Institute of Physiological Sciences, Department of Pharmacology, University of Lund, S-221 84 Lund, Sweden
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ABSTRACT |
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The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.
islets of Langerhans; nitric oxide synthase isoforms; nitric oxide synthase inhibition
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INTRODUCTION |
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FOR THE LAST 10 YEARS,
nitric oxide (NO) produced by the constitutive NO synthase (cNOS) has
been the focus of much interest because of its proposed role in both
the central and the peripheral nervous system, acting as a diffusible
inter- and intracellular messenger with multifunctional properties
(10, 22). In the peripheral nervous system, NO has
been reported to regulate gastrointestinal motility, regional blood
flow, and neuroendocrine function. Furthermore, NO biosynthesis in
excitable tissues is not only restricted to neurons. Also certain
endocrine cells such as the insulin, glucagon, pancreatic polypeptide,
and somatostatin cells of the islets of Langerhans, the somatostatin
cells in the stomach, and the renin cells of the kidney (5, 17,
23, 24, 26, 28, 32, 35) have been shown to harbor the cNOS
enzyme, which accordingly was assumed to be involved in hormone
secretory processes. With regard to the insulin producing -cells,
cNOS-derived NO has been assigned a role as a novel modulator of
insulin release stimulated by both glucose and the NO precursor
L-arginine. In this context, we emphasize that cNOS-derived
NO, which is known to be produced in small amounts in a
Ca2+-dependent pulsatile way (10, 22), is
considered an important messenger molecule, whereas NO produced by
macrophages upon cytokine activation of the inducible
Ca2+/calmodulin-independent isoform of NOS (iNOS), for
example, is produced in larger amounts in a continuous way, is
cytotoxic, and has been implicated in macrophage-induced
-cell
destruction and thus in type I insulin-dependent diabetes mellitus
(IDDM) (11, 14).
Similar to the stimulus secretion coupling in most endocrine cells, the secretion of insulin is regulated by a complex chain of events evoked in response to various nervous and hormonal factors. The major insulin secretagog, glucose, however, is metabotropic and is known to initiate insulin release through its metabolism, thereby producing ATP, acting by closure of ATP-sensitive K+ (KATP) channels. These events result in depolarization and the opening of voltage-dependent Ca2+ channels, which in turn lead to increased intracellular Ca2+ and subsequent exocytosis of insulin. This main Ca2+-dependent secretory pathway is then modulated by activation of different types of second messengers such as the cAMP protein kinase A and the phospholipase C-protein kinase C systems (43).
The putative role of cNOS in the signaling pathways of insulin release
has been widely disputed, and the results obtained have been highly
controversial. In 1992, Schmidt et al. (32) reported that
NO was a positive modulator of glucose-arginine-stimulated insulin
release in an insulinoma-derived -cell line. The same year, our data
from mouse islets (28) suggested that NO was inhibitory to
arginine-stimulated insulin release, and experiments by Jones et al.
(20) did not reveal any putative effect by NO in rat
islets. Indeed, a great number of studies using islets and
-cell
lines with various types and concentrations of NOS inhibitors and NO
donors have been performed, claiming that NO either stimulates
(13, 25, 32, 34, 35, 41) or inhibits (1-4, 7,
15-19, 26-29, 31, 38) insulin release. Much of this
confusion, however, certainly emanates from the use of various
-cell
lines with different qualitative and/or quantitative secretory reaction
patterns compared with normal
-cells and also from the fact that,
until now, there have been no studies to our knowledge directly
correlating glucose-stimulated insulin release in normal islets with
concomitant biochemical determination of the enzymatic activities of
the different islet isoforms of NOS. Moreover, as far as we are aware,
the present report is the first one showing a direct biochemical
discrimination between islet cNOS and iNOS activities during a glucose
dose-response stimulation. In this context, it is not possible, during
incubation of intact islets, to differentiate between NO produced from
cNOS vs. iNOS by measuring nitrate-nitrite (Griess reaction) or by
using an NO electrode. Furthermore, these methods are also hampered by
the fact that NO is a highly reactive molecule, which makes it
difficult to record the "true" amounts of NO evolution. Hence, in
this study, we have chosen to determine the actual NOS activities at
different time points and in the presence and absence of various test
agents known to modulate islet NO production and insulin secretion.
We now show here that 1) increased, "hyperglycemic" glucose concentrations stimulate both cNOS and iNOS activities in normal intact islets, 2) NO is indeed a negative modulator of glucose-stimulated insulin release, and 3) islet NOS activity should always be monitored when different NOS inhibitors are used because certain concentrations of such inhibitors could stimulate NO production in intact islets.
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MATERIALS AND METHODS |
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Animals. In all experiments, we used freely fed mice or rats. Normal female mice (NMRI strain; B&K, Sollentuna, Sweden) weighed 25-30 g, and rats (Sprague-Dawley; B&K) weighed 180-220 g. They were killed by cervical dislocation, and isolation of islets was performed by retrograde injection of a collagenase solution via the bile-pancreatic duct. The animal experiments were approved by the local animal welfare committee (Lund, Sweden), and the animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Incubation of intact isolated islets and determination of islet
cNOS and iNOS activities.
Preparation of isolated islets with the Gotoh method was performed as
previously described (17, 29). The freshly isolated mouse
islets were first preincubated for 30 min at 1 mmol/l glucose (17, 29) and then incubated for 60 min (in one
experimental series for 20 min) at different glucose concentrations in
the absence or presence of various modulators of islet NOS activities. Each incubation vial contained 200 islets in 2 ml of buffer, and all
incubations were performed with intact islets in a regular Krebs-Ringer
bicarbonate buffer with pH 7.40, supplemented with 10 mmol/l HEPES and
0.1% BSA (17, 29). After incubation, a sample of the
medium was withdrawn for insulin determination (29), and
the islets were thoroughly washed and collected in ice-cold buffer (200 islets in 840 µl of buffer) containing 20 mmol/l HEPES, 0.5 mmol/l
EDTA, and 1 mmol/l D,L-dithiothreitol, pH 7.2, and immediately frozen at 20°C. On the day of assay of NOS
activities, the islets were thawed and sonicated on ice, and the buffer
solution containing the islet homogenate was supplemented to contain
also 0.45 mmol/l CaCl2, 2 mmol/l NADPH, 25 U/ml calmodulin,
and 0.2 mmol/l L-arginine in a total volume of 1 ml as
previously described (17, 29). To determine iNOS activity,
both Ca2+ and calmodulin were omitted from the assay
buffer. The method is a nonisotopic modification of the Bredt and
Snyder method, and the buffer composition is essentially the same as
previously described for the assay of NOS in brain tissue using
radiolabeled L-arginine (cf. Ref. 17). The
homogenate was then incubated at 37°C under constant air bubbling,
1.0 ml/min, for 3 h. We ascertained that under these conditions
the reaction velocity was linear for at least 6 h. Aliquots of the
incubated homogenate (200 µl) were then passed through a 1-ml Amprep
CBA cation-exchange column for high-performance liquid chromatography
analysis. The amount of L-citrulline formed was then
measured in a Hitachi F-1000 fluorescence spectrophotometer (Merck) as
previously described (17, 29). NO and
L-citrulline were produced in equimolar concentrations. The
nitrogen atom of the guanidino group of L-arginine was
released as NO, which is highly reactive, and therefore the
simultaneously liberated and stable L-citrulline was
preferably measured (33). The methodology has been
described in detail previously (17, 29).
Western blot analysis. After incubation, the islets were thoroughly washed in Hanks' buffer, frozen, and then sonicated on ice (3 times for 10 s) on the day of analysis. The protein content (29) was determined according to Bradford. Homogenate samples representing 20 µg of total protein from islet tissue were run on 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrotransfer (10-15 V, 60 min; Semi-Dry Transfer Cell, Bio-Rad, Richmond, CA). The membranes were blocked in 9 mM Tris · HCl (pH 7.4) containing 5% nonfat milk powder for 40 min at 37°C. Immunoblotting with rabbit anti-mouse constitutive neuronal NOS (ncNOS; N-7155) or iNOS (N-7782) (1:2,000; Sigma, St. Louis, MO) was performed for 16 h at room temperature. The membranes were washed twice and then incubated with alkaline-phosphatase-conjugated goat anti-rabbit IgG (1:10,000; Sigma) for 90 min. Antibody binding to ncNOS and iNOS was detected by using 0.25 mM CDP-Star (Boehringer Mannheim, Mannheim, Germany) and the signal enhancer Nitro Block II (Tropix, Bedford, MA) for 5 min at room temp. The chemiluminescence signal was visualized by exposing the membranes to Dupont Cronex X-ray films for 1-5 min. Appropriate standards, i.e., molecular mass markers, were run in all analyses.
Perifusion of pancreatic islets. Groups of 100 rat islets were perifused as previously described in detail (30). The basal perifusion medium contained 1.6 mmol/l glucose, and NG-monomethyl-L-arginine(L-NMMA; 0.5 or 5.0 mmol/l) was added 10 min before the glucose concentration was raised to 16.0 mmol/l.
In vivo experiments. Glucose was dissolved in 0.9% NaCl (saline) and injected intraperitoneally into mice in a dose of 22.2 mmol/kg at 0 min. A further injection of 11.1 mmol/kg was performed at 30 min. Controls received saline. Blood sampling and determinations of plasma concentrations of insulin and glucose have previously been described (29).
Statistics. Statistical significance between sets of data was assessed by using unpaired Student's t-test or, where applicable, analysis of variance followed by Tukey-Kramer's multiple comparisons test. Results are expressed as means ± SE.
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RESULTS |
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Islet isoforms of NOS and glucose-stimulated insulin release.
To directly examine a possible relation between islet NO production and
insulin release at different glucose concentrations, we performed a
detailed dose-response study in incubated mouse islets. Figure
1A shows that the lowest total
NO production was observed at a physiological concentration of glucose
(7 mmol/l). This NO production was derived from cNOS. At 10, 12, and 20 mmol/l glucose, a gradual increase in cNOS activity was observed.
Moreover, already at 10 mmol/l glucose, we found detectable iNOS
activity. The iNOS activity was further increased at 12 and 20 mmol/l
glucose. These changes in iNOS activity were in strong agreement with
the visual impression of the immunoblots of iNOS protein (Fig.
1B). As expected, there was a clear dose-response
relationship between glucose concentrations and insulin release between
7 and 20 mmol/l glucose (Fig. 1C).
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Influence of NO donation or NOS inhibition on glucose-stimulated
insulin release.
The next series of experiments was designed to explore the effects on
glucose-stimulated insulin release by, on the one hand, either
application of NO gas in aqueous solution (Fig.
2A) or addition of the
intracellular NO donor hydroxylamine (Fig. 2B) and, on the
other, addition of two chemically different NOS inhibitors, NG-nitro-L-arginine methyl ester
(L-NAME) and 7-nitroindazole (Fig. 2B). Insulin
release from islets incubated at 20 mmol/l glucose for 60 min was
strongly suppressed by either exogenous NO or the NO donor but markedly
increased by both types of NOS inhibitors. Glucose-stimulated insulin
release from islets depolarized with 30 mmol/l K+ in the
presence of the KATP channel opener diazoxide was likewise suppressed by exogenous NO (Fig. 2A) and potentiated by NOS
inhibition (Fig. 2B).
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Islet NOS activities in relation to glucose-stimulated insulin
release in the absence and presence of a NOS inhibitor.
Figure 3A shows the effect of
another NOS inhibitor, L-NMMA, on glucose-stimulated
insulin release and iNOS, cNOS, and total NOS activities in intact
mouse islets incubated at 20 mmol/l glucose for 60 min. These islets
displayed expression of iNOS protein (Fig. 3B) and a marked
iNOS activity (Fig. 3A). L-NMMA suppressed cNOS
activity and total NOS activities and potentiated glucose-stimulated insulin release. The activity and expression of iNOS activity, however,
was unaffected by L-NMMA (Fig. 3, A and
B). After the incubation time was shortened to 20 min (Fig.
3C), there was no glucose-induced expression of islet iNOS
(Fig. 3D) and no measurable iNOS activity (Fig.
3C). cNOS activity, however, was already markedly suppressed
by L-NMMA at this time point. This suppression was accompanied by an increased insulin release (Fig.
3C). To test the specificity of the effects of
L-NMMA on NOS activities and insulin release, we
performed a series of control experiments with D-NMMA, the
enantiomer of L-NMMA. D-NMMA is reportedly
devoid of NOS inhibitory properties (22, 33). Isolated
islets were accordingly incubated with either 20 mmol/l glucose
(control) or 20 mmol/l glucose + 5 mmol/l D-NMMA for
60 min. The resulting NOS activities (pmol NO · mg
protein-1 · min-1)were as follows: for
control (n = 3) vs. D-NMMA
(n = 3): total NOS, 48.7 ± 6.6 vs. 62.6 ± 0.4; iNOS, 31.6 ± 5.5 vs. 41.8 ± 1.2; and cNOS, 17.5 ± 2.0 vs. 20.9 ± 1.3. Hence, D-NMMA did not display any inhibitory effects at all on islet NOS activities but instead tended to bring about a slight increase. Moreover, D-NMMA
had no significant effect on glucose-stimulated insulin release
[2.48 ± 0.22 (control) vs. 2.80 ± 0.15 ng · islet · h-1 (D-NMMA)].
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Effect of the KATP channel opener diazoxide on islet
NOS activities and glucose-stimulated insulin release.
To ascertain that glucose-stimulated NOS activities were really
initiated by glucose and not merely the result of the increased secretory process and/or increased amounts of insulin surrounding the
-cells, we performed the series of experiments shown in Fig. 4. Insulin release stimulated by 20 mmol/l glucose was almost completely inhibited by the KATP
channel opener diazoxide, yet there was only a slight inhibition of
total NOS activities. This inhibition was apparently due to a
suppression of the Ca2+-dependent cNOS enzyme activity
(Fig. 4) and was thus a result of the fact that diazoxide, by keeping
the KATP channels open, also prevented glucose-induced
membrane depolarization and Ca2+ influx. The
glucose-stimulated increase of the Ca2+-independent iNOS
activity, however, was unaffected (Fig. 4). These data support our
previous hypothesis (26, 27, 29) that the increased
cNOS-derived NO production is directly involved as a negative feedback
modulator of glucose-stimulated insulin release and not merely a result
of the secretory process.
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Short-term in vivo hyperglycemia and islet isoforms of NOS.
In an attempt to test whether iNOS expression could be induced after
exposing the islets to elevated glucose levels also in the in vivo
situation, we injected groups of mice with either glucose or saline
(controls) and monitored islet activities of iNOS and cNOS after ~60
min of hyperglycemia. Figure
5A shows the blood glucose
pattern during the test period, and Fig. 5B shows that
islets directly isolated ex vivo from the glucose-injected hyperglycemic animals at 75 min showed a marked elevation of iNOS enzyme activity, which was reflected in an increase in total NOS activity. cNOS activity, however, was unaffected at this time point.
Figure 5C shows that islets from glucose-injected mice, but
not those from saline-injected control mice, displayed expression of
iNOS protein. The plasma insulin levels at 30 and 75 min did not differ
between glucose- and saline-injected animals (data not shown).
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Differential effects of different concentrations of the NOS
inhibitor L-NMMA on islet NOS activities in
glucose-stimulated perifused rat islets.
Recently, Spinas et al. (34, 35) showed that 0.5 mmol/l
L-NMMA inhibited the first phase of glucose-stimulated
insulin secretion in perifused rat islets. These authors suggested that NO was involved as a promoter of glucose-stimulated insulin release, at
least during the first phase. Because their results were completely at
variance with our data, we performed a similar series of experiments in
rat islets and measured islet iNOS and cNOS enzyme activities after 10 min (first-phase insulin release) of glucose stimulation with or
without 0.5 or 5.0 mmol/l L-NMMA. Figure
6A shows that 0.5 mmol/l
L-NMMA indeed inhibited first-phase glucose-stimulated insulin secretion in perifused rat islets. However, as shown in Fig.
6B, this inhibition of insulin release was associated with an increase in islet NO production. In contrast, 5.0 mmol/l
L-NMMA strongly suppressed islet NO production, an effect
that was accompanied by a marked potentiation of glucose-stimulated
insulin release. As shown in Fig. 6B, no iNOS activity could
be recorded after 10 min of stimulation with high glucose (16.7 mmol/l), and, hence, the changes in total NOS activity could be
completely referred to changes in cNOS activity and thus in agreement
with our results from short-term incubations of mouse islets (Fig.
3C).
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DISCUSSION |
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This report provides the first direct evidence that short-time
(~60 min) exposure of pancreatic -cells to hyperglycemic glucose levels in vitro as well as in vivo will induce iNOS protein expression and activity. It is now well established that the normal healthy
-cell is equipped with ncNOS as revealed by conventional
immunocytochemistry (17, 26, 32), confocal microscopy
(5, 24), electron microscopy (24), and
biochemistry (24, 29). However, the significance of
cNOS-derived NO as a possible signaling molecule, as either a mediator
(13, 25, 32, 34, 35, 41) or a negative feedback inhibitor
(1-4, 15, 15-19, 26-29, 31, 38) of
glucose-stimulated insulin release in normal
-cell physiology, is
widely disputed. In contrast, NO derived from iNOS is now regarded as
an important cytotoxic factor in the pathogenesis of the
immunodestructive type I diabetes, whether produced from invading
macrophages or in the
-cells themselves (11, 14, 39).
In this context, note that previous immunocytochemical data from our
laboratory (5, 17, 18) did not reveal any contaminating
non-endocrine NOS-containing cells in the islets of normal mice or
rats. Furthermore, it was recently reported that transgenic mice
overexpressing iNOS in their
-cells developed severe diabetes with
ketonuria without presenting any signs of an accompanying insulitis
(37). Therefore, our present observation that short
hyperglycemic periods both in vitro and in vivo can induce expression
and a marked enzyme activity of iNOS in normal islets raises the
question of whether the NO produced during such periods might be a
contributing factor also in the pathogenesis of type II
non-insulin-dependent diabetes (NIDDM), for instance, by impairing
glucose-stimulated insulin release and/or via a progressive
"nonautoimmune" cytotoxic action.
Note that islet cNOS activity seems to be more rapidly adjusted to the glucose concentration than is iNOS activity. Thus, after 20-min incubation at high glucose, the cNOS activity was already increased to the same level as could be recorded after 60 min, whereas iNOS activity was not detectable at the 20-min time point. The rapid glucose-stimulated increase in cNOS activity is also amply illustrated in Fig. 6B, which shows that cNOS, but not iNOS, activity was increased already at 10 min after high glucose was introduced to perifused rat islets. Hence, an increase in cNOS enzyme activity and not a major increase in cNOS expression seems to be of importance during acute glucose stimulation, as is also suggested by the results of our Western blots. In contrast, in the in vivo experiments, after a large glucose dose, islet iNOS activity was still markedly increased at 75 min when blood glucose, plasma insulin, and islet cNOS activity displayed approximately the same values as the controls. Hence, at this time point there was no longer any active glucose-stimulated insulin release, and, consequently, no increase in cNOS activity could be recorded.
Our present finding, if applicable to human -cells, might also
underline the importance of a strict normoglycemic regimen in type II
diabetes and prediabetes. The intimate mechanisms behind the
glucose-stimulated iNOS activity and expression are presently unclear
and certainly remain to be elucidated. We cannot exclude the
possibility that the short time frame observed might reflect a
posttranscriptional event in which glucose acts on iNOS existing in an
inactivated and masked form. In this context, it has previously been demonstrated that iNOS in fact might exist in an inactive form in
normal untreated human platelets (9). However, caution must be exercised with regard to such an interpretation of our present
data, because minute amounts of iNOS mRNA have been found in
nonstimulated isolated mouse islets (40). The present
mechanism behind the increase in iNOS activity seems independent of
cytokine action and is possibly accomplished by a signal emanating from early glucose metabolism, because we have previously found that insulin release stimulated by glucose, but not by
-ketoisocaproic acid (KIC), was potentiated by 5 mmol/l L-NAME in
K+-diazoxide-treated islets (31), and
preliminary data in our laboratory have shown that insulinotropic
nutrients directly entering the mitochondria (e.g., leucine and KIC) do
not elicit any significant iNOS activity.
Glucose stimulation of cNOS activity, on the other hand, is at least
partly explained by the fact that glucose increases the entry of
extracellular Ca2+ into the -cell, as well as the
production of NADPH generated from glucose metabolism
(43), and both Ca2+ and NADPH are known as
essential cofactors for stimulating an increase in cNOS activity
(10, 22). This is further underlined by our observation
that diazoxide, which prevents Ca2+ entry into the cell,
inhibited the glucose-stimulated increase in cNOS activity, whereas the
Ca2+-independent iNOS activity increased to a similar level
as in the absence of diazoxide. However, although diazoxide blocks the entry of extracellular Ca2+, intracellular Ca2+
is still able to increase (42), and thus our data are
suggestive of a crucial role for Ca2+ influx mechanisms in
activating cNOS activity. Moreover, a summary of the data shown in Fig.
1 might be in accord with our hypothesis that cNOS-derived NO serves as
a physiological negative feedback signal within the
-cell to avoid
inappropriate insulin release at hyperglycemic glucose levels. In such
a scenario, the lowest NO production would be observed at a normal,
basal glucose range (i.e., for freely fed mice around 5-8 mmol/l
glucose). This is also the case; i.e., we found the lowest NO
production at 7 mmol/l glucose. In addition, in ancillary
experiments we found no difference whether the islets were incubated in
4 or 7 mmol/l glucose. Furthermore, the pattern of the dynamics of
glucose-stimulated insulin release in perifused rat islets in the
absence and presence of 5.0 mmol/l L-NAME is suggestive of
cNOS-derived NO being of some importance for the negative peak
separating the first and second phase, because the NOS inhibitor almost
abolished this peak. In contrast, the accumulated data suggest that the
importance of the slowly emerging iNOS activity for biphasic insulin
release is less prominent, iNOS possibly being a negative modulator of
-cell function within a time frame longer than 60 min. Hence, this
question needs to be further investigated in future experiments.
Also, our other experiments presented here strongly suggested that NO inhibits glucose-stimulated insulin release. Hence, the NOS inhibitor L-NMMA (but not its inactive enantiomer D-NMMA) suppressed glucose-stimulated NOS activities concomitant with an increase in insulin release. Furthermore, our data also suggest that NO exerts its major inhibitory influence on the secretory process independent of membrane depolarization events. Hence, the potentiation of glucose-stimulated insulin release after NOS inhibition by L-NAME as well as the suppression of insulin release by exogenous NO gas were both markedly exhibited also in K+-diazoxide treated islets. These data speak in favor of the inhibitory effects of NO being exerted at more distal events in the stimulus-secretion coupling of glucose-induced insulin release and thus that the previously reported influence by NO on opening of KATP channels (7) might be of less importance in this context. Interestingly, Lajoix et al. (24) very recently presented electron microscopic evidence that cNOS is mainly localized in the insulin secretory granules and, to a lesser extent, in the mitochondria and nucleus. The localization of cNOS to the secretory granules gives a morphological background to the idea of a close regulatory role for NO in the secretory process. Furthermore, the partial localization of cNOS to mitochondria, although less prominent, might be suggestive of a cNOS cytochrome c reductase activity (21) operating in certain situations and thus creating a more complex effect by the cNOS enzyme on the insulin secretory mechanisms (24).
Activating effects of NO on -cell exocytosis at low glucose as
reported by Willmott et al. (41) is not applicable to
glucose stimulation because, in contrast to basal
"non-glucose-stimulated" insulin secretion, glucose-evoked insulin
release is highly influenced by certain thiol-dependent enzymes and
regulatory proteins (6), which presumably are inactivated
by NO through S-nitrosylation processes (36).
Hence, it seems conceivable that nutrient-stimulated insulin secretion,
which has long been known to be incapacitated by a deranged balance of
intracellular reduced glutathion/oxidized glutathion ratio (GSH/GSSG)
(6), is suppressed by S-nitrosylation at
various distal sites in the stimulus-secretion coupling. The precise
nature of these sites/regulatory proteins, however, remains to be
elucidated. In this context, we have very recently found that the
insulin-releasing effect of certain agents activating the cAMP system
such as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine is
not inhibited but rather tends to be slightly potentiated by NO in the
presence of low glucose (3). This insulin-releasing pathway is reportedly thiol independent (6) and thus
escapes the strong inhibitory action of NO exerted on the
nutrient-stimulated thiol-dependent pathway. In fact, very recent
experiments in our laboratory have shown that glucagon, a classic cAMP
activator, has the ability to suppress glucose-stimulated NOS activity
(17).
Finally, our results obtained in perifused rat islets in the presence
of 0.5 mmol/l L-NMMA clearly show the danger of using NOS
inhibitors without a concomitant determination of islet NOS activity.
It is known from other tissues (8, 33) that
L-NMMA, in certain concentrations and experimental
situations, might in fact stimulate NO production. Hence, it seems
indispensable to perform analysis of islet cNOS and iNOS enzyme
activities when assessing the effects of different NOS inhibitors and
NOS inhibitor concentrations on insulin secretion. In this context, it
should be added that recent experiments in our laboratory
(18) have shown that both 1 mmol/l L-NAME and
1 mmol/l 7-nitroindazole (used in Fig. 2) do indeed inhibit islet
cNOS-derived NO production, and thus the data in Fig. 2 indicate that a
true inhibition of islet NO production results in a marked increase in
glucose-stimulated insulin release. In view of the unpredictable
behavior of different NOS inhibitors with regard to normal -cells
(also including mixed stimulatory and inhibitory effects giving "zero
result"), it seems understandable why some investigators either get
the impression that NO is a positive modulator in glucose-induced
stimulus-secretion coupling (13, 25, 32, 34, 35, 41) or
conclude that cNOS-derived NO is of negligible importance in this
respect (20). Moreover, with regard to the effects of NO
on insulin secretion in different
-cell lines, most data suggest
that NO stimulates or potentiates insulin release (13, 25,
32), thus displaying more similarity to the secretory pathways
regulating basal or cAMP-stimulated insulin release in normal
-cells
(3, 18) than the true glucose-stimulated
(nutrient-stimulated) pathway. Hence, we must draw the conclusion that
certain hitherto employed
-cell lines (e.g., RINm5F and HIT-T15),
whether claimed "glucose responsive" or not, at least with regard
to NO effects (12, 13, 25, 32), are indeed not equipped
with the appropriate, normal stimulus-secretion coupling machinery for
glucose-stimulated insulin release.
In conclusion, glucose was found to dose-dependently stimulate insulin
secretion and the activities of both cNOS and iNOS along with a
simultaneous expression of cNOS and iNOS proteins in incubated mouse
islets. Moreover, short-time hyperglycemia (~60 min) in mice induced
the expression and activity of islet iNOS. Experiments with addition of
NO gas, an NO donor, or different NOS inhibitors to incubated islets
revealed that NO presumably serves as a negative feedback inhibitor of
glucose-stimulated insulin release. This negative feedback effect was
exerted distal to the depolarization events because it was also
elicited in K+-diazoxide-treated islets. Finally, we
observed that a low concentration of L-NMMA (0.5 mmol/l),
in fact, stimulated islet NO production in parallel with an inhibition
of first-phase glucose-stimulated insulin release in perifused rat
islets. In contrast, 5.0 mmol/l L-NMMA markedly suppressed
NO production concomitant with a great potentiation of
glucose-stimulated insulin release, showing that experiments with
different NOS inhibitors always should be accompanied by measurements
of NOS activities. The results strongly suggest, but certainly do not
definitely prove, that hyperglycemic concentrations of glucose
stimulates islet NOS activities both in vitro and in vivo and that NO
might serve as a negative feedback inhibitor of the glucose-stimulated
insulin secretory process. The data also suggest that prolonged
glucose-induced stimulation of islet NOS activities results in an
NO-mediated impairment of -cell function, which might be one of
multiple factors involved in the development of NIDDM.
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ACKNOWLEDGEMENTS |
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The technical assistance of Britt-Marie Nilsson and the secretarial help of Eva Björkbom are gratefully acknowledged.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (14X-4286), the Swedish Diabetes Association, the Albert Påhlsson Foundation, the Crafoord Foundation, the Golje Foundation, and the Åke Wiberg Foundation.
Address for reprint requests and other correspondence: I. Lundquist, Institute of Physiological Sciences, Dept. of Pharmacology, BMC F13, S-221 84 Lund, Sweden (E-mail: Ingmar.Lundquist{at}farm.lu.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 20, 2002;10.1152/ajpcell.00537.2001
Received 9 November 2001; accepted in final form 24 February 2002.
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