Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase

Regulatory Peptides 122 (2004) 109 – 118 www.elsevier.com/locate/regpep

Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase Javier Jimenez-Feltstrom a,*, Ingmar Lundquist a, Stefanie Obermuller b, Albert Salehi a a

Institute of Physiological Sciences, Department of Pharmacology, University of Lund, BMC F13 S-221 84 Lund, Sweden b Department of Molecular and Cellular Physiology, University of Lund, S-221 84 Lund, Sweden Received 26 March 2004; received in revised form 19 May 2004; accepted 1 June 2004 Available online 28 July 2004

Abstract The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose – response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05 – 0.1 nmol/l insulin stimulated, 1 – 100 nmol/l had no effect, whereas concentrations z 250 nmol/l (‘‘high insulin’’), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3V-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt2cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3V-kinase and are counteracted by raising islet cyclic AMP levels. D 2004 Elsevier B.V. All rights reserved. Keywords: Isolated islets; Insulin feedback; Isoforms of islet nitric oxide synthase; Cyclic AMP; GLP-1

1. Introduction Current understanding of pancreatic h-cell physiology suggests that insulin released by the h-cells might play an important role in the regulation of its own secretion [1]. Over the last years, several papers have been published showing that h-cells are equipped with the insulin signalling machinery system and that these components can be affected by insulin [1 – 5]. Hence, an interaction between released insulin and h-cell activity might be involved in modulating the insulin secretory process. Previous studies, using mice with knockout of the insulin receptor complex and/or downstream signalling components, have suggested that insulin exerts a positive feedback on its own secretion [6,7], while other studies have * Corresponding author. Tel.: +46-46-222-7586; fax: +46-46-2224429. E-mail address: [email protected] (J. Jimenez-Feltstrom). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.06.004

argued that insulin has an inhibitory effect [4,5] or no effect [8] on the secretory process. Moreover, the intracellular events in the h-cell leading to these contradictory results are still unresolved [1]. In a series of publications, we have argued that nitric oxide (NO) derived from both the neuronal constitutive NO synthase (ncNOS) and the inducible NOS (iNOS) might have important implications for the physiology and pathophysiology of insulin release [9– 18]. It should be recalled that NO is formed from L-arginine under the influence of a family of nitric oxide synthases. The NOS enzyme appears in three major isoforms, two constitutive Ca2 +/calmodulin-dependent isoforms, i.e. ncNOS and endothelial NOS (ecNOS) as well as a third inducible Ca2 +/ calmodulin independent isoform of NOS (iNOS) [19]. Both ncNOS and iNOS have been shown to reside in the islets of Langerhans [15,20]. The expression of iNOS in islet h-cells is known to be mediated mainly by inflammatory stimuli, and there are increasing amounts

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of data suggesting that iNOS-derived NO, which is produced in high and cytotoxic amounts, is implicated in h-cell damage and dysfunction and hence the development of type 1 diabetes [21 – 24]. Surprisingly, we recently obtained results suggesting that iNOS activity could be induced during both hyperglycemic and hyperlipidemic conditions, and thus that the NO produced under these non-inflammatory circumstances might contribute to a progressive ‘‘nonautoimmune’’ cytotoxic action, and therefore could constitute an important factor in the pathogenesis of type 2 diabetes [16,17]. Moreover, the role of islet ncNOS for h-cell function is also unclear. NO, derived from ncNOS activity is produced in much lower amounts than NO from iNOS and is regarded as a putative physiological modulator of islet hormone release. However, there is no consensus as to whether it stimulates, inhibits or has no effect on insulin secretion [9– 18,25 –28]. Since it has previously been shown that insulin could stimulate NO production in different peripheral tissues such as, e.g. endothelial cells [29], adipocytes [30] and vascular smooth muscle cells [31], it occurred to us that the feedback effects of insulin on its own secretion might involve islet NOS activities. Hence, the aim of the present investigation was to directly study, using a wide range of insulin concentrations, the influence of insulin on glucose-stimulated C-peptide secretion from isolated mouse islets in relation to the activities and expression of ncNOS and iNOS.

land. Polyclonal rabbit anti-iNOS was from StressGen Biotechnologies, Victoria, BC, Canada. HRP-conjugated goat anti-rabbit IgG was from Pierce Biotechnology, Rockford, IL, USA. Cy2-conjugated anti-rabbit IgG and Cy5conjugated anti-guinea pig IgG were from Jackson Immunoresearch Laboratories, West Grove, PA, USA. Guinea pig-raised anti-insulin antibody was from Eurodiagnostica, Malmo¨, Sweden. Bovine serum albumin was from ICN Biomedicals, High Wycombe, England. The C-peptide and insulin radioimmunoassay kits were from Diagnostika, Falkenberg, Sweden. All other chemicals were from Merck, Darmstadt, Germany.

2. Materials and methods

2.4. Assay of islet NOS activities

2.1. Animals

Preincubation and incubation of freshly isolated islets were performed as stated above with the exception that each incubation vial contained 200 islets in 1.5 ml of buffer solution and was incubated for 90 min. After incubation an aliquot of the medium was taken for Cpeptide assay and then the islets were washed and collected in 200 Al buffer solution containing 20 mmol/ l HEPES, 0.5 mmol/l EDTA and 1 mmol/l DL-dithiothreitol, and immediately frozen at 20 jC. On the day of assay, the islets were sonicated on ice and the buffer solution was supplemented to also contain 0.45 mmol/ l CaCl2, 2 mmol/l NADPH, 25 U/ml calmodulin, and 0.2 mmol/l L-arginine. For the determination of iNOS activity both Ca2 + and calmodulin were omitted. The homogenate was then incubated at 37 jC under constant air bubbling, 1.0 ml/min for 2 h. Aliquots of the incubated homogenate (200 Al) were then passed through an 1 ml Amprep CBA cation-exchange column for high performance liquid chromatography (HPLC) analysis of the L-citrulline formed. The methodology has previously been described in detail [11,16] Since L-citrulline is created in equimolar concentrations to NO, and since L-citrulline is stable, whereas NO is not, L-citrulline is the preferred parameter when

Female mice of the NMRI strain (B&K, Sollentuna, Sweden) weighing 28 –32 g were used throughout the experiments. They were given a standard pellet diet (B&K) and tap water ad libitum. All animals used for preparation of pancreatic islets were killed by cervical dislocation, and isolation of the islets was performed by retrograde injection of a collagenase solution via the bilepancreatic duct [32]. The experiments were approved by the Ethical Committee for Animal Research at Lund University. 2.2. Drugs and chemicals Collagenase (CLS 4), L-arginine, calmodulin, NG-nitro-Larginine methyl ester (L-NAME), DL-dithiothreitol (DTT), wortmannin, insulin and 2V-O-dibutyryladenosine 3V:5V-cyclic monophosphate (Bt2cAMP) were obtained from Sigma, St. Louis, USA. Nicotinamide adenine dinucleotide phosphate (NADPH) was from ICN Biomedicals, Irvine, CA, USA. Glucagon-like peptide-1-(7 –36)amide (GLP-1) was purchased from Peninsula Laboratories, St. Helens, Eng-

2.3. C-peptide secretion from isolated islets Freshly isolated islets were isolated and hand-picked under a stereomicroscope at room temperature. The islets were then preincubated for 30 min at 37 jC in Krebs– Ringer bicarbonate buffer, pH 7.4, supplemented with 10 mmol/l HEPES, 0.1% bovine serum albumin and 1.0 mmol/l glucose. After preincubation, the buffer was changed and the islets were incubated with different test agents for 60 min at 37 jC unless otherwise stated. Each incubation vial contained 8– 10 islets in 1.0 ml of buffer solution except for the NOS experiments described below and was gassed with 95% O2 – 5% CO2 to obtain constant pH and oxygenation. All incubations were performed in an incubation box at 30 cycles/min. Immediately after incubation an aliquot of the medium was removed and frozen for subsequent assay of C-peptide or insulin.

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measuring NO production. Protein was determined according to Bradford [33] on samples from the original homogenate. 2.5. Western blot analysis Preincubation and incubation of freshly isolated islets were performed as stated above for the assay of islet NOS activities. After incubation, the islets were washed in Hanks’ buffer and then suspended in 150 Al of 10 mmol/l Tris lysis buffer, pH 7.4, containing 0.5% Triton X-100, 0.5 mmol/l EDTA and 0.2 mmol/l PEFA block, frozen and sonicated on ice on the day of analysis [16]. The protein content of the supernatant was determined according to Bradford [33]. Homogenate samples representing 10 Ag of total protein were run on 7.5% SDSpolyacrylamide gel (Bio-Rad, Hercules, CA, USA). After electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked in LS-buffer (10 mmol/l Tris, pH 7.4, 100 mmol/l NaCl, 0.1% Tween-20) containing 5% non-fat dry milk powder for 40 min at 37 jC. Subsequently, the membranes were incubated overnight with a polyclonal anti-iNOS (1:2000) antibody at room temperature. After three washings in LS-buffer the membranes were finally incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:50,000). Immunoreactivity was detected using an enhanced chemiluminescence reaction (Pierce). 2.6. Confocal microscopy Preincubation and incubation of freshly isolated islets were performed as stated above for the assay of islet NOS activities. The islets were then fixed with 4% formaldehyde, permeabilized with 5% Triton X-100, and unspecific sites blocked with 5% normal donkey serum (Jackson Immunoresearch Laboratories). iNOS was detected with a rabbit-raised polyclonal anti-iNOS antibody (1:100) in combination with Cy2-conjugated anti-rabbit IgG (1:150). For staining of insulin, islets were incubated with a guinea pig-raised anti-insulin (1:1000) antibody followed by an incubation with a Cy5-conjugated anti-guinea pig IgG antibody (1:150). The fluorescence was visualized with a Zeiss LSM510 confocal microscope by sequentially scanning at (excitation/emission) 488/505 – 530 nm (Cy2) and 633/>650 nm (Cy5).

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3. Results 3.1. Dose– response relationship for insulin and C-peptide secretion from isolated islets in the presence of different glucose concentrations Isolated mouse islets were incubated in the presence of increasing concentrations of glucose (1– 30 mmol/l). The dose – response curves for insulin and C-peptide were practically identical, having a basal plateau at concentrations between 1 and 7 mmol/l of glucose, showing a significant increase above basal at 8.3 mmol/l and having a maximum at 20 –30 mmol/l (Fig. 1). 3.2. Dose – response study of the effects of different concentrations of exogenous insulin on C-peptide secretion from isolated islets In Fig. 2 islets were incubated with increasing concentrations of insulin (0.01 –1000 nmol/l) in the presence of 8.3 mmol/l glucose. The results show that insulin at low concentrations (0.05 –0.1 nmol/l) markedly stimulated Cpeptide secretion (approximately 2-fold). At higher concentrations, however, insulin gradually suppressed the release of C-peptide, reaching an approximately 50% reduction from control levels at 1000 nmol/l. 3.3. Influence of the NOS-inhibitor L-NAME and the PI3kinase inhibitor wortmannin on C-peptide secretion at low and high insulin concentrations First we examined if insulin might stimulate NOS activity in the h-cells and if so, whether such a stimulation might be of importance for C-peptide-insulin releasing mechanisms. For this purpose we used the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (5 mmol/l) in the presence of a low (0.1 nmol/l) and a high

2.7. Statistics Statistical significance between sets of data was assessed using unpaired Student’s t-test or analysis of variance followed by Tukey –Kramer’s multiple comparisons test where applicable. Results are expressed as means F S.E.M.

Fig. 1. Dose – response relationship between glucose concentration and insulin and C-peptide secretion from isolated mouse islets. No effect of glucose is seen between 1 and 7 mmol/l glucose. Values are means F S.E.M for 8 – 10 batches of islets at each glucose concentration.

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333.5 F 38.93 pg/islet/h ( p < 0.05), and ncNOS activity was increased from 17.6 F 1.6 to 24.9 F 2.2 pmol NO/mg protein/min ( p < 0.01). Moreover, this high concentration of insulin induced a strong iNOS activity. Wortmannin did not influence the stimulatory effect of insulin on ncNOS activity, whereas it completely inhibited the insulin-induced activity of iNOS. This was accompanied by a marked increase in C-peptide release, which was restored to the control level (Fig. 4). 3.5. Effect of GLP-1 and cyclic AMP on C-peptide secretion in relation to insulin-induced expression and activities of NOS

Fig. 2. Dose – response effect of exogenously applied insulin on C-peptide release from isolated mouse islets. The islets were incubated in the presence of a modest stimulatory concentration of glucose, 8.3 mmol/l. Values are means F S.E.M. The number of batches used at each point is indicated within brackets. ***p < 0.001 versus control level (absence of insulin).

Since we recently observed that raising the islet cyclic AMP levels brought about a marked suppression of islet NOS activities induced by elevation of plasma lipids [18], the next series of experiments was designed to explore the effects of GLP-1 and dibutyryl cyclic AMP (Bt2cAMP) on insulin-induced activities and expression of NOS. Fig. 5A shows that addition of the cyclic AMP

(1 Amol/l) concentration of insulin at 8.3 mmol/l glucose. Fig. 3A shows that L-NAME had no significant effect on C-peptide secretion at a low concentration of insulin, whereas it markedly counteracted the suppressive effect of a high concentration of insulin on C-peptide release. In the absence of added insulin L-NAME tended to increased C-peptide release at 8.3 mmol/l of glucose (Fig. 3A). In the next series of experiments we studied the effects of 0.1 nmol/l or 1 Amol/l of insulin on C-peptide secretion at 8.3 mmol/l of glucose in the absence and presence of wortmannin (100 nmol/l). Wortmannin is a known inhibitor of PI3-kinase, which is a key element in transducing various actions of insulin on downstream effectors [5,34]. As shown in Fig. 3B, the stimulating effect of insulin at 0.1 nmol/l on C-peptide release was abolished by wortmannin. At 1 Amol/l of insulin, however, wortmannin had the opposite effect, inducing a marked increase in Cpeptide secretion which was even greater than in the controls. Wortmannin had no apparent effect on C-peptide release from islets incubated at 8.3 mmol/l glucose in the absence of insulin (Fig. 3B). 3.4. Effect of insulin on islet NOS activities in relation to C-peptide release Isolated islets were incubated at 8.3 mmol/l glucose in the absence or presence of insulin at two different concentrations (0.1 or 250 nmol/l). Fig. 4 shows that 0.1 nmol/l of insulin which markedly stimulated C-peptide release, had no significant effect on the islet activities of either ncNOS or iNOS. In contrast, in the presence of 250 nmol/l of insulin the release of C-peptide was inhibited from 499.2 F 56.74 (control) to

Fig. 3. (A) Effect of low (0.1 nmol/l) and high (1 Amol/l) concentrations of insulin in the presence or absence of the NOS inhibitor L-NAME (5 mmol/l) on C-peptide secretion. (B) Effect of 0.1 nmol/l and 1 Amol/l of insulin on C-peptide secretion in the presence or absence of the PI3-kinase inhibitor wortmannin (100 nmol/l). Glucose concentration in both series of experiments was 8.3 mmol/l. Values are means F S.E.M. The number of batches used at each point is indicated within brackets. ***p < 0.001.

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be revealed in immunolabeling studies detecting iNOS. The islets were simultaneously immunolabeled for insulin in order to investigate h-cell-specific expression of iNOS. As shown in Fig. 6B – C, no iNOS immunoreactivity was detected in control islets. In contrast, islets incubated in the presence of 250 nmol/l of insulin showed a strong immunoreactivity for iNOS in insulin immunoreactive hcells (seen as yellowish fluorescence) (Fig. 6D –F). Addition of either GLP-1 or cyclic AMP to the incubation media suppressed the expression of iNOS in insulin immunoreactive cells (Fig. 6G –L).

Fig. 4. Influence of exogenously applied insulin, 0.1 nmol/l or 250 nmol/l, on (A) Islet activities of neuronal constitutive NOS (ncNOS) and inducible NOS (iNOS), and (B) Islet C-peptide secretion. The islets were incubated for 90 min at 8.3 mmol/l glucose. The effect of the PI3-kinase inhibitor wortmannin (100 nmol/l) was tested at 250 nmol/l of insulin. NO production is expressed as the equivalent of L-citrulline formation (pmol  mg protein 1  min 1). Values are means F S.E.M for five batches of islets at each point. **p < 0.01; ***p < 0.001.

stimulating hormone GLP-1 as well as Bt2cAMP itself, in the presence of 250 nmol/l insulin, abolished the insulin-induced activity of iNOS and suppressed ncNOS activity to control levels. The changes in iNOS activity seen in Fig. 5A were in accordance with the visual impression of the immunoblots of iNOS protein (Fig. 5B). The suppressive effect of GLP-1 and Bt2cAMP on insulin-induced NOS activities were accompanied by a marked stimulation of C-peptide release from 473.5 F 34.7 to 764.4 F 53.4 pg/islet/h (GLP-1) ( p < 0.001) and to 809.2 F 46.78 (Bt2cAMP) ( p < 0.001) as seen in Fig. 5C. 3.6. Confocal microscopy study of insulin-induced as well as GLP-1 and Bt2cAMP-suppressed expression of iNOS in pancreatic islets The expression of iNOS induced by 250 nmol/l of insulin as well as its suppression by GLP-1 and cyclic AMP could

Fig. 5. ncNOS activity, iNOS activity and expression, as well as C-peptide secretion in isolated islets incubated for 90 min at 250 nmol/l insulin, in the presence or absence of 100 nmol/l GLP-1 or 2 mmol/l dibutyryl cyclic AMP (Bt2cAMP). Glucose concentration was 8.3 mmol/l. (A) Activities of ncNOS and iNOS are indicated. NO production is expressed as the equivalent of L-citrulline formation (pmol  mg protein 1  min 1). (B) Representative example of Western blots indicating islet iNOS expression. Each lane was provided with 10 Ag of islet protein. Molecular mass for iNOS is indicated. Densitometrical analysis of blots indicated a suppression of iNOS protein expression (versus 250 nmol/l insulin) when GLP-1 or Bt2cAMP were present in the incubation media (59.09 vs. 23.24 and 2.91, respectively). (C) C-peptide release during the incubation period. Values are means F S.E.M for five batches of islets at each point. *p < 0.05; **p < 0.01; ***p < 0.001.

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Fig. 6. Confocal microscopy of mouse islets. Isolated islets were incubated for 90 min in the presence of (A,B and C) 8.3 mmol/l glucose (D, E and F) 8.3 mmol/l glucose + 250 nmol/l insulin (G, H and I) 8.3 mmol/l glucose + 250 nmol/l insulin + 2 mmol/l Bt2cAMP (J, K and L) 8.3 mmol/l glucose + 250 nmol/ l insulin + 100 nmol/l GLP-1. After incubation the islets were double immunolabeled for insulin and iNOS and analysed by confocal microscopy. Insulin and iNOS stainings appear, respectively, as red (A, D, G and J) and green (B, E, H and K) fluorescence. Co-localisation of insulin/iNOS is seen as a yellowish fluorescence (C, F, I and L). Bars indicate lengths (Am).

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4. Discussion

4.1. High insulin

The results of the present study demonstrate that the dose –response curves for glucose-stimulated insulin and C-peptide release from isolated mouse islets were practically identical, and thus that measurement of C-peptide release in our mouse islet model can qualify as a reliable marker for studying the influence of insulin on its own secretion. We have recently shown that a glucose concentration as low as 10 mmol/l is able to stimulate islet NOS activities [16]. To avoid interference by glucose on NOS activation we therefore selected a modest concentration of glucose (8.3 mmol/l) that stimulated insulin release having a negligible effect on NOS activities [16]. In addition, the tonic impact of endogenously released insulin was minimized. In support of the contradictory findings previously reported concerning the feedback actions of insulin, i.e. inhibitory [4,5], stimulatory [3,34] or absence of an effect [8], we found that all these effects could be reproduced depending on the concentration of added insulin. Hence, a very low concentration of added insulin, 50 – 100 pmol/l, which is within the range of basal circulating insulin levels in the freely fed NMRI mouse [11– 13], was found to stimulate C-peptide release. This positive feedback action can, at least in part, explain why different types of knockout mice with disruption of the insulin signalling pathways in their h-cells display an impaired insulin response to glucose [2,6,7]. Furthermore, gradually increasing the concentrations of added insulin showed that 1 – 100 nmol/l of insulin had no apparent effect on glucose-stimulated C-peptide release, whereas concentrations z 250 nmol/l were suppressive. From a physiological point of view, such a pattern of insulin action seems meaningful in that low insulin might be expected to exert a positive feedback on the secretory process to prevent hyperglycemia, whereas high insulin exerts a negative feedback to prevent hypoglycemia. It should be recalled that during stimulation by high glucose, the insulin concentration surrounding the h-cells has been estimated to approximately 100– 200 nmol/l [35], i.e. concentrations of the same magnitude as we find inhibitory to C-peptide release. In this context, it could be argued that accumulation of such high insulin concentrations in the in vivo situation might be relevant only to the exocytotic part of the polarized h-cell [36,37] and that the receptive, non-exocytotic part is challenged by much lower insulin levels. Such an arrangement in the h-cell microenvironment would favour an appropriate action of both low and high insulin. Hence, considering that the exocytotic sites of the h-cell release large amounts of insulin into a small volume of intercellular fluid, the levels of insulin affecting the h-cell might well be of such a magnitude to also exert a regulatory influence on the secretory process in the in vivo situation.

In a series of reports during the last 10 years [9 –18] we have presented evidence suggesting that NO, emanating either from the intracellular NO donor hydroxylamine, addition of gaseous NO, or directly derived from h-cell ncNOS activity, is an important negative modulator of glucose-stimulated insulin secretion, operating as a feedback inhibitor of the acute release of insulin. In view of several recent observations showing that insulin might stimulate NOS activity in other tissues [29 – 31], we now studied the possible influence of insulin on h-cell NOS activities by testing the effect of the NOS inhibitor L-NAME on glucosestimulated C-peptide release both at low (stimulatory) and high (inhibitory) concentrations of added insulin. The data obtained here, at a modest insulin stimulating concentration of glucose (8.3 mmol/l), showed an expected slight increase in C-peptide release induced by the NOS inhibitor in the absence of added insulin but also a marked secretory reversal of the suppressive action of high insulin thus suggesting that NO might be involved in the action of insulin. Moreover, it should be emphasized that we have very recently shown that glucose itself dose-dependently increases islet ncNOS activity [16], thus implicating the action of ncNOS-derived NO as a negative feedback mechanism to avoid excessive and uncontrolled release of insulin stimulated by high glucose. Our present results showed that addition of high insulin concentrations indeed increased islet ncNOS activity but in addition also induced the expression and a strong activity of iNOS. In this context, it should be recalled that we have previously shown [16] that a marked increase in glucose-stimulated ncNOS activity could be demonstrated after a 20 min incubation, whereas no iNOS activity or expression was detectable at that time period. After 60 min, however, we observed increased activities of both ncNOS and iNOS. Accordingly, in the present study we used an incubation time of 60 –90 min to detect possible changes in both ncNOS and iNOS activities. From the results of our earlier studies, we hypothesized [9– 18] that the inhibitory effect of NO on the secretory process might be exerted through S-nitrosylation [38,39] of the glutathione system, and thus a derangement of the reduced glutathioneoxidised glutathione balance or, alternatively, through Snitrosylation of some regulatory proteins implicated in glycolysis and/or the pentose shunt that operate independent of membrane depolarisation and mitochondrial metabolism [16,40]. Indeed, recent data have emphasized the role of protein S-nitrosylation as a physiological signal in other tissues [38,39] and studies of the cultured h-cell line hTC3 have suggested that glucokinase could qualify as such a regulatory protein [41]. Since reportedly [1– 3,5,7,8,42,43] there are significant interactions in the h-cell between glucose and the insulin signalling pathway, we probed this pathway by using the PI3-kinase inhibitor wortmannin. Wortmannin reversed the inhibitory action of high insulin on C-peptide release

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without having any influence on islet ncNOS activity. Surprisingly, however, inhibition of PI3-kinase totally abolished the prominent iNOS activity induced by high insulin thus suggesting an important role for PI3-kinase as a stimulator of islet iNOS activity. Moreover, our present results suggest that not only ncNOS-derived NO [13,14,16] but also iNOS-derived NO in certain situations might inhibit glucose-stimulated insulin/C-peptide release in short-term incubations when NO is produced in sufficiently high amounts. This is in accordance with our recent data obtained in islets isolated from lipid-infused rats showing that these islets displayed a very strong iNOS activity and even a modest decrease of ncNOS activity accompanied by a marked reduction of glucose-stimulated insulin release [17,18]. However, it should be recalled that the major part of the ncNOS enzyme has been shown to be associated with the insulin secretory granules [27] and thus is more conveniently situated to control insulin release than the iNOS enzyme, which is located in the cytoplasm [19]. Hence, considering this cellular location of the iNOS enzyme, it seems conceivable that very high levels of iNOS-derived NO are required to suppress the insulin secretory machinery. In this context it should be noted that wortmannin has been reported to inhibit islet phosphodiesterase activity, through its effect on PI3-kinase, resulting in an increase of cyclic AMP content, thus suggesting that PI3-kinase inhibits insulin release by activating phosphodiesterase [44]. Moreover, exogenous insulin has been shown to hyperpolarize hcells by activation of the KATP channels and that this action of insulin could be reversed by wortmannin and thus again making PI3-kinase as a likely mediator [5]. In recent studies, we have reported that both hyperglycemia [16] and hyperlipidemia [17] are capable of inducing islet iNOS expression and activity. We also very recently found that raising islet cyclic AMP levels, by including either Bt2cAMP or adenylate cyclase activating agents such as GLP-1 into the incubation medium, could counteract this iNOS induction [18]. This anti-iNOS action by cyclic AMP was also found to hold true when islet iNOS activity was induced by high insulin. Thus both GLP-1 and Bt2cAMP effectively suppressed the expression and activity of iNOS in parallel with increased C-peptide release. All these changes in iNOS could be verified by Western blots and confocal microscopy imaging. Hence, the stimulating effect of insulin on iNOS activity might, at least in part, be explained by its activating action on phosphodiesterase [44]. It should be noted that GLP-1 and Bt2cAMP also significantly suppressed islet ncNOS activity, which might further explain the marked increase in C-peptide release. 4.2. Low insulin The mechanisms behind the increase in glucose-stimulated C-peptide after addition of low insulin remain unclear at present. Since wortmannin, in the present study, inhibited glucose-stimulated increase in C-peptide secretion there is

reason to believe that PI3-kinase is somehow involved as a promoter of insulin release in the presence of low insulin, although PI3-kinase independent effects have also been suggested [42]. Whether the effects of low insulin implicate presently unknown components of the downstream insulin signalling system or discrete changes of the insulin receptor complex remains to be elucidated. That insulin signalling might positively contribute to its own release is supported by the analysis of the hIRKO mouse, an animal model having a h-cell-specific insulin receptor knockout. These mice exhibit a selective loss of first-phase insulin secretion in response to glucose [7]. Interestingly, it was very recently reported [45] that the h-cells are not responsive to glucose unless a certain amount of insulin is present within the islets. It should be noted that previous data [3,34] have suggested that a positive feedback action of insulin might be due to release of intracellular Ca2 + stores mediated by IRS-1 and PI3-kinase and subsequent activation of PKC. Further, it is known from studies of adipocytes [30] that the metabolic effects of insulin in these cells are transduced through activating PI3-kinase and/or MAP-kinase involving different regulatory phosphatases with intricate actions that might take part in exerting the effect of low insulin. The dual effect of insulin on its own secretion that we have observed is difficult to explain but one should have in mind that hormones may have opposite effects on target tissues depending on its concentration. It is well known that the h-cells express two different isoforms of the insulin receptor and that their affinity for insulin differs [1,46]. Considering that one might speculate whether the dual effect we observed might be mediated through the two different receptor isoforms, respectively. It is therefore possible that an inhibitory pathway is activated at concentrations above 1 nmol/l insulin (by one of the receptor isoforms), which negates the stimulatory effects (by the other receptor isoform) seen at lower concentrations. Of course, we cannot rule out that the same receptor may very well exert both the stimulatory and inhibitory effects observed in the present study. It should be considered that wortmannin, the pharmacological tool used in the present study, has complex effects and as very recently reported [47] at the concentration used here might inhibit PI3 K class Ia and also PI3 K class II as well as other kinases that might play a role in the secretory process [47]. It is thus conceivable that low insulin activates PI3 K class Ia, while high insulin might activate PI3 K class II. Hence, future studies are needed to further clarify these mechanisms. Our present results may possibly explain, at least partly, the dynamics of insulin secretion. It is tempting to speculate that during the first phase of insulin release a positive insulin feedback contributes to the rapid increase seen during the initial stages of insulin release. Negative feedback may eventually take over to suppress insulin secretion when the concentration, in the microenvironment of the hcells, reaches higher levels. The latter effect may be mediated, at least in part, through stimulation of ncNOS-derived

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NO production. Indeed, we have previously shown that the NOS inhibitor L-NAME blunts the negative peek seen between the first and the second phase of insulin release [16]. 4.3. Concluding remarks In conclusion, we now report for the first time, that in freshly isolated mouse islets exogenous insulin at z 250 nmol/l stimulates ncNOS activity as well as the expression and activity of iNOS in parallel with an inhibitory action on glucose-stimulated C-peptide release. This effect on iNOS is most likely transduced by the action of PI3-kinase, since it was abolished by the PI3-kinase inhibitor wortmannin, and possibly exerted through its ability to activate phosphodiesterase. Such a mechanism is supported by our observation that directly raising islet cyclic AMP levels through the action of GLP-1 or Bt2cAMP resulted in a marked suppression of islet NOS activities. In addition we found that a low concentration of insulin (50 – 100 pmol/l) stimulated Cpeptide release suggesting a positive feedback at a low secretory rate of the h-cell. This effect was abolished by wortmannin and exerted independent of islet NOS activities. It is possible but currently far from proven that our findings might have clinical implications in type 2 diabetes (and possibly even in some cases of type 1 diabetes) in that endogenous and/or exogenous hyperinsulinaemia in certain situations can be expected to have negative effects on h-cell function through the induction of cytotoxic levels of NO derived from islet iNOS activity. Acknowledgements The skillfull technical assistance of Britt-Marie Nilsson is gratefully acknowledged. The study was supported by grants from the Swedish Science Council (4286), the Swedish Diabetes Association, the Albert Pa˚hlsson, Crafoord, Golje, Magnus Bergvall Foundations and the Diabetes Program, University of Lund, Sweden. References [1] Leibiger IB, Leibiger B, Berggren PO. Insulin feedback action on pancreatic beta-cell function. FEBS Lett 2002;532:1 – 6. [2] Kulkarni RN. Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function. Biochem Soc Trans 2002;30:317 – 22. [3] Aspinwall CA, Lakey JR, Kennedy RT. Insulin-stimulated insulin secretion in single pancreatic beta cells. J Biol Chem 1999;274: 6360 – 5. [4] Persaud SJ, Asare-Anane H, Jones PM. Insulin receptor activation inhibits insulin secretion from human islets of Langerhans. FEBS Lett 2002;510:225 – 8. [5] Khan FA, Goforth PB, Zhang M, Satin LS. Insulin activates ATPsensitive K(+) channels in pancreatic beta-cells through a phosphatidylinositol 3-kinase-dependent pathway. Diabetes 2001;50: 2192 – 8.

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