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

Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet ß 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 ß cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes.

Islet-specific monoamine oxidase A and B expression depends on MafA transcriptional activity and is compromised in type 2 diabetes.

Lack or dysfunction of insulin producing ß cells results in the development of type 1 and type 2 diabetes mellitus, respectively. Insulin secretion is controlled by metabolic stimuli (glucose, fatty acids), but also by monoamine neurotransmitters, like dopamine, serotonin, and norepinephrine. Intracellular monoamine levels are controlled by monoamine oxidases (Mao) A and B. Here we show that MaoA and MaoB are expressed in mouse islet ß cells and that inhibition of Mao activity reduces insulin secretion in response to metabolic stimuli. Moreover, analysis of MaoA and MaoB protein expression in mouse and human type 2 diabetic islets shows a significant reduction of MaoB in type 2 diabetic ß cells suggesting that loss of Mao contributes to ß cell dysfunction. MaoB expression was also reduced in ß cells of MafA-deficient mice, a mouse model for ß cell dysfunction, and biochemical studies showed that MafA directly binds to and activates MaoA and MaoB transcriptional control sequences. Taken together, our results show that MaoA and MaoB expression in pancreatic islets is required for physiological insulin secretion and lost in type 2 diabetic mouse and human ß cells. These findings demonstrate that regulation of monoamine levels by Mao activity in ß cells is pivotal for physiological insulin secretion and that loss of MaoB expression may contribute to the ß cell dysfunction in type 2 diabetes.

Microphthalmia transcription factor regulates pancreatic ß-cell function.

Precise regulation of ß-cell function is crucial for maintaining blood glucose homeostasis. Pax6 is an essential regulator of ß-cell-specific factors like insulin and Glut2. Studies in the developing eye suggest that Pax6 interacts with Mitf to regulate pigment cell differentiation. Here, we show that Mitf, like Pax6, is expressed in all pancreatic endocrine cells during mouse postnatal development and in the adult islet. A Mitf loss-of-function mutation results in improved glucose tolerance and enhanced insulin secretion but no increase in ß-cell mass in adult mice. Mutant ß-cells secrete more insulin in response to glucose than wild-type cells, suggesting that Mitf is involved in regulating ß-cell function. In fact, the transcription of genes critical for maintaining glucose homeostasis (insulin and Glut2) and ß-cell formation and function (Pax4 and Pax6) is significantly upregulated in Mitf mutant islets. The increased Pax6 expression may cause the improved ß-cell function observed in Mitf mutant animals, as it activates insulin and Glut2 transcription. Chromatin immunoprecipitation analysis shows that Mitf binds to Pax4 and Pax6 regulatory regions, suggesting that Mitf represses their transcription in wild-type ß-cells. We demonstrate that Mitf directly regulates Pax6 transcription and controls ß-cell function.

NANOG reporter cell lines generated by gene targeting in human embryonic stem cells.

BACKGROUND: Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency, but the precise role and regulation of NANOG are not well defined. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate the study of NANOG expression and regulation in viable hESC cultures, we generated fluorescent NANOG reporter cell lines by gene targeting in hESCs. In these reporter lines, the fluorescent reporter gene was co-expressed with endogenous NANOG and responded to experimental induction or repression of the NANOG promoter with appropriate changes in expression levels. Furthermore, NANOG reporter lines facilitated the separation of hESC populations based on NANOG expression levels and their subsequent characterization. Gene expression arrays on isolated hESC subpopulations revealed genes with differential expression in NANOG(high) and NANOG(low) hESCs, providing candidates for NANOG downstream targets hESCs. CONCLUSION/SIGNIFICANCE: The newly derived NANOG reporter hESC lines present novel tools to visualize NANOG expression in viable hESCs. In future applications, these reporter lines can be used to elucidate the function and regulation of NANOG in pluripotent hESCs.