Georgios Panagiotidis

Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues

1 Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH‐groups. 2 The NO synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME) dose‐dependently increased l‐arginine‐induced insulin release. d‐Arginine did not influence l‐arginine‐induced insulin secretion. However, d‐NAME which reportedly has no inhibitory action on NO synthase, modestly increased l‐arginine‐induced insulin release, but was less effective than l‐NAME. High concentrations (10 mm) of d‐arginine as well as l‐NAME and d‐NAME could enhance basal insulin release. 3 The intracellular NO donor, hydroxylamine, dose‐dependently inhibited insulin secretion induced by l‐arginine and l‐arginine + l‐NAME. 4 Glucose‐induced insulin release was increased by NO synthase inhibition (l‐NAME) and inhibited by the intracellular NO donor, hydroxylamine. Sydnonimine‐1 (SIN‐1), an extracellular donor of NO and superoxide, induced a modest suppression of glucose‐stimulated insulin release. SIN‐1 did not influence insulin secretion induced by l‐arginine or the adenylate cyclase activator, forskolin. 5 The intracellular ‘hydroperoxide donor’ tert‐butylhydroperoxide in the concentration range of 0.03–3mm inhibited insulin release stimulated by the nutrient secretagogues glucose and l‐arginine. Low concentrations (0.03–30 μm) of tert‐butylhydroperoxide, however enhanced insulin secretion induced by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). 6 Islet guanosine 3’:5′‐cyclic monophosphate (cyclic GMP) content was not influenced by 10 mm l‐arginine or tert‐butylhydroperoxide at 3 or 300 μm but was markedly increased (14 fold) by a high hydroxylamine concentration (300 μm). In contrast, islet adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) content was increased (3 fold) by l‐arginine (10 mm) and (2 fold) by tert‐butylhydroperoxide (300 μm). 7 Our results strongly suggest that NO is a negative modulator of insulin release induced by the nutrient secretagogues l‐arginine and glucose. This effect is probably not mediated to any major extent by the guanylate cyclase‐cyclic GMP system but may rather be exerted by the S‐nitrosylation of critical thiol groups involved in the secretory process. Similarly the inhibitory effect of tert‐butylhydroperoxide is likely to be elicited through affecting critical thiol groups. The mechanism underlying the secretion‐promoting action of tert‐butylhydroperoxide on IBMX‐induced insulin release is probably linked to intracellular Ca2+‐perturbations affecting exocytosis. 8 Taken together with previous data the present results suggest that islet production of low physiological levels of free radicals such as NO and H2O2 may serve as important modulators of insulin secretory processes.

Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse.

We examined the effects of a single intravenous injection of homologous islet amyloid polypeptide (IAPP) on the plasma levels of glucagon, insulin and glucose in the freely fed mouse. It was observed that IAPP suppressed basal glucagon levels concomitant with a decrease of the blood glucose concentrations. Basal plasma insulin levels were not affected. IAPP did not appreciably modulate the plasma concentration of glucose, insulin or glucagon after an intravenous glucose load. Further, IAPP inhibited the insulin secretory response to beta 2-adrenoceptor stimulation. IAPP also lowered the plasma glucagon levels following beta 2-adrenoceptor stimulation, whereas no apparent effect on plasma levels of glucose was observed. The data suggest that IAPP suppresses glucagon secretion and lowers blood glucose levels in the freely fed mouse. It might also exhibit a negative feedback inhibition on beta 2-adrenoceptor-induced insulin secretion, but has little influence on glucose-induced insulin release. Since IAPP is co-secreted with insulin, it is not inconceivable, that in the freely fed mouse, IAPP may act to amplify the blood glucose lowering effect of insulin through a direct suppression of glucagon secretion via the islet microcirculation.

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets.

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10(-10) and 10(-8) M, on glucagon release. Similarly, glucagon release stimulated by L-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10(-10) and 10(-8) M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and L-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.

Inhibition of islet nitric oxide synthase increases arginine-induced insulin release.

NG-Nitro-L-arginine, an inhibitor of nitric oxide (NO) synthase, markedly (+50%) increased the L-arginine-induced insulin release from isolated mouse islets but did not itself influence insulin secretion. An abundance of mouse islet cells were positively stained for the enzyme NADPH diaphorase, which reportedly is a marker for NO synthase. The data suggest that the NO synthase activity in mouse islet tissue may inhibit insulin secreting processes and that L-arginine has a dual action on insulin release.

Effect of L-dopa administration on islet monoamine oxidase activity and glucose-induced insulin release in the mouse.

Previous studies have shown that the amine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is rapidly converted to its corresponding amine, dopamine, in islet β-cells. In the present investigation, we studied the effect of acute L-DOPA administration on islet monoamine oxidase (MAO) activity and on glucose-induced insulin secretory response in mice. It was observed that at 2 min after intravenous L-DOPA administration, there was a marked increase (+ 35%) in islet MAO activity, with serotonin as substrate. At 7 min, MAO activity towards dopamine was enhanced by 32% and that towards serotonin and phenylethylamine (PEA) was decreased by 23 and 25%, respectively. The inhibitor of L-aromatic amino acid decarboxylase, benser-azide, abolished L-DOPA-induced changes of MAO activity, suggesting that the formed dopamine, and not L-DOPA itself, was responsible for the observed effects. At 60 min, no effect by L-DOPA administration on islet MAO activity was noticed. L-DOPA (125 or 250 pmol/kg), given together with glucose, induced a decrease in glucose-induced insulin response. L-DOPA (125 pmol/kg), given 7 min before glucose, totally suppressed glucose-induced insulin response. This inhibition was eliminated through pretreatment with benserazide. Enhancement of glucose-stimulated insulin response, after deposition of horseradish peroxidase (HRP) in p-cell vacuolar system, was suppressed by L-DOPA. We conclude that acute L-DOPA-induced dopamine accumulation in pancreatic islets is accompanied by rapid changes in MAO activity, concomitant with an inhibitory effect on glucose-stimulated insulin response. Increased hydrogen peroxide production, following increased MAO activity, may possibly augment the inhibitory effect of dopamine accumulation on insulin release.

Interaction of the islet nitric oxide system with L‐arginine‐induced secretion of insulin and glucagon in mice

1 Several recent in vitro studies have suggested that production of nitric oxide (NO) from the islet NO system may have an important regulatory influence on the secretion of insulin and glucagon. In the present paper we have investigated, mainly with an in vivo approach, the influence and specificity of the NO synthase (NOS) blocker NG‐nitro‐L‐arginine methyl ester (L‐NAME) on L‐arginine‐induced secretion of insulin and glucagon. 2 In freely fed mice, L‐NAME pretreatment (1.2 mmol kg−1) influenced the dynamics of insulin and glucagon release following an equimolar dose of L‐arginine, the specific substrate for NOS activity, in that the NOS inhibitor enhanced the insulin response but suppressed the glucagon responses. This was reflected in a large decrease in the plasma glucose levels of the L‐NAME pretreated animals. 3 L‐NAME pretreatment did not influence the insulin and glucagon secretory responses to the L‐arginine‐enantiomer D‐arginine, which cannot serve as a substrate for NOS activity. 4 Replacing L‐NAME pretreatment by pretreatment with D‐arginine or L‐arginine itself, which both carry the same cationic charge and are devoid of NOS inhibitory properties, did not mimic the effects of L‐NAME on L‐arginine‐induced hormone release. 5 Fasting the animals for 24 h totally abolished the L‐NAME‐induced potentiation of L‐arginine stimulated insulin release suggesting that the sensitivity of the β‐cell secretory machinery to NO‐production is greatly changed in the fasting state. However, the L‐NAME‐induced suppression of L‐arginine stimulated glucagon release was unaffected by starvation. 6 In isolated islets from freely fed mice, L‐arginine (5 mM) stimulated insulin release was greatly enhanced and glucagon release markedly suppressed by the presence of the NOS inhibitor L‐NAME in the incubation medium. These effects were abolished in isolated islets taken from 24 h fasted mice. 7 Our present results, which showed that the NOS inhibitor L‐NAME markedly enhances insulin release but suppresses glucagon release induced by L‐arginine in the intact animal, give strong support to our previous hypothesis that the islet NO system is a negative modulator of insulin secretion and a positive modulator of glucagon secretion. Additionally, we observed that the importance of the β‐cell NO‐production for secretory mechanisms, as evaluated by the effect of L‐NAME on L‐arginine‐induced insulin release, was greatly changed after starvation, an effect less prominent with regard to glucagon release.

The relationship of islet amyloglucosidase activity and glucose-induced insulin secretion

We have previously presented evidence for the involvement of islet acid amyloglucosidase, a lysosomal glycogen-hydrolyzing enzyme, in certain insulin secretory processes. In the present investigation, we studied whether differential changes in islet amyloglucosidase activity could be related to the insulin secretory response to glucose. It was observed that the dose-response curve for glucose-induced insulin response in vivo was shifted to the left by pretreatment of mice with purified fungal amyloglucosidase. In enzyme-pretreated mice, the ED50 was 2.1 mmol/kg glucose as compared with 5.7 mmol/kg in saline-pretreated controls (p <0.005). Also, the maximal insulin response to glucose was enhanced by amyloglucosidase pretreatment. Parenteral administration to mice (four injections during 2 days) of the pseudotetrasaccharide acarbose, a recognized inhibitor of intestinal α-glucosidases, surprisingly induced a marked increase in the activities of islet acid amyloglucosidase (+120%; p <0.001) and acid α-glucosidase (+ 45%; p <0.01) without affecting the activities of other lysosomal enzymes such as acid phosphatase and N-acetyl-P-D-glucosaminidase. No effect on the microsomal neutral α-glucosidase was recorded. Moreover, in these mice, the insulin secretory response to glucose was enhanced both at a maximal dose of glucose 11.1 mmoV kg and at a dose in the ED25,TED50, range, 3.3 mmol/kg (p <0.005). Direct addition of acarbose to islet homogenates strongly suppressed acid amyloglucosidase activity, the EC50, being approximately 1 μM. Acid α-glucosidase activity was also strongly inhibited, whereas the activities of acid phosphatase and N-acetyl-β-D-glucosaminidase were unaffected. Neutral α-glucosidase was slightly suppressed. Furthermore, addition of 10 mM acarbose to incubated islets markedly suppressed the insulin response to 16.7 mM glucose. We conclude that there exists a remarkable parallelism between islet acid amyloglucosidase activity and the insulin secretory response to glucose. The data lend further support to our hypothesis that certain lysosomes or lysosomal enzymes are involved in insulin secretory processes induced by glucose.

Effects of adrenergic and cholinergic stimulation on islet monoamine oxidase activity and insulin secretion in the mouse

It has been shown that the pancreatic beta-cell monoamines are located in the secretory granules, and that they have an inhibitory influence on insulin secretion. Monoamines are inactivated by the enzyme, monoamine oxidase. We now studied in vivo the relation between adrenergic and cholinergic stimulation, insulin secretion and islet monoamine oxidase activity in the mouse. Monoamine oxidase was assayed with three different substrates, serotonin, dopamine and beta-phenylethylamine. The alpha 2-adrenoceptor agonist, clonidine, induced a moderate inhibition (12-18%) of islet monoamine oxidase activity, accompanied by reduced plasma insulin and elevated plasma glucose levels. The alpha 1-adrenoceptor agonist, phenylephrine, did not induce any changes in these parameters. A marked insulin release following the injection of a maximal dose of the beta 2-adrenoceptor agonist, terbutaline, was accompanied by an increase (30-50%) in islet monoamine oxidase activity. The largest increase in monoamine oxidase activity was observed with serotonin as substrate (50%). These effects on insulin secretion and monoamine oxidase activity could not be blocked by clonidine. Similarly, injection of the non-selective alpha-adrenoceptor agonist, adrenaline, which unlike clonidine does not penetrate the blood-brain barrier, had no effect on insulin release induced by a maximal dose of the nonselective beta-adrenoceptor agonist, isoprenaline. Adrenaline, however, markedly suppressed the insulin release induced by a maximal dose of glucose. Cholinergic muscarinic stimulation by a maximal insulin releasing dose of carbachol did not affect islet monoamine oxidase activity. The results suggest that beta 2-adrenoceptor stimulation of islet monoamine oxidase activity reduced the monoamine content and thereby facilitated the release of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose modulation of islet monoamine oxidase activity in lean and obese hyperglycemic mice

Islet beta-cell monoamines are known to influence the insulin-releasing mechanisms. These amines are localized in the insulin-secretory granules and are inactivated by the enzyme monoamine oxidase (MAO), a hydrogen peroxide (H2O2)-generating enzyme. The activity of islet MAO may consequently be of importance for insulin secretion. In the present investigation, we studied the relation between islet MAO activity and plasma levels of insulin and glucose in obese (ob/ob) hyperglycemic mice and their lean littermates. In addition, the effect of glucose on the MAO activity of in vitro-cultured islets was studied. MAO activity was assayed with serotonin, dopamine (DA), and beta-phenylethylamine (PEA) as substrates. After an overnight fast in adult (age, 6 months) lean mice, islet MAO activity was increased by 35% to 70%. Plasma levels of glucose and insulin were markedly decreased as expected. However, fasting in adult obese mice either did not affect islet MAO activity (PEA and DA) or induced a slight decrease (serotonin) of approximately 25% (P < .05). Plasma glucose levels in adult obese mice were not significantly affected by the overnight fast. However, a correlation analysis based on individual adult obese mice (fed and fasted) showed a negative correlation between plasma glucose concentration and islet MAO activity with PEA (r = -.65, P < .02) and DA (r = -.66, P < .02), respectively. Further, a positive correlation (r = +.58, P < .05) was found between glucose level and islet MAO activity when using serotonin as substrate. There was no difference in islet MAO activity with PEA and DA as substrates in fed obese versus fed lean mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of β2-adrenoceptor stimulation and glucose on islet monoamine oxidase activity and insulin secretory response in the mouse

Changes in the content of monoamines such as dopamine (DA) and serotonin (5-HT) in the insulin granules are known to influence insulin release. The monoamines are inactivated by monoamine oxidase (MAO), a hydrogen peroxidegenerating enzyme, which may be of importance for the redox state of the β-cell. We studied the action of two different insulin secretagogues, the pzadrenoceptor agonist terbutaline and glucose, on islet MAO activity and the plasma levels of insulin and glucose. MAO was assayed with 5-HT, DA, and p-phenylethylamine as substrates. At 6 min (but not at 2 or 30 min) after terbutaline injection, marked increases of islet MAO activity and the plasma insulin levels were recorded. The plasma glucose levels were of the same magnitude at all time points. Injection of glucose moderately suppressed enzyme activity at 2 min. This occurred concomitantly with the peak increase in plasma levels of insulin and glucose. At 60 min, when the plasma levels of glucose and insulin were restored to basal, a slight increase in MAO activity was observed. At 2 min. after injection of different doses of glucose mixed with a maximal dose of terbutaline, the insulin secretory response was either increased (submaximal glucose dose) or unaffected (maximal dose of glucose) by the β2-adrenoceptor stimulator. However, when a maximal dose of glucose was given at 6 min after terbutaline, i.e., when islet MAO activity was increased, the insulin response to glucose was suppressed. Starvation for 24 h induced an increase in islet MAO activity. Moreover, this fasting period suppressed the glucose induced insulin response, whereas insulin release induced by β2-adrenoceptor stimulation was unaffected. The results suggest that both glucose and terbutaline influence insulin secretion through their action on islet MAO. Glucose inhibits and terbutaline increases the enzyme activity. In addition to reducing the islet monoamine content, an increased MAO activity also generates hydrogen peroxide, which might negatively modulate the insulin release induced by secretagogues that depend on a reductive redox state of the β-cell (thiol dependent; e.g., glucose) and facilitate the release by non-thiol-dependent (e.g., terbutaline) secretagogues.