Journal List > J Nutr Health > v.48(1) > 1081430

J Nutr Health. 2015 Feb;48(1):9-18. Korean.
Published online February 28, 2015.  https://doi.org/10.4163/jnh.2015.48.1.9
© 2015 The Korean Nutrition Society
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.
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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.
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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.
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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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Tables


Table 1
PCR primer sets and expected sizes of PCR products used in the experiment
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Table 2
PCR condition of each primer sets
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Table 3
The contents of total polyphenol and flavonoid of Poncirus trifoliata water extract (PF-W)
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Table 4
The half maximal inhibitory concentration (IC50) of Poncirus trifoliata water extract (PF-W)
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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).

References
1. Spiegelman BM, Flier JS. Adipogenesis and obesity: rounding out the big picture. Cell 1996;87(3):377–389.
2. Kopelman PG. Obesity as a medical problem. Nature 2000;404(6778):635–643.
3. Visscher TL, Seidell JC. The public health impact of obesity. Annu Rev Public Health 2001;22:355–375.
4. Chaput JP, St-Pierre S, Tremblay A. Currently available drugs for the treatment of obesity: Sibutramine and orlistat. Mini Rev Med Chem 2007;7(1):3–10.
5. Collins P, Williams G. Drug treatment of obesity: from past failures to future successes? Br J Clin Pharmacol 2001;51(1):13–25.
6. Li M, Cheung BM. Pharmacotherapy for obesity. Br J Clin Pharmacol 2009;68(6):804–810.
7. Vickers SP, Cheetham SC, Headland KR, Dickinson K, Grempler R, Mayoux E, Mark M, Klein T. Combination of the sodium-glucose cotransporter-2 inhibitor empagliflozin with orlistat or sibutramine further improves the body-weight reduction and glucose homeostasis of obese rats fed a cafeteria diet. Diabetes Metab Syndr Obes 2014;7:265–275.
8. Bae JS, Kim TH. Pancreatic lipase inhibitory and antioxidant activities of Zingiber officinale extracts. Korean J Food Preserv 2011;18(3):390–396.
9. Kumar P, Bhandari U, Jamadagni S. Fenugreek seed extract inhibit fat accumulation and ameliorates dyslipidemia in high fat diet-induced obese rats. Biomed Res Int 2014;2014:606021.
10. Wang H, Eckel RH. Lipoprotein lipase: from gene to obesity. Am J Physiol Endocrinol Metab 2009;297(2):E271–E288.
11. Braun JE, Severson DL. Regulation of the synthesis, processing and translocation of lipoprotein lipase. Biochem J 1992;287(Pt 2):337–347.
12. Fernández-Sánchez A, Madrigal-Santillán E, Bautista M, Esquivel-Soto J, Morales-González A, Esquivel-Chirino C, Durante-Montiel I, Sánchez-Rivera G, Valadez-Vega C, Morales-González JA. Inflammation, oxidative stress, and obesity. Int J Mol Sci 2011;12(5):3117–3132.
13. Moreno DA, Ilic N, Poulev A, Brasaemle DL, Fried SK, Raskin I. Inhibitory effects of grape seed extract on lipases. Nutrition 2003;19(10):876–879.
14. Baek J, Lee J, Kim K, Kim T, Kim D, Kim C, Tsutomu K, Ochir S, Lee K, Park CH, Lee YJ, Choe M. Inhibitory effects of Capsicum annuum L. water extracts on lipoprotein lipase activity in 3T3-L1 cells. Nutr Res Pract 2013;7(2):96–102.
15. Lee E. Antihyperlipidemic and antioxidant effects of Poncirus trifoliata. Korean J Plant Resour 2006;19(2):273–276.
16. Shim WS, Back H, Seo EK, Lee HT, Shim CK. Long-term administration of an aqueous extract of dried, immature fruit of Poncirus trifoliata (L.) Raf. suppresses body weight gain in rats. J Ethnopharmacol 2009;126(2):294–299.
17. Oh SD, Kim M, Min BI, Choi GS, Kim SK, Bae H, Kang C, Kim DG, Park BJ, Kim CK. Effect of achyranthes bidentata blume on 3T3-L1 adipogenesis and rats fed with a high-fat diet. Evid Based Complement Alternat Med 2014;2014:158018.
18. Lee SM, Kang YH, Kim DJ, Kim KK, Lim JG, Kim TW, Choe M. Comparison of antioxidant and α-glucosidase inhibition activities among water extracts and sugar immersion extracts of green pepper, purslane and shiitake. J East Asian Soc Diet Life 2014;24(1):101–108.
19. Feng Z, Hai-ning Y, Xiao-man C, Zun-chen W, Sheng-rong S, Das UN. Effect of yellow capsicum extract on proliferation and differentiation of 3T3-L1 preadipocytes. Nutrition 2014;30(3):319–325.
20. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev 1998;56(11):317–333.
21. Lee GW, Park SM, Yoo YC, Cho YH. Effect of ponciri fructus extracts fermented with ganoderma lucidum on the collagen synthesis and expression of matrix metalloproteinase-1. Korean J Biotechnol Bioeng 2013;28(2):106–114.
22. Klinger SC, Glerup S, Raarup MK, Mari MC, Nyegaard M, Koster G, Prabakaran T, Nilsson SK, Kjaergaard MM, Bakke O, Nykjær A, Olivecrona G, Petersen CM, Nielsen MS. SorLA regulates the activity of lipoprotein lipase by intracellular trafficking. J Cell Sci 2011;124(Pt 7):1095–1105.
23. Hirayama S, Bujo H, Yamazaki H, Kanaki T, Takahashi K, Kobayashi J, Schneider WJ, Saito Y. Differential expression of LR11 during proliferation and differentiation of cultured neuroblastoma cells. Biochem Biophys Res Commun 2000;275(2):365–373.
24. Kim EY, Baik IH, Kim JH, Kim SR, Rhyu MR. Screening of the antioxidant activity of some medicinal plants. Korean J Food Sci Technol 2004;36(2):333–338.
25. Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727–747.
26. Kim EJ, Choi JY, Yu M, Kim MY, Lee S, Lee BH. Total polyphenols, total flavonoid contents, and antioxidant activity of Korean natural and medicinal plants. Korean J Food Sci Technol 2012;44(3):337–342.
27. Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. ScientificWorldJournal 2013;2013:162750.
28. Goldberg IJ, Merkel M. Lipoprotein lipase: physiology, biochemistry, and molecular biology. Front Biosci 2001;6:D388–D405.
29. Wu LG, Hamid E, Shin W, Chiang HC. Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu Rev Physiol 2014;76:301–331.
30. Söllner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE. SNAP receptors implicated in vesicle targeting and fusion. Nature 1993;362(6418):318–324.
31. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev 2001;81(1):153–208.
32. Hermey G. The Vps10p-domain receptor family. Cell Mol Life Sci 2009;66(16):2677–2689.
33. Nielsen MS, Jacobsen C, Olivecrona G, Gliemann J, Petersen CM. Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase. J Biol Chem 1999;274(13):8832–8836.