Phosphoenolpyruvate Cycling via Mitochondrial Phosphoenolpyruvate Carboxykinase Links Anaplerosis and Mitochondrial GTP with Insulin Secretion

Pancreatic β-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 ¹³C-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.β-Cells in pancreatic islets of Langerhans make and release insulin in response to changes in blood glucose levels. The mechanisms by which high concentrations of glucose stimulate insulin release from islets remain unclear. The canonical explanation for GSIS² is that glucose metabolism increases mitochondrial ATP production, thereby raising the cytosolic ATP:ADP ratio that triggers the closure of ATP-sensitive K⁺ channels. This, in turn, depolarizes the membrane and stimulates the opening of voltage-dependent Ca²⁺ channels with increased Ca²⁺ influx promoting the exocytosis of insulin. Although K_ATP channels certainly have an important role in β-cells, K_ATP-independent signals are implicated to play a fundamental role in GSIS. In particular, β-cells are known to have notably elevated rates of anaplerotic flux of the carbon from glucose into the mitochondria and back out to pyruvate (pyruvate cycling) that is tightly correlated with insulin secretion (1–4).Recently, mtGTP synthesis was identified as a novel K_ATP-independent mitochondrial signal for insulin secretion (5). mtGTP is synthesized as a product of glucose metabolism by the GTP-specific isoform of the matrix enzyme SCS. mtGTP synthetic rates are determined by the rate of TCA cycle flux as well as by the ratio of activities of the ATP-specific and GTP-specific isoforms of SCS. The mtGTP signal is trapped within the matrix of the mitochondria, suggesting that another GTPase in the matrix transmits the mtGTP signal to the cytosol. Because both mtGTP synthesis and anaplerotic flux correlate with insulin secretion, we investigated whether the GTP-dependent mitochondrial isoform of PEPCK, an enzyme that lies at the intersection of anaplerosis and mtGTP metabolism (see Fig. 1A), is important for GSIS.Open in a separate windowFIGURE 1.PEP cycle. PEP is produced during glycolysis and is further metabolized to pyruvate by PK. Pyruvate that enters the TCA cycle by pyruvate dehydrogenase will generate GTP via direct synthesis by SCS-GTP. Anaplerotic pyruvate entry by PC will generate oxaloacetate. PEPCK-M will then consume oxaloacetate and GTP to produce PEP, GDP, and CO₂. PEP is then transported out of the mitochondrial matrix by an anion transporter (Ex) in exchange for another metabolite depending on the transporter. Mitochondrial PEP, thus, contributes to the PEP pool that is determined by the rate of appearance (ν_PEPCK-M+1+ ν_Glyc+1) of PEP minus the rate of disappearance (ν_PK+1). One turn of the PEP cycle will result in the net exchange of one ion into the mitochondrial matrix. Unidirectional fluxes are indicated by ν followed by the enzyme with the forward direction being +1 and the reverse −1. GDP in turn can be reused by SCS-GTP. PDH, pyruvate dehydrogenase.