Dataset Information

Amino Acid Metabolomic Profiles in Bovine Mammary Epithelial Cells under Essential Amino Acid Restriction.

ABSTRACT: Mammary epithelial cells (MECs) in culture are a useful model for elucidating mammary gland metabolism and changes that occur under different nutrient disponibility. MECs were exposed to different treatments: 100% EAA for 8 h and 24 h restriction (R); 2% EAA for 8 h and 24 h R; 2% EAA for 8 h and 24 h + 100% EAA for 8 h and 24 h restriction + re-feeding (R + RF). Western blotting and protein quantification was performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) software identified the amino acids (AAs) and signaling pathways. The chi-squared test, multiple classification analysis, and analysis of variance were used for the purification and identification of data. Intracellular casein levels were not affected. The KEGG analysis revealed that the important pathways of metabolism of AAs, which were involved in processes related to metabolism and biosynthesis of phenylalanine, tyrosine, and tryptophan (fumarate, acetyl-CoA, and tricarboxylic acid (TCA) cycle), were affected by both R and R + RF treatments, mainly through the glutamic-oxaloacetic transaminase-2 enzyme. Additionally, metabolic processes mediated by the mitochondrial malate dehydrogenase, S-adenosylmethionine synthetase, and asparagine synthase proteins positively regulated the carbohydrate pathway, pyruvate, and TCA cycles, as well as the metabolism of alanine, aspartate, and glutamate metabolism (carbohydrate and TCA cycle). We hypothesized that MECs have the capacity to utilize alternative pathways that ensure the availability of substrates for composing milk proteins.

SUBMITTER: Lopez-Diez L

PROVIDER: S-EPMC8151660 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description: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.

Dataset Information

Amino Acid Metabolomic Profiles in Bovine Mammary Epithelial Cells under Essential Amino Acid Restriction.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets