School of Animal Science , Yangtze University , Jingzhou 434020 , China.
The Key Laboratory of Dairy Science of Education Ministry , Northeast Agricultural University , Harbin 150030 , China.
J Agric Food Chem. 2019 Sep 18;67(37):10513-10520. doi: 10.1021/acs.jafc.9b04648. Epub 2019 Sep 10.
Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected β-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.
氨基酸可以刺激乳脂合成，但其中的分子机制仍很大程度上未知。在本研究中，我们研究了 cAMP 反应元件结合蛋白调节的转录共激活因子 2（CRTC2）在乳脂合成中的调控作用及其在氨基酸诱导的乳脂合成中的相应分子机制。我们发现亮氨酸和蛋氨酸刺激了 CRTC2，但不刺激 p-CRTC2（Ser171）的表达和核定位。在奶牛乳腺上皮细胞中，CRTC2 的敲低降低了乳脂合成和固醇调节元件结合蛋白 1c（SREBP-1c）的表达和激活，而其过表达则产生相反的效果。CRTC2 的敲低或过表达均不影响β-酪蛋白的合成和雷帕霉素的靶蛋白（mTOR）的磷酸化，这表明 CRTC2 仅调节乳脂合成。CRTC2 的敲低消除了亮氨酸和蛋氨酸对 SREBP-1c 表达和激活的刺激。CRTC2 的敲低或过表达均不影响 cAMP 反应元件结合蛋白（CREB）的蛋白水平及其磷酸化，但分别降低或增加了 p-CREB 与基因启动子的结合及其 mRNA 的表达。CRTC2 的 Ser171 突变不改变 CRTC2 对 SREBP-1c 表达和激活的刺激作用，进一步表明 CRTC2 在核内发挥作用。雷帕霉素对 mTOR 的抑制完全阻断了亮氨酸和蛋氨酸对 CRTC2 表达的刺激作用。在哺乳期的小鼠乳腺中，CRTC2 的表达显著高于干乳期和青春期，而 p-CRTC2（Ser171）没有变化，进一步支持 CRTC2 是乳脂合成的关键转录共激活因子。这些结果揭示了 CRTC2 是乳脂合成中氨基酸刺激 mTOR 介导的关键转录共激活因子。
