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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
Mol Endocrinol 2001 Jan
PMID:Growth hormone- and prolactin-induced proliferation of insulinoma cells, INS-1, depends on activation of STAT5 (signal transducer and activator of transcription 5). 1114 45

The effect of treatment with a 0.03% fatty acid (FA) cocktail on leptin-receptor-mediated STAT (signal transducers and activators of transcription) activation in the rat insulinoma cell line BRIN-BD11 was investigated. Leptin (10 nM) stimulated the tyrosine phosphorylation of STAT3 and STAT5b. Acute treatment with FAs prevented leptin-stimulated STAT3 tyrosine phosphorylation and significantly raised basal STAT5 phosphorylation. A chronic treatment (5 days) of BRIN-BD11 cells with FAs similarly attenuated leptin-stimulated STAT tyrosine phosphorylation. Chronic FA treatment also attenuated prolactin-stimulated STAT5b tyrosine phosphorylation but not interleukin-6-stimulated STAT3 tyrosine phosphorylation, suggesting that the effect is receptor/ligand specific. TaqMan analysis of gene expression following chronic FA treatment showed neither a decrease in the amount of leptin receptor (Ob-R) mRNA, nor an increase in the negative regulators of STAT signalling, SOCS3 (suppressors of cytokine signalling) or cytokine inducible sequence (CIS). These data demonstrate that FAs modulate leptin and prolactin signalling in beta-cells, implying that high levels of circulating FAs present in obese individuals affect the action of selective cytokines in beta-cell function.
J Mol Endocrinol 2001 Apr
PMID:Fatty acids inhibit leptin signalling in BRIN-BD11 insulinoma cells. 1124 Nov 66

In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels are potential mediators of repolarization, closure of Ca(2+) channels, and limitation of insulin secretion. The specific Kv channels expressed in beta-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased I(DR) by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60-70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat beta-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.
Mol Endocrinol 2001 Aug
PMID:Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion. 1146 64

Exposure of insulin-secreting cells to hypotonic solutions causes cell swelling followed by regulatory volume decrease (RVD). We have previously demonstrated that RVD is due to activation of a Cl(-) conductance. The present study investigates whether changes in cytosolic [Ca(2+)] play a role in these responses. Hypotonic swelling of RINm5F insulinoma cells caused a marked increase in cytosolic [Ca(2+)]. This effect was abolished by omission of extracellular Ca(2+), by the Ca(2+) channel blockers D600 or Gd(3+)and by 4,4'-dithiocyanatostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of the volume-sensitive anion. RVD was markedly impaired in the absence of extracellular Ca(2+), but not by D600 nor by Gd(3+). RVD was also inhibited by the maxi-K(+) (BK(Ca)) channel blockers tetraethylammonium (TEA) and iberiotoxin (IbTx), whereas the K(ATP) channel blocker tolbutamide was ineffective. Cell swelling was accompanied by activation of a K(+) conductance which was sensitive to TEA and IbTx but not to tolbutamide. It is concluded that cell swelling causes activation of the volume-sensitive anion channel, leading to depolarization and Ca(2+) entry via voltage-gated Ca(2+) channels. RVD is a Ca(2+)-dependent process, requiring low 'resting' levels of intracellular [Ca(2+)]. However, the swelling-induced increase in cytosolic [Ca(2+)] is not required for RVD to occur. RVD depends upon simultaneous activation of Cl(-) and K(+) channels. We suggest that the BK(Ca) channel is the major K(+) conductance involved in RVD.
Mol Cell Endocrinol 2001 Jul 05
PMID:Swelling-induced changes in cytosolic [Ca2++] in insulin-secreting cells: a role in regulatory volume decrease? 1147 51

It was previously reported that silencing of the expression of glutamic acid decarboxylase (GAD) in transgenic nonobese diabetic (NOD) mice completely protected islet beta-cells against development of diabetes. This suggests that the repression of GAD autoantigen by somatic gene delivery can prevent autoimmune destruction of pancreatic beta-cells. To repress GAD expression in islet beta-cells, we delivered an antisense GAD mRNA expression plasmid (pRIP-AS-GAD) using poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) as a gene carrier. In a gel retardation assay, the pRIP-AS-GAD/PEG-g-PLL complex was completely retarded above a weight ratio of 1:1.5 (plasmid: PEG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 60 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3. We also transfected pRIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) cell line. The antisense mRNA was expressed specifically in beta-cells and expression was dependent on glucose level. The repression of GAD after transfection of pRIP-AS-GAD was confirmed by immunoblot assay. In addition, in vivo expression of antisense RNA in pancreas was confirmed by RT-PCR after intravenous injection of the complex into mice. Therefore, our study revealed that the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GAD autoantigen expression.
Mol Ther 2001 Oct
PMID:Repression of GAD autoantigen expression in pancreas beta-Cells by delivery of antisense plasmid/PEG-g-PLL complex. 1159 37

We provide immunocytochemical evidence that the neuronal isoform of constitutive NO synthase (cNOS) is expressed in the rat insulinoma cell line INS-1. Furthermore, using N omega-nitro-L-arginine methyl ester (L-NAME), a pharmacological inhibitor of cNOS activity, we show that this enzyme is implicated in the modulation of insulin secretion in INS-1 cells. Indeed, in the presence of 2.8 mM glucose, L-NAME induced a specific and dose-dependent increase in insulin release, suggesting that cNOS exerts an inhibitory tone on basal insulin secretion. Moreover, L-arginine, the physiological substrate of cNOS, significantly reduced the marked enhancing effect of L-NAME on insulin release and to a lesser extent, at low concentrations, that of 10 mM KCl. L-NAME also potentiated the insulin secretion stimulated by 5.5 and 8.3 mM glucose, but in this case, its effect was not reduced by L-arginine. In conclusion, our data show that the neuronal isoform of cNOS exerts a negative modulation on insulin secretion in INS-1 cells, confirming the previous results obtained in the isolated perfused rat pancreas or pancreatic islets.
Mol Cell Endocrinol 2001 Oct 25
PMID:A constitutive nitric oxide synthase modulates insulin secretion in the INS-1 cell line. 1160 23

The non-invasive detection of insulinomas remains a diagnostic problem that is not solved by means of somatostatin receptor scintigraphy. We investigated the biokinetics and specificity of uptake and degradation of the incretin hormone glucagon-like peptide-1 (GLP-1) in a rat insulinoma cell line (RINm5F) in order to ascertain whether radiolabelled GLP-1 may be suitable for specific visualisation of insulinomas in vivo. GLP-1 (7-36)amide was radioiodinated according to the iodogen method. The specificity of the uptake of [(125)I]GLP-1(7-36)amide by RINm5F cells was investigated. Degradation products of GLP-1 (7-36)amide in the cell medium were purified by HPLC. Their masses and amino acid sequences were determined by (252)Cf-plasma desorption mass spectrometry. Lysosomal degradation was inhibited and after differential centrifugation the amount of radiotracer incorporated into lysosomes was determined. Biodistribution studies were performed in a rat insulinoma model (NEDH rats and RINm5F cells) with [(123)I]GLP-1(7-36)amide and its more stable agonist [(123)I]exendin 3. The uptake of radiotracer into insulinoma cells reached a maximum within 5 min. It was inhibited by an excess of unlabelled peptide. [(125)I]GLP-1(7-36)amide accumulated in the cells if lysosomal degradation was inhibited. Degradation products of the peptide were found in the cell medium. We determined their mass and derived their amino acid sequence. Radiolabelling of exendin 3 was more difficult than that of GLP-1 because of the lack of tyrosine in its primary structure. Biodistribution studies showed rapid blood clearance and uptake of the radiotracer into the tumour and the pancreas. It was also possible to detect insulinomas in an animal model by external scintigraphy using radioiodinated GLP-1 (7-36)amide and exendin 3. GLP-1 (7-36)amide is specifically internalised into insulinoma cells by a receptor-mediated mechanism. Our results demonstrate that GLP-1 receptor-directed scintigraphy may be a new method for the detection of insulinomas in vivo. Due to the short half-life of GLP-1, its more stable analogue exendin 3 may better suit this purpose in vivo.
Eur J Nucl Med Mol Imaging 2002 May
PMID:Use of the incretin hormone glucagon-like peptide-1 (GLP-1) for the detection of insulinomas: initial experimental results. 1197 97

The role of c-Jun in apoptosis evoked by human amylin was investigated using human and rat insulinoma beta-cell lines. Two transient increases in the levels of c-jun mRNA were detected at 30 minutes and eight hours after treatment with human amylin. The level of c-Jun protein was also up-regulated in a time-dependent manner, reaching maximal levels after eight hours of exposure. However, no c-Jun induction was detected in cells treated with vehicle only or with rat amylin, indicating that the amyloidogenic feature of the human peptide may be important for c-Jun induction. We found that c-Jun was activated by phosphorylation specifically at Ser63 at four hours, but not at Ser73, after treatment with human amylin, preceding increased c-Jun protein. Furthermore, expression of an antisense c-jun (AS-c-jun), which suppressed protein levels of both c-Jun and phosphorylated-c-Jun, caused a marked reduction in apoptotic cell death, whereas the corresponding sense c-jun (S-c-jun) had no effect on changes of either c-Jun production or apoptosis. This indicated that increased expression and activation of c-Jun is required for human amylin-induced apoptosis. Immunocytochemical studies showed a significant increase in nuclear staining for c-Jun, phosphorylated-c-Jun (Ser63) and phosphorylated-JNK, suggesting that c-Jun may be activated through activation of JNK. In addition, electrophoretic mobility-shift assays showed that the increase in expression and phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays demonstrated that c-Jun, c-Fos and ATF-2 are part of the AP-1 complex, indicating that activated c-Jun is dimerized with c-Fos or ATF-2 for control of its target gene expression. Finally, we showed that human amylin triggers AP-1-mediated transcriptional activation. Our results suggest strongly that human amylin induces apoptosis through stimulation of expression and activation of c-Jun, and that co-expression and dimerization of c-Jun and c-fos or ATF-2 may be important for activation of the downstream apoptotic process.
J Mol Biol 2002 Nov 22
PMID:Increased expression and activation of c-Jun contributes to human amylin-induced apoptosis in pancreatic islet beta-cells. 1244 Nov 6

During the process of insulitis in the pathogenesis of type I (insulin-dependent) diabetes mellitus, proinflammatory cytokines induce expression of the death receptor Fas on the surface of pancreatic beta-cells and thereby contribute to the enhanced susceptibility of beta-cells for apoptosis. The aim of this study was to compare cell-surface and intracellular Fas expression associated with cytokine-induced apoptosis in commonly used beta-cell models such as isolated islets and insulinoma lines derived from mouse and rat. The cell line NIT-1 responded to the interleukin (IL)-1beta+interferon (IFN)-gamma stimulus with translocation of Fas to the cell surface. Likewise, islet cells from non-obese diabetic (NOD) mice and BB/OK rats expressed increasing amounts of the Fas receptor on their surfaces after exposure to IL-1beta in combination with IFN-gamma and tumour necrosis factor-alpha. Moreover, islets obtained from BB/OK rats at an age near the onset of diabetes had an increased surface expression of Fas compared with young rats. In contrast, western blot analysis of cell lysates from cytokine-exposed islets and insulinoma cells revealed total Fas expression levels comparable to those of untreated controls. In conclusion, islets from BB/OK rats and NOD mice, in addition to NIT-1 insulinoma cells, responded to cytokine exposure with surface expression of the Fas receptor, whereas in cell lysates the levels of expression of Fas were found to be independent of cytokine exposure. Taken together, the findings indicate that cytokine-treated beta-cells might possess two pools of Fas protein, one of which is inducible by cytokines and accounts for surface Fas expression, whereas the other is constitutively expressed in cytoplasmic compartments. The underlying mechanisms, including possible interactions between these two sources of cellular Fas expression, need to be investigated in future studies.
J Mol Endocrinol 2003 Apr
PMID:Surface and intracellular Fas expression associated with cytokine-induced apoptosis in rodent islet and insulinoma cells. 1268 40

Peptide receptors have been found to represent excellent targets for in vivo cancer diagnosis and therapy. Recent in vitro studies have shown that many cancers can overexpress not only one but several peptide receptors concomitantly. One of the challenges for nuclear medicine in this field in the coming decade will be to take advantage of the co-expression of peptide receptors for multireceptor tumour targeting. In vitro receptor studies can reveal which peptide receptor is overexpressed in which tumour and which receptors are co-expressed in an individual tumour; such knowledge is a prerequisite for successful in vivo development. One group of tumours of particular interest in this respect is the neuroendocrine tumours, which have previously been shown often to express peptide receptors. This review summarises our investigations of the concomitant expression of 13 different peptide receptors, in more than 100 neuroendocrine tumours of the human intestine, pancreas and lung, using in vitro receptor autoradiography with subtype-selective ligands. The incidence and density of the somatostatin receptors sst(1)-sst(5), the VIP receptors VPAC(1) and VPAC(2), the CCK(1) and CCK(2) receptors, the three bombesin receptor subtypes BB(1) (NMB receptor), BB(2) (GRP receptor) and BB(3), and GLP-1 receptors were evaluated. While the presence of VPAC(1) and sst(2) was detected in the majority of these neuroendocrine tumours, the other receptors, more differentially expressed, revealed a characteristic receptor pattern in several tumour types. Ileal carcinoids expressed sst(2) and VPAC(1) receptors in virtually all cases and had CCK(1), CCK(2), sst(1) or sst(5) in approximately half of the cases; they were the only tumours of this series to express NMB receptors. Insulinomas were characterised by a very high incidence of GLP-1, CCK(2) and VPAC(1) receptors, with the GLP-1 receptors expressed in a particularly high density; they expressed sst(2) in two-thirds and sst(1) in approximately half of the cases and lacked CCK(1) and NMB receptors. All gastrinomas had sst(2) and GLP-1 receptors; they expressed GRP receptors in three-quarters of the cases and CCK(1) or VPAC(1) in approximately half of the cases. Most bronchial carcinoids had VPAC(1), while sst(1), sst(2) and CCK(2) were found in two-thirds of the cases and BB(3) in one-third of the cases. These data provide evidence for the vast biological diversity of these neuroendocrine tumours. Moreover, the results represent a basis for starting and/or optimising the in vivo targeting of these tumours by selecting the suitable radiopeptides for tumour diagnosis and/or therapy. Finally, the data strongly encourage concomitant application of several radiopeptides to permit more efficient targeting of these tumours.
Eur J Nucl Med Mol Imaging 2003 May
PMID:Concomitant expression of several peptide receptors in neuroendocrine tumours: molecular basis for in vivo multireceptor tumour targeting. 1270 37


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