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.     

Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells

In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.     

Role of putrescine in interleukin 1 beta production in human histiocytic lymphoma cell line U937

Treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and incubation with lipopolysaccharide (LPS) induces interleukin 1 beta (IL-1 beta) production in the histiocytic lymphoma cell line U937. Here we investigated the effect of treatment with both TPA and 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) on LPS-induced IL-1 beta production in U937 cells. To clarify the mechanism of IL-1 beta production, the possible role of polyamines in this process was examined. Combined treatment with TPA and 1,25(OH)2D3 for 72 h followed by incubation with LPS for 24 h caused synergistic induction of both IL-1 beta release and mRNA expression. On the other hand, TPA increased the numbers of vitamin D3 receptors, which may be one mechanism of this synergistic induction. Ornithine decarboxylase (ODC), a rate-limiting enzyme for polyamine biosynthesis, was also induced by these compounds biphasically: the first peak of ODC activity was observed at 4 h of the incubation with the two compounds and the second peak was at 4 h after the addition of LPS. To find whether these peaks were related to IL-1 beta production, DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, was added together with TPA and 1,25(OH)2D3. DFMO decreased the cellular levels of putrescine and spermidine and suppressed IL-1 beta release and IL-1 beta mRNA expression by 65%. Exogenous putrescine, but not spermidine, abrogated these kinds of inhibition. Similar results were obtained with DFMO and the polyamines during the differentiation of the cells up to the monocyte or macrophage stage. These results thus suggest that changes in either of these intracellular polyamines, especially putrescine, help to regulate the differentiation of U937 cells, resulting in partial control of the regulation of IL-1 beta production.     

Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is constitutively expressed in islet beta-cells and up-regulated by interferon-gamma

Interleukin-18 (IL-18) mRNA is expressed in islets of NOD mice during the early stages of insulitis and IL-18 has therefore been implicated as a contributing factor in immune-mediated beta-cell destruction. However, a recent study failed to show any effect of human IL-18 on the function of isolated rat islets. Since species differences have been shown between human and murine IL-18, the aims of this study were to investigate 1) if species homologous IL-18 alone or following IL-12 pre-exposure affected rat islet function, 2) if IL-18 dose-dependently modulated IL-1 beta or interferon-gamma (IFN-gamma) + tumor necrosis factor-alpha (TNF-alpha) actions on islet function, and 3) if IL-18 and IL-18 receptor (IL-18R) were expressed in rat islet beta-cells. Insulin release and nitric oxide (NO) production from isolated rat islets were measured after incubation with or without cytokines. RT-PCR was used to quantitate mRNA expression of IL-18 and the IL-18R signaling chain (IL-18R beta). There were no significant effects of 0.625-10 nM recombinant murine (rm) IL-18 alone on accumulated or glucose-challenged insulin release or NO production after 24 hours. Fifteen pg/ml of recombinant human (rh) IL-1 beta as well as 200 U/ml recombinant rat (rr) IFN-gamma + 250 U/ml rhTNF-alpha significantly increased islet NO production and inhibited both accumulated and glucose-challenged islet insulin release. However, rmIL-18 failed to modulate these effects of IL-1 beta or IFN-gamma + TNF-alpha. Although IL-12 induces IL-18R expression in Th1 and B lymphocytes, 24-hours rmIL-12 preincubation neither sensitized islets to effects of 10 nM of rm or rrIL-18 alone nor primed the islets to IL-1 beta actions on insulin release and NO production. IL-18R beta mRNA, which was expressed in human peripheral blood mononuclear cells (PBMC), was not expressed in rat insulinoma (RIN) cells or in isolated rat islets, even after exposure to IL-1 beta and/or IFN-gamma + TNF-alpha or IL-12. IL-18 mRNA was constitutively expressed in RIN cells, in FACS-purified rat beta-cells and in intact rat and mouse islets, and was up-regulated by IFN-gamma in an interferon regulatory factor-1- IRF-1) and NO - independent manner. However, IL-18 protein was undetectable in lysates and supernates of RIN cells by ECL, Western blotting and immunoprecipitation. In conclusion, we show for the first time that IL-18 but not IL-18R is expressed in rodent islet beta-cells. The physiological importance and pathological role of IL-18 originating from islet beta-cells deserve further investigation.     

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.     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet beta cells modulates insulin secretion in rats

Capsaicin-sensitive afferent neurons including transient receptor potential vanilloid subfamily 1, TRPV1, and neurohormonal peptides participate in the physiological regulation of pancreatic endocrine. However, the direct effect of capsaicin on insulin secretion remains unknown. Our present study showed that TRPV1 is expressed in islet beta cells as well as in neurons in rat pancreas, and also in rat beta cell lines, RIN and INS1. Capsaicin (10(-11)-10(-9) M) dose-dependently increased insulin secretion from RIN cells, and this effect was inhibited by either a TRPV1 inhibitor capsazepine or EDTA. Systemic capsaicin (10 mg/kg, s.c.) increased plasma insulin level 1 h after the treatment. We demonstrated for the first time that TRPV1 is functionally expressed in rat islet beta cells and plays a role in insulin secretion as a calcium channel. This study may account for the influences of capsaicin on the food intake and energy consumption as well as on the pathophysiological regulation of pancreatic endocrine.     

Activation of peroxisome proliferator-activated receptor-gamma protects pancreatic beta-cells from cytokine-induced cytotoxicity via NF kappaB pathway

Diabetes mellitus is characterized by cytokine-induced insulitis and a deficit in beta-cell mass. Ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) have been shown to have anti-inflammatory effects in various experimental models. We questioned whether activation of endogenous PPAR-gamma by either PPAR-gamma ligands or adenoviral-directed overexpression of PPAR-gamma (Ad-PPAR-gamma) could inhibit cytokine-induced beta-cell death in RINm5F (RIN) cells, a rat insulinoma cell line. Treatment of RIN cells with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) induced beta-cell damage through NF kappaB-dependent signaling pathways. Activation of PPAR-gamma by PPAR-gamma ligands or Ad-PPAR-gamma inhibited IL-1 beta and IFN-gamma-stimulated nuclear translocation of the p65 subunit and DNA binding activity. NF kappaB target gene expression and their product formation, namely inducible nitric oxide synthase and cyclooxygenase-2 were decreased by PPAR-gamma activation, as established by real-time PCR, Western blots and measurements of NO and PGE(2). The mechanism by which PPAR-gamma activation inhibited NF kappaB-dependent cell death signals appeared to involve the inhibition of I kappa B alpha degradation, evidenced by inhibition of cytokine-induced NF kappaB-dependent signaling events by Ad-I kappaB alpha (S32A, S36A), non-degradable I kappaB alpha mutant. I kappaB beta mutant, Ad-I kappaB beta (S19A, S23A) was not effective in preventing cytokine toxicity. Furthermore, a protective effect of PPAR-gamma ligands was proved by assaying for normal insulin secreting capacity in response to glucose in isolated rat pancreatic islets. The beta-cell protective function of PPAR-gamma ligands might serve to counteract cytokine-induced beta-cell destruction.     

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.