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Fatty acid-induced ORAI1 facilitates endoplasmic reticulum stress through mitochondrial dysfunction in calf hepatocytes.

C. Xu




Fatty acid-induced ORAI1 facilitates endoplasmic reticulum stress through mitochondrial dysfunction in calf hepatocytes.
B. Zhang1, M. Li2, W. Yang2, C. Xia2, H. Zhang2, C. Xu*2. 1College of Life Science and Technology, Heilongjiang Bayi Agricultural University Daqing, Heilongjiang, China, 2College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University Daqing, Heilongjiang, China.

High-producing dairy cows experience negative energy balance (NEB) during early lactation, which is associated with mobilization of nonesterified fatty acids (NEFA) from adipose depots. In non-ruminants, store-operated Ca2+ entry moiety ORAI1 regulates hepatic lipogenesis by mediating Ca2+ homeostasis. The present study aimed to determine whether fatty acids activate oxidative stress via altering intracellular Ca2+ signaling and ORAI1, thereby inducing mitochondrial dysfunction and endoplasmic reticulum (ER) stress. Calf hepatocytes from 4 healthy female calves (1 d old, 40—50 kg) were used to perform an in vitro challenge with a 1.2 mM mixture of fatty acids. including cis-9—18:1, cis-9,cis-12—18:2, 16:0, 18:0, and cis-9—16:1 for 0, 0.5, 1, 3, 6, 9 and 12 h. Furthermore, transfecting calf hepatocytes with small interfering ORAI1(siORAI1) or the ORAI1 inhibitor BTP2 for 24 h followed by a challenge with the 1.2 mM mixture of fatty acids for 6 h and sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin or the calcium ionophore ionomycin for 6 h, respectively, to determine ORAI regulation. Data were submitted for ANOVA testing using a mixed model for repeated measurements. There was a decrease (P < 0.05) in intracellular reduced glutathione (GSH) and superoxide dismutase (SOD) by 0.5 h, significantly elevated MDA and hydrogen peroxide by 1 h (P < 0.01), and significantly increase (P < 0.01) in the major ER stress proteins PERK, IRE, ATF6, and GRP78 by 6 h. Exogenous fatty acids upregulated ORAI1 mRNA and protein abundance, and VDAC1, CLPP, and CypD protein abundance (P < 0.01). In addition, flow cytometry showed that fatty acids increased mitochondrial membrane potential (P < 0.05) and induced transient opening of mitochondrial permeability transition pore (mPTP). The reactive oxygen species (ROS) inhibitor NAC blocked ORAI1 and major ER stress proteins. Furthermore, compared with controls, BTP2 or siORAI1 abrogated oxidative stress including increased GSH and SOD content, decreased MDA and hydrogen peroxide content (P < 0.01), and decreased ROS production. ER stress protein abundance also was downregulated and mitochondrial function was restored. Furthermore, increased ORAI1 expression by thapsigargin, or ionomycin induced mitochondrial dysfunction. Overall, these data suggest that ORAI1 mediates fatty acid-induced ER stress through controlling mitochondrial dysfunction and oxidative stress.

Keywords: ORAI1, mitochondria dysfunction, endoplasmic reticulum.

Biography: Prof. Dr. Chuang Xu, Chief technology officer of Heilongjiang Bayi Agricultural University, is entries “ten thousand people plan” leading talents of China, Chang Jiang scholars program young scholars of China. Prof. Dr. Chuang Xu is engaged in the researches of pathogenesis and prevention and control technology products of metabolic in dairy cows. published 30 papers in Journal of Dairy Research, BMC Veterinary Research and other journals as the first or corresponding author, authorized 4 invention patent, won 5 technological progress awards, obtained 3 software copyrights for early warning metabolic diseases in dairy cow and issued 1 industry standard of ministry of agriculture for monitoring ketosis in dairy cow