1739-P: Genetic Deletion of Mitochondrial PEP Carboxykinase (PCK2) in Pancreatic α-Cells Increases Amino Acid-Stimulated Ca²⁺ Influx

Amino acids potently stimulate glucagon secretion, but their mechanisms of action remain largely unknown. To assess the effects of individual amino acids on α-cell Ca²⁺, we used GcgCreERT:GCaMP6s Ca²⁺ reporter. We focused on alanine, arginine, glutamine, and leucine, which have the potential to affect Ca²⁺ and glucagon release via their transport or metabolism. Under hypoglycemic conditions, when β-cells are inactive, alanine and arginine provoked a sustained increase in α-cell Ca²⁺, while the mitochondria fuels glutamine and leucine lowered Ca²⁺ after alanine and arginine are applied. However, when applying these four amino acids simultaneously, Ca²⁺ influx is increased above that stimulated by arginine and alanine alone, suggesting the effect of mitochondria fuels on Ca²⁺ is membrane-potential dependent. Phosphoenolpyruvate (PEP) cycle can be fueled by mitochondria fuels and potentially affects K_ATP channels via the ATP-generating enzyme pyruvate kinase (PK). To test this hypothesis, we used CRISPR to generate mice lacking the mitochondrial PEP-generating enzyme PCK2 in α-cells (GcgCreERT:Pck2^f/f:GCaMP6s). PEP cycle ablation increased Ca²⁺ influx stimulated by alanine and arginine, much more strongly when leucine and glutamine were provided as mitochondria fuels. Pharmacological activation of PK reduces amino acid-stimulated Ca²⁺ influx. Taken together, the effect of mitochondria fuels on α-cell Ca²⁺ is most likely membrane potential dependent, and α-cell PEP cycle is an inhibitory pathway that lowers Ca²⁺ influx stimulated by amino acids.