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In Vitro Supplementation of Leucine Increases Murine HC11 Cell Proliferation
Author(s) -
McGuckin Molly Mae,
Suryawan Agus,
Davis Teresa A,
Peterson Daniel G,
Manjarin Rodrigo
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.448.6
Subject(s) - ribosomal protein s6 , pi3k/akt/mtor pathway , cell growth , biology , microbiology and biotechnology , cyclin d2 , cyclin d1 , signal transduction , cell , cell cycle , p70 s6 kinase 1 , biochemistry
Mammary tissue undergoes multiples cycles of cell growth and proliferation during the lifetime of the female to meet the nutrient demand of the suckling neonate. On the basis of studies in rodents, pigs and humans demonstrating the anabolic effects of leucine (Leu) through the activation of mammalian target of rapamycin (mTOR) signaling pathway, there has been recent interest in the potential of supplementary Leu to improve skeletal muscle protein synthesis and overall nitrogen retention. To determine whether Leu supplementation also upregulates mTOR pathway in mammary tissue and increases mammary cell proliferation, a murine mammary cell line (HC11) was cultured in different concentrations of Leu (0, 1, and 2 mM). Cell proliferation was measured using flow cytometry at 0, 8, and 16 days. Reverse transcription quantitative PCR was performed to measure mRNA abundance of mammary Leu transporter LAT1 ( SLC7A5 ), the intracellular Leu sensor for the mTOR signaling pathway leucyl‐tRNA synthetase ( LARS2 ), mTOR and its downstream effector ribosomal protein S6 kinase ( RPS6K1 ), and cyclin D ( CCND1 ), a marker for cell proliferation. Guanine phosphoribosyl transferase ( HPRT ), RNA polymerase 3 ( POL3 ), and ribosomal protein L3a ( RPL3a ) were used as reference genes. Statistical analysis was performed by ANOVA using a linear mixed model to account for treatment and replication effects. After 16 d cell proliferation increased in response to 1 mM Leu compared to 0 mM ( P < 0.05), and increased further ( P < 0.05) in cells supplemented with 2 mM Leu. Gene expression of SLC7A5 , LARS2 , mTOR , RPS6K1 , and CCND1 did not differ between groups. Taken together, these results suggest that Leu supplementation increases mammary cell proliferation in vitro , but this effect is not associated with an increase in mRNA abundance of aforementioned genes. Further work is needed to elucidate the mechanisms by which dietary Leu supplementation increases mammary cell proliferation, as it may hold the potential to enhance mammary growth and subsequent milk production in animals. Support or Funding Information ARI 58982