Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca 2+ levels without impacting glucagon secretion

Objective Pancreatic islet α-cells are increasingly recognized as amino acid sensors for the organism, however the metabolic pathways that α-cells use to sense amino acids have not been identified. Building on our prior work in β-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in α-cell amino acid sensing. Methods To investigate amino acid regulation of α-cells at the cellular level, we measured intracellular Ca 2+ (GCaMP6s imaging), membrane potential (JEDI-2P imaging and patch-clamp), K ATP channel activity, and glucagon secretion. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with α-cell specific deletion of pyruvate kinase M1/M2 (PKM1/2-αKO) or mitochondrial PEP carboxykinase (PCK2-αKO). Results The mitochondrial fuels glutamine/leucine antagonized alanine/arginine-stimulated Ca 2+ influx and glucagon secretion under hypoglycemic conditions. Both pyruvate kinase and PCK2 were required for glutamine/leucine to close K ATP channels and limit amino acid-stimulated membrane depolarization. The Ca 2+ response to amino acids was blocked by pyruvate kinase activation with TEPP-46, and enhanced by α-cell deletion of pyruvate kinase or PCK2 – all without changing glucagon secretion. Finally, using diazoxide/KCl to probe the pathways downstream of membrane depolarization, we identified an essential role of the PEP cycle in homeostatically restoring intracellular Ca 2+ levels. Conclusions The α-cell mitochondrial PEP cycle senses glutamine/leucine and inhibits K ATP channels similarly to β-cells, while restricting amino acid stimulated membrane depolarization and Ca 2+ influx. However, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or intracellular Ca 2+ . Highlights Our studies identify a role for the α-cell PEP cycle in sensing amino acids under hypoglycemic conditions. Pyruvate kinase and PCK2 are required for glutamine/leucine to close α-cell K ATP channels and limit membrane depolarization and Ca 2+ influx. Glutamine/leucine oppose alanine/arginine-stimulated Ca 2+ influx and glucagon secretion. All of the amino acids tested regulate glucagon secretion, but none do so by modulating membrane depolarization or intracellular Ca 2+ levels.