Effects of Essential Amino Acid Deficiency on General Control Nonderepressible 2/Eukaryotic Initiation Factor 2 Signaling and Proteomic Changes in Primary Bovine Mammary Epithelial Cells

Abstract

We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal−Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA−Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2−99.8% viability indicated low cell death rates. Proliferation (range, 1.02−1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.

Keywords: caseins; epithelial cells; eukaryotic initiation factor 2; homeostasis; protein biosynthesis.

Figure 1

Casein expression in MECs (mean ± SD). Cells were cultured for 6 days after reaching confluence. EAAs were added at concentrations of 2% and 100% to DMEM for 8 and 24 h, followed by refeeding with 100% EAAs for 8 and 24 h. The dark gray column represents the average level of casein expression in cells not depleted (100% EAA, control) and cultured for 8, 24, and 48 h. MEC: mammary epithelial cell; SD: standard deviation; EAA: essential amino acid.

Figure 2

Total and phosphorylated GCN2 expression in MECs cultured for 6 days. EAAs were added at concentrations of 2% (starvation) and 100% (cells not depleted: control, C+) for 8 and 24 h, followed by refeeding with 100% EAAs for 8 and 24 h. Columns with different letters indicate significant differences. GCN2: general control nonderepressible 2; MEC: mammary epithelial cell; EAA: essential amino acid.

Figure 3

Total and phosphorylated eIF2 expression in MECs cultured for 6 days. EAAs were added at concentrations of 2% (starvation) and 100% (cells not depleted: control) for 8 and 24 h, followed by refeeding with 100% EAAs for 8 and 24 h. Columns with different letters indicate significant differences. eIF2: eukaryotic initiation factor 2; MEC: mammary epithelial cell; EAA: essential amino acid.