Phosphoenolpyruvate cycling via mitochondrial phosphoenolpyruvate carboxykinase links anaplerosis and mitochondrial GTP with insulin secretion.
Pancreatic beta-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical K(ATP)-dependent glucose-stimulated insulin secretion (GSIS), there are important K(ATP)-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel (13)C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.
Stark
Romana
R
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Pasquel
Francisco
F
Turcu
Adina
A
Pongratz
Rebecca L
RL
Roden
Michael
M
Cline
Gary W
GW
Shulman
Gerald I
GI
Kibbey
Richard G
RG
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002952:Citric Acid Cycle; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013389:Succinate-CoA Ligases / Q000378:metabolism
D007328:Insulin; D010728:Phosphoenolpyruvate; D006160:Guanosine Triphosphate; D010730:Phosphoenolpyruvate Carboxylase; D013389:Succinate-CoA Ligases