Interleukin-1beta-induced nitric oxide production and inhibition of insulin secretion in rat islets of langerhans is dependent upon the nitric oxide synthase cofactor tetrahidrobiopterin

Interleukin (IL)-1 beta-induced inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans is mediated in part by nitric oxide (NO). The NO synthase cofactor 5,6,7,8-tetrahydrobiopterin (BH(4)) supports NO synthesis in many cell types and IL-1 beta-induced NO generation and inhibition of insulin secretion have been previously correlated with intracellular BH(4 )levels in rat insulinoma cells. Using rat islets and the beta cell line BRIN-BD11, we have investigated whether synthesis of BH(4) limits IL-1beta-induced NO generation and inhibition of glucose-induced insulin secretion. IL-1 beta-induced NO generation by BRIN cells and islets was reduced by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of de novo BH(4) synthesis. Sepiapterin, the substrate for salvage pathway BH(4) synthesis, reversed this inhibitory effect of DAHP in islets but not BRIN cells. DAHP reversed IL-1 beta-induced inhibition of islet insulin secretion, an effect prevented by sepiapterin. We conclude that BH(4) generation is necessary for IL-1 beta-induced NO generation in rat islets and BRIN cells. While a contribution of non-NO mediators cannot be excluded, our results support the proposal that IL-1 beta-induced, NO-mediated inhibition of insulin secretion in rat islets is dependent on the NOS cofactor BH(4).     

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.     

Suppression of interleukin-1 beta-induced nitric oxide production in RINm5F cells by inhibition of glucose-6-phosphate dehydrogenase

In rat pancreatic islets and insulin-producing cell lines, IL-1beta induces expression of inducible nitric oxide synthase and NO production leading to impairment of glucose-stimulated insulin release and decreased cell survival. NADPH is an obligatory cosubstrate for iNOS synthesis of NO. We hypothesized that IL-1beta stimulates an increase in activity of NADPH-producing enzyme(s) prior to NO production and that this increase is necessary for NO production. Using rat insulin-secreting RINm5F cells, we found that (1) IL-1beta caused a biphasic change in the NADPH level (increased by 6 h and decreased after prolonged incubation in the presence of 2 ng/mL IL-1beta); (2) IL-1beta stimulated increased activity of glucose-6-phosphate dehydrogenase (G6PD) in a time- and dose-dependent manner, and G6PD expression was increased by about 80% after exposure to 2 ng/mL IL-1beta for 18 h: (3) IL-1beta-stimulated NO production was positively correlated with increased G6PD activity; (4) IL-1beta did not cause any significant change in enzyme activity of another NADPH-producing enzyme, malic enzyme; (5) IL-1beta-induced NO production was significantly reduced either by inhibiting G6PD activity using an inhibitor of G6PD (dehydroepiandrosterone) or by inhibiting G6PD expression using an antisense oligonucleotide to G6PD mRNA; and (6) IL-1beta stimulated a decrease in the cAMP level. 8-Bromo-cAMP caused decreased G6PD activity, and the protein kinase A inhibitor H89 led to a increase in G6PD activity in RINm5F cells. In conclusion, our data show that IL-1beta stimulated G6PD activity and expression level, providing NADPH that is required by iNOS for NO production in RINm5F cells. Also, inhibition of the cAMP-dependent PKA signal pathway is involved in an IL-1beta-stimulated increase in G6PD activity.     

Strain-dependent difference in inducible nitric oxide synthesis (iNOS) expression in rat pancreatic islets correlates with interferon regulating factor 1 (IRF-1) and heat shock protein 70 (HSP70) expression

Nitric oxide (NO) may be a necessary but not sufficient mediator of cytokine-mediated, selective beta-cell destruction. Previously, we have described a difference in NO-dependent IL-1beta sensitivity in vivo and in vitro of pancreatic islets from two rat strains, Brown Norway (BN) and Wistar Kyoto (WK), the latter being the more sensitive strain. Here we investigated whether strain-dependent, differential islet iNOS expression was associated with differences in islet expression of the IL-1 receptor type 1(IL-1RI) or interferon regulating factor 1 (IRF-1), and/or caused differences in HSP70 expression, a marker of cell defence against oxidative stress. Methods: isolated islets from both rat strains were exposed to increasing concentrations of IL-1beta (0-150 pg/ml) for 24 hours or for varying culture periods (4-48 hours) to 15 pg/ml of IL-1beta. Measurements: accumulated insulin and nitrite release into incubation medium, and islet mRNA and protein expression of iNOS, IL-1RI, IRF-1 and HSP70 by semi-quantitative RT-PCR and Western blotting. Results: Higher insulin and lower nitrite release into the incubation medium were seen for BN compared to WK rats islets in both dose- and time-response experiments. IRF-1 expression preceded iNOS expression and was more pronounced in WK than in BN islets. No strain differences were observed for islet expression of IL-1RI. A strain-dependent HSP70 expression in response to IL-1beta with the highest levels in WK rat islets following iNOS expression was seen. Conclusion: There was a strain-dependent difference in iNOS expression which was associated with IRF-1 and HSP70 expression.     

Interleukin-1 beta inhibits proinsulin conversion in rat beta-cells via a nitric oxide-dependent pathway.

Exposure of pancreatic beta-cells to interleukin-1 beta (IL-1 beta) alters their protein expression and phenotype. Previous work has shown that IL-1 beta inhibited proinsulin conversion in rat islets, but the mechanism of this inhibition remained unknown. To investigate this phenomenon further, we examined purified rat beta-cells for IL-1 beta-induced inhibition of proinsulin conversion and nitric oxide (NO)-dependency of this inhibitory process. Rat beta-cells were cultured for 24 h with or without IL-1 beta and the inducible-nitric-oxide-synthase (iNOS) inhibitor N(G)-methyl-L-arginine (NMA). Exposure to IL-1 beta suppressed proinsulin-1 and proinsulin-2 synthesis by more than 50 %. Conversion of both proinsulin isoforms was also delayed. The suppressive effects of IL-1 beta on proinsulin synthesis and conversion were prevented by addition of NMA. Exposure to IL-1 beta also decreased the expression of the proinsulin convertase (PC) PC2. This decrease in PC2 expression was NO-dependent. In conclusion, IL-1 beta inhibition of proinsulin conversion in rat beta-cells occurs via an NO-mediated pathway.     

Amomum xanthoides extract prevents cytokine-induced cell death of RINm5F cells through the inhibition of nitric oxide formation

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.     

Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.     

Hydrogen sulfide potentiates interleukin-1beta-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells

Hydrogen sulfide (H(2)S) and nitric oxide (NO) are endogenously synthesized from l-cysteine and l-arginine, respectively. They might constitute a cooperative network to regulate their effects. In this study, we investigated whether H(2)S could affect NO production in rat vascular smooth muscle cells (VSMCs) stimulated with interleukin-1beta (IL-1beta). Although H(2)S by itself showed no effect on NO production, it augmented IL-beta-induced NO production and this effect was associated with increased expression of inducible NO synthase (iNOS) and activation of nuclear factor (NF)-kappaB. IL-1Beta activated the extracellular signal-regulated kinase 1/2 (ERK1/2), and this activation was also enhanced by H(2)S. Inhibition of ERK1/2 activation by the selective inhibitor U0126 inhibited IL-1beta-induced NF-kappaB activation, iNOS expression, and NO production either in the absence or presence of H(2)S. Our findings suggest that H(2)S enhances NO production and iNOS expression by potentiating IL-1beta-induced NF-kappaB activation through a mechanism involving ERK1/2 signaling cascade in rat VSMCs.     

Diethylmaleate and buthionine sulfoximine, glutathione-depleting agents, differentially inhibit expression of inducible nitric oxide synthase in endotoxemic mice.

Diethylmaleate (DEM) and buthionine sulfoximine (BSO), glutathione (GSH)-depleting agents, reduced the metabolic activity and the protein level of iNOS in both macrophages and hepatocytes activated by lipopolysaccharide (LPS). In this study, we examined the effects of DEM and BSO on iNOS expression in LPS-treated mice under the assumption that the level of GSH may alter the expression of nitric oxide synthase. Serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were also determined. DEM markedly decreased the levels of hepatic GSH in response to LPS. Treatment of mice with DEM significantly reduced serum nitrite/nitrate levels and hepatic iNOS protein and mRNA induction by LPS. Although BSO inhibited the level of hepatic GSH in LPS-treated mice, the agent did not alter serum nitrite/nitrate levels and hepatic iNOS expression. DEM completely inhibited an increase of serum IL-1beta level by LPS, whereas BSO failed to inhibit it. Neither DEM nor BSO significantly affected the induction of serum TNF-alpha level by LPS. These results showed that DEM and BSO differentially affect the expression of iNOS in endotoxemic mice, suggesting the possibility that suppression of iNOS expression by DEM may be associated with the inhibition of IL-1beta but not of TNF-alpha.     

Inhibition of p38 mitogen-activated protein kinase attenuates interleukin-1beta-induced thermal hyperalgesia and inducible nitric oxide synthase expression in the spinal cord

We have reported recently that intrathecal (i.t.) injection of interleukin-1beta (IL-1beta), at a dose of 100 ng, induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in the spinal cord and results in thermal hyperalgesia in rats. This study further examines the role of mitogen-activated protein kinase (MAPK) in i.t. IL-1beta-mediated iNOS-NO cascade in spinal nociceptive signal transduction. All rats were implanted with an i.t. catheter either with or without an additional microdialysis probe. Paw withdrawal latency to radiant heat is used to assess thermal hyperalgesia. The iNOS and MAPK protein expression in the spinal cord dorsal horn were examined by western blot. The [NO] in CSF dialysates were also measured. Intrathecal IL-1beta leads to a time-dependent up-regulation of phosphorylated p38 (p-p38) MAPK protein expression in the spinal cord 30-240 min following IL-1beta injection (i.t.). However, neither the phosphorylated extracellular signal-regulated kinase (p-ERK) nor phosphorylated c-Jun NH2-terminal kinase (p-JNK) was affected. The total amount of p38, ERK, and JNK MAPK proteins were not affected following IL-1beta injection. Intrathecal administration of either selective p38 MAPK, or JNK, or ERK inhibitor alone did not affect the thermal nociceptive threshold or iNOS protein expression in the spinal cord. However, pretreatment with a p38 MAPK inhibitor significantly reduced the IL-1beta-induced p-p38 MAPK expression by 38-49%, and nearly completely blocked the subsequent iNOS expression (reduction by 86.6%), NO production, and thermal hyperalgesia. In contrast, both ERK and JNK inhibitor pretreatments only partially (approximately 50%) inhibited the IL-1beta-induced iNOS expression in the spinal cord. Our results suggest that p38 MAPK plays a pivotal role in i.t. IL-1beta-induced spinal sensitization and nociceptive signal transduction.     

蛋白激酶C和蛋白酪氨酸激酶介导脂多糖及细胞因子诱导大鼠血管平滑肌细胞一氧化氮的生成

采用Spprague-Dawley大鼠胸主动脉中膜、外膜和培养的血管平滑肌细胞(VSMCs)作材料,鉴定不同类型的血管组织经炎性介质刺激后其一氧化氮(NO)的产生来源,闻明蛋白激酶C(PKC)和蛋白酪氨酸激酶(PTK)介导大鼠VSMCs生成NO的调控机制。大鼠VSMCs经脂多糖(LPG)和细胞因子(TNF-α,IL-1β)处理后,以剂量依赖方式促进NO释放。采用Western Blot证实经刺激的VSMCs伴有iNOS表达上调。进一步实验表明PKC和PTK参与LPS和细胞因子诱导NO生成的胞内信号转导。用PKC抑制剂H7与VSMCs共培育,H7能明显减少LPS、TNF-α和IL-1β诱导细胞NO的形成。白屈菜赤碱亦可抑制NO的生成,但HAl004对VSMCs的NO生成无抑制作用,提示PKC参与NO的生成与调控。PTK抑制剂genistein和tyrphostin AG18均能抑制由LPS、TNF-α和IL-1β引发VSMCs释放NO,同时伴iNOS蛋白表达下调,而PKC抑制剂不能阻断iNOS的表达。上述观察结果提示,PKC介导LPS和细胞因子诱导细胞合成NO可能是通过iNOS翻译后加工;而PTK则以上调iNOS表达而促增NO生成。     

A pyrrolidinone derivative inhibits cytokine-induced iNOS expression and NF-kappaB activation by preventing phosphorylation and degradation of IkappaB-alpha

We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expression is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1beta-induced iNOS gene expression, NF-kappaB activation, and NF-kappaB inhibitor (IkappaB)-alpha degradation in cultured rat VSMCs. N2733 dose-dependently (10-100 microM) inhibited IL-1beta-stimulated NO production, and decreased IL-1beta-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation. Western blot analyses involving anti-IkappaB-alpha and anti-phospho IkappaB-alpha antibodies showed that IL-1beta induced transient degradation of IkappaB-alpha preceded by the rapid appearance of phosphorylated IkappaB-alpha, both of which were markedly blocked by N2733. N2733 blocked IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of a proteasome inhibitor (MG115). Immunoblot analysis involving anti-IkappaB kinase (IKK)-alpha and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1beta-induced IKK-alpha phosphorylation, whereas N2733 had no inhibitory effect on IL-1beta-stimulated p42/p44 MAP kinase or p38 MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1beta-induced NF-kappaB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-alpha activation, and subsequent phosphorylation and degradation of IkappaB-alpha in rat VSMCs.     

Glucagon decreases cytokine induction of nitric oxide synthase and action on insulin secretion in RIN5F cells and rat and human islets of Langerhans.

Nitric oxide synthase, induced by cytokines in insulin-containing cells, produces nitric oxide which inhibits function and may promote cell killing. Since glucagon was shown to prevent inducible nitric oxide synthase (iNOS) expression in rat hepatocytes it was of interest to examine the action of glucagon (and cyclic AMP) on iNOS induction in insulin-producing cells. Cultured RIN5F cells and primary rat and human islets of Langerhans were treated with interleukin 1beta (IL-1beta) or a combination of cytokines, and were co-treated or pre-treated with glucagon. In RIN5F cells, the activity of iNOS induced by IL-1beta (10 pM, 24 h), was significantly reduced by glucagon (1000 nM), which raises cyclic AMP, and by forskolin (1-10 microM), a non specific activator of adenylate cyclase. Glucagon and forskolin also decreased iNOS expression in RIN5F cells, and rat and human islets, as shown by Western blotting. The inhibitory action of IL-1beta (100 pM, 24 h) on rat islet insulin secretion was partially reversed by 1-h pre-treatment with glucagon (10-1000 nM), while the contrasting stimulatory effect of 48-h treatment with cytokines on insulin secretion from human islets was similarly prevented by glucagon (1000 nM) pre-treatment. These results suggest that glucagon inhibits iNOS expression in insulin-containing cells and imply that glucagon could modulate the inhibitory effects of cytokines.     

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.     

Requirements of calcium fluxes and ERK kinase activation for glucose- and interleukin-1β-induced β-cell apoptosis

Increasing evidence indicates that beta-cell apoptosis and impaired secretory function were partly mediated by interleukin (IL)-1beta and/or high-glucose-induced beta-cell production of IL-1beta. However, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this study, we investigated whether Ca(2+) and extracellular signal-regulated kinase (ERK) activation plays a role for IL-1beta action in rat islets. Exposure of rat islets for 4 days to 33.3 mM glucose and 140 ng/ml IL-1beta- induced beta-cell apoptosis and impaired glucose-stimulated insulin secretion. By Western blotting with phosphospecific antibodies, glucose and IL-1beta were shown to activate ERK. Ca(2+) channel blocker nimodipine or ERK inhibitor PD98059 prevented glucose- and IL-1beta-induced ERK activation, beta-cell apoptosis, and impaired function. Furthermore, treatment with Ca(2+) ionophore ionomycin, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced ERK activation in rat islet. On the other hand, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl] (BAPTA/AM) and an inhibitor of calmodulin (W7) diminished IL-1beta-induced phosphorylation of ERK. Finally, islet release of IL-1beta in response to high glucose could be abrogated by nimodipine, mibefradil, or PD98059. Together, these data suggest that glucose- and IL-1beta-induced beta-cell secretory dysfunction and apoptosis are Ca(2+) influx and ERK dependent in rat islets.