N

itric oxide (NO), initially described as

endothelium-derived relaxation factor
(EDRF) in blood vessels, has emerged as a fun-
damental messenger molecule in a variety of
cells with a wide spectrum of biological actions.
In addition to its pivotal role in the cardiovascu-
lar system, NO acts as a neurotransmitter and
inhibits platelet aggregation and proliferation of
smooth muscle cells (9). These physiological
functions are catalyzed by a constitutively
expressed NO synthase (NOS) in the vasculature
(eNOS) and in the neurons (bNOS), an enzyme
that is calcium/calmodulin dependent and
releases picomolar amounts of NO in response
to receptor stimulation (5).
In addition to conveying these physiological
processes, excess NO production by a cytokine-
inducible NOS (iNOS) participates in host
defense and immunological reactions and may
play a role as effector molecule in autoimmune
diseases. Thus, since the observation by Southern
et al. that pancreatic islet cells produce NO in
response to cytokines and that inhibition of NO
production protects these from cytokine-induced
suppression of insulin release, considerable
interest has focused on NO as a mediator of -
cell injury in insulin-dependent diabetes mellitus
(11). -Cells are particularly sensitive to damage
by NO and free radicals because of their low lev-
els of free radical-scavenging enzymes such as
Mn superoxide dismutase, catalase, and glu-
tathione peroxidase.
NO released in response to exposure of pan-
creatic islets to cytokines may be formed either in
the -cells themselves, leading to self-destruction
of these, or, alternatively, in intraislet macro-
phages or endothelial cells from which it diffuses
into -cells (Fig. 1A). Evidence for the -cell as the
source and site of action of NO has been pro-
vided by Corbett and co-workers (2), who
demonstrated that interleukin (IL)-1 was able to
induce large amounts of NO in rodent -cells and
-cell lines but not in -cells. Furthermore, trans-
genic mice overexpressing iNOS in pancreatic -
cells develop insulin-dependent diabetes (14).
Alternatively, NO could be produced by residual
macrophages or endothelial cells within the islets
or by inltrating macrophages (1). There is in vitro
evidence that rat islets are lysed by activated rat
macrophages, a process that is L-arginine depen-
dent and preventable by blockade of the NOS
with N
G
-monomethyl-L-arginine (L-NMMA) (6).
As pancreatic islets are densely capillarized, the
endocrine cells are surrounded by numerous
endothelial cells. Endothelial lining cells can, on
stimulation by cytokines, be transformed into
cytotoxic effector cells and thus contribute to
local tissue destruction. In fact, primary cultures
of rat islet capillary endothelial cells were found
to respond with a marked increase of iNOS
mRNA expression and subsequent NO produc-
tion on exposure to IL-1, tumor necrosis factor
(TNF)-, and interferon- (13).
NO is the rst gas known to act as a biological
messenger in mammals. It has a very short half-
life in vivo and rapidly decomposes by conden-
sation with molecular oxygen to form nitrite and
nitrate. Before its decomposition, the highly reac-
tive NO molecule reacts with specic molecular
targets, thereby exerting its messenger and cyto-
toxic effects. Preferential molecular targets of NO
are proteins containing iron-sulfur clusters or
heme-iron prosthetic groups such as, e.g., guany-
lyl cyclase. By binding to the iron atom of this
0886-1714/99 5.00 1999 Int. Union Physiol. Sci./Am.Physiol. Soc. News Physiol. Sci. Volume 14 April 1999
49
The Dual Role of Nitric Oxide in
Islet -Cells
Giatgen A. Spinas
In pancreatic islets, nitric oxide (NO) produced on exposure to cytokines mediates
-cell injury leading to diabetes mellitus. On the other hand, L-arginine-derived
NO may participate in the signal transduction pathway of physiological insulin
secretion. This review focuses on the dual role of NO as a mediator of
physiological and pathophysiological processes in pancreatic islets.
G. A. Spinas is Professor, Division of Endocrinology and
Diabetes, Department of Internal Medicine, University Hos-
pital, CH-8091 Zrich, Switzerland.
ARTICLES
NEWS IN PHYSIOLOGICAL SCIENCES
NO is the rst gas
known to act as a
biological messenger
in mammals.
enzyme, guanosine 3,5-cyclic monophosphate
(cGMP) is synthesized. cGMP is an important
second messenger in endothelial and neuronal
cells. Also in insulin-producing cells, NO aug-
ments cGMP formation (2). On the other hand, by
binding to the iron-containing Krebs cycle
enzyme aconitase (which converts citrate to isoc-
itrate), NO inhibits enzyme activity leading to
decreased glucose oxidation, oxygen consump-
tion, and adenosine 5-triphosphate (ATP) gener-
ation in rat islets (15). Apart from binding to sus-
ceptible iron groups in enzymes, NO inhibits
ribonucleotide reductase, activates cyclooxyge-
nase and stimulates prostaglandin E
2
synthesis,
induces adenosine 5-diphosphate-ribosylation of
glyceraldehyde-3-phosphate dehydrogenase
resulting in decreased glycolysis, and directly
damages islet cell DNA. DNA strand breaks were
observed in islet cells incubated with activated
macrophages and sodium nitroprusside. In addi-
tion to directly reacting with protein prosthetic
groups, NO can also combine with oxygen to
eventually produce potent cellular killers such as
the highly toxic hydroxyl radical, OH, and perox-
ynitrite (ONOO), which is a powerful oxidant.
Studies with rat islet cells and monolayer cultures
of human islets exposed to various cytokines
have, indeed, revealed that NO and oxygen free
radicals may act in concert to kill the -cell.
Although NO appears to play a major role in -
cell destruction in rodent autoimmune diabetes,
its importance as an effector molecule of -cell
injury in human insulin-dependent diabetes mel-
litus is less evident. Human islets and human -
cells are clearly more resistant to the inhibitory
effect of NO than rodent islets (4). Thus various
combinations of cytokines are required for the
induction of iNOS, and the effects on islet func-
tion are less dramatic. Furthermore, blockade of
NOS does not protect human islets from
cytokine-mediated impairment of -cell function.
The increased resistance to damage by NO, free
oxygen radicals, and cytokines is caused by
higher expression of the antioxidant enzymes
catalase and superoxide dismutase and of the
heat shock protein HSP 70 in human islets (3). In
addition, human islets possess a higher capacity
for DNA repair compared with rodent cells,
emphasizing the importance of -cell repair and
defense mechanisms for the development of
autoimmune diabetes in different species.
Unlike in rodents, evidence is still lacking
whether human -cells are able to produce NO
on stimulation with cytokines, nor has it con-
vincingly been shown whether human macro-
phages can be induced to synthesize and release
sufcient amounts of NO to exert toxic effects on
-cells. Thus, although NO is considered to play
an important part as an effector molecule in -
cell destruction in rodents, any putative role of
NO in the pathogenesis of human autoimmune
diabetes needs to be claried.
Besides its role as an effector molecule of -
cell destruction, several lines of evidence indi-
cate that NO derived from breakdown of L-argi-
nine may be involved in the signal transduction
News Physiol. Sci. Volume 14 April 1999 50
. . .evidence is still
lacking whether
human -cells are able
to produce NO. . . .
FIGURE 1. Hypothetical dual role of nitric oxide (NO) in pancreatic islets. A: cytokine-induced NO production by inltrating
macrophages or by the -cell itself causes -cell injury resulting in insulin-dependent diabetes. iNOS, inducible NO synthase;
IL, interleukin. B: glucose-induced NO production by a constitutively expressed endothelial NO synthase (eNOS) in -cells or
endothelial cells within the islets stimulates insulin release in a paracrine manner.
pathway of physiological insulin secretion. More
than 30 years ago it was shown that L-arginine
mediates protein-induced insulin secretion and
potentiates D-glucose-induced insulin release.
Furthermore, L-arginine deciency is associated
with insulinopenia and a failure to secrete insulin
in response to glucose. Evidence that NO could
be involved in insulin secretion was provided by
Laychock et al. in 1991. These authors demon-
strated that sodium nitroprusside increased
cGMP in rat islets and stimulated insulin secre-
tion, whereas on the other hand, inhibition of
NOS reduced glucose- and arginine-induced
cGMP release (8). These data suggested a role for
NO in the regulation of cGMP levels in -cells.
Along the same line, Schmidt et al. (10) found
that -cell-derived HIT-T15 cells constitutively
expressed NOS and produced NO and insulin in
the presence of D-glucose. Insulin production
was preventable by L-NMMA and N
G
-nitro-L-
arginine, two potent inhibitors of the enzymatic
conversion of L-arginine to NO. In these same
experiments, NO formation was paralleled by an
increase in cGMP, again supporting the notion
that L-arginine-derived NO acts principally by
stimulating guanylate cyclase. Blockade of the
enzyme resulted in reduced glucose-stimulated
insulin secretion both in vivo and in vitro (10).
Sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis and protein immunoblot analysis of
HIT-T15 cells with antisera to puried NOS from
rat cerebellum revealed the presence of a consti-
tutive NOS in these -cell-derived cells. Further-
more, isolated islets of Langerhans displayed
reduced nictotinamide adenine dinucleotide
phosphate (NADPH) diaphorase, which was
merely located at the islet periphery. Although
these data were suggestive of an involvement of
L-arginine-derived NO in the signal transduction
pathway mediating insulin release, it was argued
that the insulinoma-derived HIT-T15 -cell line
may not represent a valid model of -cells and
that the presence of NADPH activity in the islets
does not convincingly prove the constitutive
expression of NOS in islets and islet -cells,
respectively.
Thus the role of NO in physiological insulin
secretion remained controversial. A series of stud-
ies looking into accumulated insulin release in
cultured islets or using perfused rat pancreas
argued against a stimulatory effect of NO on
insulin release. Moreover, incubation of islets
with a NO inhibitor or perfusion of rat pancreata
with N
G
-nitro-L-arginine methyl ester (L-NAME)
and chronic administration of L-NAME to anes-
thetized rats did not result in a detectable inhibi-
tion of insulin release. However, because NO is
likely to act rapidly and affect the early phase of
insulin secretion, a possible stimulatory effect
may be masked in experimental settings with cul-
tured islets or perfused pancreata, because inhi-
bition of NO exerts additional systemic effects on
vasoconstriction, increases capillary blood ow,
and interferes with neuronal mechanisms.
To circumvent these problems and to study the
direct effects of NO on -cell function, we peri-
fused rat islets in the presence or absence of L-
NMMA (12). Ambient glucose was raised from
nonstimulatory levels (1.6 mM) to 16 mM, and
insulin was determined at 1-min intervals in the
perifusate. In the presence of L-NMMA, but not of
News Physiol. Sci. Volume 14 April 1999
51
. . .the role of NO in
physiological insulin
secretion remained
controversial.
FIGURE 2. Effects of N
G
-monomethyl-L-arginine (L-NMMA;
A), N
G
-monomethyl-D-arginine (D-NMMA; B), and carboxy-
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide
PTIO) (C) on glucose-stimulated insulin secretion from peri-
used rat islets. Batches of 50 islets were perifused in the pres-
ence or absence of 0.5 mM L-NMMA or D-NMMA or 10 mM
carboxy-PTIO. Arrows indicate the beginning and end of the
rst phase. Traces show control (), L-NMMA (, A)-, D-
NMMA (, B)-, and carboxy-PTIO (, C)-exposed islets.
Reproduced from Ref. 12 with permission. Copyright
Springer-Verlag.
its inactive D-enantiomer, the early phase of glu-
cose-stimulated insulin secretion was blunted
(Fig. 2. A and B), suggesting that endogenously
produced NO is involved in the secretagogue-
induced insulin secretion under physiological
conditions. This was further supported by the
nding that addition of the NO scavenger car-
boxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-
oxyl 3-oxide (PTIO) to the incubation medium
also resulted in a blunted early insulin peak (Fig.
2C). The inhibitory effect was only seen during
the rst 10 min after glucose stimulation and was
strongly dependent on the concentration of the
NOS inhibitor, with 0.51 mM being most effec-
tive. This illustrates that the stimulatory effect of
NO on insulin secretion is subtle and pertains
mainly to the early phase of glucose-stimulated
insulin release. Such a narrow time/concentration
window is consistent with a physiological role of
NO as mediator of secretagogue-induced signal
transduction in rat islets and explains why the
stimulatory effect may be masked in experimental
settings measuring accumulated insulin or apply-
ing higher concentrations of the inhibitor.
If endogenously produced (L-arginine derived)
NO participates in the signal transduction path-
way of insulin secretion, the question arises
whether NO is produced in the -cell itself or in
other cell types of the islet organ. Data are still
conicting as to whether pancreatic islets, partic-
ularly the endocrine cells, constitutively express
NOS. NOS immunoreactivity and/or NADPH
diaphorase staining in rat and mouse islet cells
has been described by some investigators,
whereas others failed to demonstrate constitutive
expression of NOS in human and rat islets. We
found that (glucagon producing)-islet cells, and
to some extent (somatostatin producing)-cells,
express NOS (Fig. 3A) but could not detect NOS
in the insulin-producing islet -cells. At the sub-
cellular level, NOS immunoreactivity was con-
ned to the secretory granules of the - and -
cells (Fig. 3B). This is in keeping with reports
demonstrating NOS in secretory granules of gas-
tric D-cells and synaptic vesicles of enteric neu-
rons. The presence of NOS in the secretory gran-
ules of - and -cells suggests that NO is pro-
duced in these cells and subsequently diffuses to
-cells. Therefore, the effect of NO on glucose-
stimulated insulin release under physiological
conditions could be exerted in a paracrine man-
ner by neighboring - and/or -cells or by
endothelial cells (Fig. 1B).
The mechanism by which NO triggers insulin
release remains to be elucidated. Different possi-
bilities may be envisaged. One putative mecha-
nism could be that NO exerts its action via
increasing intracellular calcium through mobi-
lization of calcium from intracellular pools such
as the endoplasmic reticulum or from mitochon-
dria. We have provided experimental evidence
that such a mechanism may be operative, i.e.,
News Physiol. Sci. Volume 14 April 1999 52
FIGURE 3. A: light microscopic immunohistochemical localization of 2 isoforms of NOS on serial semithin sections through rat
islet. The rst section was incubated with an antiserum against constitutively expressed NOS (eNOS; a) and the second against
constitutively expressed neuronal NOS (bNOS; b). eNOS and bNOS immunoreactivities are present in identical islet cells. B:
immunocytochemical localization of eNOS as revealed by double incubation of a thin section with antisera against glucagon
(small granules) and against eNOS (large granules). The -cell secretory granules exhibit small particles, i.e., glucagon
immunoreactivity, and large particles, i.e., NOS immunoreactivity, in coexistence. Courtesy of Drs. I. David and M. Reinecke,
Division of Neuroendocrinology, Institute of Anatomy, University of Zrich, Switzerland.
. . .NOS in secretory
granules of gastric D-
cells. . . .
that the rapid NO-mediated insulin secretion
could be triggered by an increase of intracellular
calcium caused by a release of calcium from the
mitochondrial calcium pool (7). NO inhibits the
mitochondrial respiratory chain by binding to
cytochrome c and/or cytochrome oxidase. As a
consequence, the mitochondrial membrane
potential decreases and Ca
2+
leaves the mito-
chondria. This is followed by restoration of the
mitochondrial membrane potential and Ca
2+
reuptake by mitochondria. To assess the mecha-
nism whereby NO stimulates insulin secretion,
we used INS-1 cells. These rat insulinoma-
derived -cells are considered a valid model of
native -cells because they have retained many
morphological, enzymatic, and secretory char-
acteristics of normal -cells, including respon-
siveness to physiological glucose stimuli. The
addition of 2 M NO gas to INS-1 cells resulted
in a rapid increase in insulin release, which was
paralleled by a reversible decrease of the mito-
chondrial membrane potential and by an inter-
mittent rise of cytosolic Ca
2+
, increasing from
115 nM before NO was added to 160 nM there-
after. Mitochondrial calcium depletion by
uncoupling and blockade of the respiratory
chain with carbonyl cyanide m-chlorophenylhy-
drazone (CCCP) and antimycin A before addition
of NO abolished the NO-induced insulin release
(Fig. 4). Conversely, the NO-induced insulin
release could be mimicked by pretreating the
cells with the mitochondrial poisons. Chelation
of intracellular, but not extracellular, calcium
prevented the NO-induced insulin release, as did
preincubation with epinephrine, which inhibits
insulin exocytosis. An intermittent rise in the
cytosolic calcium caused by mobilization from
the mitochondrial calcium pool could thus
explain the stimulatory effect of NO observed
during the early phase of glucose-stimulated
insulin release, which is then followed by the
classical glycolytic pathway operating via clo-
sure of ATP-sensitive potassium channels, cell
depolarization, and calcium inux. Besides act-
ing via the redistribution of intracellular calcium,
NO may target the insulin vesicle and modulate
synaptic vesicle docking fusion reactions, as has
been observed in synaptosomes, for example, or
act via cell surface receptors or ion channels of
the -cells.
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302305, 1996.
News Physiol. Sci. Volume 14 April 1999
53
NO inhibits the
mitochondrial
respiratory chain. . . .
FIGURE 4. Mitochondrial membrane potential changes (A),
cytosolic Ca
2+
levels ([Ca
2+
]
i
; B), and insulin secretion from INS-
1 cells (C). Mitochondrial membrane potential was assessed by
monitoring the decrease of JC-1 red uorescence (A) and [Ca
2+
]
i
by means of indo 1 uorescence (B). Measurements were done
with 3 x 10
7
cells in 2 ml under constant stirring at 37C. At the
rst two arrows, 2 M NO was added. At the third arrow, 5 M
carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added
together with 5 g/ml antimycin A (AA), and at the fourth arrow
NO was added again (B). Insulin secretion was assessed after
addition of NO (2 M) in the form of NO-saturated buffer (time
0 on the abscissa) 1 min after suspending 1.5 x 10
7
cells. , NO
treatment; , controls. Insulin accumulation was measured at
the time points indicated. Secreted insulin is expressed as per-
centage of baseline values at time 0 (mean SE of 58 experi-
ments. *P < 0.03, P < 0.05 compared with controls). Repro-
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