FIGURE 4.

siRNA-mediated knockdown of NRXN1 in INS-1E cells increases glucose-stimulated insulin secretion. INS-1E cells were transfected for 72 h with a pool of either non-targeting control or NRXN1 siRNAs. A, mRNA was isolated, and NRXN1α gene expression was determined using RT-qPCR, normalized to 18 S RNA. B, equal protein amounts of total cell lysates were immunoblotted for NRXN to confirm knockdown at the protein level. Lysates from two independent culture wells are shown for both control and NRXN1 siRNA. N.S. indicates a nonspecific band detected by the pan- NRXN antibody. C, to determine the effect of NRXN1 knockdown on insulin secretion, cells treated with control or NRXN1 siRNA were incubated for 1 h in Krebs-Ringer solution containing either 2.75 mm (black bars) or 16.7 mm glucose (white bars) and secreted insulin, shown as % of total cellular insulin-measured by RIA. D, glucose stimulation index (the ratio of insulin secretion, as % of cellular insulin, at 16.7 mm to secretion at 2.75 mm glucose) is shown for cells treated with control (left) and NRXN1 (right) siRNA. E, total lysate from siRNA-treated cells was assayed for insulin content. F, insulin 2 gene expression in siRNA-treated cells was determined by qPCR and normalized to 18 S RNA. G, INS-1E cells were transfected with a secreted alkaline phosphatase (SEAP) construct in addition to control or NRXN1 siRNA and treated with 15 mm glucose as in C. Media and cell lysates were assayed for SEAP content. The percent of total SEAP secreted is depicted. All data are represented as mean ± S.E. from six samples, and each experiment was repeated three times with similar results. Insulin secretion experiments were repeated an additional three times with similar results using a completely different pool of control and NRXN1 siRNAs. Statistical significance was determined using a Student's t test. *, p < 0.05 indicates the difference compared with non-targeting siRNA transfected controls.