Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated β-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells

Abstract

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucineenriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced β-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of β-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Leucine-Mediated SLC7A5 Promotes Milk Protein and Milk Fat Synthesis through mTOR Signaling Pathway in Goat Mammary Epithelial Cells

“…Overexpression and interference experiments were conducted using Lipofectamine 2000 (Invitrogen) and Lipofectamine RNAiMAX Reagent (Invitrogen), respectively. The GMECs were cotreated with an AA mix and siRNA-SLC7A5 or with Leu (0.8 mmol/L) and siRNA-SLC7A5 for 0, 5, 10, and 15 min, respectively . The AA premix was prepared at the following ratios: arginine 0.60 mmol/L; cystine 0.10 mmol/L; histidine 0.20 mmol/L; isoleucine 0.40 mmol/L; leucine 0.40 mmol/L; lysine 0.40 mmol/L; methionine 0.10 mmol/L; phenylalanine 0.20 mmol/L; threonine 0.40 mmol/L; tryptophan 0.05 mmol/L; tyrosine 0.20 mmol/L; valine 0.40 mmol/L.…”

“…BODIPY staining was performed according to previous research . Briefly, cells were fixed for 30 min at 4 °C using 4% paraformaldehyde.…”