Esculetin, a coumarin derivative, suppresses adipogenesis through modulation of the AMPK pathway in 3T3-L1 adipocytes

journal of functional foods 12 (2015) 509–515

Available online at www.sciencedirect.com

ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Esculetin, a coumarin derivative, suppresses adipogenesis through modulation of the AMPK pathway in 3T3-L1 adipocytes Younghwa Kim, Junsoo Lee * Department of Food Science and Technology, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea

A R T I C L E

I N F O

A B S T R A C T

Article history:

Obesity is one of the most serious health problems in both Westernized and developing coun-

Received 4 June 2014

tries. AMP-activated protein kinase (AMPK) has emerged as a major regulator of appetite,

Received in revised form 28

body weight, and cellular energy balance. In this study, we investigated the effect of escu-

November 2014

letin (ECT) on adipogenesis via activation of AMPK in 3T3-L1 cells. ECT markedly inhibited

Accepted 1 December 2014

lipid accumulation and suppressed the expression of adipogenic specific proteins includ-

Available online 25 December 2014

ing peroxisome proliferator-activated receptors (PPARγ), CCAAT/enhancer binding protein a (C/EBPα), and adipocyte fatty acid binding protein (aP2). Moreover, ECT significantly in-

Keywords:

creased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and intracellular reactive

Esculetin

oxygen species (ROS) production. However, pretreatment with compound C, a specific AMPK

Adipogenesis

inhibitor, abolished the inhibitory effects of ECT on PPARγ and C/EBPα expression. There-

3T3-L1 cells

fore, these results indicate that ECT has anti-adipogenic effects through modulation of PPARγ

AMPK

and C/EBPα via the AMPK signaling pathway. © 2014 Elsevier Ltd. All rights reserved.

Obesity

1.

Introduction

Obesity is a risk factor for numerous diseases and contributes to the development of diseases such as type II diabetes, hypertension, coronary heart disease and cancer (Visscher & Seidell, 2001). Adipocytes play a vital role in regulating adipose tissue and obesity. Differentiation of preadipocyte into adipocytes is modulated through multiple transcription factors such as CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) (Rosen, Walkey, Puigserver, & Spiegelman, 2000). These transcription factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype, including adipocyte fatty

acid binding protein (aP2), lipoprotein lipase (LPL) and leptin (Tontonoz, Hu, Graves, Budavari, & Spiegelman, 1994). The 5′-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway plays an important role in regulating energy storage and expenditure and acts as a sensor for regulating cellular energy metabolism. When activated, AMPK phosphorylates target proteins for the metabolism of lipids and carbohydrate. Acetyl-CoA carboxylase (ACC), one of the substrates of AMPK, is a central enzyme involved in fatty acid oxidation and fatty acid biosynthesis (Lim, Kola, & Korbonits, 2010; Towler & Hardie, 2007). It is also reported that activation of AMPK pathway attenuates PPARγ, C/EBPα, and sterol regulatory element binding protein-1 (SREBP-1) expression, and thus inhibits fat accumulation during adipogenesis (Choi et al.,

* Corresponding author. Department of Food Science and Technology, College of Agriculture, Life, & Environmental Sciences, Chungbuk National University, 52 Naesudong-ro, Heungdeok-gu, Cheong-ju, Chungbuk 361-763, Korea. Tel.: +82 43 261 2566; fax: +82 43 271 4412. E-mail address: [email protected] (J. Lee). http://dx.doi.org/10.1016/j.jff.2014.12.004 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

510

journal of functional foods 12 (2015) 509–515

2011; Hwang, Kwon, & Yoon, 2009). Therefore, AMPK has been considered as a target for the treatment of obesity and metabolic disorders. In trials of obesity prevention, researchers have investigated whether some food components exhibit an ability to suppress lipid accumulation. Recent reports have proposed possible anti-obesity mechanisms of natural phytochemicals involving increased energy expenditure, lipolysis, fat oxidation, decreased pre-adipocyte differentiation, as well as, proliferation and lipogenesis (Hsu & Yen, 2008). Among the natural products, coumarin compounds are attracting interest because of their beneficial effect in human health (Hoult & Miguel, 1996). Coumarin compounds comprise a very large class of phenolic substances found in plants. They are found at high levels in some essential oils including cinnamon bark oil, cassia leaf oil and lavender oil and also found in green tea, chicory and fruits such as bilberry and cloudberry (Lacy & O’Kennedy, 2004). Esculetin (6,7-dihydroxy-2H-1-benzopyran2-one) is a coumarin derivative that is present in various natural plants such as Artemisia scoparia (Redstem Wormwood), Artemesia capillaris (Capillary Wormwood), Ceratostigma willmottianum (Chinese Plumbago) and in the leaves of Citrus limonia (Chinese lemon) (Subramaniam & Ellis, 2011). Previous study has been carried out the antihyperglycemic effect of Matricariaria chamomilla L. plant which has been used as an herbal tea or supplementary food all over the world and one of the major active phytochemical esculetin present in the plant (Atsushi et al., 2008). Esculetin (ECT) has been reported to have beneficial biological and biochemical properties such as antioxidant, anti-proliferative and anti-cancer activities (Egan et al., 1990; Kok, Yeh, Chen, & Kuo, 2009; Lin et al., 2000; Wang, Hsieh, Chu, Lin, & Tseng, 2002). According to previous studies, ECT inhibited proliferation of adipocytes by initiating an apoptotic process in 3T3-L1 cells (Yang, Della-Fera, & Baile, 2006; Yang et al., 2006). However, the molecular mechanism related to the activation of AMPK in lipid metabolism is still unclear. In this study, to reveal the underlying mechanisms of the antiadipogenenic action of ECT, we investigated the effects of ECT on adipogenesis in 3T3-L1 cells through the AMPK signaling pathway.

2.

Materials and methods

2.1.

Chemicals

Oil Red O (ORO), isobutylmethylxanthine (IBMX) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hank’s balanced salt solution (HBSS), Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA (ethylenediaminetetraacetic acid) and penicillin–streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). Esculetin (Fig. 1), antibodies for PPARγ, C/EBPα, aP2, β-actin, and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pAMPK α, AMPK, pACC, and ACC were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). ECL™ detection reagents were purchased from GE Healthcare (Buckinghamshire, UK). All other reagents and solvents used were of analytical and HPLC grade.

OH

O

O

OH Fig. 1 – The structure of esculetin (6,7-dihydroxy-2H-1-benzopyran-2-one).

2.2.

Cell culture and treatment

3T3-L1 preadipocytes were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The 3T3-L1 preadipocytes were cultured as previously described with minor modifications (Chang & Polakis, 1978). Briefly, 3T3-L1 preadipocytes were maintained at 37 °C in DMEM containing 10% FBS to confluence. At 2 days post-confluence (designated day 0) adipocyte differentiation was induced with a mixture of IBMX (0.5 mM), dexamethasone (1 µM), and insulin (1 µg/ ml) in DMEM containing 10% of FBS. On day 2, this medium was replaced with DMEM containing 10% of FBS and insulin only. On day 4, the medium was replaced with DMEM containing 10% of FBS, and the cells were harvested at day 6. Esculetin (ECT) was dissolved in DMSO at 100 mM and diluted with the culture medium to obtain the needed sample concentrations (12.5, 25, 50, 100 µM) before addition to cells. The final DMSO concentration never exceeded 0.1% (v/v) in any treatment group. ECT was added to every replacement of the medium.

2.3.

Lactate dehydrogenase assay

A lactate dehydrogenase (LDH) assay was performed to assess the effect of ECT on cellular toxicity. LDH is a cytoplasmic enzyme that is released when the cell membrane is damaged. After treatment with ECT, an LDH assay was performed on the supernatant at day 6. LDH activity was determined using an LDH assay kit (Roche Diagnostic System, Montclair, NJ, USA).

2.4.

Oil Red-O staining

To monitor the levels of intracellular lipids in differentiated adipocytes, ORO staining was performed in differentiated 3T3L1 adipocytes at day 6 as described elsewhere (Tobe et al., 1987). The cells were washed with phosphate buffered saline (PBS) and fixed with 10% formaldehyde for 10 min. The fixed cells were washed three times with distilled water. ORO in isopropanol (5 mg ORO/ml) was added to each well and cells were incubated at room temperature for 20 min. The plates were rinsed three or four times with distilled water. Photomicrographs were taken after the cells were air-dried. Finally, the dye retained in the cells was eluted with isopropanol and quantified by measuring the optical absorbance at 490 nm.

2.5.

Western blot analysis

3T3-L1 cells were collected by centrifugation and washed once with phosphate-buffered saline at day 6. Washed cell pellets were lysed in cell lysis buffer (iNtRON Biotech, Seongnam, Korea). Protein concentration was measured using

511

journal of functional foods 12 (2015) 509–515

a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA). Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. After 1 h incubation in blocking solution (5% skim milk), the membranes were incubated for 1 h at a 1:1000 (v/v) dilution of primary antibodies at room temperature. The membranes were washed thrice with Tween-20/Tris (hydroxymethyl) aminomethane-buffered saline (TTBS) and incubated in a 1:1000 dilution of horseradish peroxidaseconjugated secondary antibody for 1 h at room temperature. The membranes were again washed three times with TTBS and then developed using ECL™ (GE Healthcare, Buckinghamshire, UK) detection reagents. The autoradiograms were quantified by optical densitometry using the Image J program (NIH, Washington, DC, USA).

A

2.6.

B

ROS were quantified using the DCFH-DA fluorescent probe as previously described (Wang & Joseph, 1999). Briefly, the 3T3L1 cells were seeded in 96-well black plates and adipocyte differentiation was induced with a mixture of IBMX (0.5 mM), dexamethasone (1 µM), and insulin (1 µg/ml) in DMEM containing 10% FBS at 2 days post-confluence (designated day 0). On day 1, the cells were washed three times with PBS and the culture medium was replaced with 25 µM DCFH-DA in FBSfree medium and the cells were incubated for 1 h at 37 °C. Then, the cells were washed three times with PBS and incubated in HBSS. The fluorescence intensity, corresponding to the intracellular ROS levels, was measured with a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) after 3 h at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

N.S

Cell viability (% control)

100

80

60

40

20

0

ECT (μM) ECT (μM)

0

12.5

0

12.5

25

50

25

100

100

50

140

Oil red-O staining (490 nm, % control)

Intracellular reactive oxygen species (ROS) analysis

120

a

120

a

a 100

80 b 60

40

c

20

0

2.7.

Statistical analysis

All data that are presented are expressed as mean ± standard error and are representative of three or more independent experiments. The statistical analysis was assessed by Duncan’s multiple range test. A p-value less than 0.05 was considered significant.

3.

Results and discussion

3.1.

Cytotoxicity and lipid accumulation

As shown in Fig. 2A, no cytotoxicity was observed at any concentration of ECT (from 12.5 to 100 µM). Lipid accumulation was observed and quantified in the 3T3-L1 adipocytes treated with ECT at 12.5, 25, 50 and 100 µM. ECT significantly reduced lipid accumulation during the adipocyte differentiation period compared with that of the control (Fig. 2B). It is widely reported that various natural antioxidants inhibit adipogenesis. Hsu and Yen (2008) reported that some flavonoids and phenolic acids lower the level of intracellular triglycerides and glycerol-3phosphate dehydrogenase (GPDH) in 3T3-L1 adipocytes. Also, curcumin was shown to suppress adipogenic differentiation in 3T3-L1 cells (Ahn, Lee, Kim, & Ha, 2010). The present study

ECT (μM)

0

12.5

25

50

100

Fig. 2 – Effects of esculetin (ECT) on cytotoxicity (A) and lipid accumulation (B) in differentiated 3T3-L1 cells on day 6. Each value is expressed as a mean ± standard deviation (n = 3). a–gDuncan’s multiple range test shows that means with different letters are significantly different (p < 0.05) compared with control. N.S, no significant difference between control and all treatments.

showed that ECT has inhibitory effects of adipogenesis in 3T3L1 cells.

3.2. Effect of esculetin on the expression of adipogenicspecific proteins Adipocyte differentiation involves a series of programmed changes in adipocyte-specific transcription factors, PPARγ and C/EBPα, and many adipogenic proteins, including aP2 (Gregoire, Smas, & Sul, 1998; Rosen et al., 2000). Therefore, the effects of ECT on the expression of key adipocyte differentiation markers, including PPARγ, C/EBPα and aP2, were investigated by performing Western blotting. The treatment with ECT significantly decreased the expression of PPARγ (Fig. 3A) and C/EBPα (Fig. 3B).

512

journal of functional foods 12 (2015) 509–515

A

ECT (μM)

0

12.5

25

50

c

cd

Also, ECT treatment decreased the expression of aP2 (Fig. 3C), which is the major cytosolic protein of mature adipocytes. It is reported that aP2 plays a central role in facilitating the diffusion of fatty acids in the adipocyte and mediates cellular fatty acid trafficking (Storch & Thumser, 2000). These results were consistent with many other studies on the adipogenesis inhibiting effects of phytochemicals in 3T3-L1 cells. For example, apigenin significantly decreased the expression of adipogenic protein levels (Ono & Fujimori, 2011). Therefore, these results suggest that the inhibitory effect of ECT on adipocyte differentiation might be mediated through the down-regulation of adipogenic transcription factors, such as PPARγ and C/EBPα, which are related to the downstream adipocyte-specific gene promoters.

100

PPARγ

β-actin

1.2

PPARγ expression (folds of control)

a 1.0

0.8

b

0.6

0.4

d 0.2

0.0

ECT (μM)

B

ECT (μM)

0

12.5

0

25

12.5

50

25

3.3. Effect of esculetin on ROS generation and AMPK pathways

100

50

100

C/EBP α

β-actin

C/EBPα expression (folds of control)

1.2

a 1.0

b 0.8

c 0.6

d 0.4

e 0.2

0.0

ECT (μM)

C

ECT (μM)

0

12.5

0

25

12.5

50

25

100

50

100

aP2

β-actin

1.2

aP2 expression (folds of control)

a 1.0

b

0.8

b c

0.6

0.4

d 0.2

0.0

ECT (μM)

0

12.5

25

50

100

Fig. 3 – Effects of esculetin (ECT) on PPARγ (A), C/EBPα (B) and aP2 (C) expression in differentiated 3T3-L1 cells. Each value is expressed as a mean ± standard deviation (n = 3). Blots are representative of at least three independent experiments. a–e Duncan’s multiple range test shows that means with different letters are significantly different (p < 0.05) compared with control.

To determine whether inhibition of adipocyte differentiation by ECT is regulated by AMPK activation, the protein expressions of phosphorylated AMPK were evaluated by Western blotting. ECT enhanced AMPK activation in a concentration dependent manner (Fig. 4A) and significantly increased the activation of ACC (Fig. 4B). It is reported that AMPK is involved in cellular energy homeostasis (Carling, Mayer, Sanders, & Gamblin, 2011). AMPK is a heterotrimeric complex consisting of a catalytic a subunit and regulatory β and γ subunits. Once activated at Thr172 of the catalytic a subunits, AMPK stimulates its downstream substrates to increase ATP-generating pathways, such as fatty acid oxidation and glycolysis, and to reduce the ATP-consuming pathways, including fatty acid, cholesterol, and triacylglycerol synthesis (Kahn, Alquier, Carling, & Hardie, 2005). ACC is an important rate-controlling enzyme for the synthesis of malonyl-CoA, which is involved in fatty acid biosynthesis and oxidation. Earlier studies have reported that AMPK increases phosphorylation of ACC, their substrate. The increase of inactive phosphorylated ACC, in turn, inhibited adipogenesis. (Sullivan et al., 1994). ECT increased the phosphorylation of AMPK and its downstream target ACC and that may lead to a subsequent decrease in fatty acid synthesis and fat accumulation. ROS act as subcellular messengers in complex cellular processes, such as mitogenic signal transduction, gene expression, and regulation of cell proliferation, and their formation is a natural process (Oakley, Abbott, Li, & Engelhardt, 2009). Recently, it was revealed that ROS is a one of the upstream regulators of AMPK (Murase, Misawa, Haramizu, & Hase, 2009). To assess whether ECT increases ROS generation in 3T3-L1 cells, intracellular ROS production was measured by using DCFHDA staining. As shown in Fig. 5, ECT significantly increased ROS generation. AMPK can be activated under some physiological and pathological conditions, which are characterized by accompanying increase of intracellular levels of ROS (Han, Wang, Song, Zhu, & Zou, 2010). Some natural compounds, such as ginsenoside Rc, genistein, epigallocatechin gallate, and capsaicin increased the intracellular ROS release and activated AMPK (Hwang et al., 2005; Lee et al., 2010). Particularly, capsaicin has been reported to facilitate glucose uptake by generating ROS and subsequently activating AMPK (Kim, Hwang, Park, Kwon, & Kim, 2013). Therefore, our results suggested that

513

journal of functional foods 12 (2015) 509–515

ECT (μM)

0

12.5

25

50

pAMPKα

total-AMPK

2.5

AMPK activation (pAMPKα/total AMPK, folds of control)

a 2.0

ab b

c

120

b b c

c

100

80

60

c

ECT (μM)

1.0

0.5

ECT (μM)

ECT (μM)

0

0

12.5

12.5

25

25

50

50

100

total-ACC

7.5

a

5.0

b

bc 2.5

c c

0.0

ECT (μM)

0

12.5

25

50

0

12.5

25

50

100

Fig. 5 – Effects of esculetin (ECT) on the ROS production in differentiated 3T3-L1 cells. Each value is expressed as a mean ± standard deviation (n = 3). a–cDuncan’s multiple range test shows that the means with different letters are significantly different (p < 0.05) compared with control.

100

pACC

ACC activation (pACC/total ACC, folds of control)

a

1.5

0.0

B

140

100

Fluorescence intensity (% of control)

A

100

Fig. 4 – Effects of esculetin (ECT) on the AMPK (A) and ACC (B) phosphorylation in differentiated 3T3-L1 cells. Each value is expressed as the mean ± standard deviation (n = 3). Blots are representative of at least three independent experiments. a–cDuncan’s multiple range test shows that means with different letters are significantly different (p < 0.05) compared with control.

an increase of ROS generation by ECT treatment might involve the activation of AMPK pathway during adipocyte differentiation. The activity of AMPK could be inhibited by (6-[4-(2-piperidin1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrazolo[1,5a]pyrimidine, which is also known as compound C. The pretreatment of compound C abolished the inhibitory effects

of ECT on the expression of PPARγ (Fig. 6A) and C/EBPα (Fig. 6B). These results suggested that activated AMPK by ECT participate in the inhibition of adipocyte differentiation. AMPK activation can inhibit preadipocyte differentiation. For example, 5-aminoimidazole-4-carboxamide-riboside (AICAR), an AMPK activator, inhibits adipocyte differentiation and blocks the expression of late adipogenic markers such as PPARγ and C/EBPα in adipocytes (Daval, Foufelle, & Ferre, 2006; Habinowski & Witters, 2001). It was reported that compound C could antagonize AICAR by blocking the uptake of AICAR into cells (Fryer, Parbu-Patel, & Carling, 2002). Therefore, the anti-adipogenic effect of ECT seems to be related to the inhibition of PPARγ and C/EBPα expressions via AMPK phosphorylation. In conclusion, ECT significantly reduced lipid accumulation in 3T3-L1 adipocytes by suppressing the key adipocyte differentiation markers PPARγ, C/EBPα, and aP2. ECT increased AMPK and ACC phosphorylation and intracellular ROS generation. Also, pretreatment with compound C, a potent AMPK inhibitor, abolished the inhibitory effects of ECT on the expressions of PPARγ and C/EBPα. These results suggest that ECT inhibits adipogenic differentiation via the AMPK pathway in 3T3-L1 cells and ECT may be an effective candidate for preventing obesity. Therefore, the results of the present study suggest that ECT appears to be a promising compound for the functional foods that could prove useful in the treatment of obesity. However, further studies are still needed to investigate anti-obesity mechanism of ECT using animal model.

Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A6A3A0104031).

514

A

journal of functional foods 12 (2015) 509–515

ECT (μM)

-

-

100

100

Compound C (2 μM)

-

+

-

+

REFERENCES

PPARγ

β-actin

PPAR-γ expression (folds of control)

1.5

a

a

a

1.0

0.5

b

0.0

B

ECT (μM)

-

-

100

Compound C (2μM)

-

+

-

100 +

ECT (μM)

-

-

100

100

Compound C (2μM)

-

+

-

+

C/EBPα

β-actin

C/EBPα expression (folds of control)

1.5

a

a

a 1.0

b

0.5

0.0

ECT (μM)

-

-

100

Compound C (2 μM)

-

+

-

100 +

Fig. 6 – Involvement of AMPK in the inhibitory effects of esculetin (ECT) on PPARγ (A) and C/EBPα (B) expression in differentiated 3T3-L1 cells. Each value is expressed as a mean ± standard deviation (n = 3). Blots are representative of at least three independent experiments. a–bDuncan’s multiple range test shows that the means with different letters are significantly different (p < 0.05) compared with control.

Ahn, J., Lee, H., Kim, S., & Ha, T. (2010). Curcumin-induced suppression of adipogenic differentiation is accompanied by activation of Wnt/beta-catenin signaling. American Journal of Physiology. Cell Physiology, 298, C1510–C1516. Atsushi, K., Yuka, M., Jo, Y., Isao, A., Alison, A. W., & Robert, J. N. (2008). Protective effects of dietary chamomile tea on diabetic complications. Journal of Agricultural and Food Chemistry, 56, 8206–8211. Carling, D., Mayer, F. V., Sanders, M. J., & Gamblin, S. J. (2011). AMP-activated protein kinase: Nature’s energy sensor. Nature Chemical Biology, 7, 512–518. Chang, T. H., & Polakis, S. E. (1978). Differentiation of 3T3-L1 fibroblasts to adipocytes. Effect of insulin and indomethacin on the levels of insulin receptors. The Journal of Biological Chemistry, 253, 4693–4696. Choi, Y., Kim, Y., Ham, H., Park, Y., Jeong, H. S., & Lee, J. (2011). Nobiletin suppresses adipogenesis by regulating the expression of adipogenic transcription factors and the activation of AMP-activated protein kinase (AMPK). Journal of Agricultural and Food Chemistry, 59, 12843–12849. Daval, M., Foufelle, F., & Ferre, P. (2006). Functions of AMPactivated protein kinase in adipose tissue. The Journal of Physiology, 574, 55–62. Egan, D., O’Kennedy, R., Moran, E., Cox, D., Prosser, E., & Thornes, R. D. (1990). The pharmacology, metabolism, analysis, and applications of coumarin and coumarin-related compounds. Drug Metabolism Reviews, 22, 503–529. Fryer, L. G., Parbu-Patel, A., & Carling, D. (2002). Protein kinase inhibitors block the stimulation of the AMP-activated protein kinase by 5-amino-4-imidazolecarboxamide riboside. FEBS Letters, 531, 189–192. Gregoire, F. M., Smas, C. M., & Sul, H. S. (1998). Understanding adipocyte differentiation. Physiological Reviews, 78, 783–809. Habinowski, S. A., & Witters, L. A. (2001). The effects of AICAR on adipocyte differentiation of 3T3-L1 cells. Biochemical and Biophysical Research Communications, 286, 852–856. Han, Y., Wang, Q., Song, P., Zhu, Y., & Zou, M. H. (2010). Redox regulation of the AMP-activated protein kinase. PLoS ONE, 5, e15420. Hoult, J. R. S., & Miguel, P. (1996). Pharmacological and biochemical actions of simple coumarins: Natural products with therapeutic potential. General Pharmacology: The Vascular System, 27, 713–722. Hsu, C. L., & Yen, G. C. (2008). Phenolic compounds: Evidence for inhibitory effects against obesity and their underlying molecular signaling mechanisms. Molecular Nutrition and Food Research, 52, 53–61. Hwang, J. T., Kwon, D. Y., & Yoon, S. H. (2009). AMP-activated protein kinase: A potential target for the diseases prevention by natural occurring polyphenols. New Biotechnology, 26, 17–22. Hwang, J. T., Park, I. J., Shin, J. I., Lee, Y. K., Lee, S. K., Baik, H. W., Ha, J., & Park, O. J. (2005). Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMPactivated protein kinase. Biochemical and Biophysical Research Communications, 338, 694–699. Kahn, B. B., Alquier, T., Carling, D., & Hardie, D. G. (2005). AMPactivated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism, 1, 15–25. Kim, S. H., Hwang, J. T., Park, H. S., Kwon, D. Y., & Kim, M. S. (2013). Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive oxygen species (ROS)/AMPK/p38 MAPK pathway. Biochemical and Biophysical Research Communications, 439, 66–70.

journal of functional foods 12 (2015) 509–515

Kok, S. H., Yeh, C. C., Chen, M. L., & Kuo, M. Y. (2009). Esculetin enhances TRAIL-induced apoptosis through DR5 upregulation in human oral cancer SAS cells. Oral Oncology, 45, 1067–1072. Lacy, A., & O’Kennedy, R. (2004). Studies on coumarins and coumarin-related compounds to determine their therapeutic role in the treatment of cancer. Current Pharmaceutical Design, 10, 3797–3811. Lee, M. S., Hwang, J. T., Kim, S. H., Yoon, S., Kim, M. S., Yang, H. J., & Kwon, D. Y. (2010). Ginsenoside Rc, an active component of Panax ginseng, stimulates glucose uptake in C2C12 myotubes through an AMPK-dependent mechanism. Journal of Ethnopharmacology, 127, 771–776. Lim, C. T., Kola, B., & Korbonits, M. (2010). AMPK as a mediator of hormonal signalling. Journal of Molecular Endocrinology, 44, 87– 97. Lin, W. L., Wang, C. J., Tsai, Y. Y., Liu, C. L., Hwang, J. M., & Tseng, T. H. (2000). Inhibitory effect of esculetin on oxidative damage induced by t-butyl hydroperoxide in rat liver. Archives of Toxicology, 74, 467–472. Murase, T., Misawa, K., Haramizu, S., & Hase, T. (2009). Catechininduced activation of the LKB1/AMP-activated protein kinase pathway. Biochemical Pharmacology, 78, 78–84. Oakley, F. D., Abbott, D., Li, Q., & Engelhardt, J. F. (2009). Signaling components of redox active endosomes: The redoxosomes. Antioxidants and Redox Signaling, 11, 1313–1333. Ono, M., & Fujimori, K. (2011). Antiadipogenic effect of dietary apigenin through activation of AMPK in 3T3-L1 cells. Journal of Agricultural and Food Chemistry, 59, 13346–13352. Rosen, E. D., Walkey, C. J., Puigserver, P., & Spiegelman, B. M. (2000). Transcriptional regulation of adipogenesis. Genes and Development, 14, 1293–1307. Storch, J., & Thumser, A. E. (2000). The fatty acid transport function of fatty acid-binding proteins. Biochimica et Biophysica Acta, 1486, 28–44. Subramaniam, S. R., & Ellis, E. M. (2011). Esculetin-induced protection of human hepatoma HepG2 cells against hydrogen

515

peroxide is associated with the Nrf2-dependent induction of the NAD(P)H: Quinone oxidoreductase 1 gene. Toxicology and Applied Pharmacology, 250, 130–136. Sullivan, J. E., Brocklehurst, K. J., Marley, A. E., Carey, F., Carling, D., & Beri, R. K. (1994). Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase. FEBS Letters, 353, 33–36. Tobe, K., Kasuga, M., Kitasato, H., Takaku, F., Takano, T., & Segawa, K. (1987). Differential effects of DNA tumor virus nuclear oncogene products on adipocyte differentiation. FEBS Letters, 215, 345–349. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., & Spiegelman, B. M. (1994). mPPAR gamma 2: Tissue-specific regulator of an adipocyte enhancer. Genes and Development, 8, 1224–1234. Towler, M. C., & Hardie, D. G. (2007). AMP-activated protein kinase in metabolic control and insulin signaling. Circulation Research, 100, 328–341. Visscher, T. L., & Seidell, J. C. (2001). The public health impact of obesity. Annual Review of Public Health, 22, 355–375. Wang, C. J., Hsieh, Y. J., Chu, C. Y., Lin, Y. L., & Tseng, T. H. (2002). Inhibition of cell cycle progression in human leukemia HL-60 cells by esculetin. Cancer Letters, 183, 163–168. Wang, H., & Joseph, J. A. (1999). Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology and Medicine, 27, 612–616. Yang, J. Y., Della-Fera, M. A., & Baile, C. A. (2006). Esculetin induces mitochondria-mediated apoptosis in 3T3-L1 adipocytes. Apoptosis: An International Journal on Programmed Cell Death, 11, 1371–1378. Yang, J. Y., Della-Fera, M. A., Hartzell, D. L., Nelson-Dooley, C., Hausman, D. B., & Baile, C. A. (2006). Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells. Obesity (Silver Spring), 14, 1691–1699.