}B oligo) to confirm the specificity of NF{kappa}B binding. B) Nuclear extracts were prepared from a single liver from each mouse at each time point. Lane 1 is a positive control. Lane 2 (COMP) is the competitor reaction, which contains 100-fold excess of unlabeled NF{kappa}B oligonucleotide. This confirms the specificity of NF{kappa}B binding. Results are representative of three individual experiments.

Figure 5 illustrates that the supplementation of NAC, a GSH precursor, inhibits alloxan-induced NF{kappa}B activation in pancreas in vivo. Similar to Fig. 4A, NF {kappa}B activation was observed 30 min after alloxan administration (lane 2). However, pretreatment of NAC (90 min before alloxan administration) fully inhibited the activation of NF{kappa}B (lane 3). These results clearly demonstrate that pretreatment with NAC effectively inhibited alloxan-induced NF{kappa}B activation in the pancreas compared to unsupplemented animals.



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  		Figure 5. Administration of NAC inhibits alloxan-induced NF-{kappa}B activation in pancreas. Control group received only sham saline injections. Alloxan group received alloxan only (50 mg/kg i.v.). ALX + NAC group was injected with 500 mg/kg NAC i.p. 90 min before alloxan (50 mg/kg BW i.v.). Pancreatic tissue was removed from mice 30 min after alloxan injection. Nuclear extracts were prepared from five pooled pancreas samples from each group. This figure is a representative of two individual experiments.

To verify that NF{kappa}B activation resulted in the activation of gene transcription of downstream targets, expression of proteins with the NF{kappa}B binding site in the promoter region needed to be measured. One downstream target of NF{kappa}B activation is iNOS. Induction of this protein can be directly measured by Western blot analysis. Figure 6 depicts a significant increase in pancreatic iNOS expression from animals after alloxan exposure (ALX) when compared with that of saline-injected (saline). Mice treated with NAC before alloxan exposure (ALX+NAC) show a significant reduction in iNOS protein expression. LPS, a known iNOS inducer is used as a positive control to confirm iNOS expression in vivo. Increases in NO concentration in pancreas are expected after an induction of iNOS protein expression. To determine the effect of alloxan and NAC on NO production, nitrite levels were determined in the pancreas from animals that completed the alloxan/NAC supplementation trial. Similar to the results obtained from iNOS protein expression (Fig. 6) , Fig. 7 shows that pancreatic nitrite levels in animals injected with alloxan and fed a control diet (alloxan) were significantly elevated when compared to saline-injected controls (control). Animals that were fed the supplemented 0.4% NAC diet and injected with alloxan (ALX+NAC) showed no increase in nitrite levels compared to controls (control). Thus, NO levels were high after alloxan exposure, but supplementation with NAC can inhibit this elevation.



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  		Figure 6. NAC supplementation inhibits alloxan-induced iNOS expression in the pancreas. iNOS expression was determined by Western blot analysis in saline-injected (control), alloxan-injected (alloxan), and alloxan-injected/NAC-supplemented (500 mg/kg NAC i.p. 90 min before alloxan injection). SDS-PAGE was performed as outlined in Materials and Methods. Each sample is contains five pooled pancreases from each group. Results are representative of two individual experiments.



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  		Figure 7. NAC supplementation inhibits alloxan-induced nitric oxide (NO) formation in the pancreas. Nitrite production was used as an indirect measure of NO production. Nitrite levels were assessed in saline-injected (control), alloxan-injected (alloxan), and alloxan-injected/NAC-supplemented (0.4% NAC in the diet) (ALX+NAC) animals. Pancreas was removed on day 14 after saline or alloxan injection. Values are expressed as mean ± SE (n=6). Level of significance was evaluated using Tukey's test at P < 0.05.

The ability of NAC supplementation to inhibit NF{kappa}B activation in the pancreas may be due to its ability to enhance GSH levels and subsequently inhibit alloxan-induced free radicals in the pancreas. When pancreatic homogenates are incubated with alloxan and increasing doses of GSH, there is a dose-dependent decrease in EPR signal peak height (Fig. 8 ). This demonstrates that GSH can directly decrease alloxan-induced free radical generation and may account for NAC's ability to inhibit alloxan-induced NF{kappa}B activation in the pancreas.



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  		Figure 8. The inhibitory effect of GSH on alloxan-induced free radical production in vitro. Alloxan monohydrate (10 mg/ml), PBN (14 mg/ml) and increasing concentrations of GSH (0–20 mM) are incubated in 0.5 g pancreatic tissue homogenate for 90 min at 37°C. PBN spin adducts were extracted and analyzed by EPR. Instrument settings: microwave power, 20 mW; modulation amplitude, 1 G; receiver gain, 5 x 105; time constant 100 ms, scan range, 100 G and sweep time, 50 s.

If NF{kappa}B activation is the critical step in the development of diabetes, then NAC, which is able to inhibit NF{kappa}B activation, should also prevent the onset of the disease. To determine the efficacy of NAC supplementation to inhibit alloxan-induced diabetes, the severity of hyperglycemia and weight loss were compared between NAC-supplemented and nonsupplemented groups (Fig. 9 A, B). Alloxan treatment (alloxan) caused a significant elevation in blood glucose levels and body weight loss compared to baseline saline-injected controls (control). Animals fed a NAC-supplemented diet (ALX+NAC) had significantly lower blood glucose levels compared to the nonsupplemented group (alloxan) (Fig. 9A ). NAC supplementation also reduced the degree of weight loss observed in alloxan-induced diabetes (Fig. 9B ). Therefore, NAC supplementation proved to be effective in attenuating the severity of both the hyperglycemia and weight loss associated with the development of diabetes.



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  		Figure 9. Dietary supplementation of NAC reduces alloxan-induced hyperglycemia. On day 0, animals received 200 mg/kg alloxan i.p. after an overnight fast. Fasting blood glucose levels (A) and body weights (B) were monitored for 14 days. Control ({blacktriangleup}) animals received saline injection only. Alloxan ({diamondsuit}) -treated animals were fed a control diet. NAC-supplemented animals injected with alloxan (ALX+NAC) ({blacksquare}) received 0.4% w/w of NAC in the diet for 7 days before alloxan injection and were maintained on this diet for another 2 wk. Values are expressed as mean ± SE (n=6). Alloxan-treated groups are significantly different from control groups. ALX + NAC groups are not significantly different from control group. Level of significance was evaluated using a Tukey's test at P < 0.05.

Figure 10 confirms that NAC is an effective precursor of GSH to elevate pancreatic GSH level in vivo. After 2 wk of NAC supplementation, pancreatic GSH concentration in the NAC-supplemented group is significantly increased when compared to that of the unsupplemented group.



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  		Figure 10. Effect of NAC supplementation on pancreatic GSH level. Pancreatic tissue was removed from mice after the 2 wk feeding period of a basal and NAC-supplemented diet. Due to the small size of the tissue, four pancreases were pooled for GSH analysis. Values represent the mean + SE of triplicate measures of four pooled pancreas samples. Differences in means are determined by Student's t test with significance level P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to use an in vivo model to demonstrate the role of free radicals in mediating cellular signal transduction pathways leading to the development of IDDM. In contrast to the majority of studies investigating ROS/RNS signaling in vitro using cell culture, we have been able to characterize the link between free radicals and NF{kappa}B activation in the development of diabetes in a well-established in vivo diabetogenic model. In the present study, we have systematically documented the range of alloxan toxicity and the dose responsiveness of alloxan-induced hyperglycemia. We also demonstrated the specificity of NF{kappa}B activation in the development of alloxan-induced diabetes. Exposure to the alloxan induced NF{kappa}B activation in the pancreas, not in the liver, in CD-1 mice (Fig. 4A, B ). It is well known that alloxan is specifically cytotoxic to the pancreatic ß cells. However, the precise mechanism for this selectivity is still not clear. The structure of alloxan is very similar to glucose, hence there is some selective uptake of alloxan via glut-2 transporters to glucose-metabolizing tissues such as pancreatic islets and liver, but not skeletal muscle (37 , 38) . Alloxan is also thought to produce free radicals during its metabolism (39) . The pancreatic islets are known to have much lower antioxidant defense enzymes (24) than other tissues such as liver; thus, they are highly susceptible to oxidative damage. This vulnerability to free radicals may in part account for the exquisite sensitivity of the pancreatic islets to alloxan. Since uptake of alloxan in the pancreas is known to be highly specific to islet cells, it is assumed that alloxan-induced activation of NF{kappa}B is also islet cell specific. This specificity of NF{kappa}B activation in the pancreas may be the key factor in increased free radical production and helps to explain the selective cytotoxicity of alloxan. We have also shown that the antioxidant NAC can effectively and specifically inhibit pancreatic NF{kappa}B activation and is able to attenuate the severity of the disease.

Recent breakthroughs with the discovery of ROS-induced activation of NF{kappa}B and its role in amplifying inflammatory and immune processes have launched a revolution in understanding the role of ROS in many human inflammatory and autoimmune diseases such as inflammatory bowel disease (40) , atherosclerosis (41) , and rheumatoid arthritis (42) . Although IDDM is largely considered to be an autoimmune disease, this link between ROS and cellular response is a relatively unexplored area of research, leaving a large gap in the understanding of the mechanisms that signal ß cell death. More recently, NF{kappa}B activation has been detected in rat insulinoma cell lines after exposure to IL-1 (43 , 44) . These studies confirm the possible link between NF{kappa}B activation, inflammation, and iNOS production in a cell culture model system of diabetes. Although these previous studies suggest a role for NF{kappa}B in IDDM, the current study using an in vivo model for IDDM helps us to understand the role of NF{kappa}B in a physiological progression of the disease.

In this study, free radical production was detected in the pancreas ~15 min after injection of alloxan in CD1 mice. Pancreatic activation of NF{kappa}B occurred within 30 min of alloxan injection. Typically, symptoms of hyperglycemia and weight loss appeared 24–48 h after injections of alloxan. Peak severity of hyperglycemia was observed 4–5 days after injection. Based on these observations, we can envision the sequence of events that lead to the development of diabetes. Early initiation events begin with increased free radical production, which in turn activates NF{kappa}B. This activation of NF{kappa}B both initiates and amplifies inflammatory responses through up-regulation of cytokines and proinflammatory proteins such as iNOS (17 , 45) . This amplification cascade results in increased free radical production and eventually leads to ß cell death.

If free radical production and NF{kappa}B activation are central to the development of IDDM, then agents that can inhibit these processes should inhibit the disease. We have clearly demonstrated that administration of the GSH precursor NAC inhibited alloxan-induced NF{kappa}B activation (Fig. 5) and attenuated the severity of alloxan-induced hyperglycemia and weight loss (Fig. 9) . The ability of NAC to inhibit alloxan-induced NF{kappa}B activation may be attributed to its role as a GSH precursor and the ability of GSH to decrease alloxan-induced free radical concentration (Fig. 10 and Fig. 8 ). Indeed, our in vitro EPR spin trapping experiments directly demonstrated the ability of GSH to decrease alloxan-induced free radicals. We were unable to determine the effect of NAC on alloxan-induced radicals in vivo due to constraints of spin trapping techniques. Although EPR is a method for directly detecting free radicals, it is not highly sensitive. Thus, the ability to discern differences in alloxan-induced free radical concentration between NAC-supplemented and unsupplemented animals is difficult because in vivo EPR signals are weak. Nonetheless, NAC's ability to inhibit NF{kappa}B activation attenuated the severity of diabetic symptoms in CD1 mice (Fig. 9) . However, there are other possible mechanisms by which enhanced tissue GSH may attenuate the toxicity of alloxan. Since GSH is the substrate of GSH-transferase and GSH-peroxidase, increases in tissue GSH may alter the metabolism and detoxification of alloxan as well as reduce the oxidative damage of target tissue. This possibility needs to be investigated further.

NAC supplementation also inhibited iNOS protein expression and decreased NO concentration (Figs. 6 and 7) . NF{kappa}B has been shown to stimulate the production of NO by directly activating iNOS (11) . The induction of iNOS and NO production has been implicated in mediating ß cell death and IDDM in other models for IDDM and in cell culture (46 47 48) . Although iNOS is known to be stimulated during inflammatory and immune responses, we currently are unable to determine the source of NO. Increased NO production may result from the induction of iNOS in the pancreatic islets themselves or from infiltrated immune cells. To determine the source of NO, histopathological studies need to be pursued.

To delineate the proposed role of NF{kappa}B in the sequence of events leading to inflammation in alloxan-induced IDDM, we need to establish that NF{kappa}B activation is indeed inducing the transcription of other downstream target genes, in addition to iNOS. However, trans-activation assays using transfection methods and reporter gene assays are usually used in cell culture system to answer this question. These assays are nearly impossible to perform in an in vivo system. In addition, many of the downstream targets of NF{kappa}B activation, such as cytokines, are relatively short-lived proteins, making their detection very difficult in an in vivo setting. Future studies, using more sensitive methods such as quantitative RT-PCR assay, are needed to investigate the expression of various cytokines, chemokines, and adhesion molecules in order to directly confirm the link between free radicals, NF{kappa}B, inflammation and IDDM.

Although elucidation of the detail of signal transduction pathways is somewhat limited using an in vivo model, the results of this study highlight the key molecular events in IDDM at the physiological level. The present study has helped reinforce the concept that ROS modulate disease processes via complex signal transduction pathways. We have demonstrated that NF{kappa}B activation may be a critical determinant in the progression of the disease. We have established that GSH and NAC are able to inhibit NF{kappa}B activation and concurrently decrease ROS and NO production. Excess production of both ROS and NO production has been implicated as cytotoxic agents to the ß cells. However, this observation is only one small piece contributing to the understanding of the complex pathways leading to ß cell death in IDDM. More recently, the role of ROS as signaling molecules in cell death pathways such as apoptosis has been gaining significant attention (49 , 50) . Additional studies investigating the role of ROS and NF{kappa}B in signal cascades leading to ß cell death via necrosis and apoptosis still need to be examined.

Not all antioxidants can prevent the onset of IDDM. Antioxidant intervention studies in both human and animal models have shown conflicting results. Although it is clear the excess free radical species are detrimental to the pancreatic islet cells, why does it appear that antioxidant supplementation is of limited benefit (51 52 53) ? It appears that the efficacy of antioxidant therapy in IDDM is still a question of specificity. Only those antioxidants that can effectively target critical pathways in pancreatic tissue appear to be effective. If NF{kappa}B activation is this critical step, this knowledge will act as a useful tool for screening different classes of antioxidant for their value in IDDM therapy or prevention. Identification of the pathways leading to ß cell death in IDDM is essential before effective treatment and prevention strategies can be developed. A focus on the control of cellular response pathways will bring the field to a level of understanding that is needed to develop effective preventative strategies. This research is the first step in unifying the many factors in the diabetogenic processes in a relevant in vivo system and will help move us closer to finding effective treatment or prevention strategies for IDDM.


   ACKNOWLEDGMENTS
 
We would like to thank the technical support of Dr. Denis Medeiros for his assistance in establishing the EMSA, Dr. Russ Hille for the use of EPR spectrometer in his laboratory, and Miss N'Diris Z. Barry ("Sam") for her assistance in the animal studies. Financial support from Central Ohio Diabetes Association is gratefully acknowledged.


   FOOTNOTES
 
2 Abbreviations: ALX + NAC, alloxan + NAC; BW, body weight; EMSA, electrophoretic mobility shift assay; GSH, glutathione; i.p., intraperitoneal; IDDM, insulin-dependent diabetes mellitus; iNOS, inducible nitric oxide synthase; NAC, N-acetylcysteine ; NF{kappa}B, nuclear transcription factor {kappa}B; NO, nitric oxide; PBN, {alpha}-phenyl-t-butyl-nitrone; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate.

Received for publication December 11, 1998. Revised for publication April 12, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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