Suppression of the Nuclear Factor Eny2 Increases Insulin Secretion in Poorly Functioning INS-1E Insulinoma Cells
Figure 3
Insulin content, cellular proliferation, gene expression, glucose metabolism, and response to tolbutamide after Eny2 knockdown. (a) Cellular insulin content was measured 72 hours after transfection with control and Eny2-specific siRNA, respectively. Insulin content was comparable after both treatments (). (b) INS-1E cells were seeded at low density and in equal numbers per well and were transfected with either control or Eny2-specific siRNA. After 3 days cellular viability was quantified using MTT. No difference between the two treatments was observed indicating unchanged cellular proliferation (). (c) The expression of several key components of beta cell glucose sensing as well as of the transcription factor pdx1 and insulin itself were measured by semiquantitative rtPCR. GAPDH, cyclophilin, and beta actin were used for normalization. No difference in the expression level of any of the genes was seen. (d) Glucose-dependent cellular metabolism was examined by an MTT assay (). The increase of metabolism with rising glucose concentrations was augmented after Eny2 knockdown. (e) We also tested insulin secretion in response to the sulfonylurea secretagogue tolbutamide. Tolbutamide had a minimal effect in control cells but clearly augmented insulin secretion after Eny2 suppression. *, *** unpaired -test.
