J Nutr Health.  2015 Feb;48(1):9-18. 10.4163/jnh.2015.48.1.9.

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

• Lee SM
• Kang YH
• Kim KK
• Kim TW
• Choe M

• KMID: 2327172
• DOI: http://doi.org/10.4163/jnh.2015.48.1.9

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/EBPbeta)
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/EBPbeta, 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/EBPbeta-mediated induction of SorLA expression.

Keyword

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

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
Reverse Transcription
RNA, Messenger
Transcription Factors
Water*
CCAAT-Enhancer-Binding Proteins
Lipoprotein Lipase
RNA, Messenger
Transcription Factors
Water

Figure

• 

  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.

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