Open Access, Peer-reviewed
pISSN 2288-3886
eISSN 2288-3959
    J Nutr Health. 2015 Feb;48(1):9-18. Korean.
    Published online Feb 28, 2015.
    https://doi.org/10.4163/jnh.2015.48.1.9
    © 2015 The Korean Nutrition Society
    Original Article

    A study of the lipoprotein lipase inhibitory mechanism of Poncirus trifoliata water extracts

    Sung Mee Lee,1,** Yun Hwan Kang,2,** Kyoung Kon Kim,1 Tae Woo Kim,2 and Myeon Choe1,2
      • 1Department of Bio-Health Technology, Kangwon National University, Gangwon 200-701, Korea.
      • 2Well-being Bioproducts RIC, Kangwon National University, Gangwon 200-701, Korea.
    • To whom correspondence should be addressed. tel: +82-33-250-8645, Email: mchoe@kangwon.ac.kr

      **These authors contributed equally to this work.
    Received November 04, 2014; Revised December 04, 2014; Accepted December 16, 2014.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Purpose

    Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W.

    Methods

    We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein (C/EBPβ)

    Results

    The total polyphenol and flavonoid content of PF-W was 52.15 ± 4.02 and 6.56 ± 0.47 mg/g, respectively. PF-W treatment decreased LPL content in media to 58 ± 5% of that in control adipocyte media, and increased LPL content to 117 ± 3.5% of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and time-dependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of C/EBPβ, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes.

    Conclusion

    The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through C/EBPβ-mediated induction of SorLA expression.

    Keywords
    Poncirus trifoliata; lipoprotein lipase; sortilin-related receptor; CCAAT-enhancer-binding proteins (C/EBP) β; anti-obesity

    Figures

    Fig. 1
    Effect of a water extract of the dried, immature fruit of Poncirus trifoliata (PF-W) on the viability of 3T3-L1 preadipocytes. 3T3-L1 cells were treated with PF-W (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, or 1 mg/mL) for 24 h. Cell viability was determined using the Cell Counting Kit (CCK)-8. Data are expressed as Mean ± SD of triplicate experiments.

    Fig. 2
    Effect of a water extract of the dried, immature fruit of Poncirus trifoliata (PF-W) on the lipoprotein lipase (LPL) expression and activity in 3T3-L1 adipocytes. A: LPL content of 3T3-L1 cell culture medium. B: Comparison of LPL activities in cell extract with and without 0.25 mg/mL PF-W. C: LPL mRNA expression as a function of PF-W concentration and time of exposure. D: LPL content in 3T3-L1 cell extract. Data are expressed as Mean ± SD of triplicate experiments. #: p < 0.05, ##: p < 0.01 as compared to the preadipocytes. *: p < 0.05, **: p < 0.01 compared to control adipocytes.

    Fig. 3
    Effect of a water extract of the dried, immature fruit of Poncirus trifoliata (PF-W) on the mRNA expression levels of protein transport-related biomarkers in 3T3-L1 adipocytes. 3T3-L1 cells were treated with PF-W (0.25 mg/mL) for 6 and 12 h. A: v-SNARE mRNA expression. B: Rab3A mRNA expression. C: Sortilin (SorLA) mRNA expression. D: SorLA mRNA expression. Data are expressed as Mean ± SD of triplicate experiments. #: p < 0.05, ##: p < 0.01 compared to the preadipocyte. *: p < 0.05 compared to the adipocyte.

    Fig. 4
    Effect of a water extract of the dried, immature fruit of Poncirus trifoliata (PF-W) on C/EBPβ expression in 3T3-L1 adipocytes. 3T3-L1 cells were treated with PF-W (0.25 mg/mL) for 12 h. A: C/EBPβ mRNA expression by PF-W treatment. B: C/EBPβ protein expression in cytoplasmic and nuclear protein fraction by PF-W treatment. Data are expressed as Mean ± SD of triplicate experiments. *: p < 0.05 and **: p < 0.01.

    Tables

    Table 1
    PCR primer sets and expected sizes of PCR products used in the experiment

    Table 2
    PCR condition of each primer sets

    Table 3
    The contents of total polyphenol and flavonoid of Poncirus trifoliata water extract (PF-W)

    Table 4
    The half maximal inhibitory concentration (IC50) of Poncirus trifoliata water extract (PF-W)

    Notes

    This work was supported by grants of High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (312001-03-01-HD040), Well-being Bioproducts Regional Innovation Center project (B0009702) and Kangwon National University Institute of Bioscience & Biotechnology (320130015).

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    MeSH Terms
    Adipocytes
    Biomarkers
    Blotting, Western
    CCAAT-Enhancer-Binding Proteins
    Cell Fractionation
    Endocytosis
    Enzyme-Linked Immunosorbent Assay
    Fruit
    Lipoprotein Lipase*
    Polymerase Chain Reaction
    Poncirus*
    Protein Transport
    RNA, Messenger
    Reverse Transcription
    Transcription Factors
    Water*
    Metrics
    Share
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    Tables
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    Funding Information
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