title: Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells.
abstract: Alpha-ketoglutarate (AKG), a key intermediate in the Krebs cycle, has been reported to promote protein synthesis through activating mechanistic targeting of rapamycin (mTOR) in enterocytes. The study tested the hypothesis that AKG may enhance growth and milk protein synthesis in porcine mammary epithelial cells (PMECs). PMECs were cultured for 96 h in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing prolactin (2 µg/ml) and AKG (0 or 1.5 mM). At the end of 96-h culture, the abundance of apoptosis-related proteins (caspase-3, caspase-9), milk-specific proteins (α-lactalbumin and β-casein), mTOR signaling proteins (mTOR, p-mTOR, PERK, p-PERK, eIF2a, P70S6K and p-P70S6K), and endoplasmic reticulum stress (ERS)-associated proteins (BiP and CHOP) in PMEC were determined. Addition of AKG dose-dependently enhanced cell viability in the absence or presence of prolactin, with optimal concentrations of AKG being at 1.0 and 1.5 mM, respectively. In the presence of prolactin, addition of 1.5 mM AKG: (1) decreased (P < 0.05) the abundance of caspase-3 and caspase-9 by 21 and 39 %; (2) enhanced (P < 0.05) the phosphorylation of p-mTOR and p-P70S6K by 39 and 89 %, respectively; (3) increased (P < 0.05) the production of β-casein and α-lactalbumin by 16 and 20 %, respectively; (4) attenuated (P < 0.05) the expression of CHOP by 34 % but promoted (P < 0.05) the expression of BiP by 46 %; (5) increased (P < 0.05) the secretion of lactose by 15 %, when compared to the 0 mM AKG group. Rapamycin (50 nM; an inhibitor of mTOR) attenuated (P < 0.05) the stimulatory effect of AKG on mTOR signaling and syntheses of milk protein and lactose, while relieving (P < 0.05) an inhibitory effect of AKG on expression of proteins related to ERS. Collectively, our results indicate that AKG enhances milk protein production by modulating mTOR and ERS signaling pathways in PMECs.
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|University of the Chinese Academy of Science, Beijing, 10008, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|University of the Chinese Academy of Science, Beijing, 10008, China.
authors.forename: Liuqin
authors.initials: L
authors.lastname: He
authors.affiliation: Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan, 430023, China.
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan, 410125, China.
authors.forename: Jiashun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|University of the Chinese Academy of Science, Beijing, 10008, China.
authors.forename: Yehui
authors.initials: Y
authors.lastname: Duan
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|TRS Group, Zhuzhou, 412000, China.
authors.forename: Dun
authors.initials: D
authors.lastname: Deng
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan, 430023, China.|Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA.
authors.forename: Guoyao
authors.initials: G
authors.lastname: Wu
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China.|College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan, 410125, China.|Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha, Hunan, 410128, People's Republic of China.
authors.forename: Yulong
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, 410125, China. yaokang@isa.ac.cn.|Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha, Hunan, 410128, People's Republic of China. yaokang@isa.ac.cn.
authors.forename: Kang
authors.initials: K
authors.lastname: Yao
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D008894:Milk Proteins / Q000096:biosynthesis*; D014176:Protein Biosynthesis / Q000187:drug effects*; D013552:Swine
chemical_list: D007656:Ketoglutaric Acids; D008894:Milk Proteins
keywords: Alpha-ketoglutarate; ERS; Lactation; PMECs; mTOR