Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells
Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells
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DOI：
10.3168/jds.2013-7189
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发表时间：
2014-01-01
影响因子：
3.5
通讯作者：
Hanigan, M. D.
中科院分区：
文献类型：
--
作者：
Appuhamy, J. A. D. R. N.;Nayananjalie, W. A.;Hanigan, M. D.
乳腺蛋白质合成的调节可能会改变目前营养需求模型中AA供应与乳蛋白输出之间的关系。葡萄糖和AA通过涉及哺乳动物雷帕霉素靶蛋白（mTOR）和amp活化蛋白激酶（AMPK）的细胞信号通路调节肌肉蛋白合成。本研究的目的是探讨必需AA （EAA）和乙酸或葡萄糖对mTOR和AMPK信号通路和乳蛋白合成速率的影响。在2个独立的2 × 2因子研究中，将牛乳腺上皮细胞系MAC-T置于含有0或3.5 mmol/L EAA浓度与0或5 mmol/L乙酸或0或17.5 mmol/L葡萄糖的不同培养基中。在另一组实验中，除低EAA处理的EAA水平较低（0.18 mmol/L）外，对乳原牛乳腺组织切片进行相同处理。补充EAA增强了MAC-T细胞中mTOR （Ser2448）和真核起始因子4E结合蛋白1 （4EBP1, Thr37/46）的磷酸化，并降低了真核延伸因子2 （eEF2, Thr56）的磷酸化。EAA存在时，MAC-T细胞中ATP浓度升高，AMPK磷酸化水平降低。在乳腺组织切片中，乙酸、EAA或葡萄糖在数值上降低AMPK磷酸化约16%。无论培养基中是否存在乙酸或葡萄糖，提供EAA均可增加乳腺组织切片中mTOR和4EBP1的磷酸化、细胞内总EAA浓度和酪蛋白合成率。mTOR磷酸化与AMPK磷酸化呈微负相关，而AMPK磷酸化与eEF2磷酸化呈正相关。酪蛋白合成速率与mTOR磷酸化之间的正相关关系比eEF2磷酸化与酪蛋白合成速率之间的负相关关系更强。mTOR磷酸化100%增加与酪蛋白合成率增加0.74%.h（-1）相关，而eEF2磷酸化100%增加与酪蛋白合成率下降0.33%.h（-1）相关。尽管AMPK磷酸化对细胞能量状态有响应，并且对牛乳腺上皮细胞中mtor介导的信号有负面影响，但与mtor介导的EAA对乳蛋白合成的调节相比，其对乳蛋白合成速率的影响似乎微不足道。
Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 x 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%.h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%.h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.