Primary Beta-cells Research Articles

Global profiling of double stranded RNA- and IFN-gamma-induced genes in rat pancreatic beta cells.

Viral infections and local production of IFN-gamma might contribute to beta-cell dysfunction/death in Type 1 Diabetes. Double stranded RNA (dsRNA) accumulates in the cytosol of viral-infected cells, and exposure of purified rat beta cells to dsRNA (tested in the form of polyinosinic-polycytidylic acid, PIC) in combination with IFN-gamma results in beta-cell dysfunction and apoptosis. To elucidate the molecular mechanisms involved in PIC + IFN-gamma-effects, we determined the global profile of genes modified by these agents in primary rat beta cells. FACS-purified rat beta cells were cultured for 6 or 24 h in control condition or with IFN-gamma, PIC or a combination of both agents. The gene expression profile was analysed in duplicate by high-density oligonucleotide arrays representing 5000 full-length genes and 3000 EST's. Changes of greater than or equal to 2.5-fold were considered as relevant. Following a 6- or 24-h treatment with IFN-gamma, PIC or IFN-gamma and PIC, we observed changes in the expression of 51 to 189 genes. IFN-gamma modified the expression of MHC-related genes, and also of genes involved in beta-cell metabolism, protein processing, cytokines and signal transduction. PIC affected preferentially the expression of genes related to cell adhesion, cytokines and dsRNA signal transduction, transcription factors and MHC. PIC and/or IFN-gamma up-regulated the expression of several chemokines and cytokines that could contribute to mononuclear cell homing and activation during viral infection, while IFN-gamma induced a positive feedback on its own signal transduction. PIC + IFN-gamma inhibited insulin and GLUT-2 expression without modifying pdx-1 mRNA expression. This study provides the first comprehensive characterization of the molecular responses of primary beta cells to dsRNA + IFN-gamma, two agents that are probably present in the beta cell milieu during the course of virally-induced insulitis and Type 1 Diabetes. Based on these findings, we propose an integrated model for the molecular mechanisms involved in dsRNA + IFN-gamma induced beta-cell dysfunction and death.

Use of microarray analysis to unveil transcription factor and gene networks contributing to Beta cell dysfunction and apoptosis.

The beta cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat beta cells exposed to the proapoptotic cytokines, IL-1beta and/or IFN-gamma. The picture emerging from these experiments is that beta cells are not passive bystanders of their own destruction. On the contrary, beta cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed beta cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-kappaB. NF-kappaB blocking prevents cytokine-induced beta cell death, and characterization of NF-kappaB-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of beta cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the beta cell "decision" to undergo or not apoptosis in early T1DM.

Type VIII adenylyl cyclase in rat beta cells: coincidence signal detector/generator for glucose and GLP-1

The secretory function of pancreatic beta cells is synergistically stimulated by two signalling pathways which mediate the effects of nutrients and hormones such as glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) or glucagon. These hormones are known to activate adenylyl cyclase in beta cells. We examined the type of adenylyl cyclase that is associated with this synergistic interaction. Insulin release, cAMP production, adenylyl cyclase activity, mRNA and protein expression were measured in fluorescence-activated cell sorter-purified rat beta cells and in the rat beta-cell lines RINm5F, INS-1 832/13 and INS-1 832/2. In primary beta cells, glucagon and GLP-1 synergistically potentiate the stimulatory effect of 20 mmol/l glucose on insulin release and cAMP production. Both effects are abrogated in the presence of the L-type Ca(2+)-channel blocker verapamil. The cAMP-producing activity of adenylyl cyclase in membranes from RINm5F cells is synergistically increased by Ca(2+)-calmodulin and recombinant GTP(gamma)S-activated G(s alpha)-protein subunits. This type of regulation is characteristic for type I and type VIII AC isoforms. Consistent with this functional data, AC mRNA analysis shows abundant expression of type VI AC, four splice variants of type VIII AC and low expression level of type I AC in beta cells. Type VIII AC expression at the protein level was observed using immunoblots of RINm5F cell extracts. This study identifies type VIII AC in insulin-secreting cells as one of the potential molecular targets for synergism between GLP-1 receptor mediated and glucose-mediated signalling.

AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase.

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells.

Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the beta-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.

Functional impairment and killing of human beta cells by enteroviruses: the capacity is shared by a wide range of serotypes, but the extent is a characteristic of individual virus strains

Direct infection of beta cells could explain the diabetogenic effect of enteroviruses. Primary adult human beta cells are susceptible to coxsackievirus infections, which could result in impaired beta-cell function or cell death (coxsackieviruses B3, B4, B5) or both, or no apparent immediate adverse effects (coxsackievirus A9). We extended these studies to additional enterovirus serotypes including several echoviruses, some of which have been associated clinically with the development of Type I (insulin-dependent) diabetes mellitus. The patterns and consequences of enterovirus infections were investigated in cultured adult human isolated islets. Cell type-specific infection and viability were assessed by immunocytochemical methods. Beta-cell function was studied by perifusion. Poliovirus type 1/Mahoney, coxsackievirus A13, human parechovirus 1 and several echoviruses (serotypes 6, 7, 11) were capable of causing significant functional impairment ( p<0.05) and beta-cell death. In contrast, echovirus serotypes 9 and 30 were not destructive. However, when several different field isolates of echovirus 30 were investigated, some of them were found to be clearly more destructive than the corresponding prototype strain. This was also true for echovirus 9. A strain isolated from a 6-week-old baby suffering from acute Type I diabetes was functionally more destructive than either of the echovirus 9 prototype strains. These observations indicate that the capacity of an enterovirus to kill human beta cells or impair their function is not entirely defined by the serotype, but in addition by as yet unidentified characteristics of the virus strain involved. Moreover, any serotype could potentially be diabetogenic.

Transmembrane TNF and IFNγ Induce Caspase-independent Death of Primary Mouse Pancreatic Beta Cells

Tumor necrosis factor (TNF) is important in the pathogenesis of autoimmune diabetes. It has an important role in immunological and inflammatory processes, and has also been shown to induce apoptotic cell death. We have shown that TNF+IFN n induce islet cell death in vitro . TNF exists as a biologically active transmembrane molecule (tmTNF), which is then cleaved to form soluble TNF (sTNF). We reasoned that sTNF, which has been used in previous studies, may not represent TNF in its physiological form. We compared the contributions of caspase activation and nitric oxide production to beta cell death induced by either tmTNF or sTNF together with IFN n . CHO cells transfected with a mutated TNF were used as a source of tmTNF. Either sTNF or tmTNF, together with IFN n, induced caspase-dependent cell death of the NIT-1 insulinoma cell line, as measured by DNA fragmentation and a fluorogenic caspase 3 activation assay. TNF+IFN n did not induce caspase 3 activation in primary mouse islets. Instead, iNOS gene expression was induced and cell death which was partly NO-dependent occurred. We conclude that the role of TNF in the development of type 1 diabetes is likely to be the activation of gene expression and not apoptosis. It appears that both tmTNF and sTNF act by a similar mechanism to induce beta cell death.

Functional IL-18 Is Produced by Primary Pancreatic Mouse Islets and NIT-1 Beta Cells and Participates in the Progression towards Destructive Insulitis

Background: Preclinical stages of type 1 diabetes are characterized by infiltrating T cells and by the peri- and intra-islet accumulation of pro-inflammatory mediators, such as IFNγ. Methods/Results: Using quantitative PCR we demonstrated that mRNA specific for the IFNγ-inducing cytokine IL-18 is upregulated in NIT-1 beta cells and intact mouse islets upon exposure to IL-1β, IFNγ and TNFα. The biological activity of IL-18 was blocked using caspase inhibitors and anti-IL-18 antibodies. Increased IL-18 expression was also detected in islets during advanced stages of insulitis and correlated with elevated transcripts for IFNγ and for the IL-18 receptor. Conclusion: Thus, beta cells produce bioactive IL-18 in the course of insulitis and actively contribute to the exacerbation of inflammation leading to their own demise.

Regulation of insulin gene transcription by a Ca(2+)-responsive pathway involving calcineurin and nuclear factor of activated T cells.

Immunosuppressants such as FK506 (tacrolimus), the primary cellular target of which is calcineurin, decrease beta-cell insulin content and preproinsulin mRNA expression. This study offers an explanation for this effect by establishing that calcineurin is an important regulator of insulin gene expression through the activation of a transcription factor, nuclear factor of activated T cells. Three putative nuclear factor of activated T cells binding sites were located within the proximal region of the rat insulin I gene promoter (-410 to +1 bp). Expression of nuclear factor of activated T cells in both clonal (INS-1) and primary (islet) beta-cells was confirmed by immunoblot and immunocytochemical analyses. Moreover, nuclear factor of activated T cells DNA-binding activity was detected in INS-1 and islet nuclear extracts by EMSAs. Activation of the insulin gene promoter by glucose or elevated extracellular K(+) (to depolarize the beta-cell) was totally prevented by FK506 (5-10 microM). K(+)-induced promoter activation was suppressed (>65%) by a 2-bp mutation of a single nuclear factor of activated T cells binding site in -410 rInsI. Both stimulants also activated a minimal promoter-reporter construct containing tandem nuclear factor of activated T cells consensus sequences. The effects of FK506 on K(+)-induced nuclear factor of activated T cells reporter or insulin gene promoter activity were not mimicked by rapamycin, indicating specificity toward calcineurin. These findings suggest that the activation of calcineurin by beta-cell secretagogues that elevate cytosolic Ca(2+) plays a fundamental role in maintenance of insulin gene expression via the activation of nuclear factor of activated T cells.

Hexosamines and nutrient excess induce leptin production and leptin receptor activation in pancreatic islets and clonal beta-cells.

Activation of the hexosamine biosynthesis pathway leads to insulin resistance in muscle and adipose tissue. In these tissues leptin gene expression is increased by glucosamine. In the present study we found that glucosamine rapidly activates the production of leptin and OB-Rb, which encodes the functional leptin receptor, in both primary pancreatic islets and clonal beta-cells. Secretion of leptin from clonal beta-cells into the medium was detected readily. In addition, the level of the transcripts encoding signal transducer and activator of transcription-3 and -5, both implicated in leptin signal transduction in islet beta-cells, was increased by glucosamine, although to a lesser degree than mRNA levels of leptin and OB-Rb. High glucose (16.7 mM) induced leptin biosynthesis in primary pancreatic islet cells, and the addition of 1 mM palmitate caused an additional incremental effect. The hexosamine-mediated induction of the leptin system in clonal beta-cells was associated with increased responsiveness to leptin, as demonstrated by a 2.6 +/- 0.3-fold (P < 0.01) increase in tyrosine phosphorylation of signal transducer and activator of transcription-3. These findings are the first evidence of inducible leptin production in pancreatic islets and suggest that islet cells, like skeletal muscle, demonstrate a linkage between increased nutrient availability and both leptin expression and leptin responsiveness.

Double-stranded ribonucleic acid (RNA) induces beta-cell Fas messenger RNA expression and increases cytokine-induced beta-cell apoptosis.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells. For this purpose, fluorescence-activated cell sorting-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta and IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1beta and a combination of PIC and IFN-gamma (but not PIC or IFN-gamma alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and IFN-gamma requires nuclear factor-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. Combinations of IL-1beta plus IFN-gamma, PIC plus IFN-gamma, or PIC plus IL-1beta induced a 2- to 3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity significantly decreased PIC- plus IL-1beta-induced, but not PIC- plus IFN-gamma-induced, apoptosis. In conclusion, PIC alone or in combination with cytokines modifies the expression of several genes in pancreatic beta-cells. Two of these genes, Fas and iNOS, may contribute to beta-cell death. The transcription factor nuclear factor-kappaB is required for PIC-induced iNOS expression. PIC has an additive effect on cytokine-induced beta-cell death by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. These findings suggest that viral intermediates in synergism with local cytokine production may play an important role in beta-cell apoptosis in early T1DM.

Pancreatic β-Cell Damage Mediated by β-Cell Production of Interleukin-1: A NOVEL MECHANISM FOR VIRUS-INDUCED DIABETES

Viral infection is one environmental factor that may initiate beta-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-gamma stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-gamma-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-gamma-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1beta is synthesized as an inactive precursor protein that requires cleavage by the IL-1beta-converting enzyme (ICE) for activation. dsRNA and IFN-gamma stimulate IL-1beta expression and ICE activation in primary beta-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-gamma-induced iNOS expression by primary beta-cells. In addition, poly(I-C) + IFN-gamma-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-gamma in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate beta-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a nitric oxide-dependent fashion.

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon stimulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell.

Growth Hormone- and Prolactin-Induced Proliferation of Insulinoma Cells, INS-1, Depends on Activation of STAT5 (Signal Transducer and Activator of Transcription 5)

GH and PRL stimulate proliferation and insulin production of pancreatic beta-cells. Whereas GH- and PRL-regulated transcription of the insulin gene in insulinoma cells has been shown to depend on STAT5 (signal transducer and activator of transcription 5), the signaling pathways involved in GH/PRL-induced beta-cell replication are unknown. The roles of various signaling pathways in human GH (hGH)-induced DNA synthesis were studied by analysis of the effect of specific inhibitors in both the insulin-producing cell line, INS-1, and in primary beta-cells. The mitogen-activated protein kinase kinase (MEK)-inhibitor, PD98059, as well as the mitogen-activated protein kinase p38 (MAPKp38) inhibitor, SB203580, partially inhibited hGH- induced proliferation in INS-1 cells but had no significant effect in primary beta-cells. Staurosporine, a protein kinase C (PKC) and protein kinase A (PKA) inhibitor, blocked both basal and hGH-induced proliferation in INS-1 cells, but had no inhibitory effect in primary beta-cells. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited hGH-induced proliferation neither in INS-1 cells nor in primary beta-cells, whereas the tyrosine kinase inhibitor, genistein, completely inhibited hGH- induced proliferation in both primary beta-cells and INS-1 cells. To analyze the possible role of STAT5 in hGH-induced proliferation, a dominant negative STAT5 mutant, STAT5Delta749, was expressed in INS-1 cells under the control of a doxycycline- inducible promoter by stable transfection. Two clones were found to exhibit dose-dependent, doxycycline-inducible expression of STAT5Delta749 and suppression of hGH-stimulated transcriptional activation of a STAT5-regulated PRL receptor (PRLR) promoter-reporter construct. Furthermore, induction of STAT5Delta749 expression completely inhibited hGH-induced DNA synthesis. Analysis of endogenous gene expression revealed a doxycycline-dependent inhibition of hGH-stimulated PRLR and cyclin D2 mRNA levels. Our results suggest that GH/PRL-induced beta-cell proliferation is dependent on the Janus Kinase2 (JAK2)/STAT5 signaling pathway but not the MAPK, PI3K, and PKC signaling pathways. Furthermore, the cell cycle regulator cyclin D2 may be a crucial target gene for STAT5 in this process.

Fetal pancreatic islets express functional leptin receptors and leptin stimulates proliferation of fetal islet cells.

Previous studies have demonstrated that leptin can stimulate proliferation of insulin-secreting tumor cell lines. The objective of this study was to characterize whether leptin could stimulate proliferation of primary beta-cells too. Since adult beta-cells have very limited capacity for replication, we examined the effect of leptin on islets of Langerhans obtained from fetal rats, in a tissue culture system. Leptin receptor mRNA and c-fos mRNA were measured by RT-PCR. Proliferation of fetal rat islet cells was measured by a WST-1 colorimetric assay and [3H]-thymidine incorporation assay. Leptin stimulated proliferation of serum-deprived fetal rat islet cells, as indicated by increased formation of formazan dye from a tetrazolium salt WST-1. Leptin stimulated DNA synthesis in islet cells, as indicated by increased [3H]-thymidine incorporation into DNA. The effect of leptin on islet cell proliferation was on average 39-50% of the effect obtained with 10% fetal bovine serum. Leptin increased c-fos mRNA expression by 2.8-fold in isolated fetal islets after 30 min treatment. In fetal pancreatic islets, both the common extracellular portion (OB-R) and the intact long form (OB-Rb) of the leptin receptor were readily detected by reverse transcriptase polymerase chain reaction. Functional leptin receptors are expressed in pancreatic islet cells, as early as during the fetal stage of development of these microorgans. Leptin stimulates proliferation of fetal islet cells and might play a role in determining islet cell mass at birth.

Interleukin-1β Regulates Phospholipase D-1 Expression in Rat Pancreatic β-Cells.

The cytokine interleukin (IL)-1beta induces a biphasic effect in rat pancreatic islets, with an early and transitory stimulation of insulin release followed by progressive functional suppression. To clarify the mechanisms involved in these effects, we have recently performed a differential display of messenger RNA (mRNA) by RT-PCR (DDRT-PCR) on rat beta-cells exposed for 6 or 24 h to IL-1beta. Among the different IL-1beta-induced genes, there was an early and transient increase in phospholipase D-1 (PLD1) expression. PLD1 can induce phosphatidic acid formation and subsequent activation of protein kinase C, a process which stimulates insulin release. In the present study, we characterized the regulation of PLD isoforms by IL-1beta in pancreatic beta-cells. By using different combinations of primers and RT-PCR, we observed that IL-1beta induces an early increase (2 and 6 h) in the expression of both alternatively spliced isoforms of PLD1 (PLD1alpha and 1b). Prolonged exposure to IL-1beta (12 and 24 h) caused a decrease of PLD1a mRNA expression compared with control beta-cells, and lead to a return of PLD1b mRNA to basal level. NG-methyl-L-arginine (LMA), a blocker of the inducible form of nitric oxide synthase (iNOS), prevented this late inhibitory effect of IL-1beta, suggesting that IL-1beta-induced decrease in PLD1a expression is NO-mediated. IL-1beta induced an early (2-6 h) and sustained (16-24 h) increase in PLD1a mRNA expression in insulin-producing RINm5F cells. This was paralleled by a cytokine-induced increase in PLD1 protein expression and enzyme activity. RINm5F cells, but not primary beta-cells, expressed PLD2, and the expression of this gene was not affected by IL-1beta. In conclusion, we have shown that the cytokine IL-1beta regulates PLD1 expression in primary and clonal beta-cells. The early induction of PLD1 probably contributes to the early stimulatory effects of IL-1beta on islet insulin release.

Tumor necrosis factor-alpha-activated cell death pathways in NIT-1 insulinoma cells and primary pancreatic beta cells.

Tumor necrosis factor-alpha (TNFalpha) is a potential mediator of beta cell destruction in insulin-dependent diabetes mellitus. We have studied TNF-responsive pathways leading to apoptosis in beta cells. Primary beta cells express low levels of the type I TNF receptor (TNFR1) but do not express the type 2 receptor (TNFR2). Evidence for TNFR1 expression on beta cells came from flow cytometry using monoclonal antibodies specific for TNFR1 and TNFR2 and from RT-PCR of beta cell RNA. NIT-1 insulinoma cells similarly expressed TNFR1 (at higher levels than primary beta cells) as detected by flow cytometry and radio-binding studies. TNF induced NF-kappaB activation in both primary islet cells and NIT-1 cells. Apoptosis in response to TNFalpha was observed in NIT-1 cells whereas apoptosis of primary beta cells required both TNFalpha and interferon-gamma (IFNgamma). Apoptosis could be prevented in NIT-1 cells by expression of dominant negative Fas-associating protein with death domain (dnFADD). Apoptosis in NIT-1 cells was increased by coincubation with IFNgamma, which also increased caspase 1 expression. These data show that TNF-activated pathways capable of inducing apoptotic cell death are present in beta cells. Caspase activation is the dominant pathway of TNF-induced cell death in NIT-1 cells and may be an important mechanism of beta cell damage in insulin-dependent diabetes mellitus.

Glucose Generates Sub-plasma Membrane ATP Microdomains in Single Islet β-Cells: POTENTIAL ROLE FOR STRATEGICALLY LOCATED MITOCHONDRIA

Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.

Double-stranded RNA Inhibits β-Cell Function and Induces Islet Damage by Stimulating β-Cell Production of Nitric Oxide

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.

Insulin-stimulated Insulin Secretion in Single Pancreatic Beta Cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.