Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011854 (type 1 diabetes)
 20,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++ bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1res cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range. With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain cytokine action. However, normal IL-1beta-mediated translocation of NF-kappaB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.
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PMID:Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity. 1087 Nov 93

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze beta-cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase-polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 +/- 85 and 338 +/- 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 micromol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 +/- 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 micromol/l, 24-48 h) increased levels of IA-2 mRNA (190 +/- 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a time- and dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 +/- 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.
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PMID:Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells. 1090 70

Insulin-dependent diabetes mellitus (IDDM) or type 1 diabetes is an autoimmune disease that results in destruction of the insulin-producing pancreatic islet beta cells. Several factors induce the invasion of immune cells into islets and trigger inflammation. Gene therapy approaches targeting the islet cells could be an effective treatment to prevent the onset or reverse type 1 diabetes. Allogeneic islet transplantation provides short-term treatment. However, genetically modified islets, which resist the host immune response, could provide long-term solutions. Adeno-associated virus (AAV) is emerging as a prominent vector system for delivering therapeutic genes for human gene therapy. AAV vector can transduce nondividing cells and provide long-term gene expression by integrating into host chromosome. Therefore, it is an appropriate vector system for islet cell gene therapy. To test the efficacy of AAV vector to transduce pancreatic endocrine cells, we constructed AAV vectors using plasmid pSub201. Wild-type AAV DNA analogue from plasmid psub201 was subcloned into a cloning plasmid pSP72 and AAV vectors were constructed by inserting the transgenes with heterologous promoter in place of AAV open reading frames (rep and cap). In this report we demonstrate the transduction of pancreatic islet cells with AAV vectors encoding bacterial -galactosidase enzyme or enhanced green fluorescent protein (EGFP) as reporter gene. Dispersed porcine and rat islet cells can be transduced by AAV vector, with an efficiency of 47% and 38%, respectively. In particular porcine islet insulin producing beta cells were transduced with an efficiency of 39%. Intact rat islet cells were transduced with an efficiency of 26% as estimated by FACS analysis following transduction with an AAV vector encoding EGFP. Transduction of intact rat islets with an AAV vector did not alter glucose-induced insulin secretion. AAV vector transduction was higher in transformed islet cell lines INS-1 and RIN m5F with an efficiency of 65% and 57%, respectively. These new results suggest that AAV vectors will provide an improved method of gene delivery to pancreatic islets and isolated pancreatic beta cells.
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PMID:Adeno-associated virus vector mediated gene transfer to pancreatic beta cells. 1102 93

Destruction of pancreatic islet beta-cells in type 1 diabetes appears to result from direct contact with infiltrating T-cells and macrophages and exposure to inflammatory cytokines such as interferon (IFN)-gamma, interleukin (IL)-1 beta, and tumor necrosis factor TNF-alpha that such cells produce. We recently reported on a method for selection of insulinoma cells that are resistant to the cytotoxic effects of inflammatory cytokines (INS-1(res)), involving their growth in progressively increasing concentrations of IL-1 beta plus IFN-gamma, and selection of surviving cells. In the current study, we have investigated the molecular mechanism of cytokine resistance in INS-1(res) cells. By focusing on the known components of the IFN-gamma receptor signaling pathway, we have discovered that expression levels of signal transducer and activator of transcription (STAT)-1 alpha are closely correlated with the cytokine-resistant and -sensitive phenotypes. That STAT-1 alpha is directly involved in development of cytokine resistance is demonstrated by an increase of viability from 10 +/- 2% in control cells to 50 +/- 6% in cells with adenovirus-mediated overexpression of STAT-1 alpha (p < 0.001) after culture of both cell groups in the presence of 100 units/ml IFN-gamma plus 10 ng/ml IL-1 beta for 48 h. The resistance to IL-1 beta plus IFN-gamma in STAT-1 alpha-expressing cells is due in part to interference with IL-1 beta-mediated stimulation of inducible nitric-oxide synthase expression and nitric oxide production. Furthermore, overexpression of STAT-1 alpha does not impair robust glucose-stimulated insulin secretion in the INS-1-derived cell line 832/13. We conclude that expression of STAT-1 alpha may be a means of protecting insulin-producing cell lines from cytokine damage, which, in conjunction with appropriate cell-impermeant macroencapsulation devices, may allow such cells to be used for insulin replacement in type 1 diabetes.
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PMID:Expression of the transcription factor STAT-1 alpha in insulinoma cells protects against cytotoxic effects of multiple cytokines. 1102 34

In addition to inhibiting matrix metalloproteinase-2 and matrix metalloproteinase-9 activity, recent studies suggest that tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell lines. To address this question in pancreatic islets and beta-cells, we treated rat pancreatic islets and INS-1 cells with a high-dose combination of the cytokines interleukin (IL)-1beta, tumor necrosis factor-alpha, and interferon-gamma with or without the addition of TIMP-1 and TIMP-2 protein. Using flow cytometry, we quantitated DNA fragmentation to assess cellular apoptosis and confirmed these observations with DNA laddering experiments. Next, we transfected the mouse TIMP-1 gene into INS-1 cells and performed Western immunoblotting to demonstrate expression of TIMP-1 protein. We treated TIMP-1-expressing INS-1 cells with high-dose cytokines and again used flow cytometry to assess DNA fragmentation. We also evaluated the effect of TIMP-1 on IL-1beta-induced inhibition of glucose-stimulated insulin secretion (GSIS) in freshly isolated rat pancreatic islets. Finally, we evaluated the effect of TIMP-1 on inducible nitric oxide synthase (iNOS) gene expression and nuclear factor (NF)-kappaB activity in INS-1 cells stimulated with high-dose cytokines. TIMP-1 but not TIMP-2 prevented cytokine-induced apoptosis and cytokine-mediated inhibition of GSIS in rat islets and beta-cells. TIMP-1 mediated these effects by inhibiting cytokine activation of NF-kappaB, but it did not affect nitric oxide production or iNOS gene expression. Therefore, TIMP-1 may be an ideal gene to prevent cytokine-mediated beta-cell destruction and dysfunction in models of type 1 diabetes and islet transplantation rejection.
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PMID:Tissue inhibitor of metalloproteinase-1 prevents cytokine-mediated dysfunction and cytotoxicity in pancreatic islets and beta-cells. 1133 7

Exposure of pancreatic beta-cells to cytokines, such as interleukin-1beta (IL-1beta), is thought to contribute to the beta-cell apoptosis that underlies the onset of type 1 diabetes. One important event triggered by IL-1beta is induction of nitric oxide synthase (iNOS), an enzyme that catalyzes intracellular generation of the cytotoxic free radical NO. We recently described a novel requirement for the protein kinase C (PKC) isozyme PKCdelta in this process. Our current aim, therefore, was to assess whether PKCdelta also plays a role in beta-cell apoptosis. As assessed by either annexin V staining or DNA fragmentation, IL-1beta caused INS-1 cells to undergo apoptosis. This was completely blocked by adenoviral overexpression of a dominant-negative, kinase-dead (KD) PKCdelta mutant. The corresponding PKCalpha virus was without effect. However, apoptosis caused by the cytotoxic agent streptozotocin (STZ), which acts independent of iNOS, was also inhibited by overexpression of PKCdeltaKD. STZ was additionally shown to activate the proteolytic enzyme caspase-3, a key biochemical effector of end-stage apoptosis. Moreover, STZ caused a caspase-dependent cleavage of PKCdelta, thereby releasing a COOH-terminal fragment corresponding to the kinase catalytic domain. Thus, proteolytic activation of PKCdelta seems to be important in the distal apoptotic pathway induced by STZ. That IL-1beta also activated caspase-3 and promoted PKCdelta cleavage suggests that this distal pathway also contributes in the apoptotic response to the cytokine. These data therefore support a dual role for PKCdelta in IL-1beta-mediated cell death: it is required for efficient NO generation through regulation of iNOS levels but also contributes to apoptotic pathways downstream of caspase activation.
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PMID:Inhibition of protein kinase C delta protects rat INS-1 cells against interleukin-1beta and streptozotocin-induced apoptosis. 1181 38

Proinflammatory cytokine-mediated pancreatic beta-cell dysfunction is a key pathological event in type I diabetes mellitus. Lisofylline (LSF), an anti-inflammatory agent, has been shown to protect pancreatic islets from IL-1 beta-induced inhibitory effects on insulin release. However, the mechanism of LSF action is not known. Increasing evidence suggests that the mitochondria play an important role in regulating the beta-cell insulin release capacity and the control of cellular viability. To examine the direct effects of LSF on beta-cells, insulin-secreting INS-1 cells were exposed to a combination of recombinant IL-1 beta, TNF alpha, and IFN gamma with or without LSF for 18 h. Basal and glucose-stimulated static insulin release were measured using RIA. INS-1 cell viability was determined using in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and LIVE/DEAD dual fluorescence labeling. To evaluate INS-1 mitochondrial function, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism, change in mitochondrial membrane potential, and intracellular ATP levels were assessed. Cytokine addition reduced basal (7.8 +/- 0.30 vs. 10.0 +/- 0.46 ng/ml.h; P < 0.005), glucose-stimulated insulin secretion (11.6 +/- 0.86 vs. 17.4 +/- 1.86 ng/ml.h; P < 0.005), and MTT metabolism in INS-1 cells. Over 40% of the cytokine-treated beta-cells exhibited nuclear DNA breakage, whereas the control cell death rate remained at 1-2%. Simultaneous application of LSF and cytokines to INS-1 cells restored insulin secretion, MTT metabolism, mitochondrial membrane potential, and cell viability to control levels. LSF increased beta-cell MTT metabolism as well as insulin release and glucose responsiveness. In summary, proinflammatory cytokines lead to a reduction of glucose-induced insulin secretion, mitochondrial activity, and viability in INS-1 cells. LSF at concentrations achievable in vivo protected beta-cells from the cytokine effects. The mechanism of LSF-induced protection may be by promoting mitochondrial metabolism.
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PMID:Lisofylline, a novel antiinflammatory agent, protects pancreatic beta-cells from proinflammatory cytokine damage by promoting mitochondrial metabolism. 1202 Nov 99

Cytokines released from activated antigen-presenting cells and T-lymphocytes are crucially involved in the pathogenesis of type 1 diabetes. Previous studies have shown that proinflammatory cytokines play an important role in the induction of autoimmunity and beta-cell damage. Inhibition of insulin expression has been described, but their effects on other major target autoantigens, such as the tyrosine phosphatase-like protein IA-2, is not known. In the present study, we established sensitive real-time RT-PCR to measure IA-2, insulin, and inducible nitric oxide (NO) synthase (iNOS) mRNA expression. Rat insulinoma INS-1 cells were stimulated with IL-1beta, TNF-alpha, interferon (IFN)-gamma, and IL-2 as well as with two combinations of these cytokines (C1: IL-1beta + TNF-alpha + IFN-gamma; C2: TNF-alpha + IFN-gamma). Treatment with IL-1beta, TNF-alpha, or IFN-gamma alone caused a significant down-regulation of IA-2 and insulin mRNA levels in a time and dose-dependent manner, whereas IL-2 had no effect. Exposure to cytokine combinations strongly potentiates the inhibitory effects. Incubation of cells with C1 and C2 for 24 h induces a significant inhibition of IA-2 mRNA levels by 78% and 58%, respectively. Under these conditions, an up to 5 x 10(4)-fold increase of iNOS gene expression was observed. The hypothesis that the formation of NO is involved in IA-2 regulation was confirmed by the finding that the coincubation of C1 with 4 mM L-N(G)-monomethyL-L-arginine, an inhibitor of the iNOS, partly reversed the down-regulation of IA-2. Further, incubation with the synthetic NO-donor S-nitroso-N-acetyl-D-L-penicillamine significantly decreased IA-2 mRNA level to 51% of basal levels. In conclusion, we have demonstrated for the first time that IL-1beta, TNF-alpha, and IFN-gamma exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1beta may be partly mediated by the activation of the NO pathway.
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PMID:Effect of proinflammatory cytokines on gene expression of the diabetes-associated autoantigen IA-2 in INS-1 cells. 1223 95

Recent studies into the physiology of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have added stimulation of beta-cell growth, differentiation, and cell survival to well-documented, potent insulinotropic effects. Unfortunately, the therapeutic potential of these hormones is limited by their rapid enzymatic inactivation in vivo by dipeptidyl peptidase IV (DP IV). Inhibition of DP IV, so as to enhance circulating incretin levels, has proved effective in the treatment of type 2 diabetes both in humans and in animal models, stimulating improvements in glucose tolerance, insulin sensitivity, and beta-cell function. We hypothesized that enhancement of the cytoprotective and beta-cell regenerative effects of GIP and GLP-1 might extend the therapeutic potential of DP IV inhibitors to include type 1 diabetes. For testing this hypothesis, male Wistar rats, exposed to a single dose of streptozotocin (STZ; 50 mg/kg), were treated twice daily with the DP IV inhibitor P32/98 for 7 weeks. Relative to STZ-injected controls, P32/98-treated animals displayed increased weight gain (230%) and nutrient intake, decreased fed blood glucose ( approximately 26 vs. approximately 20 mmol/l, respectively), and a return of plasma insulin values toward normal (0.07 vs. 0.12 nmol/l, respectively). Marked improvements in oral glucose tolerance, suggesting enhanced insulin secretory capacity, were corroborated by pancreas perfusion and insulin content measurements that revealed two- to eightfold increases in both secretory function and insulin content after 7 weeks of treatment. Immunohistochemical analyses of pancreatic sections showed marked increases in the number of small islets (+35%) and total beta-cells (+120%) and in the islet beta-cell fraction (12% control vs. 24% treated) in the treated animals, suggesting that DP IV inhibitor treatment enhanced islet neogenesis, beta-cell survival, and insulin biosynthesis. In vitro studies using a beta-(INS-1) cell line showed a dose-dependent prevention of STZ-induced apoptotic cell-death by both GIP and GLP-1, supporting a role for the incretins in eliciting the in vivo results. These novel findings provide evidence to support the potential utility of DP IV inhibitors in the treatment of type 1 and possibly late-stage type 2 diabetes.
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PMID:Dipeptidyl peptidase IV inhibitor treatment stimulates beta-cell survival and islet neogenesis in streptozotocin-induced diabetic rats. 1260 16

We have been investigating the potential utility of engineered cell lines as surrogates for primary islet cells in treatment of type 1 diabetes. To this end, two strategies that have emerged for procuring cell lines with resistance to immune-mediated damage are 1) selection of cytokine-resistant cell lines by growth of INS-1 insulinoma cells in iteratively increasing concentrations of interleukin (IL)-1beta + gamma-interferon (IFN-gamma), and 2) stable overexpression of the anti-apoptotic gene bcl-2 in INS-1 cells. Herein, we show that bcl-2-overexpressing cells are resistant to the cytotoxic effects of reactive oxygen and nitrogen species (ROS/RNS), but are only modestly protected against high concentrations of IL-1beta + INF-gamma, whereas the converse is true in cytokine selected cells. We also found that the combination of bcl-2 expression and cytokine selection confers a broader spectrum of resistance than either procedure alone, such that the resultant cells are highly resistant to cytokines and ROS/RNS, with no impairment in glucose-stimulated insulin secretion. INS-1-derived cells with combined bcl-2 expression and cytokine selection are also more resistant to damage induced by coculture with mitogen-activated peripheral blood mononuclear cells. Surprisingly, application of the cytokine selection procedure to bcl-2-overexpressing cells does not result in impairment of nuclear factor-kappaB translocation, iNOS expression, and NO production, as clearly occurs upon application of the selection procedure to cells without bcl-2 overexpression. Further investigation of the diverse pathways involved in the development of cytokine and ROS/RNS resistance may define simplified and specific strategies for preservation of beta-cell mass.
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PMID:Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy. 1276 53


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