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  • 1
    ISSN: 1432-0428
    Keywords: Islet amyloid polypeptide ; human pancreatic islets ; islet transplantation ; immune histochemistry ; hyperglycaemia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Human islets of Langerhans were transplanted to the subcapsular space of the kidneys of nude mice which were either normoglycaemic or made diabetic with alloxan. After 2 weeks, the transplants were processed for light and electron microscopical analyses. In all transplants, islet amyloid polypeptide (IAPP)-positive cells were found with highest frequency in normoglycaemic animals. IAPP-positive amyloid was seen in 16 out of 22 transplants (73%), either by polarisation microscopy after Congo red staining or by immune electron microscopy. At variance with previous findings of amyloid deposits exclusively in the extracellular space of islets of non-insulin-dependent diabetic patients, the grafted islets contained intracellular amyloid deposits as well. There was no clear difference in occurrence of amyloid between diabetic and non-diabetic animals. The present study indicates that human islets transplanted into nude mice very soon present IAPP-positive amyloid deposits. This technique may provide a valuable model for studies of the pathogenesis of islet amyloid and its impact on islet cell function.
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  • 2
    ISSN: 1432-0428
    Keywords: Glucose ; streptozotocin ; pancreatic islets ; insulin secretion ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary There have previously been divergent data published regarding the effects of glucose on the diabetogenic effects of streptozotocin. In order to further explore this issue, two separate sets of experiments were performed. In the first, mouse pancreatic islets were maintained in culture for 3 days at different glucose concentrations (5.6,11.1 and 28 mmol/l) and then exposed to streptozotocin. After another 3 days in culture at 11.1 mmol/l glucose, the B cell function was evaluated by measurement of glucose-stimulated insulin release, the number of islets recovered after culture, and the islet DNA and insulin contents. In the second group of experiments islets were first maintained in culture at 11.1 mmol/l glucose, then treated with streptozotocin and subsequently cultured for 6 days at the different glucose concentrations given above. It was found that islets maintained in a medium containing 28 mmol/l glucose before or after streptozotocin exposure showed less signs of damage than islets cultured in 11.1 mmol/l glucose. A similar, but less pronounced, de creased sensitivity to streptozotocin was found in islets precultured in 5.6 mmol/l glucose, in comparison with those islets cultured in 11.1 mmol/l glucose. Culture at 5.6 mmol/l glucose just after streptozotocin treatment did not induce any improvement in islet survival or function. It is suggested that the increased damage induced by streptozotocin to islets precultured at 11.1 mmol/l glucose, in comparison with 5.6 mmol/l glucose, can be related to the fact that an increased metabolic activity of B cells render them more susceptible to the toxin. The improved preservation of islets cultured at 28 mmol/l glucose before or after streptozotocin treatment may reflect an additional effect of glucose, i. e. activation of defense mechanisms in the B cells against cytotoxins.
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  • 3
    ISSN: 1432-0428
    Keywords: Non-obese diabetic mice ; diabetes mellitus ; insulin release ; glucose oxidation ; pancreatic islets ; T lymphocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Pancreatic islets isolated from non-obese diabetic (NOD) mice, all of which have insulitis, exhibit an impaired glucose metabolism. In order to investigate the role of infiltrating lymphocytes for this altered metabolism, we injected 12- to 13-week-old female NOD mice with monoclonal antibodies directed against either the αβ-T cell receptor, CD4+ or CD8+ T cells. Control NOD mice were injected with normal rat IgG or with the vehicle (phosphate buffered saline) alone. Injection of the three different monoclonal antibodies markedly reduced the mononuclear cell infiltration. An intravenous glucose tolerance test showed no differences between the groups. Islet insulin release in response to glucose was similar in all groups. In contrast, islets isolated from the control NOD mice with insulitis showed a high basal (1.7 mmol/l glucose) glucose oxidation rate and a small increase in the glucose oxidation rate in response to a high glucose concentration (16.7 mmol/l glucose). The monoclonal antibodies counteracted the elevated basal glucose oxidation rate of the islets. Parallel studies of stimulated mononuclear cells suggested that the contribution of glucose oxidized by islet-infiltrating lymphocytes could only partially explain the observed alterations in NOD mouse islet metabolism. Culture of islets obtained from NOD mice in the presence of the cytokine interleukin-1 β induced a similar pattern of glucose metabolism as seen earlier in IgG or phosphate-buffered saline treated control NOD mice. In conclusion, alterations in the glucose oxidation rates seem to be an early sign of disturbance in islets isolated from NOD mice. These early alterations in glucose metabolism can be reversed in vivo by monoclonal antibodies directed against effector lymphocytes. This suggests that the infiltrating mononuclear cells can induce reversible alterations in pancreatic Beta-cell function which may precede impaired insulin secretion, Beta-cell destruction and overt diabetes mellitus.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0428
    Keywords: Key words Animal models NIDDM, insulin secretion, insulin mRNA, cytochrome b mRNA, islets of Langerhans.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Animals with NIDDM display abnormal glucose regulation of insulin secretion and biosynthesis. We tested reversibility of abnormal regulation by normoglycaemia using an islet transplantation technique. Inbred non-diabetic and neonatally STZ diabetic rats (n-STZ) were used. Transplantations insufficient to normalize the blood glucose levels (200 islets under kidney capsule) were performed from diabetic to normal (D-N) and from diabetic to diabetic (D-D), as well as from normal to normal (N-N) and from normal to diabetic (N-D) rats. Four weeks after transplantation, graft bearing kidneys were isolated and perfused with Krebs-Henseleit bicarbonate buffer to measure insulin secretion in response to 27.8 mmol/l glucose and 10 mmol/l arginine. Four weeks of normoglycaemia failed to restore glucose-induced insulin secretion from n-STZ islets (glucose induced increment: −1.7± 2.5 fmol/min in D-N, 1.2±7.1 fmol/min in D-D). In contrast to normal islets, normoglycaemia reduced insulin mRNA contents (60±24 in D-N, 496±119 in D-D; O. D.-arbitrary units). However, arginine-induced secretion was markedly enhanced by diabetic environment in both normal and n-STZ islet grafts. These results indicate that selected aspects of glucose recognition are irreversibly damaged by a long-term diabetic state or, alternatively, by a lasting effect of STZ administration. [Diabetologia (1994) 37: 351–357]
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  • 5
    ISSN: 1432-0428
    Keywords: Animal models NIDDM ; insulin secretion ; insulin mRNA ; cytochrome b mRNA ; islets of Langerhans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Animals with NIDDM display abnormal glucose regulation of insulin secretion and biosynthesis. We tested reversibility of abnormal regulation by normoglycaemia using an islet transplantation technique. Inbred non-diabetic and neonatally STZ diabetic rats (n-STZ) were used. Transplantations insufficient to normalize the blood glucose levels (200 islets under kidney capsule) were performed from diabetic to normal (D-N) and from diabetic to diabetic (D-D), as well as from normal to normal (N-N) and from normal to diabetic (N-D) rats. Four weeks after transplantation, graft bearing kidneys were isolated and perfused with Krebs-Henseleit bicarbonate buffer to measure insulin secretion in response to 27.8 mmol/l glucose and 10 mmol/l arginine. Four weeks of normoglycaemia failed to restore glucose-induced insulin secretion from n-STZ islets (glucose induced increment:-1.7±2.5 fmol/min in D-N, 1.2±7.1 fmol/min in D-D). In contrast to normal islets, normoglycaemia reduced insulin mRNA contents (60±24 in D-N, 496±119 in D-D; O.D.-arbitrary units). However, arginine-induced secretion was markedly enhanced by diabetic environment in both normal and n-STZ islet grafts. These results indicate that selected aspects of glucose recognition are irreversibly damaged by a long-term diabetic state or, alternatively, by a lasting effect of STZ administration.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-0428
    Keywords: Nitric oxide ; nitric oxide synthase ; promoter ; transcription factor ; nuclear factor κB ; pancreatic islets ; beta cells ; insulin-producing cells ; insulin-dependent diabetes mellitus ; superoxide dismutase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The 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.
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  • 7
    ISSN: 1432-0428
    Keywords: Keywords Beta cells ; chemokine ; phospholipase-D ; DDRT-PCR ; interleukin-1 ; monocyte chemoattractant protein-1 ; adenine nucleotide translocator ; CINC-1 ; CINC-3.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Aims/hypothesis. Interleukin-1β is a putative mediator of pancreatic beta-cell dysfunction and damage in Type I (insulin-dependent) diabetes mellitus. To better understand the molecular mechanisms involved in IL-1β effects, we carried out a differential display of mRNA by RT-PCR to identify novel cytokine-regulated genes. Methods. Fluorescence activated cell sorting-purified rat pancreatic beta-cells were exposed for 6 or 24 h to IL-1β. Differentially expressed cDNA bands were cloned and then identified by comparing their sequences with data from the GenBank. Differential gene expression was confirmed by RT-PCR using specific primers. Results. Interleukin-1β increased the expression of adenine nucleotide translocator-1, phospholipase D-1 and cytokine-induced neutrophil chemoattractant-1 and decreased expression of the protein tyrosine phosphatase-like protein IA-2. Interleukin-1β-induced differential expression of these genes in beta cells was confirmed by RT-PCR. In additional studies, IL-1β was shown to induce chemokines other than cytokine-induced neutrophil chemoattractant-1, including cytokine-induced neutrophil chemoattractant-3 and monocyte chemotactic protein-1. Conclusion/interpretation. Our observations indicate that IL-1β modifies the expression of several genes in pancreatic beta cells. These genes may affect both function, viability and beta-cell recognition by the immune system. Functional characterization of the mRNAs which have been identified could facilitate a better understanding of the mechanisms leading to beta-cell destruction in Type I diabetes. [Diabetologia (1999) 42: 1199–1203]
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  • 8
    ISSN: 1432-0428
    Keywords: Glucose ; Type 1 (insulin-dependent) diabetes mellitus ; insulin release ; NOD mice ; pancreatic islets ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In the early stages of Type 1 (insulin-dependent) diabetes mellitus patients present a deficient insulin response to glucose. The reasons for this defective response are unknown, but it has been suggested that it reflects a deleterious effect of excessive glucose stimulation on a reduced Beta-cell mass. Female non-obese diabetic (NOD) mice from our colony, at the age of 12–13 weeks, have a normal basal glycaemia but an impaired intravenous glucose tolerance test, insulitis and a defective insulin response to glucose. In order to characterize the potential effect of glucose on the Beta cells at that “pre-diabetic” stage, pancreatic islets were isolated from 12–13 week old female NOD mice. Immediately after isolation (day 0) the NOD islets displayed a defective insulin response to an acute stimulation with 16.7 mmol/l glucose. After seven days in culture at both 11 and 28 mmol/l glucose these islets showed an increased insulin release in response to an acute glucose stimulation. This increase was more pronounced in the islets cultured at 28 mmol/l glucose. Experiments performed in parallel, using islets obtained from a non-diabetes prone strain of mice (Naval Medical Research Institute, NMRI) showed that these islets had a similar insulin release in response to glucose both on day 0 and after seven days in culture at 11 mmol/l glucose. The insulin mRNA levels of NOD islets did not change over one week in culture at 11 or 28 mmol/l glucose, but culture at the high glucose concentration induced a decrease in the islet insulin content. The present data show that culture at high glucose concentrations does not impair the function of islets isolated from NOD mice. These observations make excessive glucose stimulation, as a single factor, an unlikely explanation for the defective insulin release observed in NOD islets in the “prediabetic” period.
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  • 9
    ISSN: 1432-0428
    Keywords: Pancreatic islets ; insulin release ; interleukin-1β ; glucose metabolism ; amino acid metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Acute exposure of pancreatic islets to interleukinl-1β results in an increase in insulin release, while an extension of the exposure time induces a functional suppression and eventually, destruction of the B-cells. We have recently suggested that the interleukin-1β induced inhibition of islet function is mediated through an impairment in oxidative metabolism. The aim of the current study was to investigate if the acute, stimulatory effects of interleukin-1β on islet function could also be related to changes in the substrate metabolism. For this purpose, rat islets were exposed for 90–120 min to 30 pmol/l human recombinant interleukin-1β (biological activity of 2.5 U/ml) and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 or 5.5 mmol/l glucose but in both the presence of 16.7 mmol/l glucose or 10 mmol/l leucine + 2 mmol/l glutamine there was a 50% increase in insulin release. Interleukin-1β exposure increased the oxidation of D-[U-14C]glucose at 5.5 mmol/l glucose by 25% and at 16.7 mmol/l glucose by 60%. Carbohydrate and amino acid metabolism were further examined in the presence of D-[5-3H] glucose, D-[6-14C]glucose, [1-14C]pyruvate, L-[U-14C]glutamine, L-[U-14C]leucine and L-[1-14C]leucine. There was no difference between control islets and interleukin-1β exposed islets in terms of D-[5-3H]glucose utilization or [1-14C]pyruvate decarboxylation, but the oxidation of D-[6-14C]glucose was increased by 64% in the interleukin-1β exposed islets. There was also an interleukin-1β induced 45–60% increase in the decarboxylation of L-[1-14C]leucine and oxidation of L-[U-14C]leucine and L-[U-14C]glutamine, all intramitochrondrial events. The stimulation of insulin release by interleukin-1β in the presence of 16.7 mmol/l glucose was abolished in islets incubated in Ca2+ depleted medium, but the rate of D-[6-14C] glucose oxidation remained elevated (47% increase at 16.7 mmol/l glucose). These data indicate an increase in substrate metabolism at the mitochondrial level during acute exposure of rat pancreatic islets to interleukin-1β. The increase in oxidative events can explain the observed interleukin-1β induced increase in insulin release during glucose stimulation. Furthermore, these findings raise the possibility that mitochondria are primary targets of interleukin-1β action in the B-cells.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-0428
    Keywords: Interleukin-1β ; interleukin 1 receptor ; insulin secretion ; pancreatic islets ; RINm5F cells ; insulin-dependent diabetes mellitus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The cytokine interleukin-1β may have an important role in the autoimmune mediated damage of pancreatic Beta cells in insulin-dependent diabetes mellitus. In the present study we have investigated the effects of an interleukin-1 receptor antagonist protein, a blocker of the type I interleukin-1 receptor, on the suppressive actions of recombinant interleukin-1β on insulin-producing cells. Brief exposure (1–2 h) of rat and mouse pancreatic islets to 10 ng/ml recombinant interleukin-1β induced an 70–80% inhibition of insulin response to glucose after 12 h. These effects were completely counteracted by co-incubation with 100 ng/ml interleukin-1 receptor antagonist protein. When rat islets were cultured for 48 h in the presence of recombinant interleukin-1β (5 ng/ml) higher concentrations of interleukin-1 receptor antagonist protein (5000 ng/ml) were required to protect Beta-cell function. Interleukin-1 receptor antagonist protein also counteracted the inhibitory effects of recombinant interleukin-1β on the growth of the rat insulinoma cell line RINm5F. These data suggest that interleukin-1 receptor antagonist protein can protect insulin-producing cells from the deleterious effects of recombinant interleukin-1β, and that these cells possess type I interleukin-1 receptors.
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