Mol Metab. 2024 Jul 01. pii: S2212-8778(24)00113-3. [Epub ahead of print] 101982
OBJECTIVE: Hepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+ /Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics.METHODS: Primary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knock-out (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were utilized to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed first through the monitoring of glucose levels in fasted mice in vivo and by subjecting the fasted mice to a pyruvate tolerance test while monitoring blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively.
RESULTS: Analysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations or level of IP3R1 expression, underscoring NCLX's pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic, and with a compromised conversion of pyruvate to glucose when provided challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX's significant contribution to hepatic glucose metabolism.
CONCLUSIONS: The study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for the regulation of hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism and sustenance of hepatic gluconeogenesis.
Keywords: Calcium signaling; Mitocondrial calcium; NCLX; gluconeogenesis; hepatic calcium signaling