Figure 8.

Knockdown of neuroligin-2 (NL-2) in dissociated rat islets inhibits insulin secretion. Dissociated rat islets were transfected with a pool of control (Cont), nontargeting siRNAs, or a pool of siRNAs targeted to neuroligin-2, cultured for 48 h, and then conditioned for 1 h in a solution containing 2.75 mm glucose. After conditioning, secreted insulin was measured by RIA after treatment with high (25 mm) glucose for 1 h. A, RNA was isolated from half of the samples, reverse transcribed to cDNA, and knockdown was analyzed by real-time qPCR analysis. Neuroligin-2 was decreased by 69% in dispersed islet cells treated with neuroligin-2 siRNAs relative to those treated with control siRNAs (n = 6; P = 0.0001). B, Protein was isolated from the other half of the samples, and total cellular insulin content was measured in the cell lysate. No significant difference (N.S.) in insulin content was detected between cells treated with control siRNAs and neuroligin-2 siRNAs. An equal amount of lysis buffer was used to prepare each sample. C, Under high glucose conditions (25 mm), a 43% decrease in secreted insulin was observed in neuroligin-2 siRNA treated dispersed islet cells relative to control siRNA treated cells (n = 12; P = 0.0007). At 2.75 mm glucose, there was no significant change in insulin secretion between cells treated with control siRNA and those treated with neuroligin-2 siRNA (not shown).