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N Welsh

Publications and source records attributed to N Welsh.

At least 55 records · Page 3Linked to original sources
PubMed1994

A role for tyrosine kinase activation in interleukin-1 beta induced nitric oxide production in the insulin producing cell line RINm-5F.

The aim of this investigation was to study the putative role of protein phosphorylation in interleukin-1 beta (IL-1 beta) induced signal transduction in insulin producing cells. For this purpose, insulin producing RINm-5F cells were exposed to IL-1 beta for 7 hours with or without different agonists and antagonists to protein kinases and phosphatases and the production of nitrite was subsequently determined. It has been shown earlier that IL-1 beta will stimulate the production of nitrite in such cells. It was found that EDTA, TPA and staurosporine did not affect IL-1 beta induced nitrite production. However, the tyrosine kinase antagonist tyrphostin inhibited, whereas sodium orthovanadate, okadaic acid and cyclosporin A, all inhibitors of protein phosphatases, potentiated IL-1 beta induced nitrite release to the medium. The tyrosine kinase antagonist genistein potentiated at a low concentration and inhibited at a high concentration the IL-1 beta effect. It is concluded that protein phosphorylation events, mediated either by protein kinases or phosphatases on both tyrosine and serine/threonine residues, may mediate or antagonize IL-1 induced signal transduction in insulin producing cells.

Alkaloids↗
PubMed1994

Protective action by hemin against interleukin-1 beta induced inhibition of rat pancreatic islet function.

We have presently investigated the putative protective role of hemin against the inhibitory actions of the cytokine interleukin-1 beta (IL-1 beta) on isolated rat pancreatic islets. For this purpose, islets were isolated from adult rats, pre-cultured for 3-7 days in RPMI 1640 medium + 10% fetal calf serum and then exposed to IL-1 beta (5 ng/ml), hemin for 1, 7 or 24 h after which islet nitrite production, aconitase activity, glucose oxidation rates, glucose-stimulated insulin release and medium insulin accumulation were determined. It was found that hemin did not prevent IL-1 beta induced nitrite production. On the other hand, hemin partially counteracted the IL-1 beta induced decrease in aconitase activity, glucose oxidation, insulin release and medium insulin accumulation. This protective effect was present at a hemin concentration of 10 microM and most pronounced at 100 microM. Furthermore, hemin induced the synthesis of a 31 kDa protein, which was shown to be heme oxygenase as demonstrated by Western blot analysis. Finally, the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), which protects against IL-1 beta by decreasing nitric oxide production, was found to act additively in combination with hemin in alleviating the IL-1 beta effects. It is proposed that the beneficial effects of hemin against IL-1 beta could be related to scavenging of nitric oxide and/or an increased resistance to nitric oxide production.

Aconitate Hydratase↗
PubMed1994

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.

Adolescent↗
PubMed1994

Liposomal delivery of antioxidant enzymes protects against hydrogen peroxide- but not interleukin-1 beta-induced inhibition of glucose metabolism in rat pancreatic islets.

The aim of the present investigation was to evaluate the putative involvement of oxygen free radicals in interleukin-1 beta (IL-1 beta)-induced suppression of islet glucose oxidation. Isolated adult rat pancreatic islets were exposed for 1 h to liposomally encapsulated superoxide dismutase (SOD; 10 mg/ml), catalase (CAT; 10 mg/ml) and glutathione peroxidase (GPX; 5 mg/ml), after which IL-1 beta (25 U/ml) or hydrogen peroxide (H2O2; 0.1 mM) was added, and the incubation was continued overnight. The following day, samples were taken from the incubation media for nitrite determinations, and islet glucose oxidation rates were measured. The CAT activity increased fourfold after addition of CAT-containing liposomes. It was found that IL-1 beta induced a marked increase in islet nitrite production, as an index of nitric oxide formation, and that this was paralleled by a decrease in islet glucose oxidation rates. H2O2-treated islets exhibited a modest decrease in glucose oxidation rates and a minor increase in the release of nitrite to the media. Treatment of islets with liposomes containing the antioxidant enzymes SOD, CAT and GPX, either alone or in combination, did not decrease the effect of IL-1 beta. However, the H2O2-induced decrease in glucose oxidation rates was counteracted by the combination of the antioxidants. It was concluded that, provided the intracellular delivery of the antioxidant enzymes to the islet cells was effective, oxygen free radicals probably do not play a decisive role in IL-1 beta suppression of islet glucose metabolism.

Animals↗
PubMed1994

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.

Adult↗
PubMed1994

Nitric oxide and pancreatic beta-cell destruction in insulin dependent diabetes mellitus: don't take NO for an answer.

A major surge of interest has recently focused upon nitric oxide (NO) as a mediator of autoimmune destruction of beta-cells in insulin-dependent diabetes mellitus (IDDM). It has been proposed that insulin producing cells in response to cytokines are induced to produce self destructing amounts of NO, and that endothelial cells or islet infiltrating macrophages may induce beta-cell death by releasing cytotoxic levels of NO within the islet. Recent findings in this field are presently discussed and we conclude that although NO might have a role in rodent IDDM, any putative role of NO in the pathogenesis of human IDDM remains to be clarified.

Animals↗
PubMed1993

Interleukin-10 stimulates rat pancreatic islets in vitro, but fails to protect against interleukin-1.

Rat pancreatic islets were cultured for 42 h in medium supplemented without or with 0.1-10 ng/ml of human interleukin-10 (IL-10). Medium insulin accumulation was increased by 50% after culture with 0.1 ng/ml IL-10. All concentrations of IL-10 increased the islet insulin content, but did not affect the islet DNA content. IL-10 did neither affect basal and glucose-stimulated insulin secretion nor (pro)insulin and total protein biosynthesis rates. Addition of 25 U/ml of interleukin-1 beta during the last 24 h of culture, led to a marked inhibition of glucose-stimulated insulin release and a 5-6 fold increase in nitrite production by rat insulinoma cells. These events could not be counteracted by any concentration of IL-10. The present data suggest that IL-10 might in some aspects stimulate pancreatic beta-cell function, but it fails to block the inhibitory action of IL-1 beta.

Animals↗
PubMed1993

Interleukin-1-induced expression of nitric oxide synthase in insulin-producing cells is preceded by c-fos induction and depends on gene transcription and protein synthesis.

The cytokine interleukin 1 beta (IL-1 beta) induces the expression of an isoform of nitric oxide synthase (NOS) in insulin-producing cells which is similar to that expressed in activated macrophages. In the present study we show that IL-1 beta-induced expression of NOS mRNA in these cells is preceded by expression of c-fos mRNA. Moreover, the stimulatory effects of recombinant IL-1 beta on NOS mRNA expression are prevented by co-incubation with an inhibitor of gene transcription (actinomycin D) or an inhibitor of protein synthesis (cycloheximide). These data suggest that IL-1 beta-induced NOS mRNA expression may be mediated by transcription of immediate early response genes, and that c-fos may be one of these genes.

Amino Acid Oxidoreductases↗
PubMed1993

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

Adenosine Triphosphate↗
PubMed1993

Predominance of stimulatory effects of interleukin-1 beta on isolated human pancreatic islets.

The aim of the present study was to characterize the effects of human recombinant interleukin-1 beta (rIL-1 beta) on human pancreatic islets. For this purpose, islets isolated from adult cadaveric donors were exposed to rIL-1 beta (1 or 3 ng/mL) for different periods of time. In some experiments, rat pancreatic islets were exposed in parallel to the cytokine. After 48 h of culture in the presence of rIL-1 beta, the human islets showed an increased insulin release during short term incubations in the presence of 1.7 or 16.7 mM glucose. There was also a 3- to 4-fold increase in insulin accumulation into the culture medium, but rIL-1 beta did not affect human islet glucose metabolism. These stimulatory effects of rIL-1 beta on human islets were already present after an acute (2-h) exposure to the cytokine, and this functional stimulation was blocked by an IL-1 receptor antagonist protein. After exposure of human islets to rIL-1 beta for 6 days, there was no effect of the cytokine on either glucose metabolism or insulin release compared to those in control islets. Rat islets exposed for 48 h in culture to the same concentrations of rIL-1 beta, however, showed a 40-60% decrease in insulin accumulation into the medium, glucose-induced insulin release, and glucose oxidation. Moreover, while there was no effect of rIL-1 beta on nitrite production by human islets, there was a 7- to 11-fold increase in nitrite production by rat islets. Nitrite is an end product of the highly reactive radical nitric oxide (NO), and there are data to suggest that NO is an important mediator of the suppressive and cytotoxic actions of IL-1 on rat islets. The present observations suggest that human islets are less sensitive to the inhibitory effects of human rIL-1 beta than rat islets, and that this is due to a lack of induction of NO synthesis by the cytokine in human islet cells.

Adult↗
PubMed1992

Interleukin-1 beta 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-1 beta (IL-1 beta) on rodent insulin-producing cells observed in vitro are probably mediated through formation of 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 NG-monomethyl-L-arginine than to inhibition by NG-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-1 beta markedly increased the mRNA levels of the inducible macrophage form of nitric oxide synthase in HIT cells.

Amino Acid Oxidoreductases↗
PubMed1992

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

The aim of this investigation was to further characterize the process of interleukin-1 beta (IL-1 beta) induced nitric oxide production in isolated pancreatic islets. It was found that both IL-1 beta 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-1 beta 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-1 beta for 24 h and subsequently used for nitrite production, aconitase activity and glucose oxidation determinations. The islets responded to IL-1 beta 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-1 beta 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-1 beta in mouse islets suppresses insulin secretion by a non-mitochondrial mechanism.

Aconitate Hydratase↗
PubMed1992

Adaptive response in beta-cell function in pancreatic islets isolated from partially pancreatectomized rats.

Before clinical onset of insulin-dependent diabetes mellitus a decreasing pancreatic beta-cell mass maintains glucose homeostasis. We currently aimed to study the function of pancreatic islets isolated 2 weeks after a 60% partial pancreatectomy (P) or after a sham operation (S) on adult rats. Experiments on the islets were subsequently performed acutely (day 0) and after 1 week (day 7) of tissue culture in medium RPMI 1640 (11.1 mM glucose) + 10% calf serum. There was no difference in the body weight 2 weeks after surgery. The pancreatic remnant weight of the P rats was 35% less than the pancreatic weight in the S rats. The islet DNA content was 25% higher in the islets of the P rats on day 0, indicating a stimulated islet growth. However, this difference did not remain after culture for 7 days. Islet proinsulin mRNA content and (pro)insulin biosynthesis rates were slightly increased in the islets of P rats on day 0, which could be due to the increased islet mass. The islet insulin content was not different on day 0, but was higher after culture in the islets of the P rats. The islet rates of glucose oxidation and insulin release were markedly higher in the P rats on day 0, suggesting a selective effect on these processes. A higher glucose oxidation rate was, however, not evident on day 7. The relative fraction of insulin-positive cells was slightly lowered in the islets of the P rats on day 0.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1992

Lithium increases DNA replication, polyamine content, and insulin secretion by rat pancreatic beta-cells.

The impact of long-term lithium exposure on the replication, polyamine content, and insulin production of rat pancreatic beta-cells was examined. Fetal rat pancreatic islets enriched in beta-cells were isolated and cultured for 3 days in the presence of different concentrations of LiCl. It was found that lithium dose dependently stimulated beta-cell replication, evoking a 40% increase in beta-cell replication at 1 mM, which was further increased to 70% at 10 mM of the ion. In contrast, the islet contents of insulin mRNA and insulin and insulin biosynthesis rates remained unaltered. The long-term insulin accumulation in the culture medium was nevertheless increased in LiCl-treated groups. In addition, neither the mitogenic effect nor the increased insulin accumulation in the medium by lithium was further augmented by growth hormone, which itself stimulated these functions. Lithium was also found to elevate the islet content of polyamines. Treatment with enzymatic inhibitors of polyamine synthesis failed to preclude the mitogenic and secretagogic impact of lithium, suggesting that the increased contents of polyamines did not convey the lithium effect. We conclude that lithium treatment stimulates rat beta-cell replication and long-term insulin secretion in vitro. These direct effects of the ion on the beta-cell may contribute to the antidiabetic effect of lithium encountered in animal models and patients.

Animals↗
PubMed1992

Interleukin-1 beta increases the activity of superoxide dismutase in rat pancreatic islets.

The suppressive effects of interleukin-1 beta (IL-1 beta) on the function of pancreatic islets may be related to induction of gene transcription and protein synthesis. Presently, the effects of human recombinant IL-1 beta (rIL-1 beta) on the activities of superoxide dismutase (SOD) and the expression of corresponding genes were studied in rat pancreatic islets. Islets that were exposed to rIL-1 beta for 48 h showed a 2.6-fold greater activity of mitochondrial manganese containing SOD (MnSOD) than control islets. The cytosolic copper- and zinc-containing SOD (CuZnSOD) was, however, less affected by rIL-1 beta. Also, brief exposure of the islets to rIL-beta induced an increase in SOD activities. Hence, 12 h after a 1-h exposure of the islets to rIL-1 beta, there was a 1.4-fold increase in the activity of both MnSOD and CuZnSOD. The early induction of SOD by rIL-1 beta was inhibited by an interleukin-1 receptor antagonist protein and actinomycin-D, which is a blocker of gene transcription. This suggests that the effects of rIL-1 beta on the islet SOD activities are dependent on binding to membrane receptors and activation of gene transcription. Northern blot analysis showed a 4-fold increase in islet MnSOD mRNA content after a 90-min incubation and a 10-fold increase after a 180-min incubation with rIL-1 beta. Thus, the enhanced MnSOD activity in the islets reflects increased gene expression. To evaluate a possible role for free oxygen radicals as mediators of the early action of rIL-1 beta on the pancreatic B-cells, isolated islets were exposed to rIL-1 beta only or to rIL-1 beta plus various free radical scavengers. None of the scavengers, single or in combinations, could counteract the suppressive action of rIL-1 beta on islet insulin secretion. The present data suggest that rIL-1 beta induces increased activity of SOD, in particular MnSOD, in pancreatic islets. This may be due to a direct action of rIL-1 beta that is mediated by an increase in gene transcription.

Actins↗
PubMed1991

Increased glucose oxidation and contents of insulin and ATP in polyamine-depleted rat insulinoma cells (RINm5F).

In order to elucidate the role of polyamines in the replication and insulin production of insulin-secreting cells, we have investigated the impact of partial polyamine depletion on the proliferation, metabolism, insulin synthesis and ultrastructure of clonal rat insulinoma cells (RINm5F). For this purpose RINm5F cells were exposed for 4 days to the specific ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). This resulted in a profound decrease in ODC activity and cytoplasmic polyamine contents. The polyamine content of cell nuclei was, however, not altered by DFMO. Addition of small amounts of putrescine during culture elevated the intracellular content of this diamine and suppressed ODC activity. The decrease in polyamine contents was accompanied by a pronounced inhibition of the cellular proliferative activity. The rates of glucose utilization, oxygen uptake and activity of the pentose cycle were decreased in DFMO-treated cells, whereas the glucose oxidation rate, oxidation/utilization ratio, ATP content and ATP/ADP ratio were increased. Insulin mRNA content and synthesis of proinsulin, insulin and total protein were not altered by DFMO. In contrast, there was a sizeable increase in the cellular insulin content, despite a lowered total protein content. Electron-microscopic analysis revealed an accumulation of insulin-secretion granules in the DFMO-treated cells. In addition, short-term insulin release was increased after DFMO exposure, but was not rendered glucose-sensitive. It is concluded that polyamines are necessary for the maintenance of rapid insulinoma-cell replication and that DFMO-treated RINm5F cells acquire an enhanced substrate oxidation and increased content of insulin and ATP.

Adenosine Diphosphate↗