Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

Ragnar Henningsson, Albert Salehi, and Ingmar Lundquist

Institute of Physiological Sciences, Department of Pharmacology, University of Lund, S-221 84 Lund, Sweden


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 beta -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 beta -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 beta -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 beta -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 beta -cell lines with different qualitative and/or quantitative secretory reaction patterns compared with normal beta -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.


    MATERIALS AND METHODS
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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).

In experiments where NOS activities were not measured, islet incubations and determination of insulin release were performed as previously described (29). In experiments with exogenous NO, the gas was dissolved in helium-saturated buffer, and the islet incubation vials were gassed with air instead of 95% O2-5% CO2 as recently described (16). Radioimmunoassay of insulin was performed as previously described (29). Protein was determined according to Bradford on samples from the original homogenate (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.


    RESULTS
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ABSTRACT
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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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Fig. 1.   Dose-response relationship between glucose concentration and isoforms of islet nitric oxide (NO) synthase (NOS) activities and expression, as well as insulin release in freshly isolated mouse islets incubated in vitro for 60 min. Means ± SE for 4-8 incubated batches of islets on each point are shown. A: activities of constitutive NOS (cNOS) and inducible NOS (iNOS) are indicated. NO production is expressed as the equivalent of L-citrulline formation (pmol · mg protein-1 · min-1) (22). B: representative example of Western blots run in the same assay indicating islet iNOS and cNOS expression after incubation at different glucose concentrations. Islet protein (20 µg) was used on each lane. Molecular mass for iNOS (130 kDa) and cNOS (150 kDa), respectively, is indicated by arrows. C: insulin release presented as insulin (ng) secreted during the incubation period. *P < 0.05; **P < 0.01; ***P < 0.001: probability level of random difference vs. 7 mmol/l glucose controls.

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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Fig. 2.   Influence of NO and different NOS inhibitors on insulin release from mouse islets incubated at 20 mmol/l glucose. A: effects of NO gas (200 µmol/l) in the absence and presence of 250 µmol/l diazoxide (to keep the ATP-sensitive K+ channels open) and 30 mmol/l K+ (to depolarize islets). B: effects of 1 mmol/l of the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole and the intracellular NO donor hydroxylamine (300 µmol/l). One set of experiments with L-NAME was conducted in the presence of diazoxide-K+ (Diaz + K+). Means ± SE for 8-10 batches of islets in each group are shown. **P < 0.01; ***P < 0.001 vs. controls.

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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Fig. 3.   iNOS, cNOS, and total NOS activities, as well as insulin release in islets incubated at 20 mmol/l glucose for 60 (A) or 20 min (C) in the absence and presence of 5 mmol/l of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Means ± SE for 4-11 batches of islets in each group are shown. *P < 0.05; **P < 0.01 vs. controls. Note that insulin release is expressed as ng insulin · islet-1 · 20 min-1 in C. Basal cNOS activity at 7 mmol/l glucose (n = 6) was 14.92 ± 2.28 pmol NO · mg protein-1 · min-1. No iNOS activity could be recorded at 7 mmol/l glucose. Insulin release was 0.19 ± 0.02 ng insulin · islet-1 · h-1. Western blots for iNOS protein after incubation of the islets for 60 (B) or 20 min (D) are shown. Lane I, glucose (G) alone; lane II, G + L-NMMA. Islet protein (20 µg) was used on each lane. Molecular mass for iNOS (130 kDa) is indicated by arrows.

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 beta -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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Fig. 4.   iNOS, cNOS, and total NOS activities, as well as insulin release in mouse islets incubated at 20 mmol/l glucose for 60 min in the absence and presence of 250 µmol/l diazoxide. Means ± SE for n = 4 batches of islets in each group are shown. *P < 0.05, **P < 0.01 vs. controls. Basal cNOS activity at 7 mmol/l glucose (n = 4) was 12.35 ± 2.33 pmol NO · mg protein-1 · min-1. No iNOS activity could be recorded at 7 mmol/l glucose. Insulin release was 0.29 ± 0.04 ng insulin-1 · islet · h-1.

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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Fig. 5.   In vivo administration of glucose. Effects on blood glucose (A), islet iNOS, cNOS, and total NOS activities (B), and Western blots of islet iNOS protein (C). Mice were injected intraperitoneally (ip) with glucose at 0 min (22.2 mmol/kg) and at 30 min (11.1 mmol/kg) or with saline (controls). Blood glucose levels were measured at different time intervals. After 75 min, the pancreas was removed and NOS activities were registered in freshly isolated islets. For determination of iNOS expression, 20 µg of islet protein were used on each lane. Lane I, saline-injected control mice; lane II, glucose-injected mice. Molecular mass for iNOS (130 kDa) is indicated by arrow. There were 8-10 mice in each group. Means ± SE are shown. *P < 0.05, ***P < 0.001 vs. controls.

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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Fig. 6.   Effect of the NOS inhibitor L-NMMA on glucose-stimulated insulin release and activities of NOS isoforms in isolated rat islets. A: dynamics of insulin release after stimulation with 16.0 mmol/l glucose in the absence and presence of 0.5 or 5.0 mmol/l L-NMMA. Basal glucose was 1.6 mmol/l. Means ± SE for n = 6 in each group are shown. B: iNOS, cNOS, and total NOS enzyme activities recorded in rat islets incubated for 20 min in the absence and presence of 0.5 or 5.0 mmol/l L-NMMA. The initial 10 min was thus performed at 1.6 mmol/l glucose. High glucose, 16 mmol/l, was present during the following 10 min of incubation. This design mimicked the time period between 50 and 70 min in the perifusion experiment shown in A and thus was representative of NOS activities during the first phase of glucose-stimulated insulin release. Means ± SE for n = 4 in each group are shown. *P < 0.05; ***P < 0.001.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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This report provides the first direct evidence that short-time (~60 min) exposure of pancreatic beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 alpha -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -cells (3, 18) than the true glucose-stimulated (nutrient-stimulated) pathway. Hence, we must draw the conclusion that certain hitherto employed beta -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 beta -cell function, which might be one of multiple factors involved in the development of NIDDM.


    ACKNOWLEDGEMENTS

The technical assistance of Britt-Marie Nilsson and the secretarial help of Eva Björkbom are gratefully acknowledged.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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