Islet Nitric Oxide Production Research Articles

In vitro immunomodulatory and cytoprotective effects of Origanum vulgare methanol extract

Event Abstract Back to Event In vitro immunomodulatory and cytoprotective effects of Origanum vulgare methanol extract Milica Vujicic1*, Ivana Stojanovic1, Ivana Nikolic1, Vassiliki Kontogianni2, Pantelis Charisiadis2, Andreas G. Tzakos2 and Stanislava Stosic-Grujicic1 1 Institute for Biological Research "Sinisa Stankovic", Department of Immunology, Serbia 2 University of Ioannina, Department of Chemistry, Greece In addition to being a spice, Origanum vulgare is recognized as a source of natural antioxidants and exerts anti-fungal, anti-bacterial and anti-inflammatory actions. In this study we focused on the immunomodulatory effects of methanol fraction of Origanum vulgare (oregano extract – OE) and on its influence on cellular viability. The methanol OE was prepared from ground oregano leaves by sequential extraction of four solvents of gradually increasing polarity (hexane, ethyl acetate, dichloromethane and methanol). Our results indicate that 48-hour treatment of spleen mononuclear cells with OE (25 - 100 g/ml) did not alter their basic or concanavalin A-triggered proliferation (MTT assay), as well as IFN-γ and IL-4 secretion determined by ELISA. However, OE treatment decreased IL-17 secretion implying a potential inhibitory effect on Th17 cells. Additionally, OE seemed to affect macrophage cytocidal function since it down-regulated production of nitric oxide (NO) in peritoneal cells (PC) (measured by Griess reaction) without an effect on PC viability. However, PC-related IL-1 and TNF- remained unchanged after OE treatment. Finally, apoptosis measurement by histone-DNA ELISA showed that OE rescued isolated pancreatic islets from cytotoxic effect of cytokines. Furthermore, OE fully preserved beta cell function judging by impaired insulin secretion. The mechanism of OE-exerted protection of pancreatic islets was NO-independent since OE did not change islet NO production. In conclusion, OE alters secretory pattern of immune cells and protects pancreatic islets from cytokine-mediated destruction. The observed immunomodulatory and islet cytoprotective effects of OE are being further explored for potential application for diabetes treatment. Acknowledgements This work was supported by the Project of Ministry of Education and Science, Republic of Serbia (No.: 173013) Keywords: Origanum, antioxidant, Diabetes Mellitus, immunomodulatory, cytoprotective, beta cells, Th17 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Vujicic M, Stojanovic I, Nikolic I, Kontogianni V, Charisiadis P, Tzakos AG and Stosic-Grujicic S (2013). In vitro immunomodulatory and cytoprotective effects of Origanum vulgare methanol extract. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00386 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Ms. Milica Vujicic, Institute for Biological Research "Sinisa Stankovic", Department of Immunology, Belgrade, Serbia, micijana@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Milica Vujicic Ivana Stojanovic Ivana Nikolic Vassiliki Kontogianni Pantelis Charisiadis Andreas G Tzakos Stanislava Stosic-Grujicic Google Milica Vujicic Ivana Stojanovic Ivana Nikolic Vassiliki Kontogianni Pantelis Charisiadis Andreas G Tzakos Stanislava Stosic-Grujicic Google Scholar Milica Vujicic Ivana Stojanovic Ivana Nikolic Vassiliki Kontogianni Pantelis Charisiadis Andreas G Tzakos Stanislava Stosic-Grujicic PubMed Milica Vujicic Ivana Stojanovic Ivana Nikolic Vassiliki Kontogianni Pantelis Charisiadis Andreas G Tzakos Stanislava Stosic-Grujicic Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Imidazoline‐induced amplification of glucose‐ and carbachol‐stimulated insulin release includes a marked suppression of islet nitric oxide generation in the mouse

The role of islet nitric oxide (NO) production in insulin-releasing mechanisms is unclear. We examined whether the beneficial effects of the imidazoline derivative RX 871024 (RX) on beta-cell function might be related to perturbations of islet NO production. Experiments were performed with isolated islets or intact mice challenged with glucose or carbachol with or without RX treatment. Insulin was determined with radioimmunoassay, NO generation with high-performance liquid chromatography and expression of inducible NO synthase (iNOS) with confocal microscopy. RX treatment, in doses lacking effects on basal insulin, greatly amplified insulin release stimulated by the NO-generating secretagogues glucose and carbachol both in vitro and in vivo. RX also improved the glucose tolerance curve. Islets incubated at high glucose levels (20 mmol L(-1)) displayed increased NO production derived from both neuronal constitutive NO synthase (ncNOS) and iNOS. RX abrogated this glucose-induced NO production concomitant with amplification of insulin release. Confocal microscopy revealed abundant iNOS expression in beta cells after incubation of islets at high but not low glucose levels. This was abolished after RX treatment. Similarly, islets cultured for 24 h at high glucose levels showed intense iNOS expression in beta cells. This was abrogated with RX and followed by an amplified glucose-induced insulin release. RX effectively counteracts the negative impact of beta-cell NO generation on insulin release stimulated by glucose and carbachol suggesting imidazoline compounds by virtue of NOS inhibitory properties being of potential therapeutic value for treatment of beta-cell dysfunction in hyperglycaemia and type 2 diabetes.

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the β-cells over time.

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

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 N(G)-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.

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release.

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.

Arginine-induced insulin release is decreased and glucagon increased in parallel with islet NO production.

Nitric oxide (NO) produced by islet constitutive NO synthase (cNOS) is a putative modulator of islet hormone secretion. We show here for the first time that the release of insulin induced by L-arginine or L-homoarginine is inhibited and that of glucagon stimulated in parallel with the rate of islet NO production. It was found that L-homoarginine was approximately 25-30% less potent than L-arginine as an insulin secretagogue but equally potent as a glucagon secretagogue. Biochemical determination of islet cNOS activity revealed that the NO production with L-homoarginine as substrate was only approximately 40% of that of L-arginine. Selective inhibition of islet cNOS potentiated insulin release during amino acid stimulation. Moreover, inhibition of cNOS suppressed glucagon release, more so with L-arginine than with L-homoarginine as secretagogue, reflecting the relative rates of their NO production. In K+-depolarized islets, inhibition of cNOS enhanced the insulin response to L-arginine by 50% and that to L-homoarginine by 23%, largely corresponding to their relative NO production. The intracellular NO donor hydroxylamine dose dependently inhibited insulin but increased glucagon secretion in K+-depolarized and amino acid-stimulated islets. We conclude that both amino acids have a dual action on insulin release, since their stimulatory effects are reduced in parallel with the rates of their NO production. Furthermore, the greater NO production induced by L-arginine relative to L-homoarginine corresponds to NO-mediated increases in glucagon release. These NO effects are mainly exerted independently of membrane depolarization events.

Islet constitutive nitric oxide synthase: biochemical determination and regulatory function.

Recent immunohistochemical findings suggested that a constitutive nitric oxide synthase (cNOS) resides in endocrine pancreas. Here we provide direct biochemical evidence for the presence of cNOS activity in isolated islets. The regulating influence of this nitric oxide synthase (NOS) activity for islet hormone release was also investigated. We observed that cNOS activity could be quantitated in islet homogenates by monitoring the formation of L-citrulline from L-arginine using an Amprep CBA cation-exhange minicolumn before derivatization with o-phthaldialdehyde and subsequent high-performance liquid chromatography analysis. The islet NOS was dependent on both Ca2+ and calmodulin and suppressed by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). This effect was enantiomerically specific. Islet insulin release induced by a mixture of L-arginine and glucose was enhanced by L-NAME, whereas L-arginine-induced glucagon release was inhibited. The effect of L-NAME on insulin release was dose dependently potentiated by increasing glucose concentrations, suggesting that glucose is an important regulator of islet NO production. Complementary in vivo studies showed similar results, i.e., the insulin secretory response to a mixture of glucose and L-arginine was extremely enhanced by pretreatment with L-NAME, whereas L-arginine-stimulated glucagon response was suppressed. Finally, in isolated islets, the intracellular nitric oxide (NO) donor hydroxylamine suppressed insulin release and increased glucagon release. In summary, the islets of Langerhans contain a constitutive, Ca2+/calmodulin-dependent isoform of NOS. Islet NO suppressed insulin but enhanced glucagon secretion. The data also suggest a negative feedback by NO on glucose-induced insulin release. The islet NO system is a novel and important regulatory factor in insulin and glucagon secretion.

TNF-α and IFN-γ potentiate the deleterious effects of IL-1β on mouse pancreatic islets mainly via generation of nitric oxide

Cytokines may be important mediators of β-cell damage in early insulin-dependent diabetes mellitus. In order to further characterize the mechanism(s) of action of cytokines on insulin-producing cells, mouse pancreatic islets were exposed for 48 h to IL-1β, IFN-γ or TNF-α, alone or in combinations. The three cytokines induced islet nitric oxide (NO) production, an effect most marked when islets were exposed to the three cytokines together. In parallel with NO production, IL-1β + IFN-γ + TNF-α impaired islet function, as judged by decreased islet DNA and insulin content, decreased glucose metabolism and decreased glucose-induced insulin release. Aminoguanidine, an inhibitor of NO production, prevented all the above described suppressive effects of the cytokines, with exception of depletion in islet insulin content. In parallel experiments, insulin-producing RIN cells were exposed for 6 h to the same cytokines. Both IL-1β and TNF-α, but not IFN-γ, induced NO production and expression of the mRNA encoding for the inducible form of the enzyme NO synthase (iNOS). These effects were most pronounced when combinations of IL-1β + IFN-γ or IL-1β + IFN-γ + TNF-α were used. As a whole, the data suggest that combinations of cytokines induce higher amounts of NO generation by mouse pancreatic islets than each of the cytokines isolated. An important source of islet NO production are probably the β-cells, as pointed by data obtained with an insulinoma cell line. Most of the deleterious effects of the cytokines of mouse islets are prevented by blocking NO production, suggesting that NO is the main mediator of cytokine-induced β-cell damage. However, some effects of cytokines, like early stimulation of insulin release and depletion of islet insulin content, can be dissociated from NO generation, suggesting that other mechanisms are also involved in the deleterious effects of cytokines on pancreatic β-cells.

Nicotinamide prevents interleukin-1 effects on accumulated insulin release and nitric oxide production in rat islets of Langerhans.

Nicotinamide (NA) prevents macrophage- and interleukin-1 (IL-1)-mediated beta-cell damage in vitro as well as diabetes development in animal models of insulin-dependent diabetes mellitus (IDDM). IL-1 beta-mediated inhibition of insulin release and damage to beta-cells are associated with intracellular production of nitric oxide (NO) radicals. Therefore, we studied whether NA prevented IL-1 beta-induced islet NO production, measured as nitrite release from isolated rat islets, and, if so, whether this action was associated with prevention of IL-1 beta-mediated inhibition of insulin release. NA dose- and time-dependently inhibited and delayed IL-1 beta-induced islet NO production. Light microscopy detected that 25 mM of NA protected against IL-1 beta-induced islet damage. Five to 50 mM of NA dose-dependently reduced inhibition of accumulated islet insulin release induced by 150 pg/ml of IL-1 beta. NA was not able to reverse the reduced ability of IL-1 beta-treated islets to respond to an acute glucose challenge. NO or nitrite did not interact directly with NA, because NA did not reduce sodium nitroprusside-generated nitrite. No-synthase inhibition with L-arginine depletion abolished NO production but only partially reduced IL-1 beta-induced inhibition of accumulated insulin release. Complete inhibition of IL-1 beta effects could not be obtained by adding L-arginine analogues to L-arginine-depleted medium, indicating that an NO-independent action of IL-1 beta on islet insulin release may exist.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotinamide decreases nitric oxide production and partially protects human pancreatic islets against the suppressive effects of combinations of cytokines.

It has been recently reported that human pancreatic islets in tissue culture produce nitric oxide (NO) and show a decreased function when exposed for 6 days to combinations of cytokines (interleukin-1 beta (IL-1 beta) + tumor necrosis factor-alpha (TNF-alpha) + interferon-gamma (IFN-gamma). Here we study the effects of nicotinamide (Nic; 10 or 20 mmol/l) on these deleterious effects of cytokines (50 U/ml IL-1 beta + 1000 U/ml TNF-alpha + 1000 U/ml IFN-gamma). Islets were isolated from 8 human pancreata at the Central Unit of the beta-Cell Transplant, Brussels, sent to Uppsala and, after 3-5 days in culture, exposed for 6 additional days to the cytokines and/or Nic. The cytokines induced a 6-fold increase in islet NO production (P < 0.001), and this effect was partially counteracted by Nic (50-60% decrease in NO production; P < 0.001). The cytokines severely decreased the islet insulin content and glucose-induced insulin release (16.7 mmol/l glucose; 90% decrease; P < 0.001). Both these effects of cytokines were partially counteracted by Nic, especially at the highest concentration (20 mmol/l; 2-4-fold increase compared to islets exposed to cytokines alone; P < 0.01). Nic by itself did not affect the insulin content or insulin release by control islets. In conclusion, the present data indicate that Nic counteracts the deleterious effects of cytokines on human pancreatic islets. This effect of Nic may be relevant for the beneficial effects of the drug in early IDDM.