Maf and Mitf transcription factors regulate pancreatic endocrine cell differentiation and function

Abstract: Diabetes - the imbalance in glucose homeostasis is partially caused by loss or dysfunction of pancreatic insulin producing ? cells or development of insulin resistance. In Type 1 diabetes ? cells are destroyed in the process of an autoimmune attack, whereas in Type 2 diabetes, islets produce insufficient amounts of insulin or the insulin cannot be used adequately. So far, the most promising therapy to treat Type 1 diabetes is pancreatic islet transplantation. However, this method cannot be used in a larger scale because of the severe scarcity of donors. Therefore, there is a great and urgent need to develop new methods and therapies. One of the potential sources of obtaining functional insulin cells are human embryonic stem cells (ESC) or inducible pluripotent stem cells (iPS). Hence, differentiating stem cells into functional ?-cells requires a detailed understanding of pancreas development with the focus on molecular programs underlying ?-cells formation and function. Pancreas development and differentiation of insulin and glucagon secreting cells is a complex process controlled by a variety of transcription factors. Two of these factors, MafA and MafB, have been shown to play a major role in the regulation of several genes critical to pancreas development and endocrine cell formation. MafB is essential for both ?- and ?-cell differentiation, as mice lacking MafB have fewer insulin+ and glucagon+ cells during pancreas development. In contrast, in mouse embryos lacking MafA, the development of ?-cells is normal, but adult animals develop diabetes. In adult animals these two transcription factors have a distinct expression pattern, with MafB being expressed in ?-cells, whereas MafA is exclusively found in ?-cells. We have performed gene expression profiling of wild type and MafA/B mutant pancreata to identify genes important for ?-cell maturation and function. In these microarray studies several known (insulin, glucagon, Glut2, PC2) but also novel genes were shown to be differentially expressed in MafB and MafA/MafB compound mutant embryos. Gene ontology analysis revealed that the differentially expressed genes were mainly associated with mature ?-cell function. Our findings show that Neuronatin (Nnat), islet-specific zinc transporter (Slc30a8), islet-specific glucose-6-phosphatase catalytic subunit-2 protein (G6pc2) and Microphthalmia associated transcription factor (Mitf) are downregulated in embryonic and adult mutant pancreata. In contrast, the mRNA level of Retinol Binding Protein-4 (Rbp4) was upregulated in mutant tissue. Given the differences in spatio-temporal expression pattern of MafA and MafB in developing and adult pancreas, we propose that these two factors regulate islet ?-cell formation and maturation in a unique and sequential manner. In MafA deficient models studied so far pancreas development is unaffected, most likely due to compensatory functions of MafB. Therefore, to be able to study the actual role of MafA in ?-cell function we developed a ?-cell-specific deletion of MafA. Our MafA??cell mutant animals lack expression of MafA and MafB in adult ?-cells. In contrast to other MafA mutants, our animals have normal islet architecture, ?-cells mass, ?- to ?-cell ratio, and MafB expression is restricted to ?-cells. Thus we have created a system suitable for studying the true contribution of MafA to adult ?-cell function. Our gene expression experiments have shown that in MafA and MafB mutant embryonic pancreata levels of Mitf expression are reduced comparing to levels found in wild type animals. We also show that Mitf loss of function mutation alters functionality of islet ?-cell. In response to an intraperitoneal glucose challenge, but also during non-fasted conditions, Mitf mutant mice have lower blood glucose levels than wild type animals. Mutant islets secrete more insulin upon exposure to high glucose concentrations and Mitf mutant animals have higher circulating insulin levels in fasted conditions. Additionally, Mitf directly regulates the expression of genes regulating blood glucose levels and ?-cell formation, which is significantly higher in Mitf mutant than in wild type animals. Thereby, we demonstrate that Mitf is an important factor in modulation of ?-cell function.