Molecular Mechanisms of Cellular Stress and Inflammation Unit, Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD 21224, USA.
Drug Design and Synthesis Section Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD 21224, USA.
J Hepatol. 2017 Nov;67(5):1009-1017. doi: 10.1016/j.jhep.2017.05.023. Epub 2017 Jul 17.
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.
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.
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.
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.
内质网（ER）钙稳态的破坏与肥胖有关，然而，先前的方法在纵向监测 ER 钙波动方面具有挑战性。我们最近描述了一种基于高斯荧光素酶（GLuc）的报告蛋白的开发，该蛋白对 ER 钙耗竭有反应（GLuc-SERCaMP），并研究了高脂肪饮食对 ER 钙稳态的影响。
用游离脂肪酸棕榈酸处理基于 GLuc 的报告细胞系。用 GLuc-SERCaMP 报告蛋白肝内注射的大鼠喂食自助餐厅饮食或高脂肪饮食。检查肝脏和血浆中已知的脂肪变性标志物，并将其与 SERCaMP 活性的血浆水平进行比较。
棕榈酸在体外诱导 GLuc-SERCaMP 释放，表明 ER 钙耗竭。消耗自助餐厅饮食或高脂肪丸与大鼠肝 ER 钙稳态的改变相关，表现为 GLuc-SERCaMP 释放增加。自由获得高脂肪丸也导致微粒体钙 ATP 酶活性降低和肝脂肪变性标志物增加。除了 GLuc-SERCaMP，我们还鉴定了具有类似 ER 钙耗竭反应的内源性蛋白（内源性 SERCaMPs）。我们在高脂肪饮食时证明了一种内源性 SERCaMP（被认为是一种肝酯酶）的释放。在给予丹曲林期间观察到 GLuc-SERCaMP 和内源性 SERCaMP 的衰减。
在这里，我们描述了一种用于高脂肪饮食的体外和体内模型的报告基因的使用。我们的结果支持这样一种理论，即饮食脂肪摄入与肝脏内质网钙水平的降低相关，并表明高脂肪饮食改变了内质网蛋白质组。简介：在给予自助餐厅饮食或高脂肪丸的大鼠中观察到 ER 钙失调，传感器释放的波动与脂肪摄入相关。在给予丹曲林时，观察到传感器释放的衰减以及食物摄入的衰减，丹曲林是一种稳定内质网钙的药物。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深入了解。
