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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 18, 2001 as doi:10.1096/fj.00-0855fje. |
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B decoy inhibits pancreatic activation of NF-B and prevents diabetogenesis by alloxan in mice1
2
* Department of Oral Biology, and
Department of Human Nutrition, The Ohio State University, Columbus, Ohio 43210, USA
2Correspondence: 350 E Campbell Hall, Department of Human Nutrition, The Ohio State University, 1787 Neil Ave., Columbus, OH 43210, USA. E-mail: bray.21@osu.edu bray.21{at}osu.edu
SPECIFIC AIMS
In this study, we tested the hypothesis that nuclear factor 
B
(NF-B) activation is the key event involved in the initiation of
diabetogenesis using alloxan-induced diabetes as a model. Our specific
aims are to determine whether pancreatic NF-B activation induced by
alloxan can be blocked by intravenous (i.v.) injection of NF-B decoy
(i.e., synthetic oligodeoxynucleotides of NF-B DNA binding site) and
to monitor whether inhibition of NF-B activation by the decoy can
prevent the alloxan-induced diabetes in mice.
PRINCIPAL FINDINGS
1. Pretreatment with NF-B-specific oligodeoxynucleotides
(NF-B decoy) blocked alloxan-induced NF-B activation in the
pancreas in vivo
Using the methods previously reported by Miagkov et al., we
synthesized a NF-B decoy (5'-CTGGGGACTTTCCAT-3') as
phosphorothioate. A scrambled sequence of the decoy (SCR,
5'-TAACCGACTTTGCAT-3') was prepared as control substance.
Double-stranded NF-B decoy or SCR was dissolved in TransIT In vivo
Gene Delivery System (Panvera, WI) for i.v. injection. It has been
shown that NF-B decoy binds specifically to NF-B protein whereas
the SCR does not. The role of NF-B activation was tested by i.v.
injection of 0.3 ml of NF-B decoy or SCR solution into CD-1 mice
24 h before they were injected with 50 mg/kg of alloxan.
For NF-B activation analysis, mice were killed 30 min after
injection of alloxan and the pancreas was immediately removed. NF-B
activation was determined using an electrophoretic mobility shift
assay. 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-B probe.
Figure 1
shows that injection of alloxan induced NF-B activation in the
pancreas at 30 min after the injection (lane 1). Addition of cold
NF-B competitor in nuclear extract abolished the binding of NF-B
in vitro (lane 5), confirming the NF-B band. No NF-B activation
was detected in the pancreas of saline-injected animals (lane 2).
Pretreatment with NF-B decoy (2 nM in 0.3 ml) completely blocked
alloxan-induced pancreatic NF-B activation (lane 4). Injection of
SCR had no effect on alloxan-induced NF-B activation (lane 3).
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2. Pretreatment with NF-B decoy blocked alloxan-induced
pancreatic cell death
To determine the effect of pretreatment of NF-B decoy on
alloxan-induced pancreatic cell death, organs from alloxan-injected or
saline-injected animals were removed on day 10 after the injection,
fixed in neutral buffered, and embedded in paraffin. Serial sections (5
µm thick) were cut and stained with hematoxylin and eosin (H&E). H&E
sections showed that alloxan injection induced severe necrosis in
pancreatic islet cells. Fragmented and broken nuclei and cytoplasm of
dead cells exhibiting characteristic appearance of coagulative necrosis
were apparent in these islets. Pretreatment with SCR had no effect on
the histological changes induced by alloxan whereas pretreatment with
0.25, 0.5, and 2 nM of NF-B decoy dose-dependently rescued the islet
cells from necrosis, with 2 nM of NF-B decoy completely preventing
the induction of necrosis induced by alloxan.
3. Pretreatment with NF-B decoy blocked alloxan-induced decrease
in insulin production in ß-cells
To determine the effect of pretreatment of NF-B decoy on
alloxan-induced changes in ß-cell function, immunocytochemical
labeling of insulin was performed on pancreatic sections. In
saline-injected control animals, the cytoplasm of all of the pancreatic
ß-cells was found positive (stained brown) for insulin (Fig. 2A
). Ten days after alloxan injection, fewer than 50% of the
ß-cells were still producing insulin (Fig. 2B
).
Pretreatment with SCR did not affect the decrease of insulin production
induced by alloxan (Fig. 2C
). Pretreatment with NF-B
decoy, on the other hand, dose-dependently revived the ability of
ß-cells to produce insulin (Figs. 2D
–F
).
Pretreatment with the highest does of NF-B decoy (2 nM) completely
prevented the loss of insulin production induced by alloxan (Fig. 2F
). To determine whether these changes observed at
the cellular level reflect changes in insulin levels in the blood,
serum plasma levels were measured by ELISA on day 10 after alloxan
injection. Alloxan injection reduced the serum insulin level from
408.2 ± 101.5 pg/ml (n=5) to 29.8 ± 19.8 pg/ml
(n=5) (P<0.05). Pretreatment with NF-B decoy
(2 nM) restored serum insulin levels to normal (379.4±96.2 pg/ml).
Pretreatment with SCR did not change alloxan-induced decrease in serum
insulin levels.
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4. Pretreatment with NF-B decoy blocked alloxan-induced
hyperglycemia
Finally, fasting blood glucose levels (hyperglycemia) as a
clinical index for diabetes were measured from whole blood. Blood
samples were analyzed on day 0, 1, 3, 5, 7, and 10 after animals
received either saline or alloxan. Control fasting blood glucose levels
were between 5 and 10 mmol/l. Blood glucose levels rose to 22 mmol/l on
day 1 after the alloxan injection, and sustained hyperglycemia (>22
mmol/l) was observed at all time points measured. Pretreatment with SCR
1 day before the alloxan injection did not affect the development of
hyperglycemia induction by alloxan. Animals pretreated with 0.25 nM of
NF-B decoy injection developed an attenuated hyperglycemic response,
with the peak value of serum glucose levels below 22 mmol/l.
Pretreatment with 0.5 nM of NF-B decoy abolished the increase of
serum glucose level on day 1 after the alloxan injection and reduced
the peak glucose level to below 17 mmol/l. Pretreatment with 2 nM of
NF-B decoy completely prevented the development of hyperglycemia.
5. Administration of NF-B decoy after alloxan injection did not
block alloxan-induced diabetes
To investigate whether treatment with NF-B decoy after alloxan
injection could have a therapeutic effect; animals were injected with
the NF-B decoy 1 day after the alloxan injection. Alloxan-induced
loss of insulin production was not prevented by this treatment. This
treatment also failed to rescue pancreatic cell death and reduce
hyperglycemia.
CONCLUSION AND SIGNIFICANCE
The results of the present study show that i.v. injection of
NF-B decoy blocked NF-B activation in the pancreas and prevented
the induction of diabetes by alloxan. The entire spectrum of
alloxan-induced pathological changes—pancreatic cell death, loss of
insulin producing cells, decrease in serum insulin levels, and
hyperglycemia—were abrogated when animals were pretreated with the
NF-B decoy before they received alloxan injection. This evidence
demonstrates that the activation of NF-B in the pancreas is required
for the induction of diabetes by alloxan.
The use of NF-B decoy to probe the function of NF-B activation
has been used in numerous in vitro studies. NF-B decoy is quickly
becoming a favored NF-B blocker over other NF-B antagonists
because it potently blocks the activity of NF-B regardless of how
the NF-B is composed. In vivo, local application of NF-B decoy
has been successfully used to treat animal models of arthritis,
tumor-induced cachexia, and cerebral angiopathy. The effects of
systemically injected NF-B decoy have been tested only recently. The
present study, therefore, is among the first to demonstrate the
effectiveness of the systemically injected NF-B decoy in an animal
model.
The role of NF-B activation in the pathogenesis of diabetes in
humans has only begun to be investigated. Activation of NF-B in
leukocytes has been correlated with the quality of glycemic control in
diabetic patients. Activation of NF-B has also been implicated in
the pathogenesis of interstitial cystitis associated with diabetes.
Therefore, although the results of the present study are obtained in an
animal model of chemically induced diabetes, it is likely that the
activation of NF-B also plays a pivotal role in the pathogenesis of
human diabetes. In fact, an emerging concept for pathogenesis of
numerous degenerative diseases (including diabetes, cancer, arthritis,
cardiovascular disease, macular degeneration, various neurodegenerative
disorders, and aging) is that oxidative stress plays a central role.
The present results, therefore, suggest that activation of NF-B may
also be critical for both the pathogenesis and the potential treatment
of all of these diseases. Another significant implication of the
present study is that the ability to block pancreatic activation of
NF-B may be used as a criterion for preclinical testing of potential
antidiabetic drugs in animal models.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0855fje ; to cite this
article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0855fje 
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