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PubMed1989

Relationship between hormonal, GTP and Ins(1,4,5)P3-stimulated Ca2+ uptake and release in pancreatic acinar cells.

Electrically permeabilized rat pancreatic acini were used to evaluate the contributions of GTP and Ins(1,4,5) P3 to hormone-stimulated Ca2+ uptake and release from intracellular pools. Treatment of permeabilized acini with Ca2+-mobilizing hormones, GTP or GTP[S] resulted in stimulation of an ATP-dependent, VO4(2-)-sensitive Ca2+ uptake into a non-mitochondrial intracellular pool. GTP and GTP[S] also augmented the hormone-mediated stimulation of Ca2+ uptake. Including oxalate in the uptake medium increased Ca2+ uptake into this pool but did not modify the stimulation of Ca2+ uptake induced by hormones or GTP. Ins(1,4,5)P3 released all the extra Ca2+ accumulated as a result of hormone, GTP or GTP[S] stimulation. Hence, these stimuli activated the Ca2+ pump localized in the membrane of the hormone and Ins(1,4,5)P3-sensitive Ca2+ pool. Including 2,3-diphosphoglyceric acid (PGA) [an inhibitor of Ins(1,4,5)P3 hydrolysis] in the incubation medium blunted the GTP and GTP[S]-stimulated Ca2+ uptake. In the presence of PGA, the hormones inhibited Ca2+ accumulation, and GTP and GTP[S] augmented this effect. Accordingly, PGA stabilized the Ins(1,4,5)P3-evoked Ca2+ release from intracellular pools. Only in the presence of PGA was it possible to demonstrate hormonally-evoked Ca2+ release from permeabilized cells. GTP, and more importantly GTP[S], augmented the hormone-evoked Ca2+ release. Hormones and Ins(1,4,5)P3 in the presence or absence of GTP or GTP[S] released Ca2+ from the same intracellular pool. The extent of Ca2+ release caused by the combination of hormones and GTP or GTP[S] was similar to that evoked by Ins(1,4,5)P3 alone. Taken together, these results suggest that GTP or GTP[S] facilitates stimulation of phospholipase C by hormones. Such stimulation results in stimulation of protein kinase C and increased levels of Ins(1,4,5)P3 and is sufficient to explain the effects of GTP and GTP[S] on Ca2+ uptake and release from pancreatic acinar cells.

Adenosine Triphosphate↗
PubMed1994

Bovine testis and human erythrocytes contain different subtypes of membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphomonoesterases.

1. We have purified membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatases from bovine testis and human erythrocytes by chromatography on several media, including a novel 2,3-bisphosphoglycerate affinity column. 2. The enzymes have apparent molecular masses of 42 kDa (testis) and 70 kDa (erythrocyte), as determined by SDS/PAGE, and affinities for Ins(1,4,5)P3 of 14 microM and 22 microM respectively. 3. The two enzymes hydrolyse both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and are therefore type I Ins(1,4,5)P3 5-phosphatases [nomenclature of Hansen, Johanson, Williamson and Williamson (1987) J. Biol. Chem. 262, 17319-17326]. 4. On chromatofocusing, the partially purified testicular enzyme migrates as two peaks of activity, with pI values of about 5.8 and 5.5. The erythrocyte enzyme exhibits only the latter peak. 5. The testis 5-phosphatase is labile at 37 degrees C, but its activity can be maintained in the presence of 50 mM phorbol dibutyrate (PdBu). After PdBu treatment, a third form of the enzyme, with pI about 6.2, appears on chromatofocusing, but without change in its Km or Vmax. 6. Consideration of the properties of these enzymes and of the 5-phosphatases from other tissues suggests that type I Ins(1,4,5)P3 5-phosphatases are of two well-defined subtypes. We propose that these be termed type Ia [typified by the testis enzyme: approximately 40 kDa, higher affinity for Ins(1,4,5)P3] and Type Ib [typified by the erythrocyte enzyme: approximately 70 kDa, lower affinity for Ins(1,4,5)P3].

Animals↗
PubMed2000

Nerve growth factor- and epidermal growth factor-regulated gene transcription in PC12 pheochromocytoma and INS-1 insulinoma cells.

PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA and p75NTR and the epidermal growth factor receptor (EGF). In PC12 cells, NGF treatment initiates a signaling cascade that ultimately leads to a change of the genetic program of the cell. We have investigated the role of NGF in regulating gene transcription in PC12 and INS-1 cells, in order to define if there are NGF-regulated genes per se. Furthermore, to distinguish between growth factor stimulation via receptor tyrosine kinases in general and NGF-specific changes in gene transcription, we analyzed the effects of EGF on gene transcription. First, we tested the biological activities of fusion proteins consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the phosphorylation-dependent activation domains of the transcription factors Elk1, CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells. We found a striking increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is a major substrate for the extracellular signal-regulated protein kinase (ERK). This effect was observed in NGF- as well as in EGF-treated PC12 cells. In INS-1 cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF, but not by EGF. The effects of NGF and EGF on gene transcription were subsequently studied with plasmids containing reporter genes under control of the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences. NGF stimulated Egr-1 promoter activities in PC12 and INS-1 cells, although the effect was much more pronounced in PC12 cells than in INS-1 cells. EGF also stimulated Egr-1 promoter activity in both PC12 and INS-1 cells. Stimulation of c-jun promoter activity by NGF was observed only in PC12 cells. Deletion mutagenesis demonstrated the importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within the c-jun promoter for basal and NGF-mediated transcriptional induction. Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but not in INS-1 cells and EGF did not show any effects on these promoters. We conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of the ERK subtype of mitogen-activated protein kinases in the nucleus and a subsequent activation of Egr-1 gene transcription. The NGF-induced transcription of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific, indicating that NGF can trigger different gene expression programs dependent on the signaling pathways present in a particular cell type. EGF is clearly able to activate gene transcription, suggesting that the differences in the biological activities of EGF and NGF cannot be explained by the inability of EGF to stimulate gene transcription.

Activating Transcription Factor 2↗
PubMed1998

A1 adenosine receptor-mediated Ins(1,4,5)P3 generation in allergic rabbit airway smooth muscle.

The signal transduction pathway for A1 adenosine receptor in airway smooth muscle from allergic rabbits was studied by investigating the effect of the selective A1 adenosine-receptor agonist N6-cyclopentyladenosine (CPA) on tissue levels of inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3] measured by protein binding assay. CPA caused a rapid, transient, and concentration-dependent elevation of Ins(1,4,5)P3 in airways from allergic rabbits. The agonist also produced a concentration-dependent contraction of the airway preparations from these animals. Both the Ins(1,4,5)P3 and contractile responses generated by CPA were attenuated by the phospholipase C (PLC) inhibitor U-73122, indicating the coupling of these responses to PLC. The CPA-induced Ins(1,4,5)P3 production observed in the allergic rabbit tissues was also inhibited by the adenosine-receptor antagonist 8-( p-sulfophenyl)-theophylline, suggesting that the effect was mediated by A1 adenosine receptors. On the other hand, the A2 adenosine-receptor agonist CGS-21680 was ineffective in altering the tissue concentration of Ins(1,4,5)P3, indicating that A2 adenosine receptors may not be involved in the activation of PLC in the allergic rabbit airway smooth muscle. In this preparation, the Gi-Go inhibitor pertussis toxin (PTX) attenuated the CPA-induced Ins(1,4,5)P3 accumulation, providing evidence that the generation of Ins(1,4,5)P3 by A1 adenosine-receptor stimulation is coupled to a PTX-sensitive G protein(s). The results suggest that activation of A1 adenosine receptors in allergic rabbit airway smooth muscle causes the production of Ins(1,4,5)P3 via a PTX-sensitive G protein-coupled PLC, and this signaling mechanism may be involved, at least in part, in the generation of contractile responses. It is hypothesized that this process may contribute to adenosine-induced bronchoconstriction in allergic asthma.

Adenosine↗
PubMed1999

Antibodies against the PH domain of phospholipase C-delta1 inhibit Ins(1,4,5)P3-mediated Ca2+ release from the endoplasmic reticulum.

The pleckstrin homology domain (PH domain) is now well known as a structural module for the binding of inositol compounds. In the present study, polyclonal antibodies against the peptide KVKSSSWRRERFYK, derived from the N-terminal of the PH domain of phospholipase C-delta1 (PLC-delta1), were raised in rabbits. These were then tested for their ability to inhibit the binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to the binding proteins including the receptor molecule. The Fab fragment of the antibodies but not the whole molecule inhibited the binding of Ins(1,4,5)P3 not only to PLC-delta1 but also to the Ins(1,4,5)P3 receptor, indicating that the antibodies raised recognized the binding site for Ins(1,4, 5)P3 in the receptor. Rat basophilic leukemic cells were permeabilized with saponin and assayed for Ins(1,4,5)P3-mediated Ca2+ release. Pretreatment of permeabilized RBL cells with the Fab fragment of the antibodies diminished the release of Ca2+ caused by Ins(1,4,5)P3, and further absorption experiments using a variety of synthetic peptides suggested that the tripeptide KVK is the epitope of the antibodies. Structural information about KVK will help in screening for Ins(1,4,5)P3 antagonists.

Animals↗
PubMed1996

Ins(1,4,5)P3 during myocardial ischemia and its relationship to the development of arrhythmias.

Reperfusion of globally ischemic rat hearts in vitro causes release of inositol(1,4,5) trisphosphate (Ins(1,4,5)P3) which is associated with the development of reperfusion arrhythmias. Both of these responses require the presence of a receptor agonist, either norepinephrine or thrombin, and both responses are inhibited by the aminoglycoside, gentamicin and the polyamine, spermine. In the current study, the role of Ins(1,4,5)P3 in the development of arrhythmias under ischemic conditions was addressed. Arrhythmias [ventricular premature beats, ventricular tachycardia and ventricular fibrillation (VF)] occurring over 25 min subsequent to coronary artery ligation were shown to be independent of endogenous norepinephrine or adrenergic receptor stimulation but were effectively inhibited by gentamicin (0.15-1.5 mM, 95% VF in controls compared with 0% VF, at 1.5 mM, P < 0.01) and spermine (5 mM, 40% VF, P < 0.01). Depletion of Ca2+ stores, including Ins(1,4,5)P3-sensitive Ca2+ stores, with thapsigargin (300 nM) reduced the incidence of ischemic arrhythmias (40% VF, P < 0.01). [3H]-Inositol-labeled right atria incubated under conditions of simulated ischemia retained the ability to respond to norepinephrine by releasing inositol phosphates. Under ischemic conditions, gentamicin (1.5 mM) caused a reduction in [3H]Ins(1,4,5)P3 without any effect on the other inositol phosphates. Similar effects of gentamicin were observed under ischemic conditions in the absence of norepinephrine (95 +/- 8 cpm/mg, mean +/- S.E.M., n = 4, v 29 +/- 4, P < 0.0] for 1.5 mM gentamicin). Agonist independent release of [3H]Ins(1,4,5)P3 under ischemic conditions required extracellular Ca2+ suggesting the operation of a Ca(2+)-activated phospholipase C. In agreement with this, release of [3H]Ins(1,4,5)P3 could be initiated by Ca2+ overload under normoxic conditions and this was inhibited by gentamicin. These findings show that Ca2+ overload can enhance release of Ins(1,4,5)P3 under ischemic conditions and provide evidence that this release is involved in the genesis of arrhythmias under these conditions.

Animals↗
PubMed2005

Differential change of Ins-P3-Ca2+ signaling during culture of rat hepatocytes.

Decrease of alpha-adrenergic responses during primary culture of rat hepatocytes was studied. Activation of glycogen phosphorylase by phenylephrine was decreased in the early stage of the culture (within 6 h), however, Ins-P3 production was almost intact until 12 h of the culture and then declined. alpha-Adrenoceptor-mediated Ca2+-mobilization and Ins-P3-induced Ca2+ release from microsomal fractions were decreased in the early stage of the culture, similar to the above change of phosphorylase activation. We found that decrease of Ins-P3-binding sites in the early stage of the culture was the cause of differential change of Ins-P3-Ca2+ signaling during the culture of hepatocytes. Similar changes described above were also observed in vasopressin-induced responses. However, the changes of Ins-P3-Ca2+ signaling did not occur in a high-cell density culture of rat hepatocytes. In conclusion, the loss of phenylephrine- and vasopressin-induced responses in cultured liver cells appear to be due to change of Ins-P3-binding sites as well as decreased Ins-P3 production due to reduction of receptor numbers.

Animals↗
PubMed1996

The lysosomal Ca2+ pool in MDCK cells can be released by ins(1,4,5)P3-dependent hormones or thapsigargin but does not activate store-operated Ca2+ entry.

In several cell types, Ca2+ release from intracellular Ca2+ stores by Ins(1,4,5)P3 elicits Ca2+ influx from the extracellular space into the cytoplasm, termed store-operated Ca2+ entry (SOCE). In MDCK cells, the Ins(1,4,5)P3-sensitive Ca2+ store giving rise to SOCE essentially overlaps with the thapsigargin (TG)-sensitive store. Recent evidence suggests that in MDCK cells lysosomes form a Ca2+ pool that is functionally coupled with the Ins(1,4,5)P3-sensitive Ca2+ store: Ca2+ can be selectively released from lysosomes by glycyl-L-phenylalanine naphthylamide, an agent inducing lysosomal swelling with subsequent and reversible permeabilization of the vesicular membranes. This compartment is also depleted by Ins(1,4,5)P3-dependent agonists or TG, indicating that it is part of a larger, Ins(1,4,5)P3-sensitive Ca2+ pool. Here we show that whereas SOCE is triggered by Ca2+ release from the entire Ins(1,4,5)P3-sensitive Ca2+ pool, selective Ca2+ release from lysosomes alone is unable to trigger SOCE. This finding is consistent with measurements of the store-operated cation current, a direct parameter for store-operated Ca2+ and Na+ entry into MDCK cells. Hence it is proposed that the Ins(1,4,5)P3-sensitive Ca2+ pool is composed of different intracellular compartments that do not uniformly stimulate Ca2+ entry into the cell.

Animals↗
PubMed1998

Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells.

Nutrients and certain growth factors stimulate pancreatic beta-cell mitogenesis, however, the appropriate mitogenic signal transduction pathways have not been defined. In the glucose-sensitive pancreatic beta-cell line, INS-1, it was found that glucose (6-18 mM) independently increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically relevant concentration range (6-18 mM glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mM glucose + 10 nM IGF-I. Glucose metabolism and phosphatidylinositol 3'-kinase (PI 3'-kinase) activation were necessary for both glucose and IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mM glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3'-kinase to IRS-2 and pp60. Glucose and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3-18 mM) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced beta-cell proliferation. In contrast, p70(S6K) was activated with increasing glucose concentration (between 6 and 18 mM), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus, glucose and IGF-I-induced beta-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated protein kinase but dependent on IRS-mediated induction of PI 3'-kinase activity and downstream activation of p70(S6K). The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes beta-cell signaling mechanisms are rather unique in being tightly linked to glycolytic metabolic flux.

Cell Division↗
PubMed1994

Inositol polyphosphates are not increased by overexpression of Ins(1,4,5)P3 3-kinase but show cell-cycle dependent changes in growth factor-stimulated fibroblasts.

NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.

3T3 Cells↗
PubMed1995

Suppression of ventricular arrhythmias during ischemia-reperfusion by agents inhibiting Ins(1,4,5)P3 release.

BACKGROUND: Reperfusion following myocardial ischemia causes a rapid and transient release of inositol (1,4,5)triphosphate [Ins(1,4,5)P3]. The aim of this study was to test whether this increased Ins(1,4,5)P3 release was important for the development of ventricular arrhythmias and whether agents that inhibit this signal transduction pathway, such as aminoglycoside antibiotics, suppress arrhythmias. METHODS AND RESULTS: In perfused rat hearts, ventricular tachycardia (VT), ventricular fibrillation (VF), and accumulation of Ins(1,4,5)P3 were measured during early reperfusion. A number of different compounds, including neomycin, gentamicin, streptomycin, spermine, reserpine, and prazosin, were effective in inhibiting the reperfusion-induced Ins(1,4,5)P3 release and the onset of VT and VF in parallel. A strong correlation existed between Ins(1,4,5)P3 content, measured at 2 minutes of reperfusion, and the incidence of reperfusion VT and VF. In addition, intravenous gentamicin suppressed the onset of arrhythmias under ischemic and reperfusion conditions in vivo. CONCLUSIONS: Our results are consistent with the view that Ins(1,4,5)P3 release plays a pivotal role in mediating arrhythmias during early reperfusion. Agents inhibiting Ins(1,4,5)P3 release are antiarrhythmic and may have potential use clinically.

Alkaloids↗
PubMed1997

Prolactin regulation of islet-derived INS-1 cells: characteristics and immunocytochemical analysis of STAT5 translocation.

The major changes in pancreatic islet function during pregnancy and after exposure to lactogens are an increase in beta-cell proliferation and enhanced insulin secretion. In this study we examined INS-1 cells as a potential model for further inquiry into PRL signaling in beta-cells. Proliferation of beta-cells, insulin secretion, and quantitative immunocytochemical analysis of STAT5 translocation were studied. PRL treatment of INS-1 cells resulted in a 2- to 4-fold increase in cell proliferation compared to that in the control group. In contrast, there was no effect of PRL treatment on HIT cell proliferation and only a very small effect on RIN cell proliferation. A significant effect on INS-1 cell proliferation was observed at 10 ng/ml and reached a maximum at 200 ng/ml. PRL treatment resulted in enhanced insulin secretion from INS-1 cells. There was a time-dependent increase in insulin secretion, which when corrected for cell number was 1.5-fold greater in the PRL-treated cells. The effects of PRL on cell division and insulin secretion were glucose dependent. The presence of the JAK family of tyrosine kinases and the transcription factor STAT5 in INS-1 cells was examined by immunocytochemical techniques. Although all members of the JAK family of kinases were detected, the staining intensity of JAK-2 was noticeably more intense. Initial studies of STAT5 translocation were performed using PRL-dependent Nb2 lymphoma cells, in which PRL treatment resulted in a nearly complete translocation of cytoplasmic STAT5 to the nucleus. Under control conditions there was a near-equal fluorescence intensity of STAT5 staining in the nucleus and cytoplasm of INS-1 cells. PRL treatment resulted in a time-dependent increase in STAT5 staining in the nucleus, with a corresponding decrease in the cytoplasm. The STAT5 staining intensity in the nucleus remained elevated for the duration of PRL treatment. This effect was reversible upon removal of PRL from the medium. Besides PRL, both GH and FBS induced a similar translocation of STAT5 to the nucleus. Although present in RIN cells, no detectable changes in STAT5 were observed in RIN cells after exposure to PRL, GH, or FBS. INS-1 cells should provide a good model for further inquiry into the intracellular signaling pathways used by PRL and how these events alter islet function.

Animals↗
PubMed2002

Hepatocyte nuclear factor-1alpha modulates pancreatic beta-cell growth by regulating the expression of insulin-like growth factor-1 in INS-1 cells.

Maturity-onset diabetes of the young type 3 (MODY3) is characterized by impaired insulin secretion. Heterozygous mutations in the gene encoding hepatocyte nuclear factor (HNF)-1alpha are the cause of MODY3. Transgenic mice overexpressing dominant-negative HNF-1alpha mutant in pancreatic beta-cells and HNF-1alpha knockout mice are animal models of MODY3. These mice exhibit defective glucose-stimulated insulin secretion and have reduced beta-cell mass and beta-cell proliferation rate. Here we examined the effect of HNF-1alpha on beta-cell proliferation by overexpressing a human naturally occurring dominant- negative mutation P291fsinsC in INS-1 cells under the control of doxycycline-induction system. INS-1 cells overexpressing P291fsinsC showed apparent growth impairment. The proliferation rate estimated by [(3)H]thymidine incorporation was significantly reduced in P291fsinsC-expressing INS-1 cells compared with noninduced or wild-type HNF-1alpha-overexpressing INS-1 cells. Growth inhibition occurred at the transition from G1 to S cell cycle phase, with reduced expression of cyclin E and upregulation of p27. cDNA array analysis revealed that the expression levels of IGF-1, a major growth factor for beta-cells, and macrophage migration inhibitory factor (MIF), a cytokine expressed in pancreatic beta-cells, were reduced in P291fsinsC-HNF-1alpha-expressing INS-1 cells. Although MIF seemed to have proliferative function, blockade of MIF action by anti-MIF antibody stimulated INS-1 cell proliferation, excluding its direct role in the growth impairment. However, addition of IGF-1 to P291fsinsC-expressing INS-1 cells rescued the growth inhibition. Our data suggest that HNF-1alpha is critical for modulating pancreatic beta-cell growth by regulating IGF-1 expression. IGF-1 might be a potential therapeutic target for the treatment of MODY3.

Animals↗
PubMed2003

[Role of nuclear factor of kB in tumor necrosis factor alpha-mediated INS-1 cell apoptosis].

OBJECTIVE: To investigate the effect of cytokine tumor necrosis factor alpha(TNFalpha) on pancreatic beta cells apoptosis and the effect of transcription factor nuclear factor of kappa B (NF-kB) on TNFalpha-mediated apoptosis. METHODS: INS-1 cells, a pancreatic beta cell line, were cultured,and recombinant DNA technique, transfection and reinfection technology were used to obtain INS-1/IkBDeltaN cells expressing inhibitor of kB (IkB)alphaDeltaN, mutant IkBalpha (inhibitor of NF-kB). DNA fragmentation assay and fluorescence analysis using a kB-luc reporter gene were applied to observe the NF-kB activity and beta cell apoptosis. RESULTS: NF-kB activity induced by IL-Ibeta was detectable in INS-1 cells but not in INS-1/IkBDeltaN cells. After incubation of the cells with IL-1beta (10 micro g/L), TNFalpha (100 micro g/L) and interferon (IFN)gamma (100 000 U/L) for 48 hours, the combination of TNFalpha and IFNgamma induced apoptosis in INS-1 cells but not in INS-1/IkBDeltaN cells. No apoptosis was observed after incubation of INS-1 cells with IL-1beta or IFNgamma or IL-1beta plus IFNgamma. CONCLUSION: Apoptosis is one of the TNFalpha-induced beta-cell death forms. NF-kB may play an important role in the TNFalpha-mediated beta-cell apoptosis. Inhibition of NF-kB activation protects beta-cells from TNFalpha-induced apoptosis.

Animals↗
PubMed2004

Polymorphism of INS VNTR is associated with glutamic acid decarboxylase antibodies and postprandial C-peptide in patients with onset of diabetes after 35 years of age.

Variability in the number of tandem repeats of the insulin gene (INS VNTR) is probably involved in the genetic regulation of insulin secretion. The aim of this study was to investigate the association of INS VNTR polymorphism with the presence of glutamic acid decarboxylase antibodies (GADA) and C-peptide levels in patients with the onset of diabetes after 35 years of age. We investigated 117 patients, median of age 63 (range 40-83) years, median of diabetes duration 8 (range 1-30) years; 31 GADA-positive and 86 GADA-negative subjects. INS VNTR polymorphism was typed indirectly using - 23HphI (T/A) polymorphism, which is in complete linkage disequilibrium with INS VNTR. The I/I, I/III and III/III genotypes were found in 22 (71 %), 8 (26 %), 1 (3 %) GADA-positive individuals and in 39 (45 %), 35 (41 %), 12 (14 %) GADA-negative individuals, respectively. The Class I allele and the genotype I/I were significantly associated with the presence of GADA (OR=2.72, CI 95 %=1.29-5.73 and OR=2.95, CI 95 %=1.22-7.13). The presence of Class III allele was significantly associated with a higher level of postprandial C-peptide in GADA-positive subjects, even when regarding the duration of diabetes. Our results of INS VNTR polymorphism in patients with the onset of diabetes after 35 years of age confirm the association of Class I INS VNTR with autoimmune diabetes and the protective effect of Class III INS VNTR on the insulin secretion in GADA-positive subjects.

Adult↗
PubMed1992

The effect of ischaemia-reperfusion on [3H]inositol phosphates and ins(1,4,5)P3 levels in cardiac atria and ventricles--a comparative study.

In this study incorporation of [3H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P3 levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [3H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2-3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[3H]Ins(1,4,5)P3 assay system, the Ins(1,4,5)P3 levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles. Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P3 levels as well as incorporation of [3H]inositol into ventricular InsP2 and InsP3. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P3 levels and also [3H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P3 levels as well as the incorporation of [3H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.

Animals↗
PubMed1994

Ins 1,4,5-P3 and Ca2+ signaling in quiescent neonatal cardiac myocytes.

Activation of alpha 1-adrenergic receptors in neonatal cardiac myocytes results in changes in contractile activity and the induction of hypertrophic growth. The biochemical mechanisms responsible for these diverse effects are not yet established, but presumably involve the associated alpha 1-adrenergic stimulation of phosphatidylinositol (PI) hydrolysis, with concomitant generation of Ins 1,4,5-P3 and diacylglycerol. This study examined whether alpha 1-adrenergic generation of Ins 1,4,5-P3 in intact, quiescent, neonatal cardiac myocytes resulted in a Ca2+ signal. Stimulation of myocytes with norepinephrine in the presence of propranolol caused accumulation of inositol mono-, bis and trisphosphates. However, alpha 1-adrenergic stimulation did not alter cytosolic free Ca2+ levels in 85% of the myocytes examined. Direct generation of Ins 1,4,5-P3, by photolysis of microinjected caged Ins 1,4,5-P3, was also unable to alter cytosolic free Ca2+ levels, despite the presence of Ins 1,4,5-P3 receptors. Taken together, these data indicated that alpha 1-adrenergic stimulation did not initiate Ca2+ signaling because Ins 1,4,5-P3-induced Ca2+ mobilization was not operative in quiescent neonatal cardiac myocytes. Normal excitation-contraction Ca2+ handling mechanisms were present in these cells, as illustrated by depolarization- and caffeine-induced Ca2+ transients. Analysis of these same myocytes following 48 h in the presence of norepinephrine and propranolol showed a 40% increase in the ratio of protein to DNA and a 350% increase in release of atrial naturietic factor, compared to control cells, indicating the normal operation of alpha 1-adrenergic-induced hypertrophic growth. Therefore, the assumption that Ca(2+)-dependent processes will be activated by receptor signaling pathways coupled to enhanced phosphatidylinositol turnover in cardiac cells must be avoided. In addition, the data presented in this study clearly indicated that an increase in cytosolic free Ca2+ was not necessary for the induction of alpha 1-adrenergic-mediated cardiac hypertrophy.

Animals↗
PubMed2005

Glucagon production of the rat insulinoma cell line INS-1-A quantitative comparison with primary rat pancreatic islets.

The rat insulinoma cell line INS-1 is the most commonly used clonal cell model in pancreatic beta-cell research. Considering the multihormonality of many insulinomas we examined as to how INS-1 cells comply with the notion of resembling a pure beta-cell line. Glucagon immunoassays revealed that INS-1 cells secrete glucagon in a similar range as islets. By immunohistochemistry we detected a cytoplasmic glucagon signal in INS-1 cells which colocalized with C-peptide. Cellular content of preproglucagon-mRNA and glucagon protein in INS-1 cells was less than two percent of the respective values in islets, which probably reflects differences in the intracellular metabolism and/or secretory pathways. Taken together, it is obvious that INS-1 cells do not represent an exclusively insulin producing beta-cell line.

Animals↗