Preprint Article Version 1 This version is not peer-reviewed

Supplementing L-Isoleucine Increases Medium Protein and Alters the Expression of Genes and Proteins Involved in Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells

Version 1 : Received: 5 June 2019 / Approved: 7 June 2019 / Online: 7 June 2019 (14:55:03 CEST)

How to cite: Conejos, J.R.; Lee, J.; Park, J.; Moon, J.; Lee, H. Supplementing L-Isoleucine Increases Medium Protein and Alters the Expression of Genes and Proteins Involved in Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells. Preprints 2019, 2019060063 (doi: 10.20944/preprints201906.0063.v1). Conejos, J.R.; Lee, J.; Park, J.; Moon, J.; Lee, H. Supplementing L-Isoleucine Increases Medium Protein and Alters the Expression of Genes and Proteins Involved in Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells. Preprints 2019, 2019060063 (doi: 10.20944/preprints201906.0063.v1).

Abstract

The objective of this study was to determine the effects of supplementing L-isoleucine (L-Ile) on milk protein synthesis, using an immortalized bovine mammary epithelial (MAC-T) cell line. In this case, the cells were treated with 0, 0.3, 0.6, 0.9, 1.2 and 1.5 mM of supplemental Isoleucine (Ile), and the most efficient time for protein synthesis for each amino acid was determined by measuring the cell, medium and total protein at 0, 24, 48, 72 and 96 h. Confirmatory tests showed that 48h incubation time and 0.6 mM dosage of L-Ile are considered as the optimal time and dosage. The mechanism of milk protein synthesis was elucidated through proteomics analysis to clarify the metabolic pathway. When the L-Ile was supplemented, extracellular protein (medium protein) reached a peak at 48h, whereas in the case of the intracellular cell protein, it was shown to have reached to its peak at 24h in all L-Ile dosage treatments. In total, it is noted that there were 63 upregulated and 52 downregulated proteins. The results of the protein pathway analysis showed that the L-Ile group stimulated insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase cascade, insulin/IGF pathway-protein kinase B signaling cascade, p53 pathway, de novo purine biosynthesis, Wnt signaling pathway, glycolysis, pentose phosphate pathway, and ATP synthesis which are pathways involved and related to protein and energy metabolism. Together, these results demonstrate that L-Ile supplementation was effective in stimulating β-casein synthesis by stimulating genes and pathways which are significantly related to protein and energy metabolism.

Subject Areas

beta casein; MAC-T cells; Ile; milk protein synthesis; omics; proteomics

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