Project Summary Type 2 diabetes (T2D) is an extremely prevalent disease in the United States, affecting approximately 1 in 10 adults, and it is estimated that 640 million people will be diagnosed with diabetes by 2040. While T2D is associated with β-cell failure, defects in both insulin and glucagon secretion from the pancreatic islet contribute to dysregulated blood glucose. As current therapies that target β-cells to increase insulin secretion have limited success, innovative ideas that focus on novel mechanisms for regulation of blood glucose are necessary to create new therapeutics to combat T2D. Glucagon secretion by α-cells has strong control over the magnitude of insulin secretion, making α-cells an attractive target for new therapies. The second messenger cAMP is a strong determinant of glucagon secretion. Glucose has been shown to regulate α-cell cAMP intrinsically and extrinsically. Preliminary studies show that leucine strongly reduces α-cell cAMP independently of islet paracrine signaling, KATP channel effects, or calcium. Furthermore, in a similar fashion, glucose and leucine can dampen amino acid-stimulated glucagon secretion. Glucose and leucine are both strongly anaplerotic, and preliminary data suggests that the mitochondrial enzyme phosphoenolpyruvate carboxykinase (PCK2), an essential effector of mitochondrial anaplerosis-cataplerosis, plays an important role in the regulation of amino acid-dependent glucagon secretion. We hypothesize that anaplerotic fuels such as leucine and glucose will decrease cAMP through PCK2 to inhibit glucagon release. To study this hypothesis we will: 1) Determine the intrinsic vs. paracrine (via β/δ-cells) effects of leucine on α-cell cAMP and 2) Determine whether α-cell PCK2 mediates the inhibitory effects of anaplerotic fuels. These aims will be studied by TIRF and lightsheet microscopy in combination with novel genetic mouse models and biosensors, to examine role of anaplerotic fuels on α-cell cAMP and glucagon secretion. Successful completion of this project will characterize new connections between anaplerosis and cAMP signaling, train the PI in state of the art techniques for the study of metabolism, and potentially unlock a new pathway to target for the treatment of T2D.