Nils Welsh

The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic beta cells

SummaryThe radical nitric oxide (NO) is a possible mediator of pancreatic beta-cell damage in insulin-dependent diabetes mellitus (IDDM). NO is produced by the enzyme nitric oxide synthase (NOS), in a reaction where arginine is the main substrate. There are different isoforms of NOS, but in the context of immune mediated beta-cell damage the inducible form of NOS (iNOS) is the most relevant. The beta-cell iNOS is similar and encoded by the same gene on chromosome 17 as the iNOS expressed in macrophages and other nucleated cells. iNOS activation depends on gene transcription and de novo enzyme synthesis, and NO seems to induce a negative feedback on iNOS expression. While iNOS mRNA is induced by interleukin-1Β (IL-1Β) alone in rodent insulin-producing cells, a combination of two (IL-1Β + interferon γ) (IFN-γ) or three (IL-1Β + IFNγ + tumour necrosis factor α) cytokines is required for iNOS activation in human pancreatic islets. The promoter region of the murine iNOS gene has at least 25 binding sites for different transcription factors, and the nuclear transcription factor κB is necessary for cytokine-induced iNOS transcription in both rodent and human pancreatic islets. The nature of other transcription factors relevant for iNOS regulation in these cells remains to be determined. Induction of iNOS is paralleled by induction of several other cytokine-dependent genes in beta cells, including argininosuccinate synthetase, cyclooxygenase and manganese superoxide dismutase. Some of these genes may contribute to beta-cell damage, while others are probably involved in beta-cell defence and/or repair. Regulation of iNOS and other related genes in beta cells is complex, and differs in several aspects from that observed in macrophages. There are also important differences in iNOS regulation between rodent and human pancreatic islets. A detailed knowledge of the molecular regulation of these genes in beta cells may be instrumental in the development of new approaches to prevent beta-cell destruction in early IDDM.

Cytokines suppress human islet function irrespective of their effects on nitric oxide generation.

Cytokines have been proposed as inducers of beta-cell damage in human insulin-dependent diabetes mellitus via the generation of nitric oxide (NO). This concept is mostly based on data obtained in rodent pancreatic islets using heterologous cytokine preparations. The present study examined whether exposure of human pancreatic islets to different cytokines induces NO and impairs beta-cell function. Islets from 30 human pancreata were exposed for 6-144 h to the following human recombinant cytokines, alone or in combination: IFN-gamma (1,000 U/ml), TNF-alpha (1,000 U/ml), IL-6 (25 U/ml), and IL-1 beta (50 U/ml). After 48 h, none of the cytokines alone increased islet nitrite production, but IFN-gamma induced a 20% decrease in glucose-induced insulin release. Combinations of cytokines, notably IL-1 beta plus IFN-gamma plus TNF-alpha, induced increased expression of inducible NO synthase mRNA after 6 h and resulted in a fivefold increase in medium nitrite accumulation after 48 h. These cytokines did not impair glucose metabolism or insulin release in response to 16.7 mM glucose, but there was an 80% decrease in islet insulin content. An exposure of 144 h to IL-1 beta plus IFN-gamma plus TNF-alpha increased NO production and decreased both glucose-induced insulin release and insulin content. Inhibitors of NO generation, aminoguanidine or NG-nitro-L-arginine, blocked this cytokine-induced NO generation, but did not prevent the suppressive effect of IL-1 beta plus IFN-gamma plus TNF-alpha on insulin release and content. In conclusion, isolated human islets are more resistant to the suppressive effects of cytokines and NO than isolated rodent islets. Moreover, the present study suggests that NO is not the major mediator of cytokine effects on human islets.

Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro.

The aim of the present investigation was to study whether prolonged exposure of isolated rat islets to the long chain fatty acid sodium palmitate leads to uncoupling of respiration. It was found that culture of islets in the presence of palmitate abolished glucose-sensitive insulin release and decreased insulin contents. This was paralleled by decreased ATP contents, increased respiration, and decreased islet cell mitochondrial membrane potential. Using electron microscopy, an increase in the β-cell mitochondrial volume in islets exposed to palmitate was observed. The addition of the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, at a concentration that decreased mitochondrial membrane potential to a similar extent as palmitate, diminished the glucose-induced insulin release. In addition, islet generation of reactive oxygen species, but not of nitric oxide, was increased in response to a long-term palmitate exposure. It is concluded that long-term exposure to a long chain fatty acid induc...

Interleukin-1β induces the expression of an isoform of nitric oxide synthase in insulin-producing cells, which is similar to that observed in activated macrophages

The suppressive and cytotoxic effects of interleukin‐1β(IL‐β) on rodent insulin‐producing cells observed in vitro are probably mediated through formation or nitric oxide (NO). In this study we demonstrate that IL‐1‐induced NO formation in isolated rat islets and insulin‐producing HIT cells is more sensitive to inhibition by N G‐monomethyl‐l‐arginine than to inhibition by N G‐nitro‐l‐arginine, thus suggesting that IL‐1‐exposed insulin producing cells express an isoform of nitric oxide synthase similar to that present in activated macrophages, Furthermore, IL‐1β markedly increased the mRNA levels or the inducible microphage form of nitric oxide synthase in HIT cells.

Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (Δψm) in pancreatic RINm5F cells: prevention by Bcl-2

The mechanisms of cytokine-induced beta-cell death are poorly characterised. In rat insulin-producing RINm5F cells, the combination of interleukin-1beta, interferon-gamma and tumour necrosis factor-alpha presently induced disruption of the mitochondrial membrane potential (Deltapsi(m)) as demonstrated by reduced JC-1 fluorescence. The reduction of Deltapsi(m) was maximal after 8 h and was preceded by increased formation of reactive oxygen species (ROS), as assessed by dichlorofluorescein-diacetate (DCFH-DA) fluorescence. A nitric oxide synthase-, but not a ROS-inhibitor, prevented cytokine-induced loss of Deltapsi(m). Overexpression of the anti-apoptotic protein Bcl-2 increased both JC-1 and DCFH-DA fluorescence, which was paralleled by protection against cytokine-induced apoptosis and necrosis. It is concluded that cytokines induce a nitric oxide-dependent disruption of Deltapsi(m) and that this may be a necessary event for both beta-cell apoptosis and necrosis. Bcl-2 may prevent beta-cell death by counteracting mitochondrial permeability transition.

Cytokines activate the nuclear factor κB (NF-κB) and induce nitric oxide production in human pancreatic islets

We studied the ability of cytokines to activate the nuclear transcription factor NF‐κB in human pancreatic islets and the putative role of NF‐κB for cytokine‐induced NO production. Brief exposure (20 min) of human islets of Langerhans to a combination of interleukin‐lβ + interferon‐γ + tumor necrosis factor‐a induced a 2.6‐fold increase in nuclear NF‐κB activity in gel shift analysis. This increase was prevented by the NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), which also counteracted NO production by human islets exposed for 14 h to the cytokine combination. High concentrations of interleukin‐lβ alone (150 and 250 U/ml) increased NF‐κB nuclear binding but failed to induce NO formation in human islets. The present data are the first to demonstrate that cytokines activate NF‐κB in primary adult human pancreatic islets and suggest that activation of NF‐KB may be a necessary but not sufficient signal for cytokine‐induced iNOS expression in human islets of Langerhans.

Detailed transcriptome atlas of the pancreatic beta cell

BackgroundGene expression patterns provide a detailed view of cellular functions. Comparison of profiles in disease vs normal conditions provides insights into the processes underlying disease progression. However, availability and integration of public gene expression datasets remains a major challenge. The aim of the present study was to explore the transcriptome of pancreatic islets and, based on this information, to prepare a comprehensive and open access inventory of insulin-producing beta cell gene expression, the Beta Cell Gene Atlas (BCGA).MethodsWe performed Massively Parallel Signature Sequencing (MPSS) analysis of human pancreatic islet samples and microarray analyses of purified rat beta cells, alpha cells and INS-1 cells, and compared the information with available array data in the literature.ResultsMPSS analysis detected around 7600 mRNA transcripts, of which around a third were of low abundance. We identified 2000 and 1400 transcripts that are enriched/depleted in beta cells compared to alpha cells and INS-1 cells, respectively. Microarray analysis identified around 200 transcription factors that are differentially expressed in either beta or alpha cells. We reanalyzed publicly available gene expression data and integrated these results with the new data from this study to build the BCGA. The BCGA contains basal (untreated conditions) gene expression level estimates in beta cells as well as in different cell types in human, rat and mouse pancreas. Hierarchical clustering of expression profile estimates classify cell types based on species while beta cells were clustered together.ConclusionOur gene atlas is a valuable source for detailed information on the gene expression distribution in beta cells and pancreatic islets along with insulin producing cell lines. The BCGA tool, as well as the data and code used to generate the Atlas are available at the T1Dbase website (T1DBase.org).

Amelioration of diabetes by imatinib mesylate (Gleevec): role of beta-cell NF-kappaB activation and anti-apoptotic preconditioning

It was recently reported that tyrosine kinase inhibitor imatinib mesylate (Gleevec®) improves Type 2 diabetes, possibly by decreasing insulin resistance. However, as both Type 2 and Type 1 diabetes are characterized by β‐cell dysfunction and death, we investigated whether imatinib counteracts diabetes by maintaining β‐cell function. We observed that imatinib counteracted diabetes in two animal models, the streptozotocin‐injected mouse and the nonobese diabetes mouse, and that this was paralleled by a partial preservation of the β‐cell mass. In addition, imatinib decreased the death of human β‐cells in vitro when exposed to NO, cytokines, and streptozotocin. The imatinib effect was mimicked by siRNA‐mediated knockdown of c‐Abl mRNA. Imatinib enhanced β‐cell survival by promoting a state similar to ischemic preconditioning, as evidenced by NF‐B activation, increased NO and reactive oxygen species production, and depolarization of the inner mitochondrial membrane. Imatinib did not suppress islet cell death in the presence of an NF‐B inhibitor, suggesting that NF‐B activation is a necessary step in the antiapoptotic action of imatinib. We conclude that imatinib mediates β‐cell survival and that this could contribute to the beneficial effects observed in diabetes.—Hägerkvist, R., Sandler, S., Mokhtari, D., Welsh, N. Amelioration of diabetes by imatinib mesylate (Gleevec®): role of β‐cell NF‐B activation and anti‐apoptotic preconditioning. FASEB J. 21, 618–628 (2007)

Interleukin-1β induces nitric oxide production and inhibits the activity of aconitase without decreasing glucose oxidation rates in isolated mouse pancreatic islets

Summary The aim of this investigation was to further characterize the process of interleukin-lβ (IL-lβ) induced nitric oxide production in isolated pancreatic islets. It was found that both IL-1β and nitroprusside increased islet nitrite production. This effect was paralleled by inhibition of islet aconitase activity and glucose oxidation rates. Neither trifluoroperazinen or aminopterin could prevent the IL-1β induced increase in nitrite production, aconitase inhibition and decrease in glucose oxidation rates. In a second series of experiments, isolated mouse pancreatic islets were exposed to IL-1β for 24 h and subequently used for nitrite production, aconitase activity and glucose oxidation determinations. The islets responded to IL-1β with an increased nitrite production and a decreased activity of aconitase, whereas the islet glucose oxidation rates were not decreased. It is concluded that IL-1β in both rat and mouse islets induces nitric oxide formation and that this induction leads to the inhibition of the Krebs cycle enzyme aconitase. In rat islets this probably leads to an inhibited insulin secretion, whereas IL-1β in mouse islets suppresses insulin secretion by a non-mitochondrial mechanism.

Role of p38 mitogen-activated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis

The signaling pathways mediating nitric oxide production and apoptosis in pancreatic beta-cells are not fully understood. We investigated cytokine-induced protein phosphorylation events in insulin-producing cells and evaluated their role in inducible nitric oxide synthase (iNOS) induction and cell death. Interleukin-1beta (IL-1beta), but not interferon-gamma (IFN-gamma), induced phosphorylation of p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and mitogen- and stress-activated protein kinase 1 (MSK1) in rat insulin-producing RINm5F cells. This was paralleled by an increased phosphorylation of the transcription factors activating transcription factor-2 (ATF-2) and cAMP-responsive element-binding protein (CREB). The p38 inhibitor SB203580 prevented cytokine-induced phosphorylation of CREB and MSK1, but not of ATF-2. IFN-gamma induced the phosphorylation of signal transducer and activator of transcription 1. The combination of IL-1beta and IFN-gamma increased both apoptosis and necrosis in rat islet cells. SB203580, but not the extracellular signal-regulated kinase inhibitor PD98059, partially prevented cytokine-induced apoptosis, an effect that was not associated with reduced nitrite production or lowered iNOS expression. In conclusion, cytokine-induced p38 activation participates in beta-cell apoptosis, possibly by a nitric oxide-independent mechanism or by enhancing the sensitivity to nitric oxide.