Abstract：
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.
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.
摘要：
目的：肝Ca2+信号已被确定为驱动糖异生的关键因子。线粒体参与激素诱导的Ca2+信号及其对代谢活性的贡献仍然存在，然而,知之甚少。此外，控制线粒体Ca2+外排信号的分子机制仍未解决。本研究探讨了Na+/Ca2+交换剂的作用，NCLX,在调节肝脏线粒体Ca2+流出中，并检查其在激素肝Ca2信号传导中的生理意义，糖异生,和线粒体生物能学。
方法：来自AAV介导的条件性肝特异性和总线粒体Na/Ca2交换剂的原代小鼠肝细胞，NCLX,敲除（KO）小鼠模型用于荧光监测培养的肝细胞中嘌呤能和胰高血糖素/加压素依赖性线粒体和胞质肝Ca2反应。分离的肝线粒体和透化的原代肝细胞用于分析Ca2流出的离子依赖性。利用条件性肝特异性NCLXKO模型，首先通过体内监测禁食小鼠的葡萄糖水平，并在监测血糖的同时对禁食小鼠进行丙酮酸耐受试验，评估糖异生率。此外,通过葡萄糖氧化酶测定法和海马呼吸测定法，在体外评估了两种基因型的培养原代肝细胞的胰高血糖素依赖性葡萄糖产生和细胞生物能量学，分别。
结果：对来自NCLXKO和WT小鼠的分离的肝线粒体和培养的原代肝细胞中Ca2+反应的分析显示，NCLX是肝细胞中线粒体钙挤出的主要机制。然后，我们确定了NCLX在胰高血糖素和加压素诱导的Ca2振荡中的作用。与以前的研究一致，胰高血糖素和加压素触发WT肝细胞中的Ca2+振荡，然而,NCLX的缺失导致线粒体的选择性消除，但不是细胞溶质，Ca2+振荡或IP3R1表达水平，强调NCLX在线粒体Ca2+调节中的关键作用。随后的体内研究显示，肝脏NCLX在糖异生中的作用，与禁食时保持血糖水平正常的WT小鼠相反，条件性肝特异性NCLXKO小鼠表现出更快的葡萄糖水平下降，变得低血糖，并且在禁食条件下挑战提供时丙酮酸向葡萄糖的转化受损。同时在体外评估显示受损的胰高血糖素依赖性葡萄糖生产和受损的生物能量在KO肝细胞，从而强调NCLX对肝脏葡萄糖代谢的显著贡献。
结论：研究结果表明，NCLX是肝细胞中主要的Ca2+流出机制。NCLX对于调节激素诱导的线粒体Ca2+振荡是必不可少的，线粒体代谢和肝糖异生的维持。
方法：来自AAV介导的条件性肝特异性和总线粒体Na/Ca2交换剂的原代小鼠肝细胞，NCLX,敲除（KO）小鼠模型用于荧光监测培养的肝细胞中嘌呤能和胰高血糖素/加压素依赖性线粒体和胞质肝Ca2反应。分离的肝线粒体和透化的原代肝细胞用于分析Ca2流出的离子依赖性。利用条件性肝特异性NCLXKO模型，首先通过体内监测禁食小鼠的葡萄糖水平，并在监测血糖的同时对禁食小鼠进行丙酮酸耐受试验，评估糖异生率。此外,通过葡萄糖氧化酶测定法和海马呼吸测定法，在体外评估了两种基因型的培养原代肝细胞的胰高血糖素依赖性葡萄糖产生和细胞生物能量学，分别。
结果：对来自NCLXKO和WT小鼠的分离的肝线粒体和培养的原代肝细胞中Ca2+反应的分析显示，NCLX是肝细胞中线粒体钙挤出的主要机制。然后，我们确定了NCLX在胰高血糖素和加压素诱导的Ca2振荡中的作用。与以前的研究一致，胰高血糖素和加压素触发WT肝细胞中的Ca2+振荡，然而,NCLX的缺失导致线粒体的选择性消除，但不是细胞溶质，Ca2+振荡或IP3R1表达水平，强调NCLX在线粒体Ca2+调节中的关键作用。随后的体内研究显示，肝脏NCLX在糖异生中的作用，与禁食时保持血糖水平正常的WT小鼠相反，条件性肝特异性NCLXKO小鼠表现出更快的葡萄糖水平下降，变得低血糖，并且在禁食条件下挑战提供时丙酮酸向葡萄糖的转化受损。同时在体外评估显示受损的胰高血糖素依赖性葡萄糖生产和受损的生物能量在KO肝细胞，从而强调NCLX对肝脏葡萄糖代谢的显著贡献。
结论：研究结果表明，NCLX是肝细胞中主要的Ca2+流出机制。NCLX对于调节激素诱导的线粒体Ca2+振荡是必不可少的，线粒体代谢和肝糖异生的维持。
