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N Welsh

Publications and source records attributed to N Welsh.

At least 19 recordsLinked to original sources
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↗
PubMed1991

GTP-binding proteins may stimulate insulin biosynthesis in rat pancreatic islets by enhancing the signal-recognition-particle-dependent translocation of the insulin mRNA poly-/mono-some complex to the endoplasmic reticulum.

We aimed to elucidate the putative role of GTP-binding proteins in the regulation of insulin biosynthesis. For this purpose, freshly isolated rat islets were incubated in the presence of liposomes containing GDP, guanosine 5'-[beta-thio]diphosphate (GDP[S]), GTP, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG), guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG) and ATP, and the effects of the liposomal delivery of these substances on rates of biosynthesis of insulin and total protein were determined. Insulin biosynthesis during a 1 h incubation at 1.67 mM-glucose was stimulated by ATP- and GTP[S]-containing liposomes as compared with control liposomes. At 16.7 mM-glucose, only the GTP[S]-containing liposomes stimulated insulin biosynthesis. No inhibition of islet protein and insulin synthesis was observed with GDP-, GDP[S]-, p[CH2]ppG- and p[NH]ppG-containing liposomes. By determining the subcellular distribution of insulin mRNA, it was found that the mRNA content associated with microsomes was increased and that associated with the cytosolic mono-/poly-somes decreased when the islets were incubated with GTP[S]-containing liposomes, resulting in an approximate doubling of the ratio of microsomal to polysomal-associated insulin mRNA. ATP-containing liposomes produced no effects on the association of insulin mRNA with microsomes. By using photoaffinity labelling and immunoprecipitation techniques, specific binding of GTP[35S] to the alpha-subunit of the signal-recognition particle (SRP) receptor in islet homogenates containing physiological concentrations of GTP and GDP was demonstrated. These findings suggest that the GTP-binding subunit(s) of the SRP receptor, and possibly also of other GTP-binding proteins involved in this process, may regulate insulin biosynthesis by stimulating the translocation of insulin mRNA to the endoplasmic reticulum and by increasing preproinsulin-peptide translocation into the lumen of the reticulum.

Adenosine Triphosphate↗
PubMed1991

Decreased mitochondrial gene expression in isolated islets of rats injected neonatally with streptozotocin.

The aim of the present study was to evaluate the possible role of the expression of the mitochondrial genome for the regulation of insulin production in the pancreatic Beta cell. For this purpose, islets of Langerhans were isolated from adult control rats and rats injected neonatally with streptozotocin and the islet contents of specific mitochondrial DNAs and RNAs together with nuclear-encoded RNAs were determined. The contents of mitochondrial cytochrome b mRNA, the mitochondrial 12 S rRNA and insulin mRNA were all 30-40% lower in islets isolated from the streptozotocin-treated rats as compared to islets from control rats. In contrast, the nuclear mRNA coding for the mitochondrial adenine nucleotide translocator was not decreased in the streptozotocin-treated rats. Contents of mitochondrial DNA, as assessed by the Southern blotting technique, were markedly decreased in the streptozotocin islets. Sequence analysis of mitochondrial DNA from streptozotocin islets and control islets however, did not reveal any differences in nucleotide sequences. In control islets the contents of mitochondrial cytochrome b mRNA increased in response to a high glucose concentration during a 4-h incubation period. Serum deprivation or the addition of theophylline or 4-phorbol 12-myristate 13-acetate failed to affect the cytochrome b mRNA contents in vitro. It is concluded that islets of streptozotocin-treated rats contain low contents of mitochondrial DNA and RNA. Since a lower mitochondrial RNA content may result in a diminished oxidative capacity, it is conceivable that a deficiency of this messenger may contribute to the development of insulin deficiency.

Animals↗
PubMed1991

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure.

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.

Animals↗
PubMed1991

Interleukin-1 beta-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase.

The aim of this study was to characterize the dynamics and functional relevance of interleukin-1 beta (IL-1 beta)-induced nitric oxide production in isolated pancreatic islets. Thus, islets were isolated from adult rats, precultured for 3-5 days in medium RPMI-1640 plus 10% fetal calf serum, and then exposed to IL-1 beta for different time periods, after which islet nitrite production and aconitase activity were determined. IL-1 beta (5 ng/ml) did not increase islet nitrite production during the first hour of incubation. Moreover, the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (Meth-arg; 5 mM) failed to prevent the initial (90 min) IL-1 beta-induced increase in islet insulin release. After 4, 7, and 24 h, however, nitrite production was increased by 50%, 93%, and 139%, respectively. Islet aconitase activity and glucose oxidation rates were decreased by 70% after incubation for 24 h with IL-1 beta. Both Meth-arg and N alpha-p-tosyl-L-lysine chloromethyl ketone (0.1 mM), a protease inhibitor, could completely counteract the IL-1 beta-induced increases in nitrite production and inhibition of aconitase activity and glucose oxidation rates. In a separate series of experiments, islets were incubated for 60 min with or without IL-1 beta and the RNA synthesis inhibitor actinomycin-D (5 micrograms/ml) and subsequently incubated for another 9 h without any additions. The presence of actinomycin-D during the 1-h IL-1 beta incubation period prevented the IL-1 beta-induced rise in nitrite production and the IL-1 beta-induced inhibition of aconitase activity and insulin release. It is concluded that IL-1 beta-induced nitric oxide production is a late event which requires gene transcription and does not mediate the initial stimulatory effects of IL-1 beta on beta-cell function. However, the gradually augmented rate of nitric oxide production may inhibit the enzyme aconitase, leading to a suppressed mitochondrial activity and a defective insulin release in response to nutrient secretagogues.

Aconitate Hydratase↗
PubMed1991

Liposomal delivery of purified heat shock protein hsp70 into rat pancreatic islets as protection against interleukin 1 beta-induced impaired beta-cell function.

Recently it has been demonstrated that heat shock protein 70 (hsp70) is induced in pancreatic islet cells during prolonged exposure to interleukin 1 beta (IL-1 beta). It is unclear whether this represents a cellular defense against the noxious action of IL-1 beta or whether hsp70 is involved in the suppressive action of the cytokine. To assess the role for hsp70 in isolated islets exposed to IL-1 beta, hsp70 was purified and introduced into cells of isolated rat pancreatic islets via the liposome technique. Delivery of hsp70 was efficient according to immunoblot analysis, but delivered hsp70 disappeared within 16 h. Hsp70-containing liposomes did not affect protein synthesis, insulin secretion, or islet insulin mRNA content. However, when hsp70 liposome-incubated islets were further exposed to IL-1 beta (25 U/ml) for 16 h, these islets released more insulin in response to glucose stimulation and contained more insulin mRNA than islets incubated with control liposomes and subsequently exposed to the cytokine. No protective effect of liposomes containing bovine serum albumin or ovalbumin were observed. We conclude that hsp70 may protect against IL-1 beta-induced impairment of pancreatic beta-cell function.

Animals↗
PubMed1991

Influence of protease on inhibitory and stimulatory effects of interleukin 1 beta on beta-cell function.

To elucidate the putative role of proteases in the action of interleukin 1 beta (IL-1 beta) on pancreatic beta-cells, we studied the effects on islet function of different protease inhibitors when added together with recombinant IL-1 beta to isolated rat pancreatic islets. It was found that the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) counteracted the acute stimulatory effects of IL-1 beta on islet glucose oxidation, insulin release, and biosynthesis. TLCK also partially or completely counteracted the long-term inhibitory effects of IL-1 beta on islet glucose oxidation, insulin biosynthesis, content, and release. This protease inhibitor also counteracted IL-1 beta-induced beta-cell cytotoxicity as assessed by DNA content measurements. Of the other group-specific protease inhibitors investigated, only N-tosyl-L-phenylalanine chloromethyl ketone, N alpha-p-tosyl-L-arginine methyl ester, and chloromercuriphenylsulfonic acid were found to partially protect against IL-1 beta action. We concluded that protease activation, putatively a serine protease, may be an early and perhaps primary event in the action of IL-1 beta on beta-cells.

Animals↗
PubMed1991

Exhibition of specific alterations in activities and mRNA levels of rat islet glycolytic and mitochondrial enzymes in three different in vitro model systems for attenuated insulin release.

We studied the possible relationships between the functional status of the beta-cell and activities or mRNA contents of enzymes involved in the catabolism of glucose. Three different in vitro models with attenuated insulin response were used: rat islets cultured at a low glucose concentration, rat islets incubated in vitro with streptozocin, and fetal rat islets. The fetal and streptozocin-administered islets were compared with adult islets cultured in RPMI-1640 containing 11 mM glucose, and the effects of the in vitro glucose concentrations (3.3, 11, and 28 mM) were assessed on adult islets only. Cellular mRNA levels for the mitochondrial DNA-encoded cytochrome b and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by Northern-blot analysis. Enzymatic activities of high-Km (glucokinase) and low-Km (hexokinase) glucose-phosphorylating enzymes and succinate-cytochrome c reductase were also determined. Islets cultured at 3.3 mM glucose displayed a decreased activity of glucokinase compared with islets cultured at 28 mM glucose (23.3 +/- 12%), whereas there was no difference in hexokinase activity or the level of GAPDH mRNA. The activity of succinate-cytochrome c reductase was similar in islets cultured at the different glucose concentrations. The level of cytochrome b mRNA increased at 28 mM glucose compared with islets cultured at 11 mM glucose (140 +/- 14%). Islets incubated with streptozocin and subsequently cultured for 7 days at 11 mM glucose exhibited a decreased level of cytochrome b mRNA (65 +/- 5%) and no differences in the activities of glucokinase, hexokinase, succinate-cytochrome c reductase, or the level of GAPDH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Interleukin-1 beta increases the biosynthesis of the heat shock protein hsp70 and selectively decreases the biosynthesis of five proteins in rat pancreatic islets.

Prolonged exposure to high concentrations of human recombinant interleukin-1 beta (rIL-1 beta) has been reported to exert both suppressive and cytotoxic effects on pancreatic beta-cells during culture in vitro. In order to investigate the molecular mechanism(s) underlying the actions of rIL-1 beta on the beta-cell, we have exposed isolated rat pancreatic islets for 3 or for 24 h to 25 U/ml of rIL-1 beta. Subsequently the biosynthesis of heat shock proteins, as assessed by western blot analysis, and total protein biosynthesis patterns were studied, using one and two-dimensional gel electrophoresis of [35S]methionine labelled islet proteins from different subcellular compartments. It was found that rIL-1 beta exerted no specific effects on protein synthesis when added during a 3 h incubation period. However, after a 24 h incubation period, the synthesis of a group of acidic proteins with the approximate molecular weight of 35 kD was specifically inhibited in the rIL-1 beta treated islets. This alteration was predominantly associated with the endoplasmic reticulum fraction. The cytokine also inhibited the synthesis of four cytosolic proteins with the molecular weights 75, 85, 95 and 120 kD. In contrast, rIL-1 beta increased the expression of the heat shock protein hsp70 both in the microsomal and cytosolic fractions, in contrast to the islet nuclei in which no increase was found. These results show that exposure of pancreatic islets to rIL-1 beta is accompanied by specific alterations in the protein synthesis of the islet cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A role for polyamines in glucose-stimulated insulin-gene expression.

The aim of the present study was to evaluate the possible role for polyamines in the glucose regulation of the metabolism of insulin mRNA of pancreatic islet cells. For this purpose islets were prepared from adult mice and cultured for 2 days in culture medium RPMI 1640 containing 3.3 mM- or 16.7 mM-glucose with or without the addition of the inhibitors of polyamine biosynthesis difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG). Culture at the high glucose concentration increased the islet contents of both insulin mRNA and polyamines. The synthesis of total RNA, total islet polyamines and polyamines associated with islet nuclei was also increased. When the combination of DFMO and EGBG was added in the presence of 16.7 mM-glucose, low contents of insulin mRNA, spermine and spermidine were observed. Total islet polyamine synthesis was also depressed by DFMO + EGBG, unlike islet biosynthesis of polyamines associated with nuclei, which was not equally decreased by the polyamine-synthesis inhibitors. Total RNA synthesis and turnover was not affected by DFMO + EGBG. Finally, actinomycin D attenuated the glucose-induced enhancement of insulin mRNA, and cycloheximide counteracted the insulin-mRNA attenuation induced by inhibition of polyamine synthesis. It is concluded that the glucose-induced increase in insulin mRNA is paralleled by increased contents and rates of polyamine biosynthesis and that an attenuation of the increase in polyamines prevents the increase in insulin mRNA. In addition, the results are compatible with the view that polyamines exert their effects on insulin mRNA mainly by increasing the stability of this messenger.

Animals↗