Effects of fatty acids on inducing endoplasmic reticulum stress in bovine mammary epithelial cells
Effects of fatty acids on inducing endoplasmic reticulum stress in bovine mammary epithelial cells
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DOI：
10.3168/jds.2019-18080
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发表时间：
2020-09-01
影响因子：
3.5
通讯作者：
Yonekura, Shinichi
中科院分区：
文献类型：
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作者：
Sharmin, Mst Mamuna;Mizusawa, Moeko;Yonekura, Shinichi
脂肪酸在不同细胞内质网应激诱导的细胞凋亡调控中起重要作用。目前，脂肪酸对牛乳腺上皮细胞（MEC）的影响尚不清楚。本研究检测了牛MEC活力，并测量了脂肪酸处理后未折叠蛋白反应（UPR）相关基因和蛋白的表达。为了评估脂肪酸的作用，我们用100至400 μ M的饱和脂肪酸（棕榈酸和硬脂酸）和不饱和脂肪酸（棕榈油酸、油酸、亚油酸和亚麻酸）和1至5mm的短链和中链脂肪酸（乙酸、丙酸、丁酸和辛酸）处理MAC-T细胞（MEC的一个品系）。随后，我们使用实时荧光定量PCR检测了uprr相关基因的表达。棕榈酸刺激XBP1s、ATF4、ATF6A和C/EBP同源蛋白（CHOP）的表达。硬脂酸增加XBP1s和CHOP的表达，降低ATF4和ATF6A的表达。Western blot分析和3-（4,5-二甲基噻唑-2-酰基）-2,5-二苯基溴化四唑（MTT）检测结果显示，棕榈酸和硬脂酸通过增加内质网应激标记蛋白的表达，降低了MAC-T细胞的活力，诱导了内质网应激，如磷酸化磷酸化样内质网激酶、磷酸化eif2a、cleaved CASP-3和CHOP。在不饱和长链脂肪酸中，棕榈油酸增加了ATF4和ATF6A的表达。油酸增加XBP1s、ATF4和ATF6A的表达。亚油酸和亚麻酸在最高剂量下增加了XBP1s、ATF4和ATF6A的表达，但降低了XBP1s和ATF6A的表达。虽然棕榈油酸、油酸和亚油酸降低CHOP表达，但只有棕榈油酸增加了MAC-T细胞的活力。因此，不饱和长链脂肪酸不会引起严重的内质网应激。乙酸、丙酸和丁酸降低了ATF4、ATF6A和CHOP的表达，增加了XBP1s的表达。辛酸虽然增加了ATF4和ATF6A的表达，但降低了XBP1s和CHOP的表达。虽然脂肪酸处理没有增加内质网应激蛋白的水平，但丁酸和辛酸降低了细胞活力，可能是因为早期分化。这些结果表明，与不饱和脂肪酸相比，饱和脂肪酸通过诱导严重内质网应激在MEC活力中发挥重要作用。此外，乙酸和丙酸（短链和中链脂肪酸）可降低内质网应激。因此，本研究反映了血清脂肪酸浓度在维持奶牛泌乳生理中发挥重要作用的新认识。
Fatty acids play important roles in the regulation of endoplasmic reticulum (ER) stress-induced apoptosis in different cells. Currently, the effects of fatty acids on bovine mammary epithelial cells (MEC) remain unknown. Our study examined bovine MEC viability and measured unfolded protein response (UPR)-related gene and protein expressions following fatty acid treatments. To evaluate the role of fatty acids, we treated MAC-T cells (a line of MEC) with 100 to 400 mu M of saturated (palmitic and stearic acid) and unsaturated (palmitoleic, oleic, linoleic, and linolenic acid) fatty acids and 1 to 5 mM of short- and medium-chain fatty acids (acetic, propionic, butyric, and octanoic acid). Thereafter, we determined UPR-related gene expression using quantitative real-time PCR. Palmitic acid stimulated expression of XBP1s, ATF4, ATF6A, and C/EBP homologous protein (CHOP). Stearic acid increased expression of XBP1s and CHOP and decreased expression of ATF4 and ATF6A. Results of Western blot analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that palmitic and stearic acid reduced MAC-T cell viability and induced extreme ER stress by increasing the protein expression of ER stress markers, such as phospho-PKR-like endoplasmic reticulum kinase, phospho-eIF2a, cleaved CASP-3, and CHOP. Among unsaturated long-chain fatty acids, palmitoleic acid increased expression of ATF4 and ATF6A. Oleic acid increased expression of XBP1s, ATF4, and ATF6A. Linoleic and linolenic acids increased expression of XBP1s, ATF4, and ATF6A but decreased expression of XBP1s and ATF6A at the highest dose. Although palmitoleic, oleic, and linoleic acid decreased CHOP expression, only palmitoleic acid increased MAC-T cell viability. Therefore, unsaturated long-chain fatty acids did not induce severe ER stress. Acetic, propionic, and butyric acids decreased expression of ATF4, ATF6A, and CHOP and increased XBP1s expression. Although only octanoic acid increased ATF4 and ATF6A expressions, it lowered expression of XBP1s and CHOP. Although fatty acid treatment did not increase the levels of ER stress proteins, butyric and octanoic acids reduced cell viability, possibly because of early differentiation. These results suggest that saturated fatty acids play important roles in MEC viability by inducing severe ER stress compared with unsaturated fatty acids. In addition, acetic and propionic acids (short- and medium-chain fatty acids) reduced ER stress. Therefore, the present study reflects the new insight that serum fatty acid concentration plays an important role in maintaining the lactation physiology of dairy cows.