PMID- 29550128 OWN - NLM STAT- MEDLINE DCOM- 20190327 LR - 20190327 IS - 1525-3198 (Electronic) IS - 0022-0302 (Linking) VI - 101 IP - 6 DP - 2018 Jun TI - Increasing the availability of threonine, isoleucine, valine, and leucine relative to lysine while maintaining an ideal ratio of lysine:methionine alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription. PG - 5502-5514 LID - S0022-0302(18)30255-8 [pii] LID - 10.3168/jds.2017-13707 [doi] AB - Amino acids not only serve as precursors for protein synthesis but also function as signaling molecules that can regulate the mammalian target of rapamycin (mTOR) pathway. Methionine and Lys are the most-limiting AA for milk production and a ratio of approximately 3:1 Lys:Met in the metabolizable protein has been determined to be ideal. Besides Met and Lys, recent studies have evaluated Ile, Leu, Val, and Thr as potentially limiting for milk protein synthesis. The objective of this experiment was to determine if varying the ratio of Lys:Thr, Lys:Ile, Lys:Val, and Lys:Leu while maintaining an ideal ratio of Lys:Met and fixed ratio of other essential AA (IPAA) elicits changes in intracellular metabolites, gene transcription related to protein synthesis, and phosphorylation status of mTOR pathway proteins. Immortalized bovine mammary epithelial cell line (MAC-T) cells were incubated for 12 h (n = 5 replicates/treatment) with IPAA (2.9:1 Lys:Met; 1.8:1 Lys:Thr; 2.38:1 Lys:His; 1.23:1 Lys:Val; 1.45:1 Lys:Ile; 0.85:1 Lys:Leu; 2.08:1 Lys:Arg) or IPAA supplemented with Thr, Ile, Val, and Leu to achieve a Lys:Thr 1.3:1 (LT1.3), Lys:Ile 1.29:1 (LI1.29), Lys:Val 1.12:1 (LV1.12), or Lys:Leu 0.78:1 (LL0.78). Compared with IPAA, metabolomics via gas chromatography-mass spectrometry revealed that increases in availability of Thr, Ile, Val, and Leu led to greater concentrations of essential AA (Leu, Ile, Thr), nonessential AA (Gly, Glu, Gln, Ser, Pro, Asp), and various metabolites including uric acid, phosphoric acid, N-acetylglutamic acid, and intermediates of glycolysis and the tricarboxylic acid cycle. Compared with other treatments, LV1.12 led to greater phosphorylation status of serine/threonine kinase B (Akt), mTORC1, and ribosomal protein S6 and lower phosphorylation of alpha subunit of eukaryotic translation initiation factor 2. In addition, LV1.12 upregulated abundance of CSN2 and both the abundance and promoter methylation of CSN1S1. Although LI1.29 led to the second highest response in mTORC1 phosphorylation status, it resulted in the lowest phosphorylation of Akt and eEF2 and mRNA abundance of CSN2 and various AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, SLC43A1). Overall, data indicate that an increase in Val at an ideal ratio of Lys:Met could further enhance milk protein synthesis by altering intracellular concentrations of essential AA and metabolites that could play a regulatory role, increasing phosphorylation status of mTORC1 and key signaling proteins, and upregulation of AA transporters. CI - The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association(R). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). FAU - Dong, X AU - Dong X AD - Chongqing Academy of Animal Science, Chongqing 402460, China; Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Animal Nutrition Institute of Sichuan Agricultural University, Ya'an, 625014, Sichuan Province, China. FAU - Zhou, Z AU - Zhou Z AD - Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801; Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634. FAU - Wang, L AU - Wang L AD - Chongqing Academy of Animal Science, Chongqing 402460, China; Southwest University, Rongchang, Chongqing 402460, China. FAU - Saremi, B AU - Saremi B AD - Evonik Nutrition and Care GmbH, 63457 Hanau-Wolfgang, Germany. FAU - Helmbrecht, A AU - Helmbrecht A AD - Evonik Nutrition and Care GmbH, 63457 Hanau-Wolfgang, Germany. FAU - Wang, Z AU - Wang Z AD - Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Animal Nutrition Institute of Sichuan Agricultural University, Ya'an, 625014, Sichuan Province, China. FAU - Loor, J J AU - Loor JJ AD - Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801. Electronic address: jloor@illinois.edu. LA - eng PT - Journal Article DEP - 20180315 PL - United States TA - J Dairy Sci JT - Journal of dairy science JID - 2985126R RN - 0 (Peptide Fragments) RN - 04Y7590D77 (Isoleucine) RN - 2ZD004190S (Threonine) RN - AE28F7PNPL (Methionine) RN - GMW67QNF9C (Leucine) RN - HG18B9YRS7 (Valine) RN - K3Z4F929H6 (Lysine) RN - W36ZG6FT64 (Sirolimus) SB - IM MH - Amino Acid Sequence MH - Animals MH - Cattle/genetics/*metabolism MH - Female MH - Isoleucine/metabolism MH - Leucine/metabolism MH - Lysine/*metabolism MH - Mammary Glands, Animal/*metabolism MH - Methionine/*metabolism MH - Peptide Fragments MH - Sirolimus/*metabolism MH - Threonine/metabolism MH - *Transcription, Genetic MH - Valine/metabolism OTO - NOTNLM OT - essential amino acid ratio OT - lactation OT - metabolomics OT - milk protein synthesis EDAT- 2018/03/20 06:00 MHDA- 2019/03/28 06:00 CRDT- 2018/03/19 06:00 PHST- 2017/08/18 00:00 [received] PHST- 2018/02/05 00:00 [accepted] PHST- 2018/03/20 06:00 [pubmed] PHST- 2019/03/28 06:00 [medline] PHST- 2018/03/19 06:00 [entrez] AID - S0022-0302(18)30255-8 [pii] AID - 10.3168/jds.2017-13707 [doi] PST - ppublish SO - J Dairy Sci. 2018 Jun;101(6):5502-5514. doi: 10.3168/jds.2017-13707. Epub 2018 Mar 15.