Journal: Experimental Diabetes Research

Article Title: Suppression of the Nuclear Factor Eny2 Increases Insulin Secretion in Poorly Functioning INS-1E Insulinoma Cells

doi: 10.1155/2012/460869

Figure Lengend Snippet: Insulin secretion in response to exendin-4 and forskolin after Eny2 knockdown: levels of cAMP. (a, b) INS-1E cells were stimulated with 50 nmol/l exendin-4 72 hours after transfection with either control or two different Eny2-specific siRNAs. Insulin secretion in response to exendin-4 was augmented by the two specific siRNAs, both in absolute (a) and relative terms (b) ( n = 5). (c) Insulin secretion after stimulation with 50 nmol/l exendin-4 and 10 μ mol/l forskolin, which led to comparable secretory responses. (d) INS-1E cells were stimulated with different concentrations of exendin-4 for 5 minutes. The concentration of cellular cAMP was measured by a competitive ELISA. (e, f) Time course of cellular cAMP levels after stimulation with 50 nmol/l exendin-4 in absence (e) and presence (f) of 100 μ mol/l IBMX. In both setups the knockdown of Eny2 did not affect cellular cAMP levels. * P < 0,05, ** P < 0,01, *** P < 0,001; one way ANOVA with posttests and unpaired t -test.

Article Snippet: For measurement of cAMP formation, INS-1E cells were treated with siRNA, seeded in 24-well plates and cultured for 72–96 h. Cells were then washed twice with KRBH buffer, preincubated with the same buffer at 37°C for 30 min, washed once again followed by incubation with 0.5 mL KRBH buffer containing 16.7 mM glucose with or without different concentrations of exendin-4 and 100 μ M IBMX (3-isobutyl-1-methylxanthine (Sigma)). cAMP was extracted after 5–15 minutes by adding 0.1 mol/l HCl/0.5% Triton X-100 to the cells.

Techniques: Transfection, Concentration Assay, Competitive ELISA