Mitochondrial dysfunction and endoplasmic reticulum stress in calf hepatocytes are associated with fatty acid-induced ORAI calcium release-activated calcium modulator 1 signaling.

Abstract

The store-operated Ca2+ entry (SOCE) moiety ORAI calcium release-activated calcium modulator 1 (ORAI1) located in the endoplasmic reticulum (ER) participates in key cellular functions such as protein folding, transport, and secretion, and lipid metabolism. We used an in vitro approach to test whether exogenous fatty acids alter ORAI1 signaling and to explore potential consequences on mitochondrial dysfunction and ER stress. First, hepatocytes isolated from 4 healthy female calves (1 d old, 40-50 kg) were challenged with a 1.2 mM mixture of oleic, linoleic, palmitic, stearic, and palmitoleic acids for 0.5, 1, 3, 6, 9, and 12 h to measure oxidative stress [intracellular reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and hydrogen peroxide] and ER stress (protein abundance of PERK, IRE, ATF6, and GRP78). Concentrations of GSH and SOD decreased at 0.5 h, and MDA and hydrogen peroxide increased at 1 h; ER stress proteins increased at 6 h. To determine whether ER stress was caused by oxidative stress, primary calf hepatocytes were treated with the same 1.2 mM fatty acid mix or the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) for 6 h. We found that NAC prevented an increase in ER stress protein abundance. Next, the role of ORAI1 on ER stress was measured by transfecting hepatocytes with small interfering (si)ORAI1 or the ORAI1 inhibitor BTP2, followed by a challenge with 1.2 mM fatty acids for 3 h. Without inhibiting ORAI1, exogenous fatty acids upregulated ORAI1 mRNA and protein abundance, oxidative stress, ER stress proteins, and protein abundance of marker indicators of an opened mitochondrial permeability transition pore (mPTP). Inhibition with BPT2 or silencing via siORAI1 abrogated oxidative stress, including increased GSH concentration and SOD activity, decreased MDA, hydrogen peroxide, and ROS concentration; ER stress protein abundance was downregulated, and mitochondrial function was restored. Last, changes in markers of mPTP opening were evaluated by culturing hepatocytes for 6 h with the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin or the calcium ionophore ionomycin. We detected an increase in VDAC1, CLPP, and CypD protein abundance, all of which indicated opening of the mPTP. Overall, data from these in vitro studies suggest that ORAI1 mediates ER stress induced by high concentrations of fatty acids, in part through alleviating mitochondrial dysfunction caused by oxidative stress.

• Authors: Bingbing Zhang, Ming Li, Wei Yang, Juan J Loor, Yusheng Liang, Shuang Wang, Yingying Zhao, Han Guo, Xinru Ma, Liyun Yu, Chuang Xu
• Journal: Journal of dairy science (J Dairy Sci) Vol. 103 Issue 12 Pg. 11945-11956 (Dec 2020) ISSN: 1525-3198 [Electronic] United States
• PMID: 32981726 (Publication Type: Journal Article)
• Copyright: © 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Chemical References

• Fatty Acids
• ORAI1 Protein
• Reactive Oxygen Species
• Calcium

Topics

• Animals
• Calcium (metabolism)
• Cattle
• Endoplasmic Reticulum (metabolism)
• Endoplasmic Reticulum Stress
• Fatty Acids (adverse effects)
• Female
• Hepatocytes (metabolism)
• Lactation
• Lipid Metabolism
• Liver (metabolism)
• Mitochondria (metabolism)
• ORAI1 Protein (genetics, metabolism)
• Oxidative Stress
• Reactive Oxygen Species (metabolism)
• Signal Transduction