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Insulinomas originate from pancreatic β cells and it is the most widely known tumor. Indomethacin is a nonsteroidal anti-inflammatory drug, which is used for blocking the production of some natural substances that cause inflammation and decrease pain. In this study, I aimed to investigate the effects of indomethacin on rat insulinoma INS-1 cells. The relationship between cell death and insulin metabolism was determined with the administration of indomethacin. The cell viability by WST-1; the apoptosis and necrosis levels by ELISA kits; malondialdehyde levels by spectrophotometer; and beclin, intracellular insulin, insulin secretion, KCa3.1, insulin receptor (IR), glucose transporter type 2 (GLUT2), activating transcription factor 2 (ATF2), Elk1, c-Jun, Akt and phosphorylated ATF2, Elk1, c-Jun, Akt, intracellular betacellulin and betacellulin secretion levels by Western blot analysis investigated. The Ins1, Ins2, IR, GLUT2, ATF2, Elk1, c-Jun, Akt, and Betacellulin gene expression levels were determined by the real-time quantitative reverse transcription-polymerase chain reaction method. Apoptotic cell death was observed with the administration of indomethacin. The insulin secretion and Ins1, Ins2 gene expression levels decreased. The insulin receptor and GLUT2 levels increased, while KCa3.1 (KCNN4) levels decreased with the administration of indomethacin to insulinoma INS-1 cells. A decrease was observed in the total c-Jun, phosphorylated ATF2, Elk1, c-Jun, and Akt levels. Betacellulin secretion levels increased. In insulinoma INS-1 cells, apoptotic cell death occurred in the following manner: (i) indomethacin might decrease insulin secretion by reducing KCa3.1, (ii) might inactivate the JNK/ERK pathway with the inactivity of transcription factors.

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. Biotechnol. Bioeng. 2013; 110: 619–627. © 2012 Wiley Periodicals, Inc.

Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin II. Our laboratory has previously shown that pancreatic ACE2 levels decrease as diabetes progresses and that pancreatic ACE2 gene therapy counteracts development of hyperglycemia in diabetic animal models. As a disintegrin and metalloprotease 17 (ADAM17) is an enzyme that can cleave enzymatically active ACE2 from the cell to the extracellular environment by so-called shedding, we hypothesize that increased levels of ADAM17 in pancreatic beta-cells lead to reduced cellular ACE2 levels. To this end, 832/13 cells (rat insulinoma cells derived from pancreatic beta-cells) were transfected with expression plasmids for ACE2, ADAM17, and a hydrolytically inactive E406A ADAM17 mutant. Using fluorogenic enzyme substrates, we measured shed ACE2 activity after a 24 h incubation as well as cell-associated ACE2 and ADAM17 activities. For a wide range of ACE2 expression levels, including the ACE2 expression level observed in pancreatic islets, a 4.6-fold incr...

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.

Although transforming growth factor-beta (TGF-β) stimulates pancreatic islet cells to synthesize and secret insulin, the mechanism underlying this effect is not known. To investigate this question, we examined the insulin promoter activity focusing on a transcription factor, pancreatic and duodenal homeobox gene-1 (PDX-1) that binds to the A3 element of the rat insulin promoter. Studies performed using the rat insulinoma cell line, INS-1 showed that TGF-β stimulation of endogenous insulin mRNA expression correlated with increased activity of a reporter construct containing the insulin promoter. A potential mechanism for this increase arose from, electrophoretic mobility shift assay showing that the nuclear extract from TGF-β treated cells contained higher levels of A3 binding activity. Western blot analysis confirmed that PDX-1 was increased in the nuclear extract from INS-1 cells treated with TGF-β. As expected, a mutant insulin promoter that lacked the PDX-1 binding site was not stimulated by TGF-β. In summary, the results of these studies show that TGF-β stimulates the transcription of insulin gene and this action is mediated by the transcription factor, PDX-1.

Pax4 is a paired-box transcription factor that plays an important role in the development of pancreatic β-cells. Two Pax4 cDNAs were isolated from a rat insulinoma library. One contained the full-length sequence of Pax4. The other, termed Pax4c, was identical to Pax4 but lacked the sequences encoding 117 amino acids at the COOH-terminus. Pax4 was found to inhibit the human insulin promoter through a sequence element, the C2 box, located at −253 to −244, and the islet amyloid polypeptide promoter through a sequence element located downstream of −138. The inhibitory activity of Pax4 was mapped to separate regions of the protein between amino acids 2–230 and 231–349.

We evaluated expression of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor (GluR) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting in nine established cell lines: rat CG-4 (oligodendroglial lineage) and RINm5F insulinoma cells; human CHP134, SMS-KCNR, SKNSH, and Nb69 neuroblastoma cells; and human D384Med, D425Med, and D458Med medulloblastoma cells. CG-4 expressed mRNAs encoding GluR2–7, KA-1, and KA-2 non-NMDA GluR (Yoshioka et al.: J Neurochem 64:2442–2448, 1995) and NR1 (NMDAR1) and NR2D NMDA GluR. After differentiation to oligodendrocyte-like cells, CG-4 also expressed NR2B mRNA. Rat insulinoma cells expressed GluR5, and KA-2 non-NMDA and NR1 and NR2D NMDA GluR mRNAs. The four human neuroblastoma lines all expressed mRNAs encoding GluR2–4, 6, 7 and KA-1 non-NMDA and NR1 NMDA GluR, and the three human medulloblastoma cell lines all expressed mRNAs encoding GluR1, 6 and KA-1, but none of the NMDA GluRs. Whereas CG-4 is susceptible to kainate excitotoxicity, treatment of insulinoma, neuroblastoma, and medulloblastoma lines with L-glutamate, kainate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), or NMDA failed to cause cell damage or to augment 45Ca2+ influx. Thus, despite expressing a variety of non-NMDA and NMDA GluR genes, the human neuroblastoma and medulloblastoma and rat insulinoma lines failed to assemble Ca2+-permeable NMDA or non-NMDA GluR channels. This failure confers protection against excitotoxicity and may contribute to progression of tumors of these types. © 1996 Wiley-Liss, Inc.

The action exerted on non-L-, non-N-type (Q-like) Ca2+ channels by immunoglobulins G (IgGs) obtained from two patients with Lambert-Eaton myasthenic syndrome (LEMS) was investigated in the rat insulinoma RINm5F cell line. LEMS IgGs reduced by 30–36% the whole-cell Ba2+ currents through Q-like Ca2+ channels at +10 mV without significantly modifying their voltage dependence and activation kinetics. Single- and multiple-channel recordings in cell-attached and outside-out patches of cells treated with LEMS IgGs showed no significant changes of the channel elementary properties but rather a decreased number of active channels per patch. This suggests that Q-like current depression by LEMS autoantibodies is mostly due to a down-regulation of functioning Ca 2+ channels. In agreement with previous observations, LEMS IgGs also reduced by 20–33% the dihydropyridine-sensitive (L-type) Bat+ current. The suggested down-regulation of Q-like channels by LEMS IgGs in RINm5F cells may have a functional correlation with the depressive action of LEMS autoantibodies on the PIQ-type Ca2+ channels controlling acetylcholine release from mammalian neuromuscular junctions.

Insulin gene expression in rat insulinoma (RIN) cells is extinct in RIN × fibroblast hybrids and can reappear upon loss of DNA contributed by the fibroblast parent. (Besnard et al., Exp. Cell Res. 185:101–108, 1989). In the present study, we looked for the role of 5′-flanking sequences of the human insulin gene in the negative control observed in the hybrids. RIN cells were transformed with composite genes which consisted of the coding sequence of the gpt gene placed under the control of 5′-flanking regions of the human insulin gene (Ins. gpt gene). Upon hybridization of these cells with mouse fibroblasts, the expression of both Ins. gpt and endogenous rat insulin genes were suppressed together. The results obtained indicate that cis-acting DNA elements are involved in the negative control of the gene. These elements are located in a fragment spread from -258 to +241 of the transcription origin of the human insulin gene.

In the present study electrical field-induced fusion has been applied to both normal pancreatic islet cells isolated from obese hyperglycemie mice and clonal insulin-producing cells (RINmSF) derived from a transplantable rat insulinoma. The fused cells were then punctured with microelectrodes to measure changes in membrane potential after exposure to stimulatory concentrations of D-glucose or K+. Fused cells of normal islet cellular origin revealed a resting membrane potential of −60 mV and were depolarized by 24 or 27 mV after exposure to 11 mM D-glucose or 30 mM K+. Although D-glucose induced depolarization, it was not possible to establish the existence of an oscillating burst pattern superimposed by action potentials. The resting membrane potential of the fused RINm5F cells was also −60 mV and decreased to −30 mV after exposure to 30 mM K+. As judged from the membrane potential measurements, the reconstitution of the plasma membrane subsequent to electrical breakdown is essentially the same whether the giant cells originated from normal islet cells or RINm5F cells.