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

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, knockout (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 used to analyze the ion-dependence of Ca²⁺ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their 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, 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. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when 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 regulating hormone-induced mitochondrial Ca²⁺ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.