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

The dinoflagellate toxin maitotoxin (MTX) elicited a sustained increase of [Ca2+]i in C6 glioma cells. This response was inhibited by SK&F 96365, a blocker of receptor-mediated calcium entry. In C6 cells, endothelin-1 elicited a rapid but transient increase in [Ca2+]i, followed by a smaller sustained increase. SK&F 96365 inhibited the sustained increase in [Ca2+]i. In both C6 glioma cells and RIN insulinoma cells, MTX elicited a marked influx of 45Ca2+. SK&F 96365 inhibited MTX-induced 45Ca2+ influx by 95% at 30 microM. The L-type calcium channel blocker nifedipine, even at 10 microM, inhibited MTX-induced calcium uptake by only 20% in RIN cells and by only 10% in C6 cells. MTX elicited calcium-dependent phosphoinositide breakdown in both C6 and RIN cells. In both cell lines, the MTX-induced phosphoinositide breakdown was inhibited by 90% by SK&F 96365 at 30 microM. Endothelin-1 and carbamylcholine elicited phosphoinositide breakdown in C6 cells and RIN cells, respectively. The stimulations were unaffected by the presence of SK&F 96365 up to 100 microM. In RIN insulinoma cells, MTX elicited calcium-dependent release of insulin. SK&F 96365 at 30 microM inhibited MTX-induced insulin release by 75%, whereas nifedipine, even at 30 microM, inhibited release by only 10%. The blockade of MTX-induced responses by SK&F 96365 indicates that MTX increases intracellular calcium by interacting directly with a calcium-entry system that is similar, in its sensitivity to SK&F 96365, to the calcium-entry system activated by receptors that elicit phosphoinositide breakdown. Activation of phospholipase C and hormone release by MTX also are blocked by SK&F 96365 and, thus, may be secondary to the activation of such a calcium-entry system.
Mol Pharmacol 1992 Mar
PMID:Maitotoxin effects are blocked by SK&F 96365, an inhibitor of receptor-mediated calcium entry. 131 15

The transport of L- and D-lactate into rat pancreatic islets and HIT-T15 insulinoma cells was studied by measuring uptake of 14C-labelled substrate at room temperature and by following changes in intracellular pH (pHi) in islets and HIT-T15 cells loaded with 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). Uptake of L-lactate into HIT-T15 cells was rapid, reaching equilibrium after 5 min with an apparent Km value of 4.8 mM. Transport was markedly inhibited by alpha-cyano-4-hydroxycinnamate, alpha-fluorocinnamate, quercetin and p-chloromercuribenzenesulphonate (pCMBS), and was enhanced in citrate medium. Uptake of D-lactate was less rapid, apparent equilibrium not being reached within 10 min. In contrast to HIT-T15 cells, rat pancreatic islets showed greatly reduced rates of transport of L- and D-lactate together with a correspondingly lower degree of inhibition by alpha-cyano-4-hydroxycinnamate. The addition of L- or D-lactate to HIT-T15 cells, but not dispersed islet cells, resulted in a marked and rapid intracellular acidification followed by a gradual recovery. In both HIT-T15 cells and isolated islets, the rates of transport of both L- and D-lactate in the presence of alpha-cyano-4-hydroxycinnamate were significantly greater in a depolarising K+ medium compared to the normal Na+ medium. These observations suggest that native rat islet cells have considerably reduced activity of the lactate-/H+ transport system compared to HIT-T15 insulinoma cells. There is evidence in both cell types of an additional electrogenic pathway for lactate which might play a role in coupling lactate efflux to beta-cell depolarisation.
Mol Cell Endocrinol 1992 Jul
PMID:Lactate transport in insulin-secreting beta-cells: contrast between rat islets and HIT-T15 insulinoma cells. 132 57

A human insulinoma cDNA library was constructed in expression plasmid vector pUEX1. Clone pUEX1Ins12 was selected from human insulinoma cDNA library by means of hybridization with the insulin probe and a nucleotide sequence of the insertion was determined. It codes for full size amino acid sequence preproinsulin and furthermore, contains the entire 3'-end of noncoding mRNA region and 44 nucleotides from the 5'-untranslated region. The bacterial strain pUEX3Ins8 producing preproinsulin as beta-galactosidase fusion protein was constructed.
Mol Biol (Mosk)
PMID:[Cloning, primary structure determination and expression of preproinsulin cDNA from human insulinoma in Escherichia coli]. 140 15

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and E-cadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. Insulinoma cells express E-cadherin but differ from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed alpha-cell type has converted to an immature phenotype with strong neuronal traits, reflecting the early palce of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits.
Mol Endocrinol 1992 Aug
PMID:Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas. 140 10

An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.
J Mol Endocrinol 1992 Dec
PMID:Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo. 147 9

Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1991 Jul 24
PMID:Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism. 168 21

Cell-specific expression of the insulin gene is dependent on a conserved 8-basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA-binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells.
Mol Endocrinol 1991 Feb
PMID:Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer. 171 33

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.
Mol Cell Biol 1992 Jan
PMID:Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice. 172 14

The pancreatic beta-cell is a major site of islet amyloid polypeptide (IAPP) biosynthesis, and the peptide is coreleased with insulin. We have analyzed the expression of IAPP (mRNA and protein) in various cell types in normal and transformed murine islet cell cultures by Northern blot analyses and immunocytochemistry. IAPP is primarily coexpressed with insulin in the beta-cell of GH-promoted primary rat islet cell cultures. Additionally, a small population of non-beta-cells exhibited a prominent IAPP expression, and double staining experiments showed colocalization with glucagon or somatostatin in some of these cells. IAPP mRNA was confined to the beta-cell phenotype when analyzing the phenotypically stable in vivo tumor lines, MSL-G2-IN (insulinoma) and MSL-G-AN (glucagonoma), and the transgenic mouse islet cell lines, beta-Tc and alpha-Tc. However, IAPP and insulin expression were completely uncoupled in unstable heterogeneous clones such as NHI-6F. This clone is composed of primarily glucagon-producing cells in vitro, but insulin gene expression becomes dominant after passage in vivo. Interestingly, IAPP was hyperexpressed with glucagon under in vitro conditions in this clone. We conclude that the tissue specificity of expressions of IAPP and insulin are controlled differently, and that coexpression of IAPP with hormones different from insulin may be a marker for pluripotent transformed rat islet cell clones, which are able to activate insulin gene transcription during passage in vivo.
Mol Endocrinol 1991 Jan
PMID:Islet amyloid polypeptide and insulin expression are controlled differently in primary and transformed islet cells. 185 Jan 7

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.
Mol Endocrinol 1991 Mar
PMID:Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells. 189 Sep 88


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