Large yellow tea attenuates macrophage-related chronic inflammation

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Bioactive Constituents, Metabolites, and Functions

Large yellow tea attenuates macrophage-related chronic inflammation and metabolic syndrome in high-fat diet treated mice Na Xu, Jun Chu, Min Wang, Ling Chen, Liang Zhang, Zhongwen Xie, Jinsong Zhang, Chi-Tang Ho, Daxiang Li, and Xiaochun Wan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00138 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Journal of Agricultural and Food Chemistry

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Large yellow tea attenuates macrophage-related chronic

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inflammation and metabolic syndrome in high-fat diet treated mice

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Na Xu#, †, §, Jun Chu#, ‡, Min Wang†, §, Ling Chen†, §, Liang Zhang†, §, Zhongwen Xie†, §, Jinsong

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Zhang†, §, Chi-Tang Ho§, ǁ, Daxiang Li*,†, § and Xiaochun Wan*,†, §

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†

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Agricultural University, Hefei, Anhui 230036, PR China

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‡

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R&D of Chinese Medicine, Anhui University of Chinese Medicine, Hefei, Anhui 230038, PR China

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§

State Key Laboratory of Tea Plant Biology and Utilization, School of Tea & Food Science, Anhui

Key Laboratory of Xin'an Medicine, Ministry of Education, Anhui Province Key Laboratory of

International Joint Laboratory on Tea Chemistry and Health Effects of Ministry of Education,

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Anhui Agricultural University, Hefei, Anhui 230036, PR China

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ǁ

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USA.

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# Na Xu and Jun Chu should be considered as co-first authors.

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* Correspondence: Daxiang Li, Ph.D., State Key Laboratory of Tea Plant Biology

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and Utilization, School of Tea & Food Science, Anhui Agricultural University, Hefei,

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Anhui 230036, People’s Republic of China. Email: [email protected]. Phone:

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+86-551-65786031. Fax: +86-551-65786031.

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* Correspondence: Xiaochun Wan, Ph.D., State Key Laboratory of Tea Plant Biology

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and Utilization, School of Tea & Food Science, Anhui Agricultural University, Hefei,

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Anhui 230036, People’s Republic of China. Email: [email protected]. Phone:

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+86-551-65786765. Fax: +86-551-65786765.

Department of Food Science, Rutgers University, 65 Dudley Road,New Brunswick, NJ 08901-8520,

ACS Paragon Plus Environment


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ABSTRACT: Large yellow tea is a traditional beverage in China with unique toasty

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flavor. Preliminary study using 3T3-L1 cells indicted that large yellow tea, possessed

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more potent lipid-lowering efficacy than green, black, dark and white teas. In the

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present study we further investigated its influence on metabolic syndrome in high-fat

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diet (HFD) mouse model with an emphasis on dose response. Thirty-two C57BL/6

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male mice were randomly divided into 4 groups: low-fat diet (LFD), HFD,

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HFD+2.5% large yellow tea hot-water extract (YT, equivalent to 10 cups of tea daily

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for humans), HFD+0.5% YT. Our data indicated that YT treatment for 12 weeks

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significantly reduced body weight, liver weight, and adipose tissues weight of the

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mice; lowered serum insulin and leptin; raised serum adiponectin with dose effect.

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H&E staining showed that HFD group exhibited significant enlargement of adipose

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cell sizes and the corresponding decrease of adipose cell numbers, which were

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dose-dependently attenuated in both YT groups. IHC results revealed that YT

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decreased macrophage recruitment in the liver, epididymal adipose tissue and

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subcutaneous adipose tissue, and depressed serum inflammatory cytokines including

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TNF-α, MCP-1, IFN-γ, IL-6 and IL-1β, in a dose-dependent manner. In addition, YT

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decreased serum glucose, TC, TG, LDL-C and HDL-C; as well as ameliorated

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glucose intolerance and insulin resistance independent of dose. Overall, YT would be

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a unique tea with dose-independent anti-hyperglycemic and robust lipid-lowering

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efficacies.

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KEYWORDS：Large yellow tea; Macrophages recruitment; Obesity; Steatosis;

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Insulin resistance


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■ INTRODUCTION

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The rapid worldwide prevalence of obesity affects more than 600 million obese

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people from all ages and more than 1.9 billion overweight adults.1 Obesity-induced

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insulin resistance is the major determinant of metabolic syndrome, which precedes the

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development of type II diabetes mellitus and related diseases such as atherosclerosis,

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cardiovascular diseases and nonalcoholic fatty liver diseases.2-5 Current therapeutic

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strategies of metabolic syndrome, focus on drugs and natural food supplements

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including teas.6,7

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Tea (Camellia sinensis L.) is one of the three most consumed beverages worldwide.

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The differences amongst teas arise from manufacturing processes, growth conditions,

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and geographical regions. Large yellow tea, made from “one bud and 3 to 6 leaves” of

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tea plant, is a unique tea manufactured in Anhui Province of China and is mainly

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consumed in China. Our previous studies proved that large yellow tea had significant

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anti-hyperglycemic effect, which could alleviate glucose metabolism disorder in

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high-fat diet (HFD) treated ICR mice, but had no effect on lipid metabolism disorder.8

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Based on previous reports, green tea, oolong tea, black tea, and pu-erh tea all have

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hypolipidemic effect,9 but no reports about large yellow tea. In our previous studies,

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HFD treated ICR mice is not a typical diabetic mice model, and lack long-term

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experiments, in the present study, we further examine the effectiveness of large

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yellow tea on hypolipidemic effect in a HFD treated C57BL/6 male mice model.

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Prolonged nutrient overload results in a state of chronic, low-grade inflammation in

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adipose tissue, which is a key initiator of obesity and insulin resistance development10

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accompanied by multiple leukocyte infiltration and production of pro-inflammatory

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mediators.11 The cells of innate immune system regulate these processes, in particular

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adipose tissue macrophages, which make up a large proportion of the non-adipose



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cells in adipose tissue. Meanwhile, there is a substantial increase in hepatic

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macrophages, consist of a resident macrophage population (termed Kupffer cells) and

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recruited hepatic macrophages, which migrate into the liver under obese conditions.12

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Through paracrine effects, these events promote inflammation and decrease insulin

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sensitivity in nearby insulin target cells.13, 14 Some research focused on

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anti-inflammatory effect of tea in cell model or related gene levels in mice.15 However,

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there have been no studies examining macrophages recruitment and adipose tissue

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steatosis related to anti-inflammatory effect of large yellow tea in mice.

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Obesity is characterized by adipose tissue hyperplasia (an increase in the number of

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adipocytes) and hypertrophy (an increase in the volume of adipocytes).16 Inhibition of

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proliferation and differentiation of preadipocytes may provide an approach for

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treating obesity. Thus, we chose 3T3-L1 cell line to quickly detect the effects of tea

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aqueous extracts on lipid accumulation and mature adipocyte differentiation17, which

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showed potential anti-obesity of tea in vitro. In this paper, the standard inbred line

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C57BL/6 mice were used for following in vivo studies. We found that robust effects of

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high-dose large yellow tea on fat accumulation in adipose tissues, adipose tissues

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structural deterioration, macrophage recruitment into adipose and liver tissues in HFD

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mice. Interestingly, low-dose large yellow tea showed effects on blood glucose and

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lipid, ameliorated glucose intolerance and insulin resistance, suppressed lipids

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overload and liver steatosis in mice fed with high-fat diet.

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■ MATERIALS AND METHODS

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Preparation of Tea Hot-Water Extracts. Large yellow tea sample was purchased

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from a well-known and main origin production place of Huosan in Anhui province;

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white tea was cultivated and processed in Fujian province; green tea, black tea and

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dark tea were collected from Shitai, Qimen (Keemun) and Huangsan in Anhui


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province, in 2015. The lyophilized powders of tea hot-water extracts were prepared

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according to the method reported by Gramza and Regula with slight modifications.18

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Briefly, the tea leaves (100 g) were ground and then boiled in double-distilled water

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(1000 mL), followed by stirring for 15 minutes at 70 °C (the procedure was repeated

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3 times). The yields (weight/weight) of lyophilized powder were as follow: large

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yellow tea (YT, 23.6%), green tea (GT, 24.3%), white tea (WT, 24.1%), dark tea (DT,

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16.4%), and black tea (BT, 24.9%). The HPLC-MS analysis was used to determine the contents of the major catechins,

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gallic acid (GA), caffeine, and theobromine in YT on Agilent G6300 series HPLC-MS

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system (Santa Clara, CA, USA) according to the method described by Zhang et al.19

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The main concerned components in five teas were listed in Table 1.

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3T3-L1 Cells Culture, Differentiation and Oil Red O Staining. The mouse

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3T3-L1 preadipocytes were purchased from National Infrastructure of cell line

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resource (Beijing, China), and were maintained in DMEM (pH 7.4, Gibco, Shanghai,

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China), supplemented with 10% FBS (CLARK, Shanghai, China), 100 U/mL

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penicillin and 100 mg/mL streptomycin (Gibco, Shanghai, China) at 37 oC in 5%

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CO2-humidified incubator as previously reported.20 For cell cytotoxicity assay, the

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medium was replaced by serial dilutions medium (100 μL) contained tea extract

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(0-100 μg/mL); after 12 h treatment 10 μL MTT solution (final concentration of 0.5%,

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Sigma-Aldrich, Shanghai, China) was added and incubated for 4 h. Finally measured

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spectrophotometrically in ELx800 microplate reader (Bio-Tek Industries, Inc., Atlanta,

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GA, USA) at 490 nm.

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The 3T3-L1 preadipocytes were differentiated into mature adipocytes induced by

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hormone cocktail (1 μM DEXA, 10 µg/mL insulin, and 0.5 mM IBMX,

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Sigma-Aldrich, Shanghai, China).21

Aqueous extracts of different teas were added



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and incubated the post-confluent cells (day 0), and were present throughout the

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differentiation period (8 d). Lipid accumulation in mature adipocytes was visualized

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by staining with Oil Red O stain.21 The stained cells were photographed in 100X

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amplification using fluorescent inverted microscope (LEICA DM16000B, Wetzlar,

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Germany), and the stained oil droplets were dissolved in anhydrous ethanol and

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quantified by measuring the absorbance at 510 nm. All assays were performed in

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quadruplicate.

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Experimental Mice Treatment. The 32 male C57BL/6 mice were purchased from

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Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China), housed in

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ventilated cages within pathogen-free barrier facility that maintained a 12-hour

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light/12-hour dark cycle, and allowed to have water and food ad libitum. The Anhui

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Agricultural University Standing Committee on Animals approved all mouse

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protocols. At 5-week-old, the mice were randomly divided into 4 groups: LFD

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(D12450B diet containing 3.85 kcal/g and 4.4% fat; n=8；Trophic Animal Feed

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High-Tech Co., Ltd, Nantong, China), HFD (D12451 diet containing 4.73kcal/g and

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45% fat; n=8), HFD + 2.5% YT (lyophilized powder of large yellow tea hot-water

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extracts; n=8) and HFD + 0.5% YT (n=8) groups. The lyophilized powder of large

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yellow tea hot-water extracts were mixed into feed by weight (w/w), and detailed

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composite of all diets are shown in Table 2. The body weights, water intakes and food

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intakes of mice were monitored weekly. The blood glucose of mice was monitored

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every three weeks.

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Glucose Tolerance Tests (GTT) and Insulin Tolerance Tests (ITT). At the end of

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12 weeks treatment, GTT and ITT were performed before the mice were sacrificed,

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according to Liu et al.22 Briefly, mice were fasted 12 h, either were injected with

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glucose (1 g/kg bodyweight, Sigma-Aldrich, Shanghai, China) for GTT, or with


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insulin (1.5 UI/kg bodyweight, Sigma-Aldrich, Shanghai, China) for ITT assay. The

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blood samples were taken from mice tail tip, and blood glucose levels were measured

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by a blood glucose meter (Omnitest Plus, B. BRAUN, Shanghai, China) at 0, 15, 30,

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45, 60, 90 and 120 min after injection.

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Mice Serum Index and ELISA Assay. After 12 weeks treatment, all mice were

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sacrificed by CO2. Serum samples, liver, epididymal adipose tissue (EAT), brown

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adipose tissues (BAT) and subcutaneous adipose tissues (SAT) were harvested for

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following assays. The serum samples were thawed to detect levels of insulin, leptin,

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adiponectin, tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1

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(MCP-1), interferon-γ (IFN-γ), interleukin-6 (IL-6), interleukin-1β (IL-1β),

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interleukin-10 (IL-10) and interleukin-4 (IL-4) using corresponding mouse ELISA

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kits (R&D, Shanghai, China) according to manufacturers’ instructions.

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Biological parameters of mouse serum triglycerides (TG), total cholesterol (TC),

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high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol

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(LDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were

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detected using corresponding assay kits (Nanjing Jiancheng Bioengineering Institute,

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Nanjing, China) according to manufacturers’ instructions.

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Histology Assay. Harvested tissue specimens were fixed in 4% formalin at room

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temperature, embedded in paraffin and cut serially into 5 μm thickness (Leica

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RM2255, Shanghai, China). Sections were dewaxed, hydrated and treated as

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previously described,23 followed by haematoxylin-eosin staining (BOSTER, Wuhan,

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China) and digital slice scanning (3D Histech Pannoramic Midi, Budapest, Hungary)

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for whole slide imaging. Using Quant-Center image analysis application (3D Histech),

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void fraction of liver and BAT were evaluated, and data were presented as area

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percent of blank. To assess adipocyte average diameters, numbers and population, 10



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random fields in each slide image were evaluated by Image-Pro Plus Version 6.0

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(Media Cybernetics, Rockville, MA, USA). The data were presented as average cell

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diameter, cell number per mm2, and percentage of the single group cells to total cells

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population respectively. All of these assays were performed in a blinded manner.

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Immunohistochemical stainings with Mac-2 monoclonal antibody (1:800, Bio

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Legend, San Diego, CA, USA) was used to identify macrophage in EAT, SAT and

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liver.23 Recruited macrophages crown-like structures (CLS) numbers in EAT and SAT

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were counted under a light microscope (OPTEC, Chongqing, China), and data was

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presented as numbers per mm2. In liver tissue, the Mac-2 positive area in whole slide

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imaging of each section was evaluated by detecting staining intensity with

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Quant-Center image analysis application (3D Histech), and data was presented as area

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percent of positive cell. All of these assays were performed in a blinded manner.

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Statistical Analysis. All data were present as means ± SEM. For HPLC and cell

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studies, two-sample t-test was used to assess the difference between two groups by

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Origin 9.0 statistical software (OriginLab Corporation, USA). For mouse studies,

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two-way ANOVA followed by Bonferroni post test or non-parametric Mann-Whitney

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t test were performed by GraphPad Prism 5 software (GraphPad software, La Jolla,

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CA, USA). The P value below 0.05 was considered as difference significance.

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■ RESULTS AND DISCUSSION

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Main Chemical Compounds in Hot-Water Extracts of Five Types of Tea. In

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China, tea has been used for anti-obesity prevention and considered as a crude

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medicine for more than a millennium, and has a long history of proven safety.

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However, recent studies reveal that high dose of EGCG may have cytotoxicity and

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hepatotoxicity in rodent, which suggested that crude extracts might have a better

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safety than single substance.24 Since Chinese people consumption of whole tea as


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beverage or food supplements, it is important to investigate the health benefits of

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whole tea hot-water extract. In current study, 5 kinds of tea leaves were boiled in

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water to prepare tea aqueous extracts,18 then these extracts were lyophilized under

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vacuum to obtain tea powders for the consistency of experimental materials and

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stability during storage.

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The major catechins, GA, caffeine, and theobromine in these tea powders were

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analyzed and shown in Table 1. The contents of GA (9.95±0.05 mg/g), caffeine

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(63.91±0.40 mg/g), and theobromine (0.10±0.01 mg/g) in YT were significantly less

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than those in BT, GT, WT and DT. Comparatively, YT contained less amount of total

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catechins (64.06±0.59 mg/g, GC, GCG, EC, ECG, EGC and EGCG) than GT

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(186.09±1.22 mg/g), WT (203.04±0.14 mg/g) and DT (71.16±0.68 mg/g), but higher

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than BT (28.67±0.33 mg/g). Notably, GCG level in YT (9.96±0.17 mg/g) was higher

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than other 4 teas, which was epimerized from EGCG.

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YT has Lower Cytotoxicity and the Highest Inhibitory Effect on Lipid

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Accumulation in 3T3-L1 Cell among Examined Five Types of Tea. Green tea25 and

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pu-erh tea20 could decrease proliferation and differentiation of adipocytes, but little is

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known about large yellow tea. The murine 3T3-L1 cell line was an established and

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typical model for researching the conversion of preadipocytes into adipocytes and fat

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deposition in vitro. The 3T3-L1 preadipocytes were exposed to five tea extracts

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respectively, all teas significantly inhibited cell viability beyond 50 μg/mL in a dose-

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dependent manner, as determined using MTT assay (Fig. 1A and 1C). GT, WT and

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DT but not YT and BT at 40 μg/mL were cytotoxic, therefore, this dose was used as

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the upper limit for assessing adipocytes differentiation and lipid storage in

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3T3-L1 preadipocytes (Fig. 1A and 1C).

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After 8 days induced with cocktail inducer, 3T3-L1 preadipocytes efficiently



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differentiated into morphologically distinct, fat-laden mature adipocytes with

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accumulated cytoplasmic triglycerides that stained with Oil Red O (Fig. 1E).

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Microscopic analysis and spectrophotometrical quantitative analysis demonstrated a

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marked decrease in adipocytes differentiation and lipid storage after all five tea

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extracts treatment compared to control induced cells. Surprisingly, YT possessed the

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strongest suppression effect among five teas at 20 μg/mL (Fig. 1B-1E), however, it

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also needs to recognize that such a concentration is almost impossible to reach in

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physiological circulation. These data indicate that YT is more effective than other

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four teas in attenuating the differentiation and lipid accumulation of adipocytes, and

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also suggest the potential of YT regulating obesity in vivo.

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Depending on the level of fermentation, which causes the oxidation of tea catechins,

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green tea is non-fermented tea, yellow tea is partly-fermented tea, and black tea,

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whose functions were frequently found to be nearly equivalent to green tea, is fully-

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fermented tea.26 According to the data in Table 1, fermented yellow tea and black tea

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content lower catechins than non-fermented green tea, which means except catechin

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monomor, there are other compounds responsible for the activities in fermented tea.

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Although the exact active components still remain largely enigma in black tea, some

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researchers believe that major active components in black tea extracts were

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polymerized polyphenol and their fractions.27 More notably, many reports showed

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that catechin polymers significantly inhibited pancreatic cholesterol esterase and

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lipase,28 as well as improved antiglycation and superoxide dismutase-like activities

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compared to catechin monomer.29 Thus large yellow tea may be endowed with high

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active catechin polymers during its unique processings, including moderate

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fermentation and extremely high temperature condition, these polymers may

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participate in the contribution of high activities of YT.


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YT Dose-Dependently Suppresses Fat Accumulation of Adipose Tissues in

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Mice. Having indicated more effective of YT than other teas in blocking lipid

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accumulation in vitro, we next investigated the exact function of YT on lipid

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deposition and obesity in vivo. C57BL/6 male mice were randomly grouped into 4

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groups as LFD, HFD, HFD+0.5% YT and HFD+2.5% YT. Considering one gram YT

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powder with 23.6% extraction ratio from tea leaves contained 0.29% EGCG and

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0.64% catechins, the present study used 2.5% YT contained 0.31% EGCG in mice

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diet. Based on allometric scaling, the presently used dose in mice corresponds to 10

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cups of green tea (containing 2 g tea leaves per 200 mL water) per day for an average

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person requiring 2000 kcal/d.8,

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corresponds to 2 cups of green tea daily consumption of human.

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Therefore, 0.5% YT diet used to feed mice

Obesity can arise either from increase in individual adipocyte cell size (hypertrophy)

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or from increase in total adipocyte cell number (hyperplasia) as a result of increased

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de novo adipocyte differentiation.31 After 12-weeks treatment, we tested white adipose

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tissue expansion and population change of adipocytes after YT supplement. Mice fed

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with 2.5% YT had similar tissue weight (Fig. 2A) and average adipocyte diameter

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(Fig. 2B) in EAT and SAT to those from LFD-fed mice, which were far lower than

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those from HFD-fed mice. Interestingly, the 0.5% YT diet group only restricted SAT

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expansion but not EAT (the major white fat pad), which paralleled to their body

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weight (Fig. 2B and Fig. 4A). We showed that 2.5% YT diet also increased cell

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number every mm2 both in EAT and SAT (Fig. 2C). Then 0.5% YT group only

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reduced bigger adipocyte (>100μm) hyperplasia with abnormal EAT (Fig. 2D and 2E)

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and SAT (Fig. 2F and 2G) structure, while 2.5% YT diet protected EAT (Fig. 2D and

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2E) and SAT (Fig. 2F and 2G) steatosis to normal structure like LFD group. As a

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consequence, 2.5% YT group significantly raised SAT adipocyte population ranged 40



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to 60μm, and lower numbers above 80 μm (Fig. 2F). It is interesting to note,

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high-dose YT robustly reduce fat accumulation in white adipose tissues, while

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low-dose YT has less effect on tissue structure deterioration, which proved its

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dose-dependent effects on lipids overload.

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YT Pronouncedly Suppresses Macrophages Recruitment into Adipose Tissues

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and Liver. Obesity is believed to be a low-grade and chronic inflammatory disease, 4

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and macrophage/adipocyte nexus involves migration of monocytes/macrophages to

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adipose tissue (including intramuscular fat depots) and liver with subsequent

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activation of macrophage proinflammatory pathways and cytokine secretion.14

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M1-like macrophages accumulate32 in adipose tissue as early as 3-14 days after HFD

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feeding than T- cells33 and B lymphocytes34, which begin to accumulate at 4-22 weeks.

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Therefore, as the most abundant and central immune cell in obesity-associated

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inflammation, macrophages likely represent the final effect cell secreting the

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predominance of the cytokines that cause insulin resistance.4

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These recruited macrophages accumulate in expanding adipose tissue and their

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transcripts surround dead adipocytes, forming so-called crown-like structures (CLS),

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which were distributed differential in abdominal fat depots of diet-induced obesity

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mice.35 Using immunohistochemistry staining with macrophage-specific monoclonal

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antibody Mac-2, we showed that both YT groups strikingly depressed HFD-induced

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macrophages recruitment into EAT (Fig. 3A and 3B). Although macrophages in SAT

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gained same variation tendency as in EAT after YT treatment, there were no

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significant difference between all four group mice due to lower CLS density (Fig. 3A

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and Fig. S1). Considering the differences in biological behavior of fat found in

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different anatomical locations, SAT differs from visceral fat by immune cell

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composition and a lower prevalence of CLS both in lean and obese mice.11 Although


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EAT is well developed in the mouse, which associates with an increased risk for

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metabolic dysregulations and cardiovascular diseases, increased SAT appears to pose

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little or no risk.36 It seems that SAT is a metabolic sink to buffer an energy surplus,37

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and a depot buffering the energy excess.38 Our results are consistent with these reports

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that SAT had the lowest density of CLS, so YT had limited effect on suppress

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macrophages recruit in SAT (Fig.3 A and Fig.S1).

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Unlike gather to form CLS in adipose tissue, macrophages in liver are diffused or

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clustered among hepatocytes. As expected, high fat diet raised macrophages migrated

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into liver, and YT reduced macrophages number in a dose-dependent manner (Fig. 3C

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and 3D). Tissue macrophages consist of heterogeneous populations with differential

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functions, such as M1 (classically activated macrophages are highly pro-inflammatory)

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and M2 classifications (alternatively activated macrophages are anti-inflammatory).39

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The overall macrophage-induced inflammatory state in tissue is determined by the

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balance between these different macrophage subpopulations.4 Although we proved

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that YT reduced Mac2+ mark macrophages in HFD mice tissues (Fig.3), the results

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could not confirm if the balance is toward anti-inflammatory macrophage phenotype,

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or if YT causes a polarization spectrum transform from M1 to M2 phenotypes. The

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exact functional subpopulations influenced by YT need further investigation.

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YT Decrease Systemically Inflammation Level in Mice. Since macrophages and

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adipocytes could be sources of pro-inflammatory cytokines that activate inflammatory

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pathways in resident and infiltrating cells,40 and macrophage-mediated chronic tissue

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inflammation is a key mechanism for insulin resistance in obesity. Then we tested

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serum inflammatory factor using ELISA assays. The results reveal that YT led to

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various degrees down-regulation of serum pro-inflammatory cytokines TNF-α,

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MCP-1, IFN-γ, IL-6 and IL-1β (Table 3). YT also elevated anti-inflammatory



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cytokine IL-4 and IL-10 in serum of HFD-fed mice (Table 3). Our results indicate that

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YT suppressed tissues (Fig. 3 and Fig. S1) and systemic inflammation levels (Table 3)

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in HFD mice, and proved YT’s anti-inflammatory effect in vivo.

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Since the pathogenesis of obesity is still not fully clear, it is difficult to prove

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lipid accumulation and inflammation state which one triggered this vicious cycle in

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obesity. Many researches agree that lipid overload in obesity can indirectly stimulate

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the pro-inflammatory state and an increase in inflammatory marker expression.41 With

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overnutrition, failure of packaging of excess lipid into lipid droplets causes chronic

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elevation of circulating fatty acids, which can reach to toxic levels within non-adipose

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tissues.Thus, excess diacylglycerols, ceramide and saturated fatty acids in obesity can

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induce chronic inflammation and have harmful effect on multiple organs and

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systems.42 Considering we investigate the prevention of obesity development, YT is

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likely to work by inhibiting lipid accumulation, reduce HFD-related lipotoxicity, and

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that depress inflammation. Of course, it is important to investigate the direct evidence

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of this hypothesis in the future.

334

YT Dose-Dependently Suppresses Body Weight Gain and Serum Hormone.

335

After 12 weeks of treatment, HFD resulted in an increased body weight gain

336

compared to LFD (Fig. 4A). The 2.5% YT group significantly attenuated mice body

337

weight gain since the 2th week, while 0.5% YT had no influence after diet intervention

338

(Fig. 4A). Food and water monitor showed that 2.5% YT significantly increased

339

energy intake and water consumption than HFD diet mice, while 0.5% YT had no

340

influence (Fig. S2).

341

Insulin can promote adipose tissue glucose uptake and control glucose homeostasis

342

in the circulation.43 Leptin is synthesized and secreted almost exclusively by

343

adipocytes, circulates in the blood, and serves as a major “adipostat” by repressing


Page 14 of 37

Page 15 of 37


344

food intake and promoting energy expenditure in the central nervous system

345

(primarily the hypothalamus).44 Unlike almost all other adipokines, adiponectin levels

346

are inversely correlated with body mass,45 which enhance glucose-stimulated insulin

347

secretion in islets from mice with diet-induced obesity.46 Here using ELISA kits, we

348

proved that YT reduced insulin and leptin level and raised adiponectin level in mice

349

serum (Fig. 4B-4D). In a word, YT dose-dependently reduced obesity and serum

350

hormone in HFD mice.

351

YT Dose-Independent Effects Associated with Serum Glucose, Serum Lipid

352

and Hepatic Lipid Overload. The blood glucose monitor showed that both 0.5% and

353

2.5% YT lowered blood glucose since 6th and 3th week, respectively (Fig. 5A).

354

Corresponding with blood glucose, two YT groups significantly ameliorated

355

HFD-induced glucose intolerance and insulin resistance by GTT (Fig. 5B) and ITT

356

assays (Fig. 5C). The concept of lipotoxicity holds that increased levels of circulating

357

fatty acids and/or lipid accumulation in muscle and liver can lead to insulin

358

resistance.47 The HFD induced hyperlipidemic mice model showed significant higher

359

serum TC, LDL-C and TG levels, but did not alter HDL-C level comparing to those in

360

animals on the normal chow diet (Fig. 6A-6D), suggesting the effectiveness of the

361

animal model. In this study, both 2.5% and 0.5% YT groups reduced TC, TG and

362

LDL-C levels in different degrees (Fig. 6A-6D).

363

Most researches believe that elevation of serum ALT and AST activities, are

364

accompanied with hepatic toxicity and oxidative stress occurred in mice. As shown in

365

Supporting Information Fig. S3, the levels of ALT and AST in the Model group (HFD)

366

were higher than those in the Control group (LFD). Dramatically, YT depressed ALT

367

and AST levels to almost the level in Control group. Meanwhile, neither HFD nor YT

368

influent AST/ALT ratio compared with LFD group (Fig. S3). In other words, both



369

low-dose and high-dose YT mitigated serum glucose and lipid levels, without

370

stimulated hepatic toxicity in HFD mice.

371

In obesity developing, the excessive fat calorie load exceeds the buffering capacity

372

and efficiency of adipose tissue storage, and “spill over” into other tissues like liver,

373

so called “ ectopic fat deposition”.36 Indeed, increased adipose tissue expansion and

374

hepatic steatosis are typical concomitant abnormalities of insulin resistance states,

375

which requires extracellular matrix remodeling.47, 36 So we observed diffuse fatty

376

infiltration and amyloidosis in liver tissues that caused the disruption in the structure

377

of hepatic lobules in HFD mice using H&E histological staining (Fig. 6G). The mice

378

receiving dietary YT showed reduction in liver weights, liver void fraction, and

379

protected liver tissue structure compared to the mice only fed with HFD (Fig. 6E-6G).

380

The green, black, oolong and pu-erh tea have been well documented on modulating

381

lipid metabolism, suppressing adipocyte differentiation and anti-inflammatory

382

action.20,25,26,48-51 The characterized mechanism of these teas include activation of

383

AMPK, blocking NFκB pathway and decreasing expression of transcription factors

384

C/EBPα and PPARγ.25,48-50 The current study focuses on the impact of tea, in the form

385

of large yellow tea, on inflammatory response triggered by obesity. Thus, according to

386

those finding from green or black tea, it is reasonable to speculate that large yellow

387

tea promotes lipid metabolism by activating AMPK to attenuate lipogenesis and

388

enhance lipolysis, inhibiting adipocyte differentiation by reducing transcription

389

factors C/EBPα and PPARγ, and transcriptional regulation of inflammatory cytokines.

390

However, these assumptions require more researches in the future.

391

Taken together, large yellow tea as a potential nature health product, lowered lipid

392

accumulation both in vitro and in vivo, prevented the symptoms of obesity and

393

metabolic syndrome in HFD-fed mice (Fig. S4). In 3T3-L1 cell model, YT strongly


Page 16 of 37

Page 17 of 37


394

inhibited lipid accumulation with lower cytotoxicity than other four teas (Fig. 1). In

395

HFD mice model, dietary YT dose-dependently declined adipose tissues fat

396

accumulation (Fig. 2), macrophages recruitment into tissues (Fig. 3 and Fig. S1),

397

serum inflammatory factor (Table 3), and serum hormone and body weight gain (Fig.

398

4). It also showed robust effects of high-dose YT on fat accumulation and

399

inflammation level in tissues and circulatory system in mice fed with high-fat diet.

400

Although 0.5% YT has no effect on losing weight (Fig. 4), it indeed alleviated

401

metabolic syndrome, especially in regulating glucose metabolism and insulin

402

resistance (Fig. 5), serum lipid level and hepatic steatosis (Fig. 6) in mice. These

403

findings indicated that low-dose YT could also generate certain significant effects

404

equivalent to high-dose YT.

405

■ ABBREVIATIONS USED

406

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BT, black tea

407

aqueous extract; DT, dark tea aqueous extract; EAT, epididymis adipose tissues; GT,

408

green tea aqueous extract; HDL-C, high density lipoprotein cholesterol; HFD, high fat

409

diet; IL-6, interleukin-6; LDL-C, low density lipoprotein cholesterol; LFD, low fat

410

diet; MCP-1, monocyte chemotactic protein-1; SAT, subcutaneous adipose tissues; TG,

411

serum triglycerides; TC, total cholesterol; TNF-α, tumor necrosis factor-α; WT, white

412

tea aqueous extract; YT, yellow tea aqueous extract; IFN-γ, interferon γ; PPAR-γ,

413

peroxisome proliferator-activated receptor γ

414

■ ACKNOWLEDGMENTS

415

This work was supported from Key University Science Research Project of Anhui

416

Province (Grant No. KJ2016A239); China Postdoctoral Science Foundation funded

417

project (Grant No. 2016M601997); Anhui Province Postdoctoral Science Foundation

418

funded project (Grant No. 2017B230); Anhui Province University Outstanding Young



419

Talent Support Program Key Projects (Grant No. gxyqZD2016139); Chang-Jiang

420

Scholars and the Innovative Research Team in University (Grant No. IRT1101); the

421

Earmarked fund for China Agriculture Research System (Grant No. CARS-19);

422

Anhui Provincial Natural Science Foundation (Grant No. 1508085MC59) and Anhui

423

Major Demonstration Project for the Leading Talent Team on Tea Chemistry and

424

Health.

425

Author Contributions

426

# N.X. and J.C. should be considered as co-first authors. X.W. conceived the study.

427

N.X., J.C., and J.Z. designed all experiments and analyzed data. N.X., J.C., M.W., and

428

L.C. performed the experiments. L.Z. guided the HPLC-MS analysis. N.X., C.H., J.Z.,

429

Z.X., D.L. and X.W. prepared the manuscript. All authors approved the final

430

manuscript.

431

Notes

432

The authors have declared no conflict of interest.

433

Supporting Information description

434

Figure S1 Dietary large yellow tea suppressed macrophages recruitment into SAT in

435

HFD mice

436

Figure S2 Average energy and water intake of mice during treatment

437

Figure S3 Dietary large yellow tea ameliorated HFD induced serum AST and ALT

438

profile in mice

439

Figure S4 Possible mechanism of large yellow tea in preventing HFD mice lipid

440

accumulation and chronic inflammation

441

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Figure captions

594

Figure 1 Large yellow tea has lower cytotoxicity and the highest inhibitory effect

595

on lipid accumulation in 3T3-L1 cells line among examined five types of teas

596

MTT assay showed effects of tea extracts (A) (C) on proliferation of 3T3-L1

597

preadipocytes. Differentiated adipocytes were stained with oil red dye, then measured

598

the absorbance at 510 nm to calculate adipocytes differentiation rate (B) (D) under tea

599

extracts treatment. (E) Oil red staining showed lipid droplets in mature adipocyte.

600

Induced, hormone cocktail; YT, large yellow tea; BT, black tea; GT, green tea; WT,

601

white tea; DT, dark tea. Aqueous extracts of different teas were ranged 0 -100 μg/mL.

602

All data are means ± SEM and all assays were performed in quadruplicate. Two

603

sample t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

604

Figure 2 Large yellow tea dose-dependently suppresses fat accumulation of

605

adipose tissues in mice

606

Five-week-old male C57BL/6J mice were randomly divided into 4 groups (n=8 per

607

group), and fed on the diets as follow: LFD (low fat diet, D12450B diet containing

608

3.85 kcal/g and 4.4% fat), HFD (high fat diet, D12451 diet containing 4.73 kcal/g and

609

45% fat), HFD + 2.5% YT (lyophilized powder of yellow tea aqueous extracts) and

610

HFD + 0.5% YT. (A) Average EAT and SAT weight of each group mice after 12-week

611

treatment. Average cell diameter (B) and cell numbers (C) in every mm2 EAT or SAT

612

of each group mice. The proportion of different diameter cells to all cells in every

613

EAT (D) and SAT (F) slice. H&E stain showed structure of EAT (E) and SAT (G). All

614

data are means ± SEM, n=8 per group. Each data was compared to corresponding data

615

from HFD mice using two way ANOVA or non-parametric Mann-Whitney t test, * P

616

< 0.05, ** P < 0.01, *** P < 0.001.

617

Figure 3 Large yellow tea pronouncedly suppresses macrophages recruitment



618

into EAT and liver

619

(A) Average Mac-2+ crown-like structures (CLS) numbers in every mm2 EAT or SAT

620

of each group mice after 12-week treatment. (B) Mac-2+ immunostaining showed

621

macrophages in EAT. (C) The proportion of Mac-2 positive area to total area in every

622

liver tissue slice. (D) Mac-2+ immunostaining showed macrophages in liver. Arrows

623

indicate Mac-2+ immunostaining macrophages. All data are means ± SEM, n=8 per

624

group. Each data was compared to corresponding data from HFD-fed mice using two

625

way ANOVA or non-parametric Mann-Whitney t test, * P < 0.05, ** P < 0.01, *** P

Large yellow tea attenuates macrophage-related chronic inflammation

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