BACKGROUND & AIMS Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. METHODS A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. RESULTS Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. CONCLUSIONS Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.

中文翻译：

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高脂肪饮食破坏大鼠肝脏内质网钙稳态

背景和目的 内质网 (ER) 钙稳态的破坏与肥胖有关，然而，纵向监测 ER 钙波动的能力对先前的方法具有挑战性。我们最近描述了响应内质网钙耗竭（GLuc-SERCaMP）的基于高斯荧光素酶（GLuc）的报告蛋白的开发，并研究了高脂肪饮食对内质网钙稳态的影响。方法 基于 GLuc 的报告细胞系用棕榈酸酯（一种游离脂肪酸）处理。肝内注射 GLuc-SERCaMP 报告基因的大鼠被喂食食堂饮食或高脂肪饮食。检查肝脏和血浆中已建立的脂肪变性标志物，并与血浆中 SERCaMP 活性水平进行比较。结果 棕榈酸酯在体外诱导 GLuc-SERCaMP 释放，表明 ER 钙耗竭。食堂饮食或高脂肪颗粒的消耗与大鼠肝内质网钙稳态的改变相关，表现为 GLuc-SERCaMP 释放增加。随意摄入高脂肪颗粒也导致微粒体钙 ATP 酶活性相应降低，肝脂肪变性标志物增加。除了 GLuc-SERCaMP，我们还鉴定了对 ER 钙耗竭具有类似反应的内源性蛋白质（内源性 SERCaMP）。我们证明了在获得高脂肪饮食期间内源性 SERCaMP（被认为是肝脏酯酶）的释放。在丹曲林给药期间观察到 GLuc-SERCaMP 和内源性 SERCaMP 的衰减。结论 在这里，我们描述了报告基因在高脂肪饮食的体外和体内模型中的使用。我们的结果支持膳食脂肪摄入量与肝脏中 ER 钙水平降低相关的理论，并表明高脂肪饮食会改变 ER 蛋白质组。总结：在喂食自助餐厅饮食或高脂肪颗粒的大鼠中观察到 ER 钙失调，传感器释放的波动与脂肪摄入量相关。在给予丹曲林（一种稳定 ER 钙的药物）期间观察到传感器释放以及食物摄入的衰减。该研究描述了一种用于肝脏研究的新技术，并提供了对可能导致肥胖和脂肪肝疾病发病机制的细胞过程的见解。传感器释放的波动与脂肪摄入量相关。在给予丹曲林（一种稳定 ER 钙的药物）期间观察到传感器释放以及食物摄入的衰减。该研究描述了一种用于肝脏研究的新技术，并提供了对可能导致肥胖和脂肪肝疾病发病机制的细胞过程的见解。传感器释放的波动与脂肪摄入量相关。在给予丹曲林（一种稳定 ER 钙的药物）期间观察到传感器释放以及食物摄入的衰减。该研究描述了一种用于肝脏研究的新技术，并提供了对可能导致肥胖和脂肪肝疾病发病机制的细胞过程的见解。