calcimycin and Insulinoma

Both the serine/threonine protein kinase Akt and the voltage-gated L-type calcium channel act as important players in glucose-stimulated insulin secretion. Akt recruits the L-type calcium channel to and maintains them in the plasma membrane. This study aimed to characterize the role of L-type calcium channels in mediation of Akt signaling in glucose-stimulated insulin secretion.. Insulin secretion was evaluated in rat pancreatic islets and INS-1 pancreatic β cells by a standard insulin radioimmunoassay.. Akt inhibition effectively abrogates not only glucose-stimulated but also potassium depolarization-stimulated insulin secretion from rat islets, the latter critically relying on the voltage-gated calcium channel-mediated Ca(2+) influx without involvement of glucose metabolism. Likewise, Akt inhibition also reduces both glucose-stimulated and potassium depolarization-stimulated insulin secretion from INS-1 cells. Importantly, pharmacological ablation of L-type calcium channels partially blocks Akt inhibition-induced reduction in glucose-stimulated insulin secretion but completely prevents that in potassium depolarization-evoked insulin release from INS-1 cells. Furthermore, Akt inhibition does not influence calcium ionophore A23187-induced insulin secretion from INS-1 cells, which occurred without involvement of L-type calcium channels.. Akt signals upstream of L-type calcium channels to optimize glucose-stimulated insulin secretion.

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Ionophores; Cell Line, Tumor; Female; Glucose; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Male; Nifedipine; Potassium Chloride; Protein Subunits; Proto-Oncogene Proteins c-akt; Radioimmunoassay; Rats; Rats, Wistar

Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.

Topics: Animals; Calbindin 1; Calbindins; Calcimycin; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; Gene Expression Regulation; Genes, Reporter; Glucose Transporter Type 1; Insulin; Insulinoma; Ionophores; Islets of Langerhans; Monosaccharide Transport Proteins; Radioimmunoassay; Rats; S100 Calcium Binding Protein G; Transfection; Tumor Cells, Cultured

To elucidate the regulatory pathway through which pancreastatin inhibits insulin secretion, RINm5F insulinoma cells were challenged with physiological and pharmacological probes known to stimulate insulin release through different mechanisms. Utilizing the electrophysiological technique of capacitance measurements as a correlate to exocytosis, pancreastatin was found to significantly diminish maximum capacitance changes evoked by glyceraldehyde, an effect which was attenuated in pertussis toxin-treated cells. In static incubations of this cell line, pancreastatin significantly inhibited insulin secretion stimulated by glyceraldehyde, carbachol and A23187, secretagogues known to directly elevate beta-cell cytosolic Ca2+. This peptide also inhibited insulin secretion stimulated by phorbol myristate acetate (PMA), but only at incubation times < or = 15 min. It was without effect on insulin secretion stimulated by mastoparan and longer incubations (30 min) with PMA, where the secretory mechanisms are not necessarily Ca(2+)-dependent. Additionally, pancreastatin had no effect on carbachol-generated inositol phosphate accumulation but inhibited simultaneously stimulated insulin secretion. All inhibitory effects of pancreastatin were pertussis toxin sensitive. These results suggest that pancreastatin inhibits insulin secretion in RINm5F cells through a G-protein regulated mechanism at a control point involved in the Ca(2+)-directed exocytotic machinery, a feature shared by other physiologic inhibitors of insulin secretion.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Carbachol; Chromogranin A; Exocytosis; Glyceraldehyde; GTP-Binding Proteins; Inositol Phosphates; Insulin; Insulin Secretion; Insulinoma; Intercellular Signaling Peptides and Proteins; Pancreatic Hormones; Pancreatic Neoplasms; Peptides; Pertussis Toxin; Phorbol Esters; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

In order to study the role of arachidonic acid (AA) in depolarization-induced insulin secretion rat insulinoma cells (RINm5F) were depleted of AA by cultivation in essential fatty acid-free medium. Within 2 weeks AA content of these cells was decreased to a non-detectable level as assessed by gas chromatography (GC). Different cell lines were obtained by supplementation of the defatted medium with oleic acid or the AA precursor linoleic acid (7 and 70 microM, each). The AA content varied in dependence from the precursor availability from 0 to about 14% of long chain fatty acids. Variation in AA content or the depletion of AA to a non-detectable level did not modulate insulin synthesis, basal and potassium-induced insulin release, cell growth (cell number and protein), membrane depolarization and increases in cytosolic Ca2+. In AA containing cells no eicosanoids was produced in the course of stimulated hormone release. The data suggest that in RINm5F cells release of AA and/or formation of oxidized metabolites from AA are not essential for functional integrity.

Topics: Alanine; Animals; Arachidonic Acid; Calcimycin; Calcium; Cell Division; Insulin; Insulin Secretion; Insulinoma; Lipoxygenase; Membrane Potentials; Pancreatic Neoplasms; Potassium Chloride; Prostaglandin-Endoperoxide Synthases; Rats; Tumor Cells, Cultured

The cell line In-R1-G9 is one of the clones from the hamster insulinoma cell line, In-111-R1, and it produces glucagon. Phorbol esters markedly enhanced glucagon secretion and the stimulatory effect was found to be correlated to their biological activity as tumor promoters. At a concentration of 200 nM, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated glucagon secretion 13-fold more than the control in 10 min. The effect of TPA was not influenced by actinomycin D, cycloheximide, colchicine or vincristine. Depletion of calcium from the incubation medium inhibited TPA-induced glucagon secretion by approximately 50% and dibucaine also suppressed glucagon secretion to 67.4%. An addition of A23187 to TPA induced 150% enhancement over the TPA-stimulated glucagon level, and the maximum secretory response was observed when the cells were stimulated with the simultaneous addition of TPA, A23187 and theophylline.

Topics: Adenoma, Islet Cell; Animals; Calcimycin; Cell Line; Clone Cells; Cricetinae; Drug Synergism; Glucagon; In Vitro Techniques; Insulinoma; Pancreatic Neoplasms; Phorbols; Tetradecanoylphorbol Acetate; Theophylline

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.

Topics: Adenoma, Islet Cell; Animals; Calcimycin; Calcium; Cell Line; Cell Membrane Permeability; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insulinoma; Kinetics; Mitochondria; Pancreatic Neoplasms; Rats; Subcellular Fractions; Sugar Phosphates

The regulation of extramicrosomal Ca2+ concentration maintained by suspensions of rat insulinoma microsomes was studied using Ca2+-selective minielectrodes. The Ca2+-transporting activity was MgATP dependent and correlated with the endoplasmic reticulum marker NADPH-cytochrome c reductase. When incubated in a high KCl medium containing Mg2+ and phosphate, the microsomes lowered [Ca2+] within less than 10 min to around 0.2 microM. They had a high Ca2+-sequestering activity since they were able to take up and retain several small Ca2+ additions. No evidence for a Na+/Ca2+ countertransport was obtained. The accumulated Ca2+ was released by the Ca2+ ionophore A23187 or upon transforming ATP into ADP using glucose plus hexokinase. The addition of ADP, at concentrations present in cells, resulted in a dose-dependent and reversible net Ca2+ efflux from the microsomes until a higher [Ca2+] steady state was reached. This was specific for ADP since GDP, UDP, CDP, IDP, and the nonhydrolyzable analogue methylene-ADP as well as AMP and cAMP did not reproduce the effect. Insulin secretory granules were unable to lower medium [Ca2+] or to take up a pulse addition of Ca2+. However, most of the large granular calcium content was released by A23187. The addition of Na+ and lowering or increasing medium pH by 0.2 pH unit did not induce Ca2+ uptake or efflux from the secretory granules. The results indicate that insulinoma endoplasmic reticulum but not insulin secretory granules may play a critical role in the regulation of cytosolic Ca2+. A variation in cellular ADP content following secretagogue addition might modulate Ca2+ fluxes across the endoplasmic reticulum and contribute in raising cytosolic Ca2+.

Topics: Adenoma, Islet Cell; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Fractionation; Cell Line; Cytoplasmic Granules; Endoplasmic Reticulum; Insulin; Insulin Secretion; Insulinoma; Kinetics; Microsomes, Liver; Organoids; Pancreatic Neoplasms; Rats; Rats, Inbred Strains