Institute of Physiological Sciences, 1 Departments of Pharmacology and 2 Pathology, University of Lund, Lund, Sweden
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ABSTRACT |
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Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.
chronic nitric oxide synthase blockade; nitric oxide and carbon monoxide; insulin and glucagon secretion; isolated islets; cyclic nucleotides
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INTRODUCTION |
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NITRIC OXIDE (NO) is today considered as a ubiquitous messenger molecule in many different organ systems (11, 28). It is also produced in the islets of Langerhans (16, 41, 47, 50) and might serve as a physiological regulator of islet hormone secretion (2-5, 7, 16, 17, 21, 26, 27, 31, 39-41, 47-50, 52). NO is a short-lived free radical gas that results from oxidation of L-arginine into L-citrulline, a reaction that is catalyzed by nitric oxide synthase (NOS; see Ref. 28). There are two main forms of NOS: a Ca2+/calmodulin-dependent constitutive (cNOS) and a Ca2+/calmodulin-independent inducible (iNOS) form (28). Furthermore, there are at least two isoforms of the constitutive enzyme, i.e., endothelial cNOS and neuronal cNOS (ncNOS; see Refs. 11 and 28). The different forms of NOS are inhibited by L-arginine analogs (53). The simplest of these compounds, NG-nitro-L-arginine, is reportedly an inhibitor of NOS and is widely used as its methyl ester analog NG-nitro-L-arginine methyl ester (L-NAME; see Ref. 53). Also, nonarginine analogs such as nitrated indazoles, e.g., 7-nitroindazole, have recently been shown to be potent NOS inhibitors (10, 53).
We and others have shown that both cNOS and iNOS occur in the islets of
Langerhans (5, 16, 19,
41, 39, 47, 49, 50). There are increasing amounts of data suggesting that
NO derived from iNOS is implicated in the autoimmune destruction and
dysfunction of -cells in insulin-dependent diabetes mellitus (16, 17, 19) and that different
cytokines induce the expression of iNOS (16,
17, 19). However, the role of islet cNOS,
which produces NO in a pulsatile way and in much lower absolute amounts than iNOS, is more controversial (2-3,
7, 17, 21, 26, 27, 31, 40, 41,
47-50, 52), especially since the
earliest studies suggested that NO was either a stimulator
(31, 50), an inhibitor (41), or
had no effect (27) on insulin secretion. In a number of
studies concerning short-term effects of blockade and stimulation of
cNOS, we have shown that NO derived from cNOS inhibits insulin
secretion induced by glucose, L-arginine, and cholinergic
stimulation, whereas it increases glucagon secretion (2-4, 26, 39,
41, 44, 47-49). In the
present study, the aim was focused on the possible effects of a
long-term NOS blockade on insulin and glucagon secretion by giving mice
the NOS blocker L-NAME in their drinking water for 4-6
wk. A few earlier studies did not reveal any effects of such a
treatment on in vivo insulin secretion (43,
55), and the results thus suggested that NO did not have
any appreciable effects on islet hormone secretion. However, the direct
effects of chronic L-NAME treatment on islet NOS activity
and NO production have never been investigated. Only general indirect
indexes of NOS blockade (e.g., increase in blood pressure and aortic
wall cGMP content) have been used (8, 43, 55). Interestingly, recent investigations on long-term
L-NAME treatment did not reveal any inhibition of NO
synthesis in the intestine or the liver because of a compensatory
increase in iNOS activity (37). Moreover, because we very
recently have discovered (24, 25) that the
islets of Langerhans express the constitutive isoform of the
CO-producing enzyme heme oxygenase (HO)-2 and because our data
suggested (24) that the CO produced could suppress islet
NOS activity and stimulate both insulin and glucagon release, we were
interested to see whether long-term blockade of NOS would affect islet
CO production and thus, in turn, the secretion of insulin and glucagon.
The effects of chronic NOS blockade were compared both in freely fed
and 24-h-fasted animals, since preliminary data in our laboratory
suggested that the islet NO-producing system might be activated by
fasting and thus could be an important factor behind the diabetic-like
condition displayed in starvation (51). To get a more
complete biochemical pattern of islet NOS activity in the present
experimental situation, we have now adapted our original assay of total
NOS activity in isolated islets (47) to include
determination of both cNOS and iNOS activity. Complementary data were
obtained by immunoblotting for both neuronal (n)cNOS and iNOS. Hence,
the present investigation was conducted to study in detail the effects
of a long-term NOS blockade on insulin and glucagon secretion in
relation to expression and activity of mouse islet cNOS and iNOS.
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MATERIALS AND METHODS |
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Animals. Female mice of the NMRI strain (B&K, Sollentuna, Sweden) weighing 25-30 g were used in all studies. Freely fed animals were fed a standard pellet diet (B&K) and tap water ad libitum before and during all experiments. In the fasting experiments, food was withdrawn for 24 h before the experiment started. These mice had free access to water during this period. The animal experiments were approved by the local animal welfare committee.
Chronic NOS blockade.
The mice were given 50 mg · kg1 · day
1 L-NAME in the drinking water for
4-6 wk. This dose has been proved to cause NOS blockade in rats
(43). Control mice were given drinking water only. The L-NAME-drinking mice displayed a normal weight gain, and
their body weights at the time of islet isolation or the performance of
in vivo experiments were similar to the controls.
Drugs and chemicals. Collagenase (CLS 4) was obtained from Worthington Biochemicals (Freehold, NJ). BSA was from ICN Biochemicals (High Wycombe, UK). 7-Nitroindazole was obtained from Calbiochem (La Jolla, CA). L-NAME, forskolin, and all other chemicals were from Sigma Chemical (St. Louis, MO). The RIA kits for insulin and glucagon determination were obtained from Diagnostika (Falkenberg, Sweden) and Euro-Diagnostica (Malmö, Sweden).
Immunocytochemistry. Pancreatic glands from freely fed L-NAME-drinking mice were dissected and divided in pieces and were processed further for immunocytochemistry of ncNOS and iNOS as previously described in detail (5, 24). In control experiments, iNOS immunoreactivity was only observed in animals injected with endotoxin (lipopolysaccharide) in, e.g., macrophages in lung and liver, in which no ncNOS immunoreactivity could be visualized. The immunocytochemical results were also tested by observers not knowing original data.
In vivo experiments. D-Glucose was dissolved in 0.9% NaCl (saline). Forskolin was dissolved in DMSO and then diluted in saline. Glucose (3.3 mmol/kg) and forskolin (7.3 µmol/kg) were injected intravenously (volume load: 5 µl/g mouse) into a tail vein. Controls received either saline (glucose) or vehicle (forskolin). Blood sampling was performed as described previously (44). The concentrations of insulin and glucagon in plasma were determined by RIA (1, 22, 42). Plasma glucose concentrations were determined enzymatically (14).
Hormone secretion in vitro. Preparation of isolated pancreatic islets from mouse was performed by retrograde injection of a collagenase solution via the bile-pancreatic duct (20). Islets were then isolated and hand picked under a stereomicroscope at room temperature. The islets were then preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer (29), pH 7.4, supplemented with 10 mmol/l HEPES, 0.1% BSA, and 1 mmol/l glucose. Each incubation vial contained 10 islets in 1.0 ml buffer solution and was gassed with 95% O2-5% CO2 to obtain constant pH and oxygenation. After preincubation for 30 min, the buffer was changed to a medium containing 1, 7, or 20 mmol/l glucose and the different test agents, and the islets were then incubated for 60 min. All incubations were performed at 37°C in an incubation box (30 cycles/min). Immediately after incubation, aliquots of the medium were removed and frozen for subsequent assays of insulin and glucagon (1, 22, 42).
Assay of islet NOS.
Freshly isolated islets were thoroughly washed and collected in
ice-cold buffer (200 islets in 840 µl buffer) containing 20 mmol/l
HEPES, 0.5 mmol/l EDTA, and 1 mmol/l DL-dithiothreitol (DTT), pH 7.2, and were immediately frozen at 20°C. For comparative purposes, small pieces of mouse cerebellum were also removed and frozen
in the same DTT-containing buffer as described above. On the day of
assay, the islets and the specimens of cerebellum were sonicated on
ice, and the buffer solution containing the islet homogenate was
reconstituted to contain, in addition to the above-mentioned compounds,
also 0.2 mmol/l L-arginine, 0.45 mmol/l CaCl2,
2 mmol/l NADPH, and 25 units calmodulin in a total volume of 1 ml. To
determine iNOS activity, both Ca2+ and calmodulin were
omitted. The buffer composition was essentially the same as previously
described for assay of NOS in brain tissue using radiolabeled
L-arginine (13). The crude homogenate was then
incubated at 37°C under constant air bubbling (1.0 ml/min) for 3 h. Aliquots of the incubated homogenate (200 µl) were then passed
through a 1-ml Amprep CBA cation-exchange column for HPLC analysis of
the L-citrulline formed, according to Carlberg
(15). Because L-citrulline is created in
equimolar concentrations to NO and because L-citrulline is
stable whereas NO is not, L-citrulline is the preferred
parameter when measuring NO production. The method has been described
in detail earlier (15, 47). cNOS activity was
calculated as total NO production minus
Ca2+/calmodulin-independent NO production. Protein was
determined by the method of Bradford (12) on samples from
the original homogenate.
Western blot analysis. Approximately 200 islets were hand picked in Hanks' buffer under a stereomicroscope and were sonicated on ice (3 × 10 s). The protein content was determined according to Bradford (12). Homogenate samples representing 20 µg of total protein from islet tissue were then processed as previously described (24). Immunoblotting with rabbit anti-mouse ncNOS (N-7155), iNOS (N-7782; 1:2,000; Sigma), and HO-2 and HO-1 (1:2,000) antibodies (StressGen Biotechnol, Victoria, Canada) was performed for 16 h at room temperature. Antibody binding to ncNOS, iNOS, HO-2, and HO-1 was detected using 0.25 mmol/l CDP-Star (Boehringer Mannheim, Mannheim, Germany) and the signal enhancer Nitro Block II (Tropix, Bedford, MA) for 5 min at room temperature. The chemiluminescence signal was visualized by exposing the membranes to Dupont Cronex X-ray films for 1-5 min.
Measurement of islet cAMP and cGMP.
The methodology has been described previously (40).
Incubation of isolated islets in the presence of the phosphodiesterase inhibitor IBMX (0.2 mmol/l) was stopped by removal of the buffer and
the addition of 0.5 ml of ice-cold 10% TCA followed by immediate freezing in a 70°C ethanol bath. Before assay, 0.5 ml of
H2O was added, and the samples were sonicated for 3 × 5 s followed by centrifugation at 1,100 g for 15 min.
The supernatants were collected and extracted with 4 × 2 ml of
water-saturated diethyl ether. The aqueous phase was removed and
freeze-dried using a Lyovac GT 2 freeze-drier. The residue was then
dissolved in 450 µl of 50 mmol/l sodium acetate buffer (pH 6.2). The
amounts of cAMP and cGMP were quantified with a
125I-labeled cAMP and 125I-labeled cGMP RIA kit
(RIANEN; Du Pont, Boston, MA). [3H]cGMP was added to the
TCA islet homogenate to determine the recovery of cAMP and cGMP during
the ether extraction. The mean recovery was 85%.
Measurement of islet HO activity.
CO production was determined with a sensitive gas chromatographic
method essentially as previously described (24,
25). Islets were isolated and hand picked under a
stereomicroscope at room temperature and then, unless otherwise stated,
thoroughly washed and collected in ice-cold phosphate buffer (0.1 mol/l, pH 7.4, ~300 islets in 200 µl buffer). Thereafter, the
islets were immediately frozen at 20°C. On the day of assay, the
islets were sonicated on ice, and methemalbumin (30 µl),
-NADPH
[100 µl; 4 mg dissolved in 1 ml phosphate buffer (0.1 mol/l)], and Hb (2 mg) were added together with phosphate buffer up to a final volume of 1 ml. Methemalbumin solution was prepared by dissolving 25 mg
hemin, 82.5 mg NaCl, and 12.1 mg Tris base in 5 ml of 0.1 mol/l NaOH
followed by the addition of 5 ml albumin solution (20 g/l) and 5 ml
distilled water. The homogenate was then incubated in a water bath at
37°C under protection from light. Aliquots (330 µl) were taken
after 6 min of incubation, which was terminated by placing the tubes on
ice. The samples were then injected in reaction tubes containing
ferricyanide-citric acid (100 µl). Nitrogen was used as a carrier gas
and to purge the reaction vessels for 4 min before the samples were
injected in them. After a reaction time of 4 min, the liberated CO was
brought to a nickel catalyst, mixed with H2, and then
brought further as methane to the detector. CO (99.9%) was used as a
standard. The amount of CO produced was calculated from the area under
the curve. Protein was determined according to the method of Bradford
(12) on samples from the original homogenate.
Statistics. Statistical significance between sets of data was assessed using unpaired Student's t-test or, where applicable, ANOVA followed by Tukey-Kramer's multiple comparisons test. Results are expressed as means ± SE.
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RESULTS |
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Effect of chronic L-NAME treatment on total NO
production (total NOS activity) from pancreatic islets and cerebellum.
The total NO production was determined in the presence of
Ca2+ and calmodulin, and the results obtained reflect the
total NO production of both cNOS
(Ca2+/calmodulin-dependent) activity and iNOS
(Ca2+/calmodulin-independent) activity. The total
production of NO in mouse islets from freely fed and 24-h-fasted mice
and from cerebellum of freely fed mice is shown in Fig.
1A. In freely fed control
mice, total islet NOS activity was 25 pmol NO · min
1 · mg protein
1, whereas islets
from freely fed NAME-drinking mice showed a considerably (more than
doubled) higher total NO production (52 pmol NO · min
1 · mg protein
1; Fig.
1A). Moreover, fasting the control mice for 24 h was
found to markedly increase islet NO production (37 pmol NO · min
1 · mg protein
1) compared with
islets of the freely fed controls. In islets from L-NAME-drinking fasted mice, the production of NO was not
further increased compared with L-NAME-drinking freely fed
mice (47 vs. 52 pmol NO · min
1 · mg
protein
1). NO production was, however, still
significantly greater in islets from fasted NAME-drinking mice (47 pmol
NO · min
1 · mg protein
1) than
in fasted control mice (37 pmol NO · min
1 · mg protein
1). In comparison, also in cerebellar specimens
of freely fed mice, NO production was considerably increased after
L-NAME drinking (Fig. 1A).
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Ca2+/calmodulin-dependent (cNOS activity)
and Ca2+/calmodulin-independent (iNOS activity)
NO production in islets from L-NAME drinking mice and
control mice.
iNOS activity was estimated as NO formation measured in the absence of
Ca2+ and calmodulin (Fig. 1, B and
C). In islets from freely fed L-NAME-drinking mice, iNOS activity attained a high level (40 ± 2.1 pmol NO
· min1 · mg protein
1), whereas
very low iNOS activity (1.1 ± 1.1 pmol NO · min
1 · mg protein
1; not different
from 0) was demonstrated in freely fed control mice (Fig.
1B). In comparison, islets of fasted control mice expressed an overt iNOS activity in their islets (15 ± 1.3 pmol NO · min
1 · mg protein
1), and in islets
of fasted L-NAME-drinking mice the iNOS activity was even
higher (26 ± 3.0 pmol NO · min
1 · mg
protein
1; P < 0.02; Fig. 1C).
Western blot analysis. ncNOS expression could be detected in islets from both control and L-NAME-drinking freely fed mice. However, the expression was clearly stronger in the control group (Fig. 1D). In comparison, the opposite pattern could be detected for iNOS expression, i.e., no iNOS expression in control islets but strong iNOS expression in islets after L-NAME drinking (Fig. 1E). Moreover, iNOS expression could also be clearly detected in islets of 24-h-fasted control mice (data not shown).
Immunocytochemical findings.
In L-NAME-drinking mice, most islet cells exhibited
significant iNOS immunofluorescence (Fig.
2B), which contrasted against the exocrine tissue. By comparison, in control mice, the islet cells
displayed a very weak tissue fluorescence comparable to that in
surrounding exocrine tissue (Fig. 2A). In control mice, most
islet cells expressed ncNOS immunofluorescence (Fig. 2C), which was clearly reduced in intensity in L-NAME-drinking
mice (Fig. 2D).
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Effects of chronic L-NAME treatment on basal plasma levels of insulin, glucagon, and glucose in freely fed and 24-h-fasted mice. L-NAME-drinking mice did not show significant differences in basal plasma levels of insulin or glucagon compared with controls, irrespective of whether they were freely fed or fasted for 24 h (data not shown). Basal plasma glucose levels, however, were increased in fasted L-NAME-drinking mice (controls 5.72 ± 0.29 vs. L-NAME drinking 8.83 ± 0.32; P < 0.001) but not in L-NAME-drinking freely fed mice (controls 11.80 ± 0.44 vs. L-NAME drinking 11.90 ± 0.74).
Effect of chronic L-NAME treatment on insulin and
glucagon release from isolated islets in the presence of low and high
glucose concentrations in freely fed and 24-h-fasted animals.
Figure 3A shows that insulin
secretion at a high glucose concentration (20 mmol/l) was sixfold
greater compared with a low glucose concentration (1 mmol/l) in both
freely fed controls and L-NAME-drinking mice. Furthermore,
there was no apparent difference compared with controls after
L-NAME drinking, either at low or high glucose
concentrations. However, insulin secretion was significantly greater at
a high concentration of glucose in islets of fasted L-NAME-drinking mice compared with islets of fasted
control mice (Fig. 3C). This difference was not seen at the
low concentration of glucose. In comparison, glucagon secretion was
higher both at low and high glucose concentrations in the
L-NAME drinking group in both freely fed (Fig.
3B) and 24-h-fasted animals (Fig. 3D).
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Effects of chronic L-NAME treatment on the in vivo
dynamics of insulin, glucagon, and glucose in plasma after an acute
glucose injection in freely fed and 24-h-fasted animals.
No significant differences were observed between freely fed
L-NAME-drinking mice and controls with regard to the
insulin and glucagon response after an intravenous glucose load (Fig.
4, A and B). There
was, however, a very slight improvement of the glucose tolerance curve
in the L-NAME group (Fig. 4C). In fasted control mice (Fig. 4, D-F) the acute insulin response after glucose
injection was markedly impaired compared with the freely fed animals,
but it was significantly increased in fasted
L-NAME-drinking mice compared with fasted controls (Fig.
4D). This was accompanied by a greatly improved glucose
tolerance curve (Fig. 4F). There was no apparent difference
in the glucagon response between fasted controls and fasted
L-NAME-drinking mice (Fig. 4E).
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Short-term in vitro effects of two different NOS inhibitors
(L-NAME and 7-nitroindazole) in the presence of 20 mmol/l
glucose on hormone release and NO production from isolated islets taken
from control or L-NAME-drinking mice.
To assess the effects of the two chemically different NOS
inhibitors L-NAME and 7-nitroindazole on islet cNOS and
iNOS activities, we measured the NO production from control islets
incubated at 20 mM glucose in the presence of these inhibitors. Figure
5A shows that islet cNOS
activity was dose dependently inhibited by 1 and 5 mM
L-NAME and that 7-nitroindazole (1 mM) was a potent cNOS inhibitor. Moreover, 7-nitroindazole markedly inhibited both cNOS and
iNOS, whereas L-NAME was a good inhibitor of cNOS but
displayed practically no effect on iNOS activity during the present
experimental conditions (Fig. 5A). In addition, it should be
noted that islet incubation with 20 mM glucose itself apparently
stimulated iNOS activity (compare with Fig. 1B showing no
iNOS activity in freshly isolated nonincubated islets).
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Addition of L-NAME to the incubation medium
(60-min incubation) increased insulin secretion from isolated islets
taken from both L-NAME-drinking and control freely fed mice
in the presence of 20.0 mmol/l glucose (Fig. 5B).
This effect was much more pronounced in islets from
L-NAME-drinking mice than in the control islets. In the
presence of 5 mmol/l L-NAME, the secretion of insulin was
almost doubled [9.64 ± 1.21 (L-NAME drinking)
vs. 5.33 ± 0.79 (control) ng insulin · islet1 · h
1]. Addition of
7-nitroindazole also markedly increased insulin secretion from islets
of both controls and L-NAME-drinking mice (Fig.
5B). Figure 5C shows that the highly increased
glucagon secretion in the L-NAME-drinking group was
inhibited by the addition of 5 mmol/l L-NAME or 1 mmol/l
7-nitroindazole to the incubation medium. Also in fasted animals,
L-NAME and 7-nitroindazole potentiated insulin secretion
from islets of both controls and L-NAME-drinking mice (Fig.
5D). However, only at 5 mmol/l of L-NAME was a
slightly greater secretion of insulin seen from islets of fasted
L-NAME-drinking mice compared with the effects of
L-NAME in fasted controls (Fig. 5D). In
contrast, at this high glucose concentration, glucagon secretion in
fasted control animals was not affected by the addition of the NOS
inhibitors (Fig. 5E). However, in islets of fasted L-NAME-drinking animals, the high rate of glucagon release
in islets could still be significantly inhibited by both
L-NAME and 7-nitroindazole (Fig. 5E).
Influence of chronic L-NAME treatment on islet
cAMP and cGMP content.
Islets from freely fed L-NAME-drinking mice incubated
in the presence of 1 mmol/l glucose for 1 h showed a fourfold
greater content of cAMP than control islets (Table
1). Incubation of islets at a high
concentration of glucose (20 mmol/l) evoked a further increase in cAMP
levels in control islets but not in islets of
L-NAME-treated mice. In fact, at this high concentration of glucose, the cAMP content was somewhat lower (30%) in islets of
L-NAME-drinking mice. cGMP levels were significantly
greater (+60%) in islets of L-NAME-drinking mice than in
islets of control mice after incubation at a low (1 mmol/l)
concentration of glucose (Table 1). At a high concentration of glucose
(20 mmol/l), islet cGMP content was increased to the same level in both
controls and L-NAME-drinking mice.
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Effect of chronic L-NAME treatment on insulin and
glucagon release stimulated by the adenylate cyclase activator
forskolin in freely fed and 24-h-fasted animals.
In view of the increased islet cAMP levels after
L-NAME drinking, the effect of the adenylate cyclase
activator forskolin was tested. Figure
6A shows that the in vivo
insulin response to forskolin was of similar magnitude in freely fed
L-NAME-drinking and control animals. In fasted animals,
however, the insulin response was markedly increased in the
L-NAME-drinking group. In comparison, there was no effect
by L-NAME drinking on the glucagon response to
forskolin in either freely fed or fasted animals (Fig. 6B).
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Effect of chronic L-NAME treatment on CO production and
protein expression of the CO-producing isoenzymes HO-2 and HO-1 in
pancreatic islets.
To elucidate a possible contribution by the newly discovered HO-2-CO
pathway having a stimulatory effect on insulin secretory mechanisms
(24, 25) in the restoration of
glucose-stimulated insulin release from islets of
L-NAME-drinking mice, we measured CO production and
expression of HO isoenzymes. In islets from freely fed
L-NAME-drinking mice, the production of CO was fivefold higher compared with control mice (Fig.
7A). Immunoblotting revealed that this increase was accompanied by an increased expression of the
constitutive HO-2 protein (Fig. 7B). In contrast, no HO-1 expression could be seen in islet tissue of either freely fed mice or
controls after L-NAME drinking (Fig. 7B).
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DISCUSSION |
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Immunoblots, immunocytochemistry, and biochemistry demonstrated the presence of a constitutive NOS isoform (ncNOS) in the islets of both control and L-NAME-drinking mice. The expression of cNOS, however, was markedly decreased after L-NAME drinking. This was in accordance with the biochemical findings of a decreased islet cNOS activity. An accidental but interesting immunocytochemical observation in this context was that the ncNOS immunofluorescence exhibited by a cell body of probably neuronal nature situated close to the islet shown in Fig. 2D (L-NAME-drinking mice) was not at all reduced by L-NAME treatment, being suggestive of a different reaction pattern to chronic L-NAME treatment in peripheral nitrinergic neurons compared with the islet endocrine cells.
No iNOS was found in freshly isolated islets of freely fed control mice, but there was a strong expression of iNOS in the islets of L-NAME-treated mice that contributed to an increase in total islet NO production. The compensatory increase in iNOS activity after chronic L-NAME drinking suggests the occurrence of a regulatory NO-producing system within the islets of Langerhans trying to assure adequate production of NO by inducing iNOS expression when cNOS activity is suppressed and thus maintain the function of NO as a negative feedback modulator of glucose-stimulated insulin release (24, 40, 47-49). One possible mechanism responsible for the compensatory induction of islet iNOS could be the decrease in cNOS-derived NO production after L-NAME drinking. It should be noted that we found no evidence of insulitis or macrophage invasion in the islets of our L-NAME-drinking mice, and thus the measured NOS activities in the present study do not involve cytokine stimulation and are to be referred to as islet endocrine cells. This is also emphasized by our immunocytochemical data.
In control mice we found, unexpectedly, that a fasting period of 24 h evoked an increase in islet total NO production due to a marked induction of iNOS expression. Because fasting is known to greatly reduce glucose-stimulated insulin release (32) and because NO is a negative modulator of the insulin response to glucose (24, 40, 47-49), the present data suggested to us that the increased total NO production in islets from fasted animals might be an important mechanism in fasting-induced impairment of glucose-stimulated insulin release (32) and thus also an essential factor in the induction of the diabetic-like condition known to occur in starvation, such as increased lipolysis and glycolysis, increased gluconeogenesis, increased peripheral insulin resistance, and impaired insulin response to glucose (51).
We now expected that chronic treatment with L-NAME would inhibit starvation-induced NO production and thus improve this "diabetic" condition in fasted mice. In fact, the starvation diabetes was modestly improved, at least with regard to glucose-stimulated insulin release and glucose tolerance, but not in the way we had anticipated, since, similar to what happened in freely fed mice, chronic L-NAME drinking induced a further increase in islet NO production (due to iNOS). This improvement might be explained, at least partially, by a previous observation (30) that 8-bromo-cGMP (8-BrcGMP) potentiated glucose-stimulated insulin release in islets from starved but not freely fed rats.
NO is known to increase soluble guanylate cyclase activity and cGMP production (28, 33). In accordance with this, islet cGMP levels, probably due to increased iNOS activity, were increased after long-term L-NAME treatment when islets were incubated in the presence of a low glucose concentration. In contrast, at a high glucose concentration, which by itself increases islet NO production (Fig. 5A and Ref. 24), no appreciable difference was seen. It is not inconceivable that the NO-evoked negative feedback effect on glucose-stimulated insulin release (24, 40, 47) might be mediated by the cGMP system. However, we have also recently shown that cGMP might be involved as a possible short-term modulator of CO-stimulated insulin and glucagon secretion (24, 25). Such a cGMP-mediated direct stimulatory mechanism is apparently not operating for NO with regard to glucose-stimulated insulin release. However, the enhanced NO production in islets of L-NAME-drinking mice to increase islet cAMP levels after incubation in low glucose is probably attributed to the cGMP-inhibited cAMP-phosphodiesterase (56). Hence, this cGMP effect upregulating the cAMP system might contribute to the enhanced insulin and glucagon secretion at low glucose in islets from L-NAME-drinking mice. At high glucose, however, the further increase in NO production might adversely affect the cAMP production, as previously described (18).
Insulin secretion after chronic L-NAME drinking. Insulin secretion is regulated by a complex chain of events exerted in response to different metabolic, nervous, and hormonal factors (59). Glucose, the major insulin secretagogue, is known to initiate insulin release through closure of ATP-sensitive K+ channels, with subsequent depolarization and opening of voltage-dependent Ca2+ channels leading to increased intracellular Ca2+ and subsequent exocytosis of insulin. This secretory process is regulated by different intracellular modulatory systems, among which the importance of cNOS-derived NO as a negative or positive modulator has been discussed (26, 27, 47, 49, 50).
Chronic NOS inhibition in the rat has been reported to have no effects on in vivo insulin secretion and, furthermore, no influence on glucose tolerance or insulin resistance (8, 43, 55). Our observation that glucose-stimulated insulin secretion was unaffected after L-NAME drinking might at first glance favor the assumption that the secretion of insulin is independent of NO production (27, 43) more than our previous suggestion that NO is an important negative modulator of glucose-induced insulin release (24, 40, 47-49). However, when studying the effects of in vitro addition of two chemically different NOS inhibitors, L-NAME and 7-nitroindazole (10, 53), on glucose-stimulated insulin secretion, we found that insulin release from islets of both controls and L-NAME-drinking mice was greatly increased in the presence of the NOS inhibitors. Moreover, we could directly show here by biochemical assay of islet cNOS and iNOS activities that the increase of glucose-stimulated insulin release in the presence of L-NAME or 7-nitroindazole was accompanied by a parallel decrease in cNOS activity. Interestingly, such a parallel decrease was not found with regard to iNOS, thus suggesting that iNOS-derived NO might be less efficient than cNOS-derived NO in suppressing insulin release, at least during short-term glucose stimulation. Indeed, the present data suggest that L-NAME is a potent inhibitor of islet cNOS, whereas 7-nitroindazole is a slightly more efficient inhibitor against iNOS than against cNOS. Moreover, the results also underline the fact emphasized in a recent review (53) that different NOS inhibitors display different inhibitory effects against cNOS and iNOS dependent on the type of tissue investigated. We have repeatedly found that NO is a negative modulator of insulin secretion induced by glucose, L-arginine, and cholinergic stimulation (2, 4, 26, 39, 40, 47-49). The mechanisms of the inhibitory effects of NO on nutrient-induced insulin secretion are still unclear but most probably are exerted through the formation of S-nitrosothiols (54), thereby impairing important regulatory thiol groups, which have long been known to be essential for glucose-induced insulin secretion (6, 23). Hence, any possible stimulating effect of NO on insulin release exerted via the cGMP system in theGlucagon secretion after chronic L-NAME drinking.
The physiological mechanisms behind the secretion of glucagon are not
so well understood. It has been known for a long time that the
glucagon-producing cells, being located in the periphery of the islets,
are richly innervated by both adrenergic and cholinergic nerves
(34, 38, 58), that adrenergic as
well as cholinergic stimulation induces an increase in glucagon
secretion (38, 45, 58), and that
Ca2+ and the cAMP system are important intracellular
messengers in mediating stimulus-secretion coupling of glucagon release
(45). We originally proposed (39) that NO
should be looked upon as a novel physiological stimulator of glucagon
secretion, and we have now demonstrated that the glucagon-producing
-cells indeed harbor the cNOS enzyme (5). Moreover, we
have previously shown that glucagon secretion induced by
L-arginine or by cholinergic stimulation is inhibited by
different types of NOS inhibitors both in vivo and in vitro
(2-4, 26, 47,
49), thus suggesting that NO is a positive modulator of
glucagon release. Furthermore, we have recently found that addition of
NO gas to isolated islets stimulates glucagon secretion
(24). In the present paper, we now show that islets taken
from L-NAME-drinking mice display a markedly increased NO
production in parallel with an increased glucagon secretion at both low
and high glucose and after stimulating the cAMP system by forskolin.
The greatest increase in glucagon release, relative to control islets,
was found at 1 mM glucose in islets from fasted animals, thus being in
accordance with the notion that glucose is a potent inhibitor of
glucagon release. Interestingly, and somewhat unexpectedly, islets
from L-NAME-drinking mice displayed a marked increase in
glucagon secretion relative to controls, even when incubated at
high (20 mM) glucose. This was especially notable in islets from
fasted L-NAME-drinking animals, where also insulin, a
well-known inhibitor of glucagon release (36), was
markedly enhanced. These findings might be explained, at least partly,
by our recent observation (24) that glucose itself is a
potent stimulator of islet NO production. In addition, NO-stimulated
glucagon release seems to be less efficiently inhibited by insulin. Our
observation that NO might be a very potent stimulator of glucagon
release also in the presence of 20 mM glucose is shown, although
indirectly, in islets from fasted L-NAME-drinking mice. The
greatly increased glucagon release from these islets was markedly reduced after in vitro addition of the NOS inhibitors
L-NAME and 7-nitroindazole. The mechanism of action of
NO to stimulate glucagon release is presently unclear. However, much
evidence suggests that the cGMP-protein kinase G system constitutes an
important transduction pathway (cf. Table 1).
Possible islet compensatory mechanisms against the increased NO production during chronic L-NAME treatment. The present results showed that chronic L-NAME treatment of mice resulted in a marked increase in islet NO production. The inhibitory action of NO on glucose-stimulated insulin release (16, 39, 40, 47), however, had been clearly compensated for during the long-term L-NAME treatment, since the insulin response to glucose was found to be essentially normal both in vitro and in vivo in freely fed mice and even increased in fasted mice. As previously discussed, this could partly be explained by iNOS-derived NO being less efficient than cNOS-derived NO as an inhibitor of glucose-stimulated insulin release. However, evidently, the islet adenylate cyclase-cAMP secretory pathway contributed to the compensatory mechanisms, since islets taken from L-NAME-drinking mice displayed a marked increase in the sensitivity and activity of the cAMP system, as documented by a great increase in islet cAMP content at low glucose and a greatly enhanced insulin response to the adenylate cyclase activator forskolin. Furthermore, the present results also suggested that a marked increase in the newly discovered (24, 25) HO-2-CO pathway for insulin release is another compensatory mechanism operating to counteract the increased NO production. We have very recently shown (24) that CO can promote insulin release, both by directly stimulating the guanylate cyclase-cGMP system and by indirectly inhibiting islet NOS activity. Moreover, additional CO-induced mechanisms to stimulate insulin release cannot be excluded. Hence, the very high production of CO in islets from L-NAME-drinking mice is most probably an important compensatory mechanism to potentiate glucose-stimulated insulin release in islets displaying increased NO formation. It is notable that the compensatory CO production was conveyed by HO-2 and not by HO-1, which previously has been found to be induced in various situations that are toxic to the islets (57). With regard to glucagon release, our data showed that chronic L-NAME treatment resulted in a pronounced hypersecretion of glucagon compared with controls in the in vitro situation both at low and high glucose and after forskolin stimulation. The glucagon hypersecretion was most probably due to the increased formation of NO in the islets, since it was virtually abolished by the addition of the NOS inhibitors L-NAME or 7-nitroindazole. Furthermore, the NO-stimulated hypersecretion of glucagon seen in isolated islets was apparently counteracted in vivo, since both basal and stimulated glucagon secretion was essentially normal in these mice, apart from an increased glucagon response to forskolin after fasting. The present data do not tell the nature of the mechanisms that inhibit the NO-stimulated increase in glucagon secretion in the vivo situation. Further studies will hopefully reveal whether in situ neural and/or endocrine mechanisms are involved.
Our accumulated data show that chronic L-NAME treatment results in an induction of islet iNOS activity and an increase in total NO production and emphasize the necessity of measuring both cNOS and iNOS activities in the islets, and certainly also in other tissues (35), when investigating the effects of both chronic and acute effects of different NOS inhibitors. Fasting, which enhanced the total NO production also in islets from control mice by inducing the expression of the iNOS protein, only slightly increased the NO production after L-NAME drinking and did not increase it above the values achieved in freely fed L-NAME-drinking mice. It is not inconceivable that the increase in islet NO production in starvation is an important factor contributing to the impaired glucose-stimulated insulin release in the fasting state. This effect is, however, improved by L-NAME drinking, since in this situation the cAMP system and the increase in HO activity compensate for this defect. The observed effects of both L-NAME drinking and fasting as well as of in vitro addition of NOS inhibitors on insulin and glucagon secretion support our idea of cNOS-derived NO as a physiological negative modulator and feedback inhibitor of glucose-stimulated insulin secretion and further strengthen our hypothesis of NO being an important positive modulator of glucagon secretion. Important compensatory mechanisms counteracting and reversing the NO-induced suppression of glucose-stimulated insulin release include an enhanced sensitivity and capacity of the islet cAMP-protein kinase A system and the newly discovered HO-2-CO pathway. ![]() |
ACKNOWLEDGEMENTS |
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The skillful technical assistance of Britt-Marie Nilsson, Maj-Britt Johansson, and Lillemor Thuresson is gratefully acknowledged.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (14X-4286 and 11205), the Swedish Diabetes Association, and the foundations of Magnus Bergvall, Crafoord, Anna-Lisa and Sven-Erik Lundgren, Albert Påhlsson, Åke Wiberg, and Thelma Zoega.
Address for reprint requests and other correspondence: R. Henningsson, Dept. of Pharmacology, Sölvegatan 10, S-223 62 Lund, Sweden (E-mail: Ragnar.Henningsson{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. §1734 solely to indicate this fact.
Received 29 September 1999; accepted in final form 23 February 2000.
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