mTORC2‐AKT‐LAT1 signalling participates in methionine‐induced β‐CASEIN expression in mammary epithelial cells of dairy cows

Abstract

AbstractThis study investigated the role of the mammalian target of rapamycin complex 2 (mTORC2)‐protein kinase B (AKT) signalling in methionine (Met)‐induced L‐type amino acid transporter 1 (LAT1) expression and milk protein production. Primary mammary epithelial cells (MECs) from mammary parenchymal tissues of three lactating cows and MAC‐T bovine MECs were cultured with or without 0.6 mM Met. Rapamycin‐insensitive companion of mTOR (RICTOR) siRNA, the mTORC1 inhibitor rapamycin and the AKT activator SC79 were used to evaluate the effects of mTORC2‐AKT signalling on Met‐induced LAT1 expression and function. Each experiment was performed three times. Data were analysed with a two‐sided unpaired t test or ANOVA with the Bonferroni multiple‐comparison test. Western blotting showed that Met stimulation increased RICTOR expression (~244.67%; p < 0.05; control, 0.15 ± 0.026; Met, 0.517 ± 0.109) and AKT‐S473 levels (~281.42%; p < 0.01; control, 0.253 ± 0.067; Met, 0.965 ± 0.019) in both primary MECs and MAC‐T cells. Rapamycin‐induced mTORC1 signalling inhibition decreased only Met‐induced β‐CASEIN expression by ~21.24% (p < 0.01; Met, 0.777 ± 0.01; Met and rapamycin, 0.612 ± 0.04) and did not affect Met‐stimulated AKT‐S473 levels, suggesting that mTORC2‐AKT activation upon Met stimulation also contributes to milk protein synthesis. LAT1 participates in Met‐induced β‐CASEIN expression. In dairy cow MECs, mTORC2 inhibition by RICTOR siRNA decreased LAT1 levels on the plasma membrane by ~45.13% (p < 0.01; control, 0.359 ± 0.006; siRICTOR, 0.197 ± 0.004). However, SC79‐induced AKT activation had the opposite effect (p < 0.01). In primary MECs and MAC‐T cells, Met stimulation increased cytosolic and plasma membrane LAT1 expression respectively (MECs, 113.98% and 58.43%; MAC‐T, 165.85% and 396.39%; p < 0.05). However, RICTOR siRNA significantly reduced Met‐induced plasma membrane LAT1 expression (~76.48%; Met, 0.539 ± 0.05; Met and siRICTOR, 0.127 ± 0.012; p < 0.05). Thus, Met increased LAT1 expression and function via mTORC2‐AKT signalling, upregulating milk protein synthesis in dairy cow MECs.