Epigallocatechin Exerts Anti-Obesity Effect in Brown Adipose Tissue
Hae-Soo Kim,a Jae-Hak Moon,a Young-Min Kim,*a and Joo-Young Huh*b

aDepartment of Food Science & Technology, Chonnam National University, Gwangju 61186, Republic of Korea,
e-mail: [email protected]
bCollege of Pharmacy, Chonnam National University, Gwangju 61186, Republic of Korea,
e-mail: [email protected]

Catechins in green tea are well-known to be effective in reducing the risk of obesity. The purpose of this study was to elucidate the effects of catechins present in green tea on adipocyte differentiation and mature
adipocyte metabolism. Treatment of 3T3-L1 mouse adipocyte during differentiation adipocytes with (ti)- epigallocatechin (EGC) and gallic acid (GA) resulted in dose-dependent inhibition of adipogenesis. Specifically, EGC increased adiponectin and uncoupling protein 1 (UCP1) transcription in mature adipocytes. Transcription levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were not significantly impacted by either of the compounds. These results suggest that the EGC is the most effective catechin having anti-obesity activity. Finally, EGC is an attractive candidate component for remodeling obesity.

Keywords: anti-obesity, (ti)-epigallocatechin, (ti)-epigallocatechin gallate, gallic acid.

Introduction
Obesity is one of the most widespread chronic diseases worldwide and results from the energy imbalance in energy intake and expenditure.[1,2] Thus, it arises due to an excessive accumulation of fat in the adipose tissue which results in metabolic diseases such as insulin resistance, type 2 diabetes mellitus, and cardiovascular disease.[3] Development of obesity involves two mechanisms: increasing adipocyte size (hypertrophy) and number (hyperplasia), both of which are considered targets for the treatment of obesity.[4] Increase in adipocyte number is achieved through adipocyte differentiation.[5] Currently, adipose tissue is classified into three types, including white (WAT), brown (BAT), and beige. WAT is inherently programmed to store energy in large lipid droplets for later use, and the accumulation of WAT in body fat leads to both hypertrophy and hyperplasia of adipocytes.[6,7] In contrast, BAT generates energy, mostly in the form of heat, and promoting BAT activities has been reported to prevent obesity.[8,9]
Therefore, BAT has been suggested as a potential target for anti-obesity therapy.[10] The phenomenon of ‘browning’ in certain WAT has very attractive target for thermogenic and fat-burning properties.[11] More re-
cently, it was shown that UCP1 is expressed in inguinal white fat (at the mitochondrial level) at sufficient levels and with potential ability to thermogenesis.[11]
Mature adipocytes are primarily involved in the regulation of energy homeostasis by buffering lipid metabolites, and in the secretion of adipokines such as leptin and adiponectin.[12] Notably, adiponectin is a well-known anti-inflammatory adipokine, which regu- lates not only local adipocyte insulin sensitivity but also whole-body metabolism via enhancement of energy expenditure by peripheral tissues (e.g., liver and muscle).[12] Two lipolytic enzymes, adipose trigly- ceride lipase (ATGL) and hormone-sensitive lipase (HSL) play a critical role in lipolysis within adipose tissues.[13]
Green tea, a natural plant with health benefits, is consumed globally and is rich in catechins, which can comprise up to 30% of dry leaf mass.[14] The most
abundant tea catechins as shown in Figure 1, (ti)- epigallocatechin gallate (EGCG), accounts for approx- imately 68–69% of tea catechins, followed by EGC (approximately 15–18%), (ti)-epicatechin gallate (ECG, approximately 5–6%), (ti)-epicatechin (EC, approxi- mately 2–5%), catechin (C, approximately under 2%), and trace of gallic acid (GA).[15] Many studies have

Figure 1. Chemical structures of the green tea derived catechins, (ti )-epigallocatechin, (ti)-epicatechin gallate, (ti)-epicatechin, (ti)- epigallocatechin, and gallic acid.

reported that green tea extracts or tea catechins stimulate fat oxidation, thermogenesis, and energy expenditure.[16] Lee et al. attributed the health benefits of tea catechins to their involvement in the loss of fat in mice and humans.[17] The current study reports the evaluation of regulatory effects of catechins on adipocyte differentiation and mature adipocyte me- tabolism.

Results and Discussion
To investigate the effects of tea catechins during adipocyte differentiation, the cell viability of 3T3-L1 pre-adipocytes was analyzed by treatment with vari- ous doses of these compounds (12.5–200 μmol/L) for 24 h. Since no cellular toxicity was observed up to 200 μmol/L for all test compounds (data not shown), doses of 20, 50, and 100 μmol/L were selected for subsequent experiments. Next, to examine the effect of tea catechins on adipogenesis, 3T3-L1 cells were treated from the start of differentiation process until the fourth day (the critical period for adipogenic determination and differentiation). Results from Day 8 ORO staining demonstrated that catechins and GA inhibited accumulation of intracellular lipids (Figure 2). Quantification of ORO staining confirmed the dose-
dependent inhibitory effect of test compounds on 3T3-L1 cell lipid accumulation (Figure 2). The degree of inhibition did not vary among the different test compounds. In addition, at a concentration of 100 μmol/L, the inhibitory effect was 40–60% (relative to fully-differentiated control cells). These data confirm that high-concentration green tea catechins inhibit adipogenesis possibly through regulation of transcrip- tional factors.
In addition to the effects of the test compounds on adipocyte differentiation, their effects on mature adipocyte metabolism were examined. Fully differ- entiated adipocytes were treated with 100 μmol/L of catechins or GA, for 24 h and gene expression of adiponectin, UCP1, ATGL, and HSL was measured. Among the catechins and GA, only EGC significantly induced adiponectin and UCP1 mRNA expression; the catechins had no effect on ATGL or HSL mRNA expression (Figure 3). These results imply that EGC effectively induces expression of adiponectin, an adipokine known to improve insulin resistance, which may be beneficial for weight-loss strategies. Further- more, EGC, by inducing UCP1 expression, leads to increased uncoupled respiration and thermogenesis, thus increasing loss of energy as heat.
Obesity is becoming an increasingly global public health issue.[11] Obese individuals are at major risk for

Figure 2. Effect of catechins (C, EC, EGC, ECG, and EGCG) and GA on lipid accumulation during adipocyte differentiation. 3T3-L1 preadipocytes were treated with various concentrations of compounds from start of adipocyte differentiation (day 0) until the fourth day. A) Images from Oil Red O (ORO) staining of differentiated adipocytes on day 8. B) Quantification of ORO staining. Values
are means ti SE of three experiments. *p < 0.05 versus control (no treatment). C: (ti)-catechin, EC: (ti )-epicatechin, EGC: (ti)- epigallocatechin, ECG: (ti)-epicatechin gallate, EGCG: (ti )-epigallocatechin gallate, and GA: gallic acid.

a range of comorbid conditions, including cardiovas- cular disease, gastrointestinal disorders, type 2 diabe- tes, joint and muscular disorders, respiratory problems, and psychological issues, which may adversely affect their daily lives, as well as increase their mortality risk.[18] Till date, several studies have explored the pharmacological effects of green tea, and several components of green tea have shown anti-inflamma- tory and antioxidant effects which could potentially contribute to the beneficial role of green tea poly- phenols in the treatment of metabolic diseases.[19]
However, there is limited evidence regarding the direct role of green tea components in modulation of
metabolic diseases such as obesity and diabetes, as EGCG has been the major focus of investigation. A recent study examining the effects of EGCG on 3T3-L1 adipocytes demonstrated results similar to those of the present study regarding the adipocyte differ- entiation marker UCP1.[17] Other studies have shown that high EGCG concentrations cause decrease in peroxisome proliferator-activated receptor gamma and adiponectin expression, along with a reduction in cell number.[20] However, viability of both 3T3-L1 pre- adipocytes and adipocytes was decreased by exposure to high concentrations of EGCG.[21,22] In this current study, we examined whether purified tea catechins

Figure 3. Effect of catechins (C, EC, EGC, and ECG) and GA on mature adipocyte gene expression. 3T3-L1 mature adipocytes were treated with 100 μmol/L of catechins. Gene expression was measured after 24 h incubation using real-time PCR. Values are means ti SE of three experiments. *p < 0.05 versus control (no treatment).

and GA exerted an anti-obesity effect on mature adipocytes, by measuring transcript levels of adiponec- tin and UCP1. Among the tested catechins, EGC induced the largest fold-change in mRNA expression of adiponectin and UCP1. The fact that EGC upregu- lates adiponectin mRNA expression implies that these compounds are potential therapeutics for metabolic disease and may exert anti-obesity and antidiabetic effects via insulin signaling enhancement. Whether such effects persist in vivo and do indeed lead to insulin sensitization should be further studied using animal models.

Conclusions
The present study demonstrated that treatment of adipocytes with catechins and GA inhibited adipocyte differentiation, and EGC significantly regulated meta- bolic gene transcription in mature adipocytes. These
results suggest that EGC indeed exerts beneficial effects regarding regulation of adipocyte differentia- tion and metabolism. Bio-transformation of EGCG, a major component in green tea, into increased EGC and GA provide effective approach to improve health benefits.[23] In addition to this, this anti-obesity effect of green tea infusion with tannase should be studied further.

Experimental Section
Materials
Catechin samples, namely, those for EGCG, EGC, EC, ECG, and C were obtained from Aktin Chemicals (Chengdu, China), and GA was purchased from Tokyo Chemical Industry (Chuo-ku, Tokyo, Japan).

Cell Culture and Differentiation

For cell culture and differentiation, the 3T3-L1 murine cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and were cultured as described previously.[24] The cells were treated with catechins and GA on day 0 and day 2 to investigate their adipogenesis. Differentiating cells were subjected to 4 days of total exposure to each of the test compounds (100 μmol/L concentration). Each compound was added at concentrations of 20, 50, and 100 μmol/L to determine morphological cell differ- entiation following staining with Oil Red O (ORO) stain containing 10% (v/v) formalin.[24] After staining, the cells were washed with distilled water and representa- tive pictures were taken under the microscope. For quantification, 100% isopropanol was added and the absorbance was measured at 490 nm. In the control experiment, an equivalent volume of hydrolysate solvent (water) was added.

Cell Viability Assay and Gene Expression Assay
Cell viability was observed using the 3-(4,5-dimeth- ylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay as described previously.[25,26] To test the effect of catechins and GA on mature adipocytes, cells on day 8 of differentiation were treated with catechins and GA (100 μM) for 24 h. Total RNA was extracted from cells using TRI Reagent (MRC, TR118, Cincinnati, OH), cDNA was synthesized using TOPscriptTM RT DryMIX (Enzy- nomics, Daejeon, Korea), and mRNA levels were measured via real-time PCR using Rotor-Gene Q (QIAGEN, Foster City, CA, USA) with 20 μL reaction buffer [cDNA transcripts, primer pairs, and reagents from the TOPreal SYBR Green PCR Kit (Enzynomics, Daejeon, Korea)]. Gene expression was normalized to 18S rRNA levels. Statistical analysis of obtained data was performed using StatView software. Mean values obtained from each group were compared using ANOVA. Significance level was set at p < 0.05.

Acknowledgement
This research was supported by the support of ‘Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012565)’, Rural Development Administration, Republic of Korea.

Author Contribution Statement

Y.M. and J.Y. conceived and designed the study. H.S. performed the experiments and analyzed the data. Y.M., J.Y., and J.H. wrote the article. J.Y. reviewed and edited the manuscript. All authors read and approved the manuscript.

References
[1]S. Smyth, A. Heron, ‘Diabetes and obesity: The twin epidemics’, Nat. Med. 2006, 12, 75–80.
[2]P. C. Jou, B. Y. Ho, Y. W. Hsu, T. M. Pan, ‘The effect of Monascus secondary polyketide metabolites, monascin and ankaflavin on adipogenesis and lipolysis activity in 3T3-L1’, J. Agric. Food Chem. 2010, 22, 12703–12709.
[3]L. F. Van Gaal, I. L. Mertens, C. E. De Block, ‘Mechanisms linking obesity with cardiovascular disease’, Nature 2006, 444, 875–880.
[4]J. Jo, O. Gavrilova, S. Pack, W. Jou, S. Mullen, A. E. Sumner, S. W. Cushman, V. Periwal, ‘Hypertrophy and/or hyper- plasia: dynamics of adipose tissue growth’, PLoS Comput. Biol. 2009, 5, e1000324.
[5]J. Y. Huh, S. A. Lee, E. B. Ma, H. J. Eom, J. Baek, Y. J. Ko, K. H. Kim, ‘The effects of phenolic glycosides from Betula platyphylla var. japonica on adipocyte differentiation and mature adipocyte metabolism’, J. Enzyme Inhib. Med. Chem. 2018, 33, 1167–1173.
[6]P. E. Scherer, ‘Adipose tissue: from lipid storage compart- ment to endocrine organ’, Diabetes 2006, 55, 1537–1545.
[7]P. R. Shepherd, L. Gnudi, E. Tozzo, H. Yang, F. Leach, B. B. Kahn, ‘Adipose cell hyperplasia and enhanced glucose disposal in transgenic mice overexpressing glut4 selec- tively in adipose tissue’, J. Biol. Chem. 1993, 268, 22243– 22246.
[8]S. Klaus, ‘Functional differentiation of white and brown adipocytes’, BioEssays 1997, 19, 215–223.
[9]J. Kopecky, G. Clarke, S. Enerbäck, B. Spiegelman, L. P. Kozak, ‘Expression of the mitochondrial uncoupling protein gene from the ap2 gene promoter prevents genetic obesity’, J. Clin. Invest. 1995, 96, 2914–2923.
[10]A. M. Cypess, C. R. Kahn, ‘Brown fat as a therapy for obesity and diabetes’, Curr. Opin. Endocrinol. Diabetes Obes. 2010, 17, 143–149.
[11]I. G. Shabalina, N. Petrovic, J. M. de Jong, A. V. Kalinovich, B. Cannon, J. Nedergaard, ‘UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic’, Cell Rep. 2013, 5, 1196–1203.
[12]M. E. Trujillo, P. E. Scherer, ‘Adipose tissue-derived factors: Impact on health and disease’, Endocr. Rev. 2006, 27, 762– 778.
[13]W. J. Shen, S. Patel, H. Miyoshi, A. S. Greenberg, F. B. Kraemer, ‘Functional interaction of hormonesensitive lipase and perilipin in lipolysis’, J. Lipid Res. 2009, 50, 2306–2313.
[14]T. Goto, Y. Yoshida, M. Kiso, H. Nagashima, ‘Simultaneous analysis of individual catechins and caffeine in green tea’, J. Chromatogr. A 1996, 749, 295–299.

[15]Z. Chen, Q. Y. Zhu, D. Tsang, Y. Huang, ‘Degradation of green tea catechins in tea drinks’, J. Agric. Food Chem. 2001, 49, 477–482.
[16]K. Diepvens, K. R. Westerterp, M. S. Westerterp-Plantenga, ‘Obesity and thermogenesis related to the consumption of caffeine, ephedrine, capsaicin, and green tea’, Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007, 292, 77–85.
[17]M. S. Lee, Y. J. Shin, S. Y. Jung, Y. H. Kim, ‘Effects of epigallocatechin-3-gallate on thermogenesis and mito- chondrial biogenesis in brown adipose tissues of diet- induced obese mice’, Food Res. 2017, 61, 1325307– 13253015.
[18]S. M. Fruh, ‘Obesity: Risk factors, complications, and strategies for sustainable long-term weight management’, J. Am. Assoc. Nurse Pract. 2017, 29, S3–S14.
[19]G. L. Tipoe, T. M. Leung, M. W. Hung, M. L. Fung, ‘Green tea polyphenols as an anti-oxidant and anti-inflammatory agent for cardiovascular protection’, Cardiovasc. Hematol. Disord. Drug Targets 2007, 7, 135–144.
[20]H. Y. Sung, C. G. Hong, Y. S. Suh, H. C. Cho, J. H. Park, J. H. Bae, W. K. Park, J. Han, D. K. Song, ‘Role of (ti)-epigalloca- techin-3-gallate in cell viability, lipogenesis, and retinol- binding protein 4 expression in adipocytes’, Naunyn- Schmiedebergs Arch. Exp. Pathol. Pharmakol. 2010, 382, 303–310.
[21]J. Lin, M. A. Della-Fera, C. A. Baile, ‘Green tea polyphenol epigallocatechin gallate inhibits adipogenesis and induces

apoptosis in 3T3-L1 adipocytes’, Obes. Res. 2005, 13, 982– 990.
[22]H. S. Moon, C. S. Chung, H. G. Lee, T. G. Kim, Y. J. Choi, C. S. Cho, ‘Inhibitory effect of (ti)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells’, Obesity 2007, 15, 2571– 2582.
[23]Y. N. Zhang, J. F. Yin, J. X. Chen, F. Wang, Q. Z. Du, Y. W. Jiang, Y. Q. Xu, ‘Improving the sweet aftertaste of green teas infusion with tannase’, Food Chem. 2016, 192, 470– 476.
[24]J. Y. Huh, I. Jung, L. Piao, M. H. Chung, ‘8-Hydroxy-2- deoxyguanosine ameliorates high-fat diet-induced insulin resistance and adipocyte dysfunction in mice’, Biochem. Biophys. Res. Commun. 2017, 491, 890–896.
[25]C. Beemelmanns, T. R. Ramadhar, K. H. Kim, J. L. Klassen, S. Cao, T. P. Wyche, Y. Hou, M. Poulsen, T. S. Bugni, C. R. Currie, Y. Clardy, ‘Macrotermycins A–D, glycosylated macrolactams from a termite-associated Amycolatopsis sp. M39’, Org. Lett. 2017, 19, 1000–1003.
[26]S. R. Kopalli, K. M. Cha, M. S. Jeong, S. H. Lee, J. H. Sung, S. K. Seo, S. K. Kim, ‘Pectinase-treated Panax ginseng ameliorates hydrogen peroxide-induced oxidative stress in GC-2 sperm cells and modulates testicular gene expression in aged rats’, J. Ginseng Res. 2016, 40, 185–195.

Received June 23, 2019
Accepted September 3, 2019