The Orai calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance in nonruminants. Alterations in ORAI1 abundance have been associated with endoplasmic reticulum (ER) stress and changes in lipid metabolism in hepatocytes, an important lipogenic organ in nonruminants. Objectives were to (1) determine abundance of ORAI1 and components of the ER stress response in mammary tissue of ketotic cows, and (2) the potential role of ORAI1 on mammary cell responses to high levels of β-hydroxybutyrate (BHB). Healthy (n = 6, plasma BHB < 0.60 mmol/L) and clinically ketotic (n = 6, plasma BHB > 2.0 mmol/L) Holstein cows (days in milk = 10.13 ± 1.90) were used for mammary gland tissue and blood sample collection. Although milk production (22.5 ± 1.26, 33 ± 1.59, kg of milk/cow per day) and dry matter intake (19.5 ± 1.05, 21.9 ± 0.95, kg/d) were lower in ketotic cows, abundance of ORAI1 protein was greater and was associated with greater mRNA abundance of ER stress proteins (PERK, IRE1, ATF6, and GRP78) and lipogenic genes (FASN, SREBP1, and ACACA). Cellular mechanisms to establish links between BHB and mammary cell responses were evaluated using the immortalized cell line bovine mammary epithelial cells (MAC-T). First, a dose response study was performed with 0, 0.6, 1.2, 1.8, 2.4, or 4.8 mM BHB for 24 h. The mRNA abundance of FASN, SREBP1, and ACACA and lipid droplet formation peaked at 1.2 mM BHB. A subsequent study involved transfecting MAC-T with small interfering Orai 1 (siORAI1) or the ORAI1 inhibitor BTP2 for 24 h followed by a challenge with 1.2 mM BHB for 24 h. Transcription and protein abundance of FASN, SREBP1, ACACA, and ER stress proteins returned to basal levels when ORAI1 was silenced or inhibited. Furthermore, the Ca2+ ionophore ionomycin (raises the intracellular level of Ca2+) also increased abundance of ORAI1, FASN, SREBP1, ACACA, and ER stress proteins. Data suggest that the mammary gland experiences ER stress during ketosis, partly due to the greater supply of BHB originating from ketogenesis in the liver. Intracellular Ca2+ signaling and ORAI1 seem to mediate in part the BHB-induced ER stress in mammary cells.

中文翻译：

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Orai钙释放激活钙调节剂1（ORAI1）在牛乳腺上皮细胞内质网应激中，受到酮体生理水平的挑战。

Orai钙释放激活钙调节剂1（ORAI1）是调节非反刍动物中细胞Ca2 +平衡的存储操纵性Ca2 +进入机制的关键组成部分。ORAI1丰度的变化与内质网应激和肝细胞脂质代谢的变化有关，肝细胞是非反刍动物中重要的生脂器官。目的是（1）确定酮奶牛乳腺组织中ORAI1的含量和内质网应激反应的组成部分，以及（2）ORAI1对高水平β-羟基丁酸酯（BHB）的乳腺细胞反应的潜在作用。健康（n = 6，血浆BHB <0.60 mmol / L）和临床酮症（n = 6，血浆BHB> 2.0 mmol / L）荷斯坦奶牛（产奶天数= 10.13±1.90）用于乳腺组织和血液样本采集。尽管产奶量（22.5±1.26、33±1.59，泌乳性奶牛的每日牛奶/牛千克kg和干物质摄入量（19.5±1.05，21.9±0.95，kg / d）较低，ORAI1蛋白的丰度更高，并且与ER应激蛋白（PERK）的mRNA丰度更高相关，IRE1，ATF6和GRP78）和脂肪生成基因（FASN，SREBP1和ACACA）。使用永生化细胞系牛乳腺上皮细胞（MAC-T）评估了建立BHB和乳腺细胞反应之间联系的细胞机制。首先，使用0、0.6、1.2、1.8、2.4或4.8 mM BHB进行剂量反应研究24小时。FASN，SREBP1和ACACA的mRNA丰度和脂质滴的形成在1.2 mM BHB时达到峰值。后续研究涉及用小干扰Orai 1（siORAI1）或ORAI1抑制剂BTP2转染MAC-T 24小时，然后用1.2 mM BHB攻击24 h。当沉默或抑制ORAI1时，FASN，SREBP1，ACACA和ER应激蛋白的转录和蛋白丰度恢复到基础水平。此外，Ca2 +离子载体离子霉素（提高了Ca2 +的细胞内水平）也增加了ORAI1，FASN，SREBP1，ACACA和ER应激蛋白的含量。数据表明，乳腺在酮症期间会遇到内质网应激，部分原因是由于肝脏中的酮症生成所产生的BHB的供应量增加。细胞内Ca2 +信号传导和ORAI1似乎部分介导了BHB诱导的乳腺细胞内质网应激。数据表明，乳腺在酮症期间会遇到内质网应激，部分原因是由于肝脏中的酮症生成所产生的BHB的供应量增加。细胞内Ca2 +信号传导和ORAI1似乎部分介导了BHB诱导的乳腺细胞内质网应激。数据表明，乳腺在酮症发生过程中会受到内质网应激，部分原因是由于肝脏中酮症产生的BHB的供应量增加。细胞内Ca2 +信号传导和ORAI1似乎部分介导了BHB诱导的乳腺细胞内质网应激。