Saturday, December 2, 2023
12/2/2023
¿ DESCRIPTION (provided by applicant): Glucose metabolism is central to regulation of ß-cell function and survival. We have found that phosphorylation of the BCL-2 family protein BAD within its helical BH3 domain imparts cell autonomous protective effects to ß-cells. The benefits of genetic and pharmacologic strategies that mimic the phospho-BAD BH3 helix include resistance to death induced by inflammatory and oxidative stress, as well as increased glucose handling and insulin secretory capacities. In the long-term, these benefits manifest in superior engraftment of donor islets in transplanted diabetic mice and an attendant increase in functional ß-cell mass. The ß-cell protective effect of phospho-BAD is dependent on the glucose-metabolizing enzyme glucokinase (GK), and is mediated by direct binding and activation of this enzyme by the phospho-BAD BH3 helix. Our proposed studies address a key question that stems from these findings: How is the ß-cell sparing effect of phospho-BAD mediated by GK-dependent glucose metabolism? We will address this question at two highly integrative molecular layers. First, using metabolic flux analyses we will identify phospho-BAD dependent changes in mitochondrial metabolism of glucose-derived pyruvate at the level of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) pathway fluxes as ß-cells are subjected to defined stress stimuli, and define their contribution to phospho-BAD's capacity to shield ß-cells from stress-induced dysfunction and death (aim 1). Second, we will interrogate a defined number of MODY-derived GK mutants for their in vitro properties as well as their effect on ß-cell survival in the presence of phospho-BAD (aim 2). These mutants, which are selected for their proximity near the binding interface of the BAD BH3 helix on GK, will reveal additional molecular aspects of GK regulation by phospho-BAD mimicry and provide a complementary experimental system to examine the effect of GK-dependent glucose metabolism on the ß-cell protective effect of BAD phosphorylation. These studies will provide an integrated picture of the pathway connecting phospho-BAD-dependent GK activation and ß-cell resistance to stress-induced dysfunction and death. In the fullness of time, understanding this pathway will yield valuable translational insights into the most effective strategies to capture and mimic the protective aspects of glucose signaling for preservation/restoration of functional ß-cell mass.
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