Diabetes mellitus is metabolic condition that is characterized by impaired function and/or mass of the pancreatic ?-cell population that results in severe hyperglycemia. Efforts supported by the NIH are aimed at ?-cell replacement or reprogramming other islet cell types into functional ?-cells (e.g. islet ?-cells). Thus, the discovery of mechanisms which control ?- and ?-cell proliferation will provide significant insigh into developing therapeutics to treat diabetes. The Stein lab has a direct interest in characterizing the transcription factors and their obligatory coregulators that govern pancreas size and islet development. In this regard, we have discovered that FoxP1, FoxP2 and FoxP4 are essential for ?-cell proliferation and will investigate the molecular impact of Pdx1 recruited Swi/Snf chromatin remodeling complex on regulating pancreas size. The role of the FoxP class of transcription factors have yet to be characterized in the islet.
In aim 1 of this proposal I hav generated, along with Dr. Chad Hunter, transgenic mice with an endocrine specific knockout of FoxP1/2/4. Our data reveals that this cohort of mice clear glucose normally, but have hypoglycemia and decreased serum glucagon levels under fasting conditions at 4 weeks of age. All endocrine cell types (?-, ?-, ?-) have reduced proliferative capability; however, the ?-cel population is the most profoundly impacted (~90% reduction in cell number). Previous reports show that only 3% of the ?-cell population is required for euglycemia and normoglucagonemia, which suggests the remaining 10% of ?-cells in the FoxP1/2/4 conditional knockout mice are dysfunctional. This question will be addressed using glucagon secretion assays from isolated islets and immunohistochemical techniques in pancreata from 4 week old experimental groups. Pdx1 is one of the first transcription factors expressed during pancreatogenesis and its transcriptional activity is required in both pancreas development and postnatal ?-cell functions. To address how the activity of Pdx1 is modulated, our lab has shown that the Swi/Snf chromatin remodeling complex interacts with (both in vivo and in vitro) and regulates Pdx1 transcriptional activity. Preliminary results have shown that RNAi- mediated knockdown of Brg1, an ATPase essential for Swi/Snf chromatin remodeling activity, negatively impacts the expression of glucose homeostasis genes. Conditional knockout of Brg1 during the early stages of pancreatogenesis results in mice with a hypoplastic pancreas, approximately 50% the size of control littermates. This conditional knockout model will be used in Aim 2 to examine how Brg1 impacts pancreas size. I will examine whether the population of cells in the Brg1 mutant that are designated to become all pancreatic cell types, known as the multipotent progenitor cells (MPCs), are decreased in number, exhibit decreased proliferation or increased apoptosis that could explain the hypoplastic pancreas. Collectively, this proposal will be of major interest to th diabetes field as it will shed light on how the Swi/Snf chromatin remodeling complex impacts pancreas mass and the FoxP1/2/4 regulators influence proper ?-cell development and function.
In this proposal, the impact of FoxP1/2/4 transcriptional regulators on islet ?-cell development/function and of Brg1, the ATPase subunit of the chromatin remodeling Swi/Snf complex, on pancreas development will be tested in vivo using independent mouse models. Following FoxP1/2/4 removal from endocrine cells and Brg1 removal from pancreatic progenitor cells, the glucagon secretion properties of the remaining ?-cell population (for FoxP1/2/4 model) and expansion of pancreatic progenitor cells (for Brg1 model) will be assessed using biochemical-, molecular-, immunohistochemical- and physiological-based assays. Taken together these studies will examine the importance of FoxP1/2/4 transcriptional regulators on ?-cell development/function and the Swi/Snf coregulator on pancreas formation.
