L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic beta-cell line and protects from pro-inflammatory cytokine-induced apoptosis

Acute effects of nutrient stimuli on pancreatic beta-cell function are widely reported; however, the chronic effects of insulinotropic amino acids, such as L-alanine, on pancreatic beta-cell function and integrity are unknown. In the present study, the effects of prolonged exposure (24 h) to the amino acid L-alanine on insulin secretory function, gene expression and pro-inflammatory cytokine-induced apoptosis were studied using clonal BRIN-BD11 cells. Expression profiling of BRIN-BD11 cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the iNOS (inducible nitric oxide synthase) inhibitor NMA (N(G)-methyl-L-arginine acetate) or the iNOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL-1beta (interleukin-1beta), TNF-alpha (tumour necrosis factor-alpha) and IFN-gamma (interferon-gamma)] was additionally assessed by flow cytometry. Culture for 24 h with 10 mM L-alanine resulted in desensitization to the subsequent acute insulin stimulatory effects of L-alanine. This was accompanied by substantial changes in gene expression of BRIN-BD11 cells. Sixty-six genes were up-regulated >1.8-fold, including many involved in cellular signalling, metabolism, gene regulation, protein synthesis, apoptosis and the cellular stress response. Subsequent functional experiments confirmed that L-alanine provided protection of BRIN-BD11 cells from pro-inflammatory cytokine-induced apoptosis. Protection from apoptosis was mimicked by NMA or DPI suggesting L-alanine enhances intracellular antioxidant generation. These observations indicate important long-term effects of L-alanine in regulating gene expression, secretory function and the integrity of insulin-secreting cells. Specific amino acids may therefore play a key role in beta-cell function in vivo.     

Can the interleukin-6 response to endotoxin be predicted? Studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines

OBJECTIVES: To evaluate whether the -174 G/C promoter polymorphism of the interleukin-6 gene, gender, the monocyte density of the endotoxin receptor CD14, or the inflammatory cytokines tumor necrosis factor-alpha or interleukin-1beta influence the interleukin-6 response of whole blood to endotoxin. DESIGN: Analysis of interleukin-6 release from endotoxin-stimulated human whole blood. SETTING: Medical research laboratory. PATIENTS: Healthy human blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The interleukin-6 -174 G/C and the tumor necrosis factor -308 G/A promoter polymorphisms were determined by real-time polymerase chain reaction assay by using specific fluorescence labeled hybridization probes. Monocyte CD14 expression was assessed by flow cytometry. After incubation of whole blood with endotoxin, plasma concentrations of interleukin-6, tumor necrosis factor-alpha, and interleukin-1beta were measured by means of chemiluminescence. The interleukin-6 concentrations were lower (p = .005) in individuals who were CG heterozygotes compared with individuals homozygous for the C or the G. The difference between C and G homozygotes was not significant (p = .67). The interleukin-6 response was enhanced in men compared with women (p = .015). There was no correlation between interleukin-6 concentrations and monocyte CD14 density. Interleukin-6 concentrations correlated with the concentrations of tumor necrosis factor-alpha (r = .59, p = .01) and interleukin-1beta (r = .47, p = .01). There was no linkage between the tumor necrosis factor -308 and the interleukin-6 -174 polymorphisms. CONCLUSIONS: The interleukin-6 response to endotoxin was influenced by gender and correlated with the concentrations of more proximal cytokine tumor necrosis factor-alpha and interleukin-1beta. The interleukin-6 -174 G/C promoter polymorphism can only partly predict the interleukin-6 response of human whole blood to endotoxin stimulation, and the results were different from previous reporter gene assays that reported higher interleukin-6 concentrations for the G allele. Tumor necrosis factor -308 G homozygotes produce the lowest tumor necrosis factor concentrations. The number of tumor necrosis factor -308 G homozygotes was not higher among interleukin-6 -174 heterozygotes, and thus this cannot account for their significantly smaller interleukin-6 production.     

Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood

OBJECTIVE: To investigate the role of endotoxin-induced inflammatory mediators in blood on the permeability of endothelial monolayers. DESIGN: Whole blood of healthy volunteers was treated with bacterial lipopolysaccharide (Escherichia coli, B55:05), and the resultant plasma was added to human umbilical venular endothelial cells (HUVEC) cultured on semipermeable membrane inserts (Transwells). SETTING: University hospital laboratory. SUBJECTS: Whole blood of healthy volunteers. INTERVENTIONS: Donor plasma was treated with excess antibodies against either tumor necrosis factor-alpha, interleukin-1beta, or both, before the incubation on HUVEC. MEASUREMENTS AND MAIN RESULTS: The permeability of HUVEC monolayers to fluorescent-labeled albumin and dextran was measured over a 6-hr period, after removal of the stimulus. The production of tumor necrosis factor-alpha and interleukin-1beta in lipopolysaccharide-treated whole blood was determined by radioimmunoassay. Individually, lipopolysaccharide (10 microg/mL), tumor necrosis factor-alpha (10 ng/mL), and interleukin-1beta (50 ng/mL) all increased endothelial permeability by about 2.5-fold. A much larger increase could be achieved by preincubation of lipopolysaccharide (10 microg/mL) in whole blood: the resultant plasma induced a ten-fold increase of the permeability. The permeability response after preincubation of lipopolysaccharide in whole blood was time- and dose-dependent. Moreover, this treatment increased the sensitivity of endothelial monolayers to lipopolysaccharide by a factor of several thousand-fold: Whereas high doses of lipopolysaccharide were required for direct stimulation of the permeability, picomolar amounts of lipopolysaccharide in whole blood induced a similar increase. Significant amounts of tumor necrosis factor-alpha and interleukin-1beta were produced in blood at similar doses of lipopolysaccharide. The addition of antibodies against tumor necrosis factor-alpha or interleukin-1beta to plasma partially but significantly abrogated the permeability increase. However, a complete inhibition could be achieved by the simultaneous addition of anti-tumor necrosis factor-alpha and anti-interleukin-1beta to plasma. CONCLUSIONS: Although lipopolysaccharide is capable of directly inducing endothelial permeability, blood-borne tumor necrosis factor-alpha and interleukin-1beta mediate lipopolysaccharide-induced endothelial permeability at low endotoxin concentrations. These findings support the idea that multifactorial inhibition of inflammatory mediators may improve survival in septic patients.     

Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide.

OBJECTIVE: Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced hyperpermeability. Induction of inducible nitric oxide synthase (iNOS), an isoform of the enzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-gamma, interleukin-1beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hydrophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N(G)-monomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiron (a superoxide radical anion scavenger) on the development of cytokine-induced hyperpermeability. DESIGN: Caco-2 monolayers were incubated under control conditions or with IFN-gamma, interleukin-1beta, or tumor necrosis factor-alpha alone, pairwise combinations of these cytokines, or all three cytokines together (cytomix; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blot analysis. The permeability of Caco-2 monolayers growing on permeable supports in bicameral chambers was assessed by measuring the apical-to-basolateral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction. RESULTS: After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Expression of iNOS mRNA was far greater in cells incubated with CM than in cells treated with IFN-gamma alone or either of the two-component IFN-gamma-containing cytokine combinations. Compared with IFN-gamma, CM resulted in a higher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and approximately 20-fold after incubation for 48 hrs with IFN-gamma alone and CM, respectively. The increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N(G)-monomethyl arginine, or Tiron. CONCLUSIONS: IFN-gamma-containing combinations of cytokines are potent inducers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an important mediator of cytokine-induced gut epithelial hyperpermeability.     

Intraislet release of interleukin 1 inhibits beta cell function by inducing beta cell expression of inducible nitric oxide synthase

Cytokines, released in and around pancreatic islets during insulitis, have been proposed to participate in beta-cell destruction associated with autoimmune diabetes. In this study we have evaluated the hypothesis that local release of the cytokine interleukin 1 (IL-1) by nonendocrine cells of the islet induce the expression of inducible nitric oxide synthase (iNOS) by beta cells which results in the inhibition of beta cell function. Treatment of rat islets with a combination of tumor necrosis factor (TNF) and lipopolysaccharide (LPS), conditions known to activate macrophages, stimulate the expression of iNOS and the formation of nitrite. Although TNF+LPS induce iNOS expression and inhibit insulin secretion by intact islets, this combination does not induce the expression of iNOS by beta or alpha cells purified by fluorescence activated cell sorting (Facs). In contrast, IL-1 beta induces the expression of iNOS and also inhibits insulin secretion by both intact islets and Facs-purified beta cells, whereas TNF+LPS have no inhibitory effects on insulin secretion by purified beta cells. Evidence suggests that TNF+LPS inhibit insulin secretion from islets by stimulating the release of IL-1 which subsequently induces the expression of iNOS by beta cells. The IL-1 receptor antagonist protein completely prevents TNF+LPS-induced inhibition of insulin secretion and attenuates nitrite formation from islets, and neutralization of IL-1 with antisera specific for IL-1 alpha and IL-1 beta attenuates TNF+LPS-induced nitrite formation by islets. Immunohistochemical localization of iNOS and insulin confirm that TNF+LPS induce the expression of iNOS by islet beta cells, and that a small percentage of noninsulin-containing cells also express iNOS. Local release of IL-1 within islets appears to be required for TNF+LPS-induced inhibition of insulin secretion because TNF+LPS do not stimulate nitrite formation from islets physically separated into individual cells. These findings provide the first evidence that a limited number of nonendocrine cells can release sufficient quantities of IL-1 in islets to induce iNOS expression and inhibit the function of the beta cell, which is selectively destroyed during the development of autoimmune diabetes.     

Role of inducible nitric-oxide synthase in regulation of whole-cell current in lung epithelial cells

Lung inflammation is associated with enhanced expression of proinflammatory cytokines and increased production of nitric oxide (NO) by inducible NO synthase (iNOS). To investigate the possible relationship between cytokine-induced expression of iNOS and epithelial ion channel function, we measured whole-cell current in A549 cells treated with a mixture of cytokines: tumor necrosis factor, interleukin-1 beta, and interferon-gamma for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donor S-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 microM GSNO increased the whole-cell current from 11.2 +/- 1.8 to 19.6 +/- 2.7 pA/pF (n = 16) in control cells, but had no effect in cytokine-treated cells (n = 9). N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of iNOS, restored activation of the current by GSNO in cytokine-treated cells, indicating a crucial role for iNOS in this process. Cells treated with cytokines showed increased levels of peroxynitrite (ONOO(-)), compared with the control, or cells that were treated with the cytokines and 1400W or superoxide dismutase/catalase. Treatment of cells with 100 microM ONOO(-) had no effect on the whole-cell current, but in contrast to untreated cells, subsequent application of GSNO did not activate the current. In conclusion, cytokine-induced expression of iNOS affects activation of the whole-cell current via NO/cGMP pathway, likely by increasing the generation of ONOO(-).     

Effect of interferon-gamma, interleukin-10, lipopolysaccharide and tumor necrosis factor-alpha on chitotriosidase synthesis in human macrophages.

Human chitotriosidase is a chitinolytic enzyme and mainly produced by activated macrophages. Recently, we observed that prolactin, which is structurally related to several cytokines and is involved in regulating monocyte/macrophage functions, upregulates chitotriosidase gene expression in human macrophages, suggesting that chitotriosidase is not only a biochemical marker of macrophage activation in lysosomal diseases and hematological disorders, but also may reflect induction of an immunological response. To confirm this hypothesis we evaluated by quantitative real-time PCR the mRNA chitotriosidase levels in human monocytes/macrophages following treatment with pro-inflammatory stimuli such as interferon-gamma, tumor necrosis factor-alpha, lipopolysaccharide, and interleukin-10, an anti-inflammatory cytokine. Stimulation of macrophages with interferon-gamma, tumor necrosis factor-alpha and lipopolysaccharide resulted in increased levels of chitotriosidase mRNA, as well as chitotriosidase activity, whereas interleukin-10 decreased chitotriosidase synthesis. This finding is consistent with the hypothesis that the production of chitotriosidase by macrophages could have biological relevance in the immune response.     

Glabridin, an isoflavan from licorice root, inhibits inducible nitric-oxide synthase expression and improves survival of mice in experimental model of septic shock

(R)-4-(3,4-Dihydro-8,8-dimethyl)-2H,8H-benzo[1,2-b:3,4-b']dipyran-3yl)-1,3-benzenediol (glabridin), a flavonoid present in licorice extract, is known to have antimicrobial, anti-inflammatory, and cardiovascular protective activities. In the present study, we report the inhibitory effect of glabridin on nitric oxide (NO) production and inducible nitric oxide (iNOS) gene expression in murine macrophages. Glabridin attenuated lipopolysaccharide (LPS)-induced NO production in isolated mouse peritoneal macrophages and RAW 264.7 cells, a mouse macrophage-like cell line. Moreover, iNOS mRNA expression was also blocked by glabridin treatment in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced nuclear factor (NF)-kappaB/Rel DNA binding activity and NF-kappaB/Rel-dependent reporter gene activity were significantly inhibited by glabridin in RAW 264.7 cells and that this effect was mediated through the inhibition of inhibitory factor-kappaB degradation and p65 nuclear translocation. Moreover, reactive oxygen species generation was also suppressed by glabridin treatment in RAW 264.7 cells. In contrast, the activity of mitogen-activated protein kinases was unaffected by glabridin treatment. In animal model, in vivo administration of glabridin increased the rate of survival of LPS-treated mice and inhibited LPS-induced increase in plasma concentrations of nitrite/nitrate and tumor necrosis factor-alpha. Collectively, these data suggest that glabridin inhibits NO production and iNOS gene expression by blocking NF-kappaB/Rel activation and that this effect was mediated, at least in part, by inhibiting reactive oxygen species generation. Furthermore, in vivo anti-inflammatory effect of glabridin suggests a possible therapeutic application of this agent in inflammatory diseases.     

Comparison of tumor necrosis factor-alpha effect on the expression of iNOS in macrophage and cardiac myocytes

Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), are elevated during cardiopulmonary bypass (CPB), heart failure, and inflammatory cardiac and systemic diseases. Elevated TNF-alpha has been linked to diminished cardiac function, decreased systemic vascular resistance, as well as renal and pulmonary dysfunction. It is understood that myocardial tissues can express TNF-alpha, which results in the induction of inducible nitric oxide synthase (iNOS) leading to a significant decline in cardiac function and other direct effects. The hypothesis of this study was to determine if TNF-alpha would stimulate iNOS and its product nitric oxide (NO) similarly in immortalized macrophage and cardiac myocytes. Cultured macrophages (RAW 264.7) and cardiac myocytes (HL-1) were placed into two treatment groups and a control. The treatments included: (1) TNF-alpha and lipopolysaccharide (LPS); and (2) LPS, TNF-alpha, interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) incubated for 8 h. The macrophage expression of iNOS increased by 365% (p < 0.01) and its product, NO, increased proportionally. The expression of iNOS in the cardiac myocyte did not increase with TNF-alpha and LPS. However, with the addition of IFN-alpha and IL-1beta iNOS increased to 140% of control (p < 0.05). Myocyte cGMP and NO did not increase significantly with TNF-alpha treatment. This study suggests that HL-1 myocyte iNOS cannot be induced by TNF-alpha, unlike macrophage iNOS. Furthermore, the resultant cardiac dysfunction, secondary to proinflammatory cytokines effects, is regulated via diverse pathways.     

Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on lipocyte contractility.

In liver injury, perisinusoidal cells known as lipocytes (Ito cells) undergo "activation," acquiring smooth muscle-like features and a contractile phenotype. To assess whether contraction of these cells is regulated by nitric oxide (NO), we examined the production of NO by lipocytes and the effect of NO on lipocyte contractility. Cultured lipocytes were exposed to cytokines and/or LPS. Single agents had little or no effect on the level of inducible NO synthase (iNOS) mRNA. However, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), or LPS in combination with interferon-gamma (IFN-gamma) stimulated iNOS mRNA, which was present within 4 h after exposure. iNOS mRNA levels were paralleled by changes in nitrite (a metabolic product of NO). Intraperitoneal administration of IFN-gamma, TNF-alpha, and LPS led to rapid induction of iNOS mRNA in lipocytes, confirming in vivo the culture findings. Ligation of the common hepatic bile duct, which induces periportal-based liver injury, stimulated iNOS mRNA in lipocytes. Transforming growth factor-beta 1 decreased IFN-gamma/TNF-alpha--stimulated iNOS mRNA and nitrite. Finally, the effect of NO on lipocyte contractility was examined. In cells incubated with IFN-gamma and TNF-alpha, the contractile response to either serum or endothelin-1 was blocked. Contraction was restored entirely by an inhibitor of NO synthase, NG-monomethylarginine. Furthermore, 8-bromoguanosine 3':5'-cyclic monophosphate and sodium nitroprusside inhibited lipocyte contractility, consistent with the effect of NO induced by cytokines. We conclude that NO is a potent modulator of lipocyte contractility and may regulate this function by autocrine (or intracrine) mechanisms. Moreover, NO may play an important role in liver injury, countering the effect of contractile agonists on lipocytes.     

Differential effect of benzydamine on pro- versus anti-inflammatory cytokine production: lack of inhibition of interleukin-10 and interleukin-1 receptor antagonist

The production and action of primary proinflammatory cytokines are strictly controlled by a series of circuits to avoid damage that they can cause if produced in excess. Interleukin-10 and interleukin-1 receptor antagonist contribute to the control of the magnitude of the inflammatory responses in vivo. Benzydamine, a non-steroidal anti-inflammatory drug that has been shown to have suppressive activity for the proinflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, was investigated for its effects on interleukin-10 and interleukin-1ra production. The drug did not modify the production of interleukin-10 and interleukin-1ra by peripheral blood mononuclear cells stimulated with lipopolysaccharide, under conditions where tumor necrosis factor-alpha and interleukin-1beta were decreased. The antiinflammatory capacity of benzydamine might thus result from its ability to reduce the production of proinflammatory cytokines, without affecting antiinflammatory factors.