High energy intake, malnourished diets containing lots of fat, and refined carbohydrates coupled with a sedentary lifestyle are believed to contribute to the global obesity epidemic [1
]. Obesity tends to result in other pathological disorders, such as diabetes, atherosclerosis, hypertension, and cancer [3
]. It is estimated that the prevalence of obesity will increase by 7% and 10% among men and women, respectively, by 2020 [6
]. As one of the most popular beverages consumed worldwide, tea has been shown in numerous clinical trials to affect body weight and fat metabolism in humans [7
] and in rodents [8
] fed high-fat diets (HFDs). Oolong tea (OT) is a kind of partially fermented Chinese tea, and it is considered to have a stronger weight loss effect than any other kinds of tea. Previous research has shown that OT leaves can remarkably decrease rats’ body weight [9
]. However, the effects of the water extract from aged OTs (EAOTs, three different storage years) on fat deposition and inflammation, as well as potential mechanisms of action, were not adequately studied. During storage, the contents of tea polyphenols, soluble sugar, and other ingredients changed [10
]. As these ingredients are the material basis of OT’s anti-obesity and anti-inflammation effects, it is reasonable to speculate that the physiological effects of OT would change with storage as well.
The aim of this study was to investigate the effects of EAOTs on anti-obesity and anti-inflammation. Additionally, we analyzed the protein expression following EAOT treatments to evaluate HFD-induced pathways of alleviating obesity and inflammation. Therefore, the present study could demonstrate new insights into the anti-obesity and anti-inflammation mechanism induced by EAOTs.
2. Materials and Methods
Dancong Oolong tea (a kind of OT, stored in 2016, 2006, 1996 separately) was purchased from Dapuxiyan Tea Group Co. Ltd. in Meizhou, Guangdong, China.
2.2. Preparation of EAOTs
Powder prepared from OT was extracted three times by placing in boiling distilled water for 30 min each time (tea/water, 1:20 w:v). These extracted solutions were combined and centrifuged, then concentrated at 60 °C. Finally, the solution was dried by lyophilization.
2.3. Determination of Ingredients in EAOTs
The water content was measured by comparing the weight difference between before and after heating in a 130 °C oven for 3 h, and the content of free amino acids was determined by the ninhydrin method. In addition, anthrone–sulfuric acid colorimetric assay was used to determine the total soluble sugar content, and the Folin-phenol method for tea polyphenols.
Male, 7-week-old C57BL/6J mice were purchased from Beijing Huafukang Bioscience Co. Ltd. (Beijing, China). All experimental procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals, and all efforts were made to minimize animal suffering. The protocols were approved by the Ethical Committee of Tea Research Institute (2015-005) at Guangdong Academy of Agricultural Sciences.
Mice were individually housed in standardized conditions for animal facilities: a 12-h light/dark cycle (off at 7 pm), 23 ± 2 °C room temperature, and 55 ± 5% relative humidity. Mice had ad libitum access to water and food (Beijing Huafukang Bioscience Co., Ltd.).
2.5. Diet-Induced Obesity
After a week of adaptation, 45 C57BL/6J were randomly divided into five groups as follows: (i) the control group (n = 9); (ii) the group fed an HFD (Model) (n = 9); (iii) the group fed an HFD and 1000 mg/kg·BW EAOTs stored in 2016 (2016) (n = 9); (iv) mice fed an HFD and 1000 mg/kg·BW EAOTs stored in 2006 (2006) (n = 9); and (v) the group fed an HFD and 1000 mg/kg·BW EAOTs stored in 1996 (1996) (n = 9). Control mice were fed a normal diet (containing protein 18%, fat 4%, carbohydrate 62%, fiber 5%, minerals 8%, and vitamins 3%, w:w; Beijing Huafukang Bioscience Co., Ltd., 1022), and mice in the other four groups were fed an HFD (45% calories from fat, 20% calories from protein, 35% calories from carbohydrate; Beijing Huafukang Bioscience Co., Ltd., H10045) for 18 weeks, during which time all mice were not administered EAOTs.
2.6. EAOT Treatment
After 18 weeks, the mice in all groups remained on the same diet as before. Mice from three EAOT treatment groups were given, by gavage, 1000 mg/kg·BW EAOTs daily for approximately 6 weeks, while the control group and the model group mice were given, by gavage, distilled water at the same time. Body weight, food, and water intake were recorded once a week. At the end of the experimental period, the mice fasted for 12 h, and then blood samples were collected by cardiac puncture technique under carbon dioxide anesthesia. Thereafter, the mice were euthanized with an anesthetic overdose, and were perfused transcardially through the ascending aorta with normal saline so as to remove any blood clots from tissues and organs. The liver was rapidly removed, weighed, and washed thoroughly with phosphate buffer saline (PBS, pH 7.4). Half hepatic lobar was preserved in 10% buffered formalin solution for histopathological examinations. The rest of the liver was homogenized in ice-cold phosphate buffer saline, then stored in liquid nitrogen for various biochemical and molecular assays. The entire procedure was carried out under cold conditions.
2.7. Lee’s Index
Lee’s index is similar to the human body mass index, and mainly reflects the proportion of body fat in the total weight. It can characterize the degree of obesity more accurately than body weight [11
]. The smaller the Lee’s index, the more slender the body. After the animals were sacrificed, their body weight and length were measured to calculate the Lee’s index.
Lee’s = [weight (g)/length (cm)]1/3
2.8. Serum Chemistry Analysis
After being kept at room temperature for 30 min, blood samples were centrifuged at 13,200 r/min for 20 min at 4 °C, and subsequently stored at −80 °C. The serum was used to measure triglycerides (TGs), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels (iMagic-V7 Automatic Analyzer, ICUBIO, Beijing, China).
2.9. Histological Examination
The liver and epididymal fat sections fixed in 10% neutral buffered formalin were dehydrated in graded concentrations of alcohols, and embedded in paraffin. Then the tissue was sectioned at 4 μm and stained with hematoxylin and eosin (H&E). The pathological changes were assessed and photographed on an Olympus BX-53 microscope (Olympus, Tokyo, Japan).
2.10. Western Blotting Analysis
Protein from each liver sample was extracted with lysis buffer at 4 °C. Then, the extracts were centrifuged at 13,200 r/min and 4 °C for 30 min, and the supernatants of these tissues were used for Western blotting analyses. The protein samples were separated on 10% SDS-PAGE gels and electrophoretically transferred onto PVDF membranes. The membranes were blocked at room temperature with 5% non-fat dry milk in TBST for 1 h, and then incubated overnight at 4 °C with the indicated primary antibodies as follows: AMP-activated protein kinase α (AMPKα), p-AMPKα (Thr172), acetyl-CoA carboxylase (ACC), p-ACC (Ser79), camitinepalmitoyl transferase 1 (CPT-1), fatty acid synthase (FAS), iNOS (Cell Signaling Technology, Danvers, MA, USA), TNF-α (Abcam, Cambridge, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA). After washing three times with TBST, the blots were hybridized with secondary antibodies conjugated to horseradish peroxidase. The proteins were visualized by enhanced chemiluminescence.
2.11. Immunohistochemistry (IHC)
The liver paraffin samples were sliced 4 μm thick and were dewaxed in xylene and rehydrated through a graded series of ethanol concentrations. Then, the slides were pretreated with a microwave antigen retrieval technique and blocked with 5% BSA. The slices were incubated overnight at 4 °C with diluted primary antibodies (Cell Signaling Technology, Danvers, MA, USA). After three washes with PBS for 5 min, the sections were incubated with secondary antibodies (Boster, Wuhan, China) for 30 min at 37 °C. Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG (Beyotime, Shanghai, China) were used for enhanced chromogenic reaction. After washing, the DAB chromogen was added for 3 min before counterstaining with hematoxylin. Images of immunostained liver tissues were taken with an Olympus BX-53 microscope (Olympus, Tokyo, Japan).
2.12. Statistical Analysis
Repeated measures analysis of variance (ANOVA) was conducted, and Tukey’s post-hoc test was used to adjust for multiple comparisons. A p-value < 0.05 was considered statistically significant, and Prism 6.0 software for Windows (GraphPad Software, La Jolla, CA, USA) was used to conduct statistical analyses. Data are presented as mean ± standard error of the mean (SEM) for effects of EAOT treatment on body weight, food intake, water consumption, and Lee’s index, and mean ± standard deviation (SD) for all other results.
In the present study, we provided evidence that EAOTs significantly improved obesity phenomena, such as body weight, Lee’s index, fatty liver, adiposity, and hyperlipidemia in mice with HFD-induced obesity. The reduction of body fat accumulation and organ index in the study also indicated that EAOTs modulated obesity in mice. Furthermore, proteins up-regulating fat consumption were higher in the treatment groups, particularly in the 2006 group, and proteins related to fat synthesis and inflammation were down-regulated in the EAOT groups. These results revealed that obesity seemed to be alleviated within six weeks when C57BL/6J mice were given EAOTs daily.
White adipose tissues (WATs) are optimized to store energy in large lipid droplets for later use [16
]. The accumulation of body fat in WATs leads to both hypertrophy and hyperplasia of white adipocytes [17
]. These changes are associated with obesity-related diseases such as type-2 diabetes and an inflammatory response [18
]. Previous studies have demonstrated that AMPK is a master energy sensor that integrates nutrients, hormones, and stress signals to maintain whole-body energy homeostasis [20
]. Activated AMPKα, namely p-AMPKα, is able to stimulate catabolism and inhibit anabolism, leading to an increase of fat consumption. Rasmus’s study has proved that AMPK in muscle is activated in response to exercise [21
]. Moreover, p-AMPK can inactivate ACC1, leading to inhibition of de novo fatty acid and cholesterol synthesis. Phosphorylation of ACC2 by p-AMPK, on the other hand, causes increases of fatty acid oxidation [22
]. In addition, the AMPK-ACC pathway is able to suppress the TG production because ACC is the rate-limiting enzyme for the synthesis of malonyl-CoA, which is a critical substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation [23
]. Various phytochemicals used to treat metabolic diseases are often able to stimulate AMPK activation [24
]. In the present study, EAOTs markedly induced AMPKα and ACC phosphorylation in the liver, and increased the p-AMPKα/AMPK and p-ACC/ACC ratio, suggesting that AMPK may be the key mechanism for the beneficial effects of EAOTs on fatty liver.
This speculation was further supported by the observation that EAOTs activated CPT-1, whose expression is inhibited by ACC in the mouse liver. The phosphorylation of ACC promoted by p-AMPKα decreased the activation of Malonyl CoA [27
], which is a fatty acid precursor, as well as an inhibitor of CPT-1. Therefore, p-AMPKα promotes the expression of CPT-1 by improving the level of p-ACC and decreasing the expression of Malonyl CoA. CPT-1 is the rate-limiting enzyme of mitochondrial fatty acid oxidation [28
], which can increase the capacity of fatty acid flux into the mitochondria. In other studies [29
], drugs affect the lipid regulatory system by activating the AMPK-ACC-CPT-1 pathway. Therefore, it is reasonable to speculate that EAOTs relieve disorders of lipid metabolism by activating the AMPK-ACC-CPT-1 pathway.
FAS is a key lipogenic enzyme in mammals, and it catalyzes all the reactions in the conversion of acetyl-CoA and malonyl CoA to palmitate [30
]. Its concentration is sensitive to nutritional and hormonal status in lipogenic tissues such as liver and adipose tissues. The nutritional regulation of FAS occurs mainly via changes in FAS gene transcription [31
]. Our study has shown that FAS expression is significantly inhibited by EAOT administration, also suggesting that EAOTs’ anti-obesity effects are partly due to the inhibition of expression of fatty acid and TG synthesis-related proteins. However, more experiments are needed to understand the effect of EAOTs on anti-obesity in other body parts.
In addition, it has been reported that AMPK activation led to decreased production of inflammatory mediators in macrophages [32
]. In our study, EAOTs stimulated AMPKα phosphorylation and decreased expression of inflammatory factor TNF-α, a potent inflammatory inducer, as well as iNOS. It appears that EAOT-induced AMPK might also contribute to the suppression of inflammatory responses, which would lead to improvements in metabolic disorders.
While the 2006 group showed better anti-obesity and anti-inflammatory effects than the other EAOT treatment groups, it is difficult to speculate which ingredients contributed to this difference, if only relying on the data in this study. As shown in Table 1
, we determined the content of free amino acids, total soluble sugar, and tea polyphenols in EAOTs. The free amino acids content was significantly lower in aged oolong tea, and the content of total soluble sugar markedly increased with the storage time. The tea polyphenols content was the highest in 2006 EAOTs, the second highest in 2016 EAOTs, and the lowest in 1996 EAOTs. After correlation analysis, these three ingredients were not related to EAOTs’ anti-obesity and anti-inflammatory effects. It may be the effect of undetermined ingredients, or the combined effect of one known ingredient and other unclear substances, that contributes to these effects. Actually, this question is exactly what we plan to research later. HPLC-MS will be used to determine and analyze more comprehensive ingredients.
In conclusion, EAOT administration has a beneficial effect, reducing body weight gain, adipose tissue weight, and adipocyte size, as well as lipid levels in serum and liver in HFD-induced obese mice. Moreover, EAOTs affect the lipid metabolic regulatory system in the liver by activating the expression of FAS and changing the activation of the AMPK-ACC-CPT-1 pathway, which is known to play a central role in lipid metabolism regulation that facilitates catabolism of fuel storage. In addition, EAOTs could have an impact on inflammation, which protects the liver function and energy metabolism indirectly. Taken together, these results suggest that, as a novel AMPK activator, EAOTs would be a potential candidate for the treatment of obesity and metabolic disorders.