MafA-Controlled Nicotinic Receptor Expression Is Essential for Insulin Secretion and Is Impaired in Patients with Type 2 Diabetes

Abstract

Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet β cells and their impact on diabetes development are only partially understood. Using a glucose-intolerant, MafA-deficient mouse model, we demonstrate that MAFA controls ANS-mediated insulin secretion by activating the transcription of nicotinic (ChrnB2 and ChrnB4) and adrenergic (Adra2A) receptor genes, which are integral parts of acetylcholine- and monoamine-signaling pathways. We show that acetylcholine-mediated insulin secretion requires nicotinic signaling and that nicotinic receptor expression is positively correlated with insulin secretion and glycemic control in human donor islets. Moreover, polymorphisms spanning MAFA-binding regions within the human CHRNB4 gene are associated with type 2 diabetes. Our data show that MAFA transcriptional activity is required for establishing β cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes.

Figure 1. β-Cell-Specific Deletion of MafA Results in Impaired Glucose Clearance and ANS-Stimulated Insulin Secretion

(A and B) 2DG-stimulated insulin secretion in adult MafA^WT, MafA^−/−, and MafA^RIP mice is shown; n = 9 or 10. (C) Glucose levels in 2DG-treated MafA^WT and MafA^RIP animals; n = 9 or 10. (D) Glucagon secretion induced by 2DG in MafA^WT and MafA^RIP mice, with saline (NaCl) treatment as a control; n ≥ 3. (E) MafA mRNA expression in the hypothalamic, cortex, and brainstem regions in MafA^RIP and MafA^WT mice. Data were normalized to the geomean of HPRT and β-actin mRNA levels. n = 4 or 5. (F and G) Immunohistochemistry staining for MafB (green), α cells (stained for glucagon; blue), and β cells (stained for insulin; red) of freshly isolated pancreatic sections from MafA^WT and MafA^RIP mice. (H) β cell area in MafA^RIP and MafA^WT mice; n = 4. (I) α cell area in MafA^RIP and MafA^WT mice; n = 4. (J and K) Islet innervation in MafA^WT and MafA^RIP mice was assessed by β-tubulin immunostaining (Tubβ; green). Autonomic nerve fibers are denoted by white arrows. Insulin is visualized in red and nuclei in blue (DAPI staining). Data are presented as mean ± SEM and were analyzed with multiple (A and C) and Student’s (B, D, E, H, and I) t test. *p < 0.05 and **p < 0.01. The scale bar represents 20 µm. See also Table S1 and Figure S1 for additional characterization of MafA^RIP animals.

Figure 2. MafA^RIP Islets Have Altered Neurotransmitter Receptor Expression

(A) qPCR measurement of nicotinic receptor (Chrn) mRNA expression in freshly isolated MafA^WT and MafA^RIP islets (n ≥ 4). (B) Quantitative analysis of Chrn protein levels (n ≥ 4). (C) qPCR measurement of Adra2A mRNA expression in freshly isolated MafA^WT and MafA^RIP islets (n ≥ 4). (D) Left panel: qPCR measurement of Adra2A mRNA expression in cultured MafA^WT and MafA^RIP islets (n ≥ 4). Right panel: quantitative analysis of ADRA2A protein levels is shown (n ≥ 4). qPCR data were normalized to the geomean of HPRT and β-actin mRNA levels (n ≥4). (E and F) Immunohistochemistry staining for ADRA2A (green) in MafA^WT and MafA^RIP pancreatic islets, with insulin (red) and nuclear counterstaining with DAPI (blue). ADRA2A expression in β cells is further illustrated by insets presented in the left corner of respective images. The insets were derived from portions of the images marked by white rectangles. Data are represented as mean ± SEM and were analyzed using the multiple t test (A and C) and ratio t tests (B and D). *p < 0.05 and **p < 0.01. The scale bar represents 20 µm. See Figure S2A for validation of the ADRA2A antibody.

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.

Figure 4. Human Nicotinic Receptor Expression in Islets Is Associated with Type 2 Diabetes and Controlled by MAF Transcription Factors

(A) Regional plot of the CHRNB4 gene showing the presence of a cluster of SNPs that infer low expression of CHRNB4 in human islets. The leading SNP (rs12910237) is indicated in purple. (B) Effect of alternate rs12910237 allele copy numbers on CHRNB4 transcript levels in islets from human donors; n = 89 (p = 1.98E–05). (C) Overview of the CHRNB4 upstream region containing SNPs affecting islet gene transcription and the risk for developing type 2 diabetes. MAF and other transcription-factor-binding sites and active islet enhancer regions are shown (Pasquali et al., 2014). Transcriptional activity in the β cell line β-TC6 and activation by MafA is indicated. (D–G) Analysis of RNA-seq data from human donor islets to show the correlation between CHRNB2 expression and MAFA and MAFB transcript levels, insulin secretion (stimulatory index), and HbA1c levels. (H–K) Analysis of RNA-seq data from human donor islets to show the correlation between CHRNB4, MAFA, and MAFB; stimulatory index; and HbA1c levels. Experiments were analyzed with linear regression and Pearson correlation analysis; p values are indicated in the respective graphs; n = 131. (L) siRNA-mediated knockdown of MAFA in EndoC-βH1 cells, showing the effect on mRNA levels of CHRNB2 (p = 0.04), CHRNB4 (p = 0.05), and ADRA2A (p = 0.055); n = 3 or 4. (M) Effect of pCMVMafA (MafA) expression on luciferase (Luc) activities of human CHRNB4 upstream reporter constructs (p-3.7kbLUC and p-7.5kbLUC) spanning sequences that contain identified risk(R) and corresponding non-risk alleles (NR) for rs12910237 or rs922691 and islet enhancer regions. Activity was assessed in HEK293 cells, which do not have endogenous MAFA activity. n = 4. Data are mean ± SEM and were analyzed using one-way ANOVA and Tukey multiple comparison tests (M) and Student’s t test (L). *p < 0.05; **p < 0.01; ****p < 0.001. Abbreviations: T2D, type 2 diabetes; TF, transcription factor. See also Figures S3 and S4 and Table S2 for additional correlation and transcriptional data.