Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca2+ levels independently of glucagon secretion

Pancreatic islet -cells are increasingly recognized as amino acid sensors for the organism. Building on our prior work in {beta}-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in -cell amino acid sensing. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with -cell specific deletion of pyruvate kinase (PKM1/2-KO) or mitochondrial PEP carboxykinase (PCK2-KO). The mitochondrial fuels glutamine/leucine antagonized alanine/arginine-stimulated Ca2+ influx and glucagon secretion under hypoglycemic conditions. Both PKM1/2 and PCK2 were required for glutamine/leucine to close KATP channels and limit amino acid-stimulated membrane depolarization. The Ca2+ response to amino acids was suppressed by pyruvate kinase activation with TEPP-46, and enhanced by -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 Ca2+ levels. In sum, the -cell mitochondrial PEP cycle senses glutamine/leucine and inhibits KATP channels similarly to {beta}-cells, while restricting amino acid-stimulated membrane depolarization and Ca2+ influx. However, defying expectations, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or intracellular Ca2+. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/656009v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@18a29f0org.highwire.dtl.DTLVardef@1c77041org.highwire.dtl.DTLVardef@9adc1dorg.highwire.dtl.DTLVardef@e91407_HPS_FORMAT_FIGEXP M_FIG Graphical abstract-cells as amino acids sensors. Arginine, alanine, and glutamine potentiate glucagon secretion, while leucine has a suppressive effect (left). Independently of glucagon secretion, glutamine and leucine suppress alanine and arginine-stimulated Ca2+ influx via the phosphoenolpyruvate (PEP) cycle, which closes KATP channels and suppresses Vm depolarization (right). C_FIG HighlightsO_LIOur studies identify a role for the -cell PEP cycle in sensing amino acids under hypoglycemic conditions. C_LIO_LIPyruvate kinase and PCK2 are required for glutamine/leucine to close -cell KATP channel and limit membrane depolarization and Ca2+ influx. C_LIO_LIGlutamine/leucine oppose alanine/arginine-stimulated Ca2+ influx and glucagon secretion. C_LIO_LIAll of the amino acids tested regulate glucagon secretion, but none do so by modulating membrane depolarization or intracellular Ca2+ levels. C_LI