PMID- 36083345 OWN - NLM STAT- MEDLINE DCOM- 20221125 LR - 20221125 IS - 1438-2199 (Electronic) IS - 0939-4451 (Linking) VI - 54 IP - 11 DP - 2022 Nov TI - Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells. PG - 1491-1504 LID - 10.1007/s00726-022-03203-y [doi] AB - Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37 degrees C in customized Dulbecco's modified Eagle medium containing 5 mmol/L D-glucose, 1 mmol/L L-phenylalanine, L-[ring-2,4-(3)H]phenylalanine, 0.1 (control), 0.25, 0.5, 1, or 2 mmol/L L-leucine, L-isoleucine or L-valine or an equimolar mixture of the three BCAAs. The culture medium also contained physiological concentrations of other amino acids found in the plasma of lactating sows. Proliferation, protein synthesis, proteolysis, beta-casein production, the mechanistic target of rapamycin (mTOR) signaling, and the ubiquitin-proteasome pathway were determined for PMECs. Cell proliferation and abundances of phosphorylated mTOR, eukaryotic translation initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase beta-1 proteins increased (P < 0.05), but abundances of ubiquitinated protein and 20S proteasome decreased (P < 0.05) when extracellular concentrations of L-leucine, L-isoleucine, L-valine, or an equimolar mixture of BCAAs were increased from 0.1 to 2 mmol/L. Compared with the control, 0.25, 0.5, 1 or 2 mmol/L BCAAs enhanced (P < 0.01) protein (including beta-casein) synthesis, while decreasing (P < 0.05) proteolysis in PMECs in a dose-dependent manner. Collectively, our results indicate that physiological concentrations of BCAAs regulate protein turnover in mammary epithelial cells to favor net protein synthesis through stimulating the mTOR signaling pathway and inhibiting the ubiquitin-proteasome pathway. CI - (c) 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature. FAU - Rezaei, Reza AU - Rezaei R AD - Department of Animal Science, Texas A&M University, College Station, Texas, 77843, USA. FAU - Wu, Guoyao AU - Wu G AUID- ORCID: 0000-0001-8058-6969 AD - Department of Animal Science, Texas A&M University, College Station, Texas, 77843, USA. g-wu@tamu.edu. LA - eng GR - H-8200/Texas A&M AgriLife Research/ PT - Journal Article DEP - 20220909 PL - Austria TA - Amino Acids JT - Amino acids JID - 9200312 RN - 0 (Amino Acids, Branched-Chain) RN - GMW67QNF9C (Leucine) RN - 0 (Caseins) RN - 04Y7590D77 (Isoleucine) RN - EC 3.4.25.1 (Proteasome Endopeptidase Complex) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - HG18B9YRS7 (Valine) RN - 0 (Ubiquitin) SB - IM MH - Swine MH - Female MH - Animals MH - *Mammary Glands, Animal MH - *Amino Acids, Branched-Chain/metabolism MH - Proteolysis MH - Leucine/pharmacology/metabolism MH - Caseins MH - Isoleucine/metabolism MH - Lactation MH - Proteasome Endopeptidase Complex/metabolism MH - TOR Serine-Threonine Kinases/metabolism MH - Epithelial Cells/metabolism MH - Valine/metabolism MH - Ubiquitin/metabolism OTO - NOTNLM OT - Isoleucine OT - Lactation OT - Leucine OT - Nutrition OT - Pigs OT - Protein turnover OT - Valine EDAT- 2022/09/10 06:00 MHDA- 2022/11/26 06:00 CRDT- 2022/09/09 11:14 PHST- 2022/03/26 00:00 [received] PHST- 2022/08/23 00:00 [accepted] PHST- 2022/09/10 06:00 [pubmed] PHST- 2022/11/26 06:00 [medline] PHST- 2022/09/09 11:14 [entrez] AID - 10.1007/s00726-022-03203-y [pii] AID - 10.1007/s00726-022-03203-y [doi] PST - ppublish SO - Amino Acids. 2022 Nov;54(11):1491-1504. doi: 10.1007/s00726-022-03203-y. Epub 2022 Sep 9.