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Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells

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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.

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Abbreviations

AKG:

Alpha-ketoglutarate

PMECs:

Porcine mammary epithelial cells

ERS:

Endoplasmic reticulum stress

mTOR:

Mammalian target of the rapamycin

UPR:

Unfolded protein response

CHOP:

C/EBP homologous protein

BiP:

Binding immunoglobulin protein

PRL:

Prolactin

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Acknowledgments

This work was supported by National Basic Research Program of China (2013CB127306), National Natural Science Foundation of China (31472107), the Chinese Academy of Sciences through its Hundred Talent Program to Kang Yao, the Hubei Hundred Talent program, and Texas A&M AgriLife Research (H-8200).

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    Authors and Affiliations

    1. 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

      Qian Jiang, Liuqin He, Jiashun Chen, Yehui Duan, Dun Deng, Guoyao Wu, Yulong Yin & Kang Yao

    2. University of the Chinese Academy of Science, Beijing, 10008, China

      Qian Jiang, Liuqin He & Yehui Duan

    3. Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan, 430023, China

      Yongqing Hou & Guoyao Wu

    4. College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan, 410125, China

      Jiashun Chen & Yulong Yin

    5. TRS Group, Zhuzhou, 412000, China

      Dun Deng

    6. Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA

      Guoyao Wu

    7. Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha, Hunan, 410128, People’s Republic of China

      Yulong Yin & Kang Yao

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    1. Qian Jiang
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    2. Liuqin He
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    Correspondence to Kang Yao.

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    This study involved the cultures of an existing cell line and did not require an Animal Use Protocol.

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    Q. Jiang and L. He made equal contributions to this study, so they are joint first authors.

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    Jiang, Q., He, L., Hou, Y. et al. Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells. Amino Acids 48, 2179–2188 (2016). https://doi.org/10.1007/s00726-016-2249-5

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