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B activation and protects against alloxan-induced diabetes in CD-1 mice
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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-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 NFB 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 NFB activation
and reduced hyperglycemia. Thus, NFB activation by ROS may initiate
a sequence of events leading to IDDM. Inhibition of NF-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
NFB activation and protects against alloxan-induced diabetes in CD-1
mice.
Key Words: free radicals • antioxidants • GSH • transcription factor • IDDM
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 B (NFB) is a transcription factor
that can be activated by a variety of stresses such as oxidants,
viruses, metals, xenobiotics, and proinflammatory cytokines (11
, 12)
. NFB is usually stored in the cytosol in its inactive
form bound to the inhibitory unit iB. Activation of
NFB in response to extracellular stimuli involves the release of
ikB, resulting in a rapid translocation of NFB to the
nucleus (13
, 14)
. ROS/RNS appear to be a key factor in
initiating NFB activation (15
16
17)
. Once activated,
NFB 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 
(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 NFB, 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 NFB activation (28
, 29
, 31)
Hence, if ROS-induced NFB activation modulates and amplifies the
processes leading to ß cell death, inhibition of NFB activation by
GSH should prevent this damage and the development of IDDM.
The objectives of this study were to determine whether free
radical-mediated NFB 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 NFB activation and the development of
alloxan-induced diabetes in CD-1 mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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, -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 NFB activation in vivo
For NFB 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. NFB activation was determined using an electrophoretic
mobility shift assay (EMSA). To determine the effect of NAC
supplementation on alloxan-induced NFB 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 NFB
(5'-AGTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, Wis.) were end-labeled
using [
-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 NFB
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 NFB 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 (=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
(=0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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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.
|
Figure 4
A is a representative EMSA radiograph that depicts the
32P-DNA/NFB 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 NFB. The presence of binding is
indicative of the translocation and activation of NFB (lane 5 and 7)
in the pancreas. The specificity of NFB binding was confirmed by
using an excess of unlabeled DNA oligo, which contains NFB binding
sites as a specific competitor (lane 6 and 8). Significant binding of
NFB was seen 30 min after alloxan injection in pancreatic nuclear
extracts. The presence of NFB in nuclear extracts disappeared by 60
min. The result demonstrates that administration of alloxan activates
NFB rapidly in the pancreas of CD1 mice. Identical EMSA experiments
were performed with liver extracts; however, no evidence of NFB
activation could be seen with alloxan administration in the liver (Fig. 4B
). These experiments illustrate both the time course of
alloxan-induced NFB activation and the specificity of NFB
activation to the pancreas in the in vivo system.
|
Figure 5
illustrates that the supplementation of NAC, a GSH precursor, inhibits
alloxan-induced NFB activation in pancreas in vivo.
Similar to Fig. 4A, NF
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
NFB (lane 3). These results clearly demonstrate that pretreatment
with NAC effectively inhibited alloxan-induced NFB activation in the
pancreas compared to unsupplemented animals.
|
To verify that NFB activation resulted in the activation of gene
transcription of downstream targets, expression of proteins with the
NFB binding site in the promoter region needed to be measured. One
downstream target of NFB 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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The ability of NAC supplementation to inhibit NFB 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 NFB activation in the pancreas.
|
If NFB activation is the critical step in the development of
diabetes, then NAC, which is able to inhibit NFB 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.
|
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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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 NFB
activation in the development of alloxan-induced diabetes. Exposure to
the alloxan induced NFB 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 NFB is also islet cell specific. This
specificity of NFB 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 NFB activation
and is able to attenuate the severity of the disease.
Recent breakthroughs with the discovery of ROS-induced activation of
NFB 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, NFB activation has been detected in rat
insulinoma cell lines after exposure to IL-1 (43
, 44)
.
These studies confirm the possible link between NFB activation,
inflammation, and iNOS production in a cell culture model system of
diabetes. Although these previous studies suggest a role for NFB in
IDDM, the current study using an in vivo model for IDDM
helps us to understand the role of NFB 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 NFB 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 NFB. This activation of NFB 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 NFB 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
NFB activation (Fig. 5)
and attenuated the severity of
alloxan-induced hyperglycemia and weight loss (Fig. 9)
. The ability of
NAC to inhibit alloxan-induced NFB 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 NFB 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)
. NFB 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 NFB in the sequence of events
leading to inflammation in alloxan-induced IDDM, we need to
establish that NFB 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 NFB 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, NFB, 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 NFB activation may be a critical determinant
in the progression of the disease. We have established that GSH and NAC
are able to inhibit NFB 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 NFB
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 NFB 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 |
|---|
|
FOOTNOTES |
|---|
B, nuclear transcription factor B; NO, nitric oxide; PBN, -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.
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B and cytokine-inducible enhancers. FASEB J 9,899-909[Abstract]
B activity by intracellular glutathione levels and by variations of the extracellular cysteine supply. FASEB J 9,246-252[Abstract]
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) animals
received saline injection only. Alloxan (
) -treated animals were fed
a control diet. NAC-supplemented animals injected with alloxan
(ALX+NAC) (
) 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.