Figure 3. MafA-Dependent Nicotinic Receptor Expression Modulates Insulin Secretion.

(A–H) Immunohistochemistry of MafA^WT and MafA^RIP islets to show expression of CHRNA4 (A and B; green), CHRNA5 (C and D; green), CHRNB2 (E and F; green), and CHRNB4 (G and H; green). β cells are stained for insulin (red) and nuclei (DAPI; blue); scale bar represents 20 µm.

(I) qPCR amplification of Chrn upstream sequences after immunoprecipitation of βTC-6 chromatin with a MAFA or rabbit IgG antibody are presented as % input. Differences in percent input for IgG reflect variations in primer efficiencies. n = 4.

(J) Induction of luciferase reporter activity of ChrnB2 (pB2LUC) and ChrnB4 (pB4LUC) luciferase reporter constructs upon co-transfection with MAFA. Empty vector control (pGl2b and pFOX) is set to one; n = 3 or 4.

(K) qPCR measurements of Chrn expression levels in MafA siRNA-treated βTC6 cells; n ≥ 3. Data were normalized to the geomean of HPRT and β-actin mRNA levels.

(L) Dynamic insulin secretion of MafA^WT and MafA^RIP islets stimulated with 10 mM glucose (G) and 100 µM nicotine (NIC), 100 µM nicotine + 100 µM oxotremorine (NIC+OXO), and 100 µM oxotremorine (OXO). The transient decrease in insulin secretion upon NIC treatment in MafA^WT islets is marked by a solid arrow. The biphasic insulin secretion induced by NIC+OXO treatment is indicated by a dashed arrow and a dotted arrow; n = 8.

(M) Dynamic insulin secretion of wild-type islets with 1 or 10 µM acetylcholine (n ≥ 5). Acetylcholine treatment is illustrated by black lines.

Data are mean ± SEM and were analyzed using one-way ANOVA and Tukey multiple comparison tests (J) or paired t test (I and K). *p < 0.05 and **p < 0.01. See Figure S2B for validation of the nicotinic receptor antibodies.