Abstract Hepatic Ca 2+ signaling is emerging as a key factor in mediating gluconeogenesis. However, the identity of the hepatic mitochondrial Ca 2+ transporter is controversial and the role of mitochondria in controlling hormonal Ca 2+ signaling and linking them to metabolic activity is poorly understood. We first interrogated the role of the mitochondrial Na + /Ca 2+ exchanger NCLX by triggering cytosolic Ca 2+ purinergic signaling in primary hepatocytes, and Ca 2+ responses in isolated mitochondria from WT, global NCLX KO, and conditional hepatic NCLX KO mice models. We monitored a higher rate of Na + -dependent mitochondrial Ca 2+ efflux in NCLX-expressing hepatocytes, indicating that it constitutes the major Ca 2+ efflux pathway. We then asked if NCLX is controlling the hormone-dependent mitochondrial Ca 2+ oscillations by employing physiological concentrations of glucagon and vasopressin. Consistent with previous studies, hormone applications triggered mitochondrial Ca 2+ oscillations in WT hepatocytes. In NCLX KO hepatocytes the cytosolic oscillations persisted, however, the mitochondrial Ca 2+ oscillations were suppressed. To further understand the metabolic role of NCLX in the hepatic system, we examined gluconeogenic function in vivo and ex vitro by monitoring hepatic glucose production. We found that blood glucose dropped faster in the conditional KO mice and their hepatic glucagon-dependent glucose production was reduced, indicating that gluconeogenesis was impaired in hepatic conditional NCLX KO mice. Taken together, our results indicate that NCLX is the primary Ca 2+ extruder in hepatocytes and is required for mediating the hormone-dependent mitochondrial Ca 2+ oscillations and gluconeogenesis. Significance Hepatic Ca 2+ signaling is crucial for gluconeogenesis, but the mitochondrial control of this process is not resolved. This study identifies the mitochondrial transporter, NCLX, as a critical link between hormonal-dependent mitochondrial Ca 2+ oscillations and gluconeogenesis. We first show that NCLX is the major hepatic mitochondrial efflux pathway. We then demonstrate that NCLX is required for glucagon-dependent mitochondrial Ca 2+ oscillations and the acceleration of mitochondrial oxidative function. Using a conditional hepatic NCLX-null mouse model, we show that NCLX is required for maintaining hepatic glucose production during fasting and in response to glucagon stimulation. Overall, the study identifies NCLX as the integrator of hepatic mitochondrial Ca 2+ signaling, required for gluconeogenesis.

NCLX controls hepatic mitochondrial Ca<sup>2+</sup>extrusion and couples hormone-mediated mitochondrial Ca<sup>2+</sup>oscillations with gluconeogenesis

NCLX controls hepatic mitochondrial Ca2+extrusion and couples hormone-mediated mitochondrial Ca2+oscillations with gluconeogenesis