Cytokine-induced apoptosis and necrosis are preceded by disruption
of the mitochondrial membrane potential (Dc
m
) in pancreatic
RINm5F cells: prevention by Bcl-2
Andreea Barbu, Nils Welsh, Johan Saldeen *
Department of Medical Cell Biology, Biomedical Centre, Uppsala University, PO Box 571, S-751 23 Uppsala, Sweden
Received 26 September 2001; accepted 8 January 2002
Abstract
The mechanisms of cytokine-induced b-cell death are poorly characterised. In rat insulin-producing RINm5F cells, the
combination of interleukin-1b, interferon-g and tumour necrosis factor-a presently induced disruption of the mitochondrial
membrane potential (Dc
m
) as demonstrated by reduced JC-1 fluorescence. The reduction of Dc
m
was maximal after 8 h and was
preceded by increased formation of reactive oxygen species (ROS), as assessed by dichlorofluorescein-diacetate (DCFH-DA)
fluorescence. A nitric oxide synthase-, but not a ROS-inhibitor, prevented cytokine-induced loss of Dc
m
.Overexpression of the anti-
apoptotic protein Bcl-2 increased both JC-1 and DCFH-DA fluorescence, which was paralleled by protection against cytokine-
induced apoptosis and necrosis. It is concluded that cytokines induce a nitric oxide-dependent disruption of Dc
m
and that this may
be a necessary event for both b-cell apoptosis and necrosis. Bcl-2 may prevent b-cell death by counteracting mitochondrial
permeability transition. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Mitochondrial permeability transition; b-Cell; Apoptosis; Necrosis; Cytokines; Nitric oxide
1. Introduction
Insulin-dependent (type-1) diabetes mellitus (IDDM)
results from destruction of the insulin-producing b-cells
in the pancreas, a process that may involve cytokines
(reviewed by Mandrup-Poulsen, 1996). Interleukin-1b
(IL-1b) and tumour necrosis factor-a (TNF-a) both
potently activate transcription factor NF-kB, a neces-
sary event for the induction of the inducible form of
nitric oxide synthase (iNOS), in insulin-producing cells
(Saldeen and Welsh, 1994; Saldeen et al., 2000). IL-1b,
especially in combination with interferon-g (IFN-g) and
TNF-a, induce the formation of high levels of nitric
oxide and reactive oxygen species (ROS) in b-cells and
neighbouring cells leading to reduced generation of ATP
and decreased b-cell function and viability (Sandler et
al., 1990; Mandrup-Poulsen, 1996). Moreover, cytokines
have been shown to induce b-cell death both by
apoptosis (reviewed by Mauricio and Mandrup-Poul-
sen, 1998) and necrosis (Saldeen, 2000).
It has been shown that many forms of apoptosis and
necrosis share a common effector phase of mitochon-
drial permeability transition (MPT) which may result
from the opening of a multiprotein complex pore
located at the contact site between the inner and outer
mitochondrial membrane (reviewed by Kroemer et al.,
1998). MPT is associated with disruption of the
mitochondrial membrane potential (Dc
m
). Various
apoptosis signalling pathways converge in the activation
of the pro-apoptotic protein Bax followed by the
translocation of this molecule to the outer mitochon-
drial membrane where it oligomerises and induces
opening of the MPT pore (Mikhailov et al., 2001). A
reduced Dc
m
, with or without MPT, may also be
observed in response to diverse molecules including
nitric oxide or its metabolite peroxynitrite (Packer and
Murphy, 1994; Kroemer et al., 1997) and ROS (Ber-
nardi, 1996; Kroemer et al., 1997; Herrera et al., 2001).
In addition to disruption of Dc
m
, MPT leads to
uncoupling of the respiratory chain with cessation of
* Corresponding author. Tel.: '46-18-471-4395; fax: '46-18-55-
6401.
E-mail address: johan.saldeen@medcellbiol.uu.se (J. Saldeen).
Molecular and Cellular Endocrinology 190 (2002) 75 Á
/
82
www.elsevier.com/locate/mce
0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
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