The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 1,000-10,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for longitudinal in vivo studies. Our lab has recently developed a novel secreted ER calcium monitoring protein (GLuc-SERCaMP), to longitudinally monitor ER calcium levels in vivo by measuring small volumes of blood. Additionally, we describe a complementary tool to measure the unfolded protein response utilizing a Nano luciferase (NLuc) reporter. This work highlights the application of both reporters in vivo. Furthermore, towards the overarching goal of monitoring ER calcium homeostasis in a disease model, we describe this use of GLuc-SERCaMP in in vitro and in vivo models of diet-induced obesity. Disruption to ER calcium homeostasis has been implicated in obesity, however, the ability to directly monitor fluctuations to ER calcium has been limited with previous techniques. GLuc-SERCaMP release revealed ER calcium depletion in the presence of free fatty acid (FFA), palmitate. Consumption of a cafeteria diet or high fat pellets further demonstrated alterations to hepatic ER calcium homeostasis in rats, as evidenced by increased GLuc-SERCaMP release. Attenuation of GLuc-SERCaMP was observed during dantrolene administration. Taken together, our results further corroborate the influence of dietary intake on ER calcium homeostasis.
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|Advisor:||Harvey, Brandon K., Randall, William|
|Commitee:||Fang, Shengyun, Harvey, Brandon K., Henderson, Mark J., Randall, William, Yang, Peixin|
|School:||University of Maryland, Baltimore|
|School Location:||United States -- Maryland|
|Source:||DAI-B 78/06(E), Dissertation Abstracts International|
|Keywords:||Calcium, ER stress, Endoplasmic reticulum, High fat diet, Liver, SERCaMP|
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