Sung Mee Lee; Yun Hwan Kang; Kyoung Kon Kim; Tae Woo Kim; Myeon Choe

doi:10.4163/jnh.2015.48.1.9

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Lee, Kang, Kim, Kim, and Choe: A study of the lipoprotein lipase inhibitory mechanism of Poncirus trifoliata water extracts∗

Research Article

J Nutr Health 2015;48(1):9-18.

Published online: 19 January 2015

DOI: https://doi.org/10.4163/jnh.2015.48.1.9

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

Sung Mee Lee^1,^∗∗, Yun Hwan Kang^2,^∗∗, Kyoung Kon Kim¹, Tae Woo Kim², Myeon Choe^1,^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, e-mail: mchoe@kangwon.ac.kr

^∗ 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).

^∗ These authors contributed equally to this work.

Received 4 November 2014 Revised 4 December 2014 Accepted 16 December 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

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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.

Table 1.

PCR primer sets and expected sizes of PCR products use in the experiment

Gene		Primer (5'-3')	Size (bp)
LPL	forward	ATC CAT GGA TGG ACG GTA AC	483
LPL	reverse	CTG GAT CCC AAT ACT TCG AC	483
v-SNARE	forward	AAT TCC ACC CCA AAG TCG AG	224
v-SNARE	reverse	ACC CAG CCT CTA GTC TCC GA	224
Rab3A	forward	CTA CCG CAA CGA CAA GAG GA	221
Rab3A	reverse	CAC ACT TGT TTC CCA CCA GC	221
Sortilin	forward	ACA AAG ACG GCT GCA TTT TG	282
Sortilin	reverse	GGC ATT TGT CTC CTG GGA TT	282
SorLA	forward	CAG CAG TCC ACC AGC TCC TA	194
SorLA	reverse	ACT GCT CAT CCT TGC TGA CG	194
C/EBPβ	forward	CTG AGC GAC GAG TAC AAG AT	445
C/EBPβ	reverse	TTG ATC CGG ATT GCA TCA AG	445
GAPDH	forward	GGA GCC AAA AGG GTC ATC AT	203
GAPDH	reverse	GTG ATG GCA TGG ACT GTG GT	203

Table 2.

PCR condition of each primer sets

Gene	Pre-denaturation	Denaturation	Annealing	Extension	Final extension
LPL	95^oC 5 min	95^oC, 30 sec	56^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
LPL	95^oC 5 min	30 cycle			72^oC 5 min
v-SNARE	95^oC 5 min	95^oC, 30 sec	55.4^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
v-SNARE	95^oC 5 min	30 cycle			72^oC 5 min
Rab3A	95^oC 5 min	95^oC, 30 sec	57.5^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
Rab3A	95^oC 5 min	30 cycle			72^oC 5 min
Sortilin	95^oC 5 min	95^oC, 30 sec	55^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
Sortilin	95^oC 5 min	30 cycle			72^oC 5 min
SorLA	95^oC 5 min	95^oC, 30 sec	59^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
SorLA	95^oC 5 min	30 cycle			72^oC 5 min
C/EBPβ	95^oC 5 min	95^oC, 30 sec	59^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
C/EBPβ	95^oC 5 min	30 cycle			72^oC 5 min
GAPDH	95^oC 5 min	95^oC, 30 sec	55^oC, 30 sec	72^oC, 30 sec	72^oC 5 min
GAPDH	95^oC 5 min	30 cycle			72^oC 5 min

Table 3.

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

Sample	Total polyphenol (mg/g, GAE¹⁾)	Total flavonoid (mg/g, QE²⁾)
PF-W	52.15 ± 4.02	6.56 ± 0.47

Values are presented as Mean ± SD from three independent experiments.

¹⁾ GAE, gallic acid equivalents

²⁾ QE, quercetin equivalents

Table 4.

The half maximal inhibitory concentration (IC₅₀) of Ponci rus trifoliata water extract (PFW)

Anti-oxidant experiments	IC₅₀ (mg/mL)
Anti-oxidant experiments	Ascorbic acid	PF-W
DPPH radical scavenging activity	0.027	1.931
ABTS radical scavenging activity	0.092	1.536
Reducing power	0.069	2.098
SOD-like activity	0.129	11.53

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