Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.

Abstract

New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.

Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

“…Cells INS-1 and INS-1E cells were a kind gift from C. Wollheim, Departments of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland [20] and were maintained in RPMI-1640 culture medium with glutamax supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Invitrogen/Gibco, Taastrup, Denmark), and 50 μmol/l β-mercaptoethanol (Sigma, St Louis, MO, USA). Cells were cultured under standard cell culture conditions at 37°C in a humidified atmosphere containing 5% CO 2 .…”

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets

“…The clonal beta cell line INS-1E [34], derived from parental INS-1 cells [35], was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 5% fetal calf serum. Adherent cultured INS-1E cells were infected with recombinant adenovirus for 90 min at 37°C, washed once, and further cultured in complete RPMI-1640 medium for 18 to 20 h before experiments.…”

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity

“…INS832/13 cells [42] (passages were grown in monolayer cultures as described previously [43]. This was done in a humidified (5% CO 2 , 95% air) atmosphere at 37°C in regular RPMI 1640 medium supplemented with 10 mmol/l HEPES, 10% FCS, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 50 µmol/l β-mercaptoethanol (referred to below as complete medium).…”