Journal of Autoimmunity (2001) 16, 449–455 doi:10.1006/jaut.2001.0507, available online at http://www.idealibrary.com on
An Inhibitor of Inducible Nitric Oxide Synthase and
Scavenger of Peroxynitrite Prevents Diabetes
Development in NOD Mice
Wilma L. Suarez-Pinzon
1
, Jon G. Mabley
2
, Ken Strynadka
3
, Robert F. Power
4
,
Csaba Szabo ´
2
and Alex Rabinovitch
1
1
Department of Medicine, University of
Alberta, Edmonton, Alberta, Canada
2
Inotek Corporation, Beverly,
Massachusetts, USA
3
Department of Pediatrics, University of
Alberta, Edmonton, Alberta, Canada
4
Department of Laboratory Medicine and
Pathology, University of Alberta,
Edmonton, Alberta, Canada
Received 4 December 2000
Revised version accepted 7 February
2001
Key words: free radicals,
insulitis, nitric oxide, NOD mice,
peroxynitrite
Peroxynitrite (ONOO
-
) is a highly reactive oxidant produced by the inter-
action of the free radicals superoxide (O
c -
2
) and nitric oxide (NO
c
). In a
previous study, we found that peroxynitrite is formed in islet -cells of
nonobese diabetic (NOD) mice. Here, we report that guanidinoethyldi-
sulphide (GED), a selective inhibitor of inducible nitric oxide synthase (iNOS)
and scavenger of peroxynitrite prevents diabetes in NOD mice. GED treatment
of female NOD mice, starting at age 5 weeks, delayed diabetes onset (from age
12 to 22 weeks) and significantly decreased diabetes incidence at 30 weeks
(from 80% to 17%). GED did not prevent pancreatic islet infiltration by
leukocytes; however, -cells that stained positive for nitrotyrosine (a marker of
peroxynitrite) were significantly decreased in islets of GED-treated mice
(1±1%) compared with vehicle-treated mice (30±9%). In addition, GED
significantly inhibited nitric oxide and nitrotyrosine formation and decreased
destruction of -cells in NOD mouse islets incubated in vitro with the
combination of proinflammatory cytokines interleukin 1-beta (IL-1), tumour
necrosis factor-alpha (TNF-) and interferon-gamma (IFN-). These findings
indicate that both superoxide and nitric oxide radicals contribute to islet -cell
destruction in autoimmune diabetes via peroxynitrite formation in the -cells.
© 2001 Academic Press
Introduction
Insulin dependent diabetes mellitus results from
destruction of the insulin-producing pancreatic islet
-cells by a response considered to be autoimmune
[1]. Pancreatic islets are infiltrated by mononuclear
cells of the immune system, mostly macrophages and
T lymphocytes and this is followed by destruction
of the -cells. Islet -cell destruction may result
from direct contact with -cell specific cytotoxic
T-lymphocytes, as well as from exposure to inflamma-
tory products of activated macrophages and
T-lymphocytes, such as cytokines, oxygen radicals,
and nitric oxide [2–5]. In addition, the proinflamma-
tory cytokines, interleukin 1-beta (IL-1), tumour
necrosis factor-alpha (TNF-), and interferon gamma
(IFN-) are cytotoxic to islet -cells via mechanisms
that may involve production of oxygen radicals
and/or nitric oxide in the -cells [4–11].
Peroxynitrite (ONOO
-
) is a highly reactive oxidant
species produced by the combination of the free
radicals, superoxide (O
c -
2
) and nitric oxide (NO
c
)[12,
13]. Peroxynitrite production has been observed in
many inflammatory conditions [14, 15] and current
evidence suggests that peroxynitrite is a more potent
and cytotoxic mediator than superoxide or nitric oxide
alone [13, 15, 16]. Also, both rodent and human islets
are highly sensitive to peroxynitrite-induced damage
[17]. In a previous study, we found that peroxynitrite
is formed in islet -cells of acutely-diabetic NOD mice
[18]. The aim of the present study was to determine
if prevention of peroxynitrite formation in -cells
of autoimmune diabetes-prone NOD mice would
prevent -cell destruction and diabetes development.
Materials and Methods
Animals
Female and male NOD mice, 4 weeks of age, were
purchased from Taconic (Germantown, NY, USA). The
mice were housed and fed under specific pathogen-
free conditions and were cared for according to the
guidelines of the Canadian Council on Animal Care.
Female NOD mice of this colony develop pancreatic
islet infiltration by immune system cells (insulitis)
Correspondence to: Alex Rabinovitch, M.D., 430 Heritage Medical
Research Centre, University of Alberta, Edmonton, Alberta,
Canada T6G 2S2. Fax: (780) 492-4666. E-mail:
alex.rabinovitch@ualberta.ca
449
0896–8411/01/040449+07 $35.00/0 © 2001 Academic Press