Insulinoma-derived Cells Research Articles

TMEM219 regulates the transcription factor expression and proliferation of beta cells.

Pancreatic beta cells replenishment is considered the next therapeutic option for type 1 diabetes; while stimulating endogenous beta cells proliferation is the "holy grail" for those patients with exhausted beta cell mass. Here we are demonstrating that the pro-apoptotic receptor TMEM219 is expressed in fetal pancreas, in beta cell precursors and in in vitro embryonic-derived endocrine progenitors. TMEM219 signaling negatively regulates beta cells at early stages and induces Caspase 8-mediated cell death. Pharmacological blockade of TMEM219 further rescued beta cell precursor and proliferation markers, and decreased cell death, both in islets and in in vitro-derived endocrine progenitors, allowing for beta cell preservation. While addressing the upstream controlling TMEM219 expression, we determined the TMEM219 miRNet; indeed, one of those miRNAs, miR-129-2, is highly expressed in human islets, particularly in patients at risk or with established type 1 diabetes. miR-129-2 mimic downregulated TMEM219 expression in islets, in in vitro embryonic-derived endocrine progenitors and in highly proliferating insulinoma-derived cells. Moreover, miR-129-2 inhibitor induced a TMEM219 overexpression in insulinoma-derived cells, which restored cell proliferation and functional markers, thus acting as endogenous regulator of TMEM219 expression. The TMEM219 upstream regulator miR129-2 controls the fate of beta cell precursors and may unleash their regenerative potentials to replenish beta cells in type 1 diabetes.

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved Förster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved Förster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Tumor-specific promoter-driven adenoviral therapy for insulinoma.

Insulinomas are the most common type of neuroendocrine (NE) pancreatic islet tumors. Patients with insulinomas may develop complications associated with hyperinsulinemia. To increase the treatment options for insulinoma patients, we have tested a conditionally replicating adenovirus that has been engineered in such a way that it can specifically express therapeutic genes in NE tumors. We used a promoter-specific adenoviral vector delivery system that is regulated by an INSM1 (insulinoma-associated-1) promoter, which is silent in normal adult tissues but active in developing NE cells and tumors. Through a series of modifications, using an insulator (HS4) and neuron-restrictive silencer elements (NRSEs), an oncolytic adenoviral vector was generated that retains tumor specificity and drives the expression of a mutated adenovirus E1A gene (Δ24E1A) and the herpes simplex virus thymidine kinase (HSV-tk) gene. The efficacy of this vector was tested in insulinoma-derived MIN, RIN, βTC-1 and pancreatic (Panc-1) cells using in vitro cell survival and in vivo tumor growth assays. Using in vitro insulinoma-derived cell lines and an invivo subcutaneous mouse tumor model we found that the INSM1 promoter-driven viruses were able to replicate specifically in INSM1-positive cells. INSM1-specific HSV-tk expression in combination with ganciclovir treatment resulted in dose-dependent tumor cell killing, leaving INSM1-negative cells unharmed. When we combined the INSM1-promoter driven HSV-tk with Δ24E1A and INSM1p-HSV-tk (K5) viruses, we found that the co-infected insulinoma-derived cells expressed higher levels of HSV-tk and exhibited more efficient tumor suppression than cells infected with INSM1p-HSV-tk virus alone. INSM1 promoter-driven conditionally replicating adenoviruses may serve as a new tool for the treatment of insulinoma and may provide clinicians with additional options to combat this disease.

Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line

We report a transient modulation of cell fate in fibroblasts briefly exposed to an extract derived from the rat insulin-producing β cell line, INS-1E. Primary fetal rat fibroblasts were reversibly permeabilized with Streptolysin O, incubated for 1 h in a 15,000 g INS-1E nuclear and cytoplasmic extract, resealed, and cultured. A first marker of change in cell fate was a reduction of cell and nuclear size within days of exposure to extract such that in some instances the fibroblasts resembled INS-1E cells. Second, two β cell transcripts, Pdx-1 and insulin, were detected in the fibroblasts for up to 4 weeks. Third, (pro)insulin labeling was detected in 5–30% of the cells for a period of 8–14 days after incubation in extract. These phenotypes were absent from fibroblasts exposed to heat-treated INS-1E extracts, a human fibroblast cell line-derived extract or buffer. The results indicate that the extract of an insulinoma-derived cell line can promote at least a transient modification of cell fate towards a β cell phenotype in non-β cells. Because they are easily accessible, cell extracts may represent a practical source of material for investigating the mechanisms of alteration of a nuclear and cellular program.

1,5-Anhydroglucitol stimulates insulin release in insulinoma cell lines

Concentrations of 1,5-anhydroglucitol (1,5-AG), which is a major circulating polyol, decrease in patients with diabetes mellitus. In both insulinoma-derived RINr and MIN6 cells, 1,5-AG stimulated insulin release within the range of 0.03-0.61 mM in a dose-dependent manner. Insulin release was maximally stimulated by 1,5-AG to levels that reached 25% and 100% greater than that of control (1,5-AG-free group) in RINr and MIN6 cells, respectively. A physiological concentration of 1,5-AG stimulated insulin release after a 5-min incubation and this action was maintained for 60 min. In addition, at approximately 1/200 the concentration of glucose, 1,5-AG had additive action with 20 mM glucose. The action of 1,5-AG on insulin secretion with other types of saccharides and polyol was similarly additive. Mannnoheptulose and diazoxide suppressed the stimulative action of 1,5-AG on insulin release. The secretagogue action of 1,5-AG seemed to be independent on an increase in the intracellular content of cAMP and ATP. These results suggest that 1,5-AG can stimulate insulin secretion through a mechanism that completely differs from that of glucose.

Establishment of a tet-on gene expression system in glucose-responsive and -unresponsive MIN6 cells.

The tetracycline (or doxycycline)-inducible gene expression (Tet-On) system developed by Gossen and Bujard (1) is useful for controlling the expression of targeted genes and for determining the roles of the gene products in cellular functions. The Tet-On system has recently been applied to the rat insulinoma cell line INS-1 to study the roles of wild-type and mutant genes in insulin secretion (2,3). We have recently established two cell lines, MIN6-m9, and MIN6-m14, derived from the mouse insulinoma-derived cell line MIN6. MIN6-m9 and MIN6-m14 cells are glucose-responsive and glucose-unresponsive, respectively. To study the roles of newly identified genes in pancreatic � -cell functions, we have established a Tet-On system in these MIN6 sublines.

Transport of 1,5-anhydro- D-glucitol into insulinoma cells by a glucose-sensitive transport system

The uptake of 1,5-anhydro- D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic β-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K m of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K m of 131 in RINr cells and 211 mM in MIN6 cells. Uptake of 1,5-AG was markedly inhibited by phloretin and cytochalasin B, but was only slightly affected by phloridzin. Uptake of 1,5-AG was markedly inhibited by glucose at physiological concentrations (1.0–10 mM), as well as by galactose and mannose. The 1,5-AG concentration required to inhibit 2-deoxyglucose uptake exceeded that of glucose by >100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic β-cells.

CCK receptor subtype in insulin-producing cells: a combined functional and in situ hybridization study in rat islets and a rat insulinoma cell line

Cholecystokinin (CCK) stimulates insulin secretion. It is, however, not established whether CCK receptors are expressed in insulin-producing cells. We therefore investigated, by in situ hybridization, whether CCK-A or CCK-B receptor mRNA could be detected in normal rat pancreatic islets and in the rat insulinoma cell line, RINm5F. The CCK-A, but not the CCK-B, receptor transcript was detected in both islets and RINm5F cells. Islet CCK-A receptors were mostly confined to the center of the islets corresponding to the distribution of the B cells. In RINm5F cells, insulin release was not significantly affected by cholecystokinin (CCK)-8-S (10 −13 to 10 −7 M), which is in contrast to the insulinotropic effect of CCK-8-S in normal rat islets. Similarly, in FURA-2AM-loaded cells, CCK-8-S (10 −11 to 10 −7 M) was without effect on the intracellular Ca 2+ concentration ([Ca 2+]ic) in RINm5F cells, whereas CCK-8-S (10 −7 M) markedly increased [Ca 2+]ic (by 366±2 nM ( P<0.001) in normal rat islet cells. We conclude that the CCK-A, but not the CCK-B, receptor subtype is expressed in both normal rat islets and in the rat insulinoma-derived cell line RINm5F. There is, however, a functional difference between normal islets and the RINm5F cells with respect to effects of CCK-8-S on insulin release and [Ca 2+]ic.

INTERLEUKIN 1β-MEDIATED INHIBITION OF ARGINASE IN RINm5F CELLS

Induction of nitric oxide synthase and generation of nitric oxide in pancreatic islet β-cells may mediate cytokine-induced dysfunction leading to insulin-dependent diabetes mellitus. Nitric oxide generation can be regulated by availability of arginine substrate which, in turn, may be affected by substrate utilization in competing pathways such as the arginase-catalysed formation of ornithine and urea. In this study we have investigated the activity of arginase in the rat insulinoma-derived cell line RINm5F and the effect on this of interleukin 1β, the nitric oxide synthase reaction intermediate NG-hydroxy-l-arginine and the nitric oxide-generating compounds 3-morpholinosydnonimine and S-nitrosoglutathione. Cytosols from RINm5F cells treated with or without interleukin 1β (0.1nM, 18h) were incubated (45min, 37°C) with [U-14C]arginine. Radiolabelled products ([14C]citrulline from nitric oxide synthase, [14C]ornithine and [14C]urea from arginase) were separated by high-performance liquid chromatography or ion-exchange chromatography. Interleukin 1β increased citrulline production (from 0.01±0.002 to 0.58±0.03 pmol/μg cell protein), indicating induction of nitric oxide synthase, and significantly decreased production of both ornithine (from 4.60±0.20 to 3.40±0.20 pmol/μg) and urea (0.93±0.05 to 0.69±0.04 pmol/μg) (P<0.001), indicating decreased activity of arginase. Arginase was significantly inhibited by NG-hydroxy-l-arginine (IC50=50 μM), S-nitrosoglutathione (500 μM: 69±7% of control) and 3-morpholinosydnonimine (1 mM: 57±7% of control) (P<0.05). We conclude that during cytokine-directed β-cell assault nitric oxide synthase-catalysed production of NG-hydroxy-l-arginine and nitric oxide may inhibit arginase thereby increasing the availability of arginine for nitric oxide production.

Involvement of MAP kinase and c-fos signaling in the inhibition of cell growth by somatostatin.

Somatostatin significantly suppressed cell growth of the mouse insulinoma-derived cell line MIN6. MIN6 cells exhibited high-affinity binding of somatostatin with 50% inhibitory concentration value of 0.9 nM. RNA blot analysis revealed that MIN6 cells expressed only SSTR3 among the five somatostatin receptors so far identified. Treatment of MIN6 cells with somatostatin significantly reduced the serum-induced c-fos expression levels. On the other hand, somatostatin (100 nM) treatment of MIN6 cells cultured in medium containing 10% serum transiently increased c-fos expression levels to 282 +/- 4.7% and then significantly decreased them to 27 +/- 7.6% of the levels before treatment. Mitogen-activated protein (MAP) kinase activity transiently increased to 656 +/- 91.2% and decreased thereafter to 39 +/- 13.3% of the activity before the addition of somatostatin (100 nM) into the medium. In addition, the stimulatory effect of somatostatin on c-fos expression and MAP kinase activity (early effect) was not altered by pertussis toxin (PTX), whereas the suppressive effect of somatostatin on c-fos expression and MAP kinase activity (late effect) was mitigated by PTX. These findings suggest that an inhibition of c-fos expression mediated by cross talk between PTX-sensitive G protein signaling and receptor tyrosine kinase signaling is one of the mechanisms by which somatostatin inhibits cell growth in MIN6 cells.

The Role of Prohormone Convertases Pc1 (PC3) and PC2 in the Cell-Specific Processing of Proglucagon

To elucidate the mechanism of the differential processing of proglucagon, we analyzed the processing products of proglucagon in three types of rodent endocrine cells and their relation to prohormone convertases PC1 (PC3) and PC2. Proglucagon gene was transfected into AtT-20 cells and GH3 cells, which are derived from pituitary tumors. InR1-G9 cells, which are insulinoma-derived cells, express an endogenous proglucagon gene. Oxyntomodulin was the predominant processing product in AtT-20 cells, which contained abundant PC1 mRNA. In contrast, glucagon was the major product in GH3 cells, which expressed PC2 mRNA. Oxyntomodulin and glucagon were produced in equal amounts in InR1-G9 cells, which expressed both PC1 and PC2 mRNAs. These findings suggest that PC1 and PC2 preferentially cleave proglucagon into oxyntomodulin and glucagon, respectively, thus contributing to the cell-specific processing of proglucagon.

Insulin gene transcription is decreased rapidly by lowering glucose concentrations in rat islet cells.

Glucose is the main physiological regulator of insulin biosynthesis and secretion. To exert an effect on biosynthesis, stimulatory concentrations of glucose act on insulin gene expression at both transcriptional and posttranscriptional levels. The relative importance of these two effects is controversial. Studies on the effect of decreasing glucose concentrations in vitro have been contradictory as to its rapidity and magnitude. We have investigated whether a decrease in glucose concentrations from a stimulatory (11 mM) to a nonstimulatory (2 mM) level regulates insulin gene expression in both isolated rat islets and INS-1 cells, an insulinoma-derived cell line that retains a number of the differentiation characteristics of native beta-cells. We show that, in the presence of a nonstimulatory concentration of glucose, insulin mRNA levels decrease rapidly in both rat islets and INS-1 cells. Low glucose decreases insulin gene expression at the transcriptional level, and this effect is mediated by DNA sequences present within 254 base pairs of the 5'-flank of the rat insulin I gene. We conclude that lowering glucose from 11 to 2 mM decreases insulin mRNA rapidly by a transcriptional effect.

Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells.

Exendin-4 purified from Heloderma suspectum venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)-amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced 125I-GLP-1, and unlabeled GLP-1 displaced 125I-exendin-4 from the binding site at rat insulinoma-derived RINm5F cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular cAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on cAMP. They each competed with the radio-labeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin secretion in isolated rat islets, and, in mouse insulinoma beta TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin-(9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP-1 receptor antagonist.

Expression of the ras-related rab3a gene in insulinoma-derived cell lines

This study was designed to search for the expression of the small-molecular-weight GTP-binding protein rab3a in endocrine pancreatic cell lines. Total RNA was isolated from five different cell lines (RINm5F, RIN 104836, β-TC1, HIT-15, and INRI-G9) and from whole rat brain. The expression of rab3a was analyzed by Northern blots. Similar as in brain two transcripts of 1300 and 1800 bp were detected in RIN-cells at low stringency conditions with the predominant signal at 1300 bp. At high stringency the stronger signal was at 1800 bp. When a 300 bp PstI fragment derived from the coding region of rab3a was utilized as probe the 1800 bp signal was predominant under each condition. Only a faint band at 1800 bp occurred in preparations from βTCI-cells and no signal at all was found in HIT-15 and INRI-G9-cells. In conclusion, rab3a is expressed in rat insulin-releasing insulinoma-derived RIN-cells with a specific 1800 bp transcription product.

Dexamethasone pretreatment of rat insulinoma cells decreases binding of glucagon-like peptide-1(7–36)amide

The effect of dexamethasone on binding of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) to rat insulinoma-derived cells (RINm5F) was investigated. Preincubation of RINm5F cells with dexamethasone (100 nmol/l) for 24 h resulted in a decrease of GLP-1(7-36)amide binding to 55.0 +/- 8.16% (mean +/- S.E.M.), incubation for 48 h to 39.1 +/- 1.76%, and for 72 h to 15.5 +/- 4.35% of maximal binding. The GLP-1(7-36)amide-induced stimulation of cyclic AMP (cAMP) production was significantly decreased to 61.03 +/- 7.4% of maximum production in cells pretreated with dexamethasone (100 nmol/l) for 48 h. The decreased binding was due to a reduction of the receptor number while the receptor affinity remained unchanged. These inhibitory effects on binding and cAMP formation induced by dexamethasone were completely abolished when the antiglucocorticoid RU 38486 (100 nmol/l) was added during preincubation with dexamethasone. RU 38486 alone had no effects. Our data suggest that the biological action of GLP-1(7-36)amide at the B-cell may be modified by glucocorticoids.

Glucagon-like peptide-1(7–36)amide: characterization of the domain responsible for binding to its receptor on rat insulinoma RINm5F cells

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) is a potent stimulator of insulin secretion. Receptors for this hormone have been found on different insulinoma-derived cell lines, e.g. the RINm5F cell line which is derived from a radiation-induced rat insulinoma. To characterize the part of the GLP-1(7-36)amide molecule that is responsible for binding to its receptor on RINm5F cells, binding studies with synthetic C-terminal (GLP-1(21-36)amide) and synthetic N-terminal (GLP-1(7-25] GLP-1 fragments were carried out. GLP-1(21-36)amide showed dose-dependent binding to the GLP-1(7-36)amide receptor but was approximately 1500 times less potent in inhibiting binding of 125I-labelled GLP-1(7-36)amide than the intact hormone. GLP-1(7-25) at concentrations up to 10 mumol/l did not inhibit binding of label. Neither fragment changed intracellular cyclic AMP concentrations, in contrast to GLP-1(7-36)amide which increased intracellular cyclic AMP. GLP-1(21-36)amide, however, acted as a weak partial antagonist of GLP-1(7-36)amide with respect to GLP-1(7-36)amide-dependent stimulation of cyclic AMP production.

Selective Expression of the Insulin I Gene in Rat Insulinoma-Derived Cell Lines

Four rat insulinoma-derived cell lines were found to express only the rat insulin I gene, although an apparently normal insulin II gene was present in each cell line. This finding was reflected by the absence of insulin II and the presence of insulin I gene transcripts in the products of run-on transcription assays, the absence of insulin II and the presence of insulin I unprocessed (pre-) mRNA, and the absence of mature insulin II and the presence of insulin I mRNA. Analysis of insulin II genes in cell lines by gene amplification indicated that the lack of insulin II gene transcription is not explained by the absence of or gross alterations in the insulin II genes per se. These studies indicate that the two nonallelic insulin genes do not function equivalently in pancreas-derived cells.

Characterization of the receptor for glucagon-like peptide-1(7–36)amide on plasma membranes from rat insulinoma-derived cells by covalent cross-linking

125I-Labelled glucagon-like peptide-1(7-36)amide was cross-linked to a specific binding protein in plasma membranes prepared from RINm5F rat insulinoma-derived cells using disuccinimidyl suberate. Consistent with the presence of a single class of binding site on the surface of intact cells, only a single radiolabelled band at Mr63,000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex. The band was not observed when 10nM glucagon-like peptide-1(7-36)amide was included in the binding assay, but 1 microM concentrations of glucagon-like peptide-1(1-36)amide, glucagon-like peptide-2 and glucagon did not decrease the intensity of labelling. No change in the mobility of the band was observed under reducing conditions, suggesting that the binding protein in the receptor is not attached to other subunits via disulphide bonds. In control incubations using plasma membranes from pig intestinal epithelial cells, which do not contain specific binding sites for glucagon-like peptide-1(7-36)amide, no cross-linked ligand-binding protein complex was observed.

Receptors for glucagon-like peptide-1(7-36) amide on rat insulinoma-derived cells.

Specific binding of 125I-labelled glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) to rat insulinoma-derived RINm5F cells was dependent upon time and temperature and was proportional to cell concentration. Binding of radioactivity was inhibited in a concentration-dependent manner by GLP-1(7-36) amide consistent with the presence of a single class of binding site with a dissociation constant (Kd) of 204 +/- 8 pmol/l (mean +/- S.E.M.). Binding of the peptide resulted in a dose-dependent increase in cyclic AMP concentrations (half maximal response at 250 +/- 20 pmol/l). GLP-1(1-36)amide was approximately 200 times less potent than GLP-1(7-36)amide in inhibiting the binding of 125I-labelled GLP-1(7-36)amide to the cells (Kd of 45 +/- 6 nmol/l). Binding sites for GLP-1 (7-36)amide were not present on dispersed enterocytes from porcine small intestine.