The authors have declared that no competing interests exist.
Conceived and designed the experiments: CH GJ XL. Performed the experiments: SF YZ NH QS GL BZ MG FH YQS XD. Analyzed the data: SF YZ XL CH. Wrote the paper: SF YZ GJ CH ZZ.
To investigate the effects of ilex kudingcha C. J. Tseng (kuding tea), a traditional beverage in China, on the metabolic disorders in C57BL/6 mice induced by high-fat diets.
For the preventive experiment, the female C57BL/6 mice were fed with a standard diet (Chow), high-fat diet (HF), and high-fat diet mixed with 0.05% ethanol extract of kuding tea (EK) for 5 weeks. For the therapeutic experiment, the C57BL/6 mice were fed high-fat diet for 3 months, and then mice were split and EK was given with oral gavages for 2 weeks at 50 mg/day/kg. Body weight and daily food intake amounts were measured. At the end of treatment, the adipocyte images were assayed with a scanning electron microscope, and the fasting blood glucose, glucose tolerance test, serum lipid profile and lipids in the livers were analyzed. A reporter gene assay system was used to test the whether EK could act on nuclear receptor transcription factors, and the gene expression analysis was performed with a quantitative PCR assay.
In the preventive treatment, EK blocked the body weight gain, reduced the size of the adipocytes, lowered serum triglyceride, cholesterol, LDL-cholesterol, fasting blood glucose levels and glucose tolerance in high-fat diet-fed C57BL/6 mice. In the therapeutic treatment, EK reduced the size of the white adipocytes, serum TG and fasting blood glucose levels in obese mice. With the reporter assay, EK inhibited LXRβ transactivity and mRNA expression of LXRβ target genes.
We observed that EK has both preventive and therapeutic roles in metabolic disorders in mice induced with high-fat diets. The effects appear to be mediated through the antagonism of LXRβ transactivity. Our data indicate that kuding tea is a useful dietary therapy and a potential source for the development of novel anti-obesity and lipid lowering drugs.
Obesity is a worldwide problem and its prevalence is increasing rapidly
Liver X receptors (LXRs) are members of the nuclear receptor family of transcription factors. Two isoforms of LXR, LXRα and LXRβ, have been identified, and they are important regulators of lipids and cholesterol homeostasis. LXRα knockout mice are healthy when fed a low-fat diet. However, LXRα knockout mice develop high cholesterol levels in the liver and enlarge fatty livers when fed a high-fat diet
LXRs are potential drug targets for obesity, dyslipidemia and atherosclerosis. Previous work has shown that the synthetic LXR agonist GW3965 lowers cholesterol levels in both serum and the liver, inhibits the development of atherosclerosis in mouse models
Green tea and kuding tea are two of the most popular beverages in China. Green tea has been well studied for its various health benefits, but there is little data on the biological activities of bitter tea. Kuding tea has been used in China for more than 2000 years as a beverage. In traditional Chinese medicine, kuding tea has also been used in the formulae for treating obesity, hypertension, cardiovascular disease, hyperlipidemia and various other diseases. Recently, several clinical studies have focused on its effects on lipid lowering, body weight reduction and blood glucose lowering in patients with metabolic syndromes. Animal studies have shown that the phenolic constituents and phenylethanoid glycosides of kuding tea exhibit significant antioxidant activities
To analyze the fingerprint profiles, EK (5 mg) was dissolved in methanol (1 ml). Filtered extracts were analyzed using an Agilent 1200 liquid chromatograph system with a UV detector at the λ max of 270 nm. Chromatographic separation was performed on Discovery C-18 reverse phase column (250×4.6 mm, 5 µm) with an injection volume of 10 µl methanol (as solvent A) and water (as solvent B). The gradient was set as follows: 0∼10 min, 5% B; 20 min, 30% B; 25 min, 50% B; 40 min, 90% B; 45 min, 95% B (flow rate 1 ml/min).
Ursolic acid and Lupeol (purity >98%) were purchased from Shanghai R&D Center for standardization of Chinese Medicines (Shanghai, China). The compounds were monitored at 210 nm using a Discovery C18 Column with methanol or acetonitrile (as solvent A) and water containing 0.1% phosphoric acid (as solvent B) in the mobile phase at a flow rate of 1.0 mL/min at 30°C for 60 min. To detect Ursolic acid, the gradient elution of HPLC was 48% A (acetonitrile) and 52% B (phosphoric acid, pH 2.5) at 0 min, 75% A and 25% B at 40 min, 85% A and 15% B at 60 min. The constant mobile phase of methanol: water (98∶2, v/v) was used for detection of lupeol.
3T3-L1 cells were grown and maintained in DMEM containing 10% fetal bovine serum (Hyclone, Logan, UT). For adipocyte differentiation, cells were grown in 12-well plates to full confluence for 2 days and then differentiation medium (DM) containing 10 µg/ml insulin (Sigma, St. Louis, MO), 0.5 µM dexamethasone (Sigma, St. Louis, MO), and 0.8 mM isobutylmethyl xanthine (IBMX, Sigma, St. Louis, MO) was added to the culture. After 4 days, the medium was changed to DMEM with 10% fetal bovine serum for differentiation at 37°C in 10% CO2. EK was dissolved in DMSO and was added to the medium at indicated concentrations. DMSO was added to the cells as the untreated control.
The cells were washed with PBS twice, fixed with 10% formalin at room temperature for 10 minutes, and then stained with oil red O (Sigma, St. Louis, MO) at 60°C for 10 minutes. Pictures were then taken using an Olympus (Tokyo, Japan) microscope.
Total RNA was extracted using a spin column (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and RNA was treated with DNase I to remove genomic DNA contamination. The first strand cDNA was synthesized using a cDNA synthesis kit (Fermentas, Madison, WI), and gene expression levels were analyzed by quantitative real-time RT-PCR using the ABI Stepone Plus Real Time PCR system (Applied Biosystems, Carlsbad, CA). The primers used in the experiments are shown in
Gene | Forward primer | Reverse primer |
β-Actin |
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LXRα |
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LXRβ |
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ABCA1 |
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ABCG1 |
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ApoE |
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Cyp7a1 |
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SREBP1 |
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FAS |
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LPL |
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C/EBPβ |
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PPARα |
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PPARγ |
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PPARβ/δ |
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C/EBPα |
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aP2 |
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ACC |
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ACO |
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UCP-2 |
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The reporter assay was carried out as previously described
The animal protocols used in this study were approved by the Shanghai University of Traditional Chinese Medicine for animal studies (Approved Nember:11002). Female C57BL/6 mice were purchased from the SLAC Laboratory (Shanghai, China). All mice were kept under controlled temperatures (22–23°C) and on a 12-hour light, 12-hour dark cycle. For the preventive experiment, C57BL/6 mice of similar ages and body weights were randomly divided into different groups, and then placed on a high fat (HF) diet (60% of calories derived from fat, Research Diets, New Brunswick, NJ; D12492), or on a high-fat diet mixed with 0.05% of EK, or on a low-calorie diet as the equivalent chow diet control (10% of calories derived from fat, Research Diets; D12450B). The diet study was started at 6 weeks of age and continued for 11 weeks. For the therapeutic experiment, the mice were placed on a high fat diet for 3 months and then the obese mice were grouped randomly. EK was given with oral gavages for 2 weeks at 50 mg/day/kg (HF +EK) while the control mice were given water with oral gavages (HF). The normal control mice were kept on the normal diet through the experiment (Chow). Twenty-four hour food intake was measured by recording the difference in weight between the food put into the cage and that remaining at the end of 24 hours in both treated groups and controls. The experimental diets did not result in any change in the daily food intake compared with controls. Serum triglyceride (TG), total cholesterol (TC), HDL cholesterol (HDL-c), and LDL cholesterol (LDL-c) levels were examined using a Hitachi 7020 Automatic Analyzer (Hitachi, Tokyo, Japan) with 100 µl of heart blood serum.
The liver samples were weighed and homogenized in tissue lysis buffer (20 mM Tris·HCl pH 7.5, 150 mM NaCl, 1% Triton) and extracted with an equal volume of chloroform. The chloroform layers were dried and dissolved in isopropyl alcohol to measure lipid levels as described above. Fecal lipids were also extracted and measured as described above.
For H&E staining, the tissue was fixed in 10% formaldehyde, embedded in OCT compound and cut into 10 µm section according to a standard protocol. The sections were stained with Hematoxylin and eosin and examined under a light microscope.
Scanning electron microscopy was used to examine the structure of fat tissue according to the previously described protocols
After 2 weeks of treatment, C57BL/6 mice were fasted overnight for 12 hours. The blood samples were collected from the tail vein for determination of baseline glucose values (0 minutes) before the injection of glucose (1 g/kg body weight). Additional blood samples were collected at regular intervals (15, 30, 60, and 90 minutes) for glucose measurement.
Data analyses were performed using the SPSS12.0 for Windows statistical program. All data were presented as means ± SE. Statistical analysis was done by one-way analysis of variance (ANOVA). Differences were considered significant when P<0.05.
To analyze the components of the ethanol extract of kuding tea, we assayed the fingerprint of EK by HPLC.
Since kuding tea has been used for the prevention and treatment of obesity and hyperlipidemia, we observed the effects of EK on the differentiation of 3T3-L1 adipocytes. We used insulin, dexamethasone, and isobutylmethyl xanthin (differentiation medium, DM) to induce 3T3-L1 pre-adipocyte differentiation. During the DM induction, EK was added to the medium from day 0 until day 6 of differentiation. The results showed that the ethanol extract of kuding tea inhibited the differentiation of 3T3-L1 adipocyte significantly, whereas the water extract of kuding tea did not alter 3T3-L1 adipocyte differentiation (
The water extract and the ethanol extract were added into the medium at the concentration of 20 µg/ml. DMSO was used as the vehicle control. The cells were stained with oil red O at day 6 of differentiation. GM: growth medium; DM: differentiation medium.
The differentiation of 3T3-L1 adipocytes involves two steps: a 4 day induction and 5–7 days of differentiation, both of which are controlled by different molecular events
(A): EK was used at the beginning of DM induction of 3T3-L1 cells and was removed during differentiation. (B): EK was only used during the differentiation of 3T3-L1 cells. The cells were stained with oil red O at day 6 of differentiation. (C): Real-time RT-PCR results of gene expression levels in 3T3-L1 adipocyte. The cell was differentiated for 6 days and then the cell was treated with EK at 20 µg/ml for 24 hours. DM: differentiation medium. β-actin was used as an internal control. Data are presented as means ± SE for 4 treatments. *P<0.05.
To confirm this, quantitative PCR was carried out to test the alterations of gene expression of related genes in the EK treated 3T3-L1 adipocyte. The results showed that EK significantly inhibited the expression of the adipocyte marker of PPARγ and aP2. The expression of fatty acid synthase (FAS) was also reduced following EK treatment. However, EK treatment did not alter the expression of ACC, CD36 and UCP2 genes (
We next examined the effects of EK on increased body weight induced by a high-fat diet in C57BL/6 mice. After 5 weeks of treatment, the body weight of the mice fed the high-fat diet was increased significantly when compared to that of the mice on the standard diet (chow). When supplemented with EK, the body weight gained was much less than in the HF control mice (
(A): Body weight gain. (B): Food intake amount. (C): Fecal TG contents. (D): Images of white and brown adipocytes under a scanning electron microscope. (E): Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) levels. (F): Effects of EK on glucose tolerance in HFD fed mice as determined by the glucose tolerance test (GTT). NS: No significance. The data were shown as mean ± SE. N = 7 for all groups. * P<0.05.
Obesity is closely associated with hyperlipidemia and insulin resistance, so we tested the serum lipid and fasting blood glucose levels in mice. Serum lipid analysis displayed that EK treatment led to a reduction of LDL-c levels under HF diet conditions, but TG, TC and HDL-c levels were not changed significantly (
To assay the effects of EK on hepatic steatosis induced by a high-fat diet, we examined the fat content and lipid profile in the liver of EK-treated mice. HE staining showed that mice fed the high-fat diet for 5 weeks had similar morphologies of hepatocyte tissues to the standard diet-fed mice (
(A-C): H&E staining (×200) of livers from the standard diet (A), HF diet (B) and HF+EK mice (C). (D-F): Oil red O staining (×400) of the liver sections from the standard diet (D), HF diet (E) and HF+EK mice (F). The sections were counterstained with hematoxylin. The quantitative results of TG (G) and TC (H) content in livers are shown. The mice were fed with a high-fat diet for 5 weeks and EK was powdered and mixed into the diet at 0.05% (wt/wt). The data were presented as mean ± SE. N = 7 for all groups. * P<0.05.
Next, we tested whether EK could affect the metabolic disorders in obese mice. After 3 months feeding of high-fat diet, C57BL/6 mice developed high body weight, serum lipids and blood glucose. Then the mice were divided into two groups and treated with 50 mg/kg/day EK for 2 weeks. EK treatment did not significantly reduce body weights (
(A): Body weights before and after treatment. (B): Food intake amount. (C): Images of white adipocytes using scanning electron microscopy. (D): Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) levels. (E): Glucose tolerance in HFD fed mice as determined by glucose tolerance test (GTT). The mice were injected glucose at 1 mg/kg for the intra-peritoneal glucose tolerance test, and glucose levels were tested at regular intervals of 15, 30, 60, and 90 minutes. NS: No significance. The data were shown as mean ± SE. N = 7 for all groups. * P<0.05.
Serum lipid analysis showed that TC, TG, LDL-c and HDL-c levels in obese mice were significantly increased when compared to those seen in standard diet fed mice. EK treatment notably lowered the TG contents, but did not change the serum TC, LDL-c or HDL-c levels (
Furthermore, we tested the fasting blood glucose levels and glucose tolerance in EK treated mice.
To test the effects of EK in vivo, we examined the gene expression in the liver of EK treated mice. As shown in
3T3-L1 adipocyte differentiation induced by DM. Real-time RT-PCR results of gene expression levels of PPARγ, PPARα, PPARδ/ß, C/EBPα, C/EBPß, UCP2, ACO, ACC and aP2 in liver of EK treated mice were compared to that of HF control mice from preventive treatment. β-actin was used as an internal control. Data are presented as means ± SE for 5 mice per group. *P<0.05.
Nuclear receptor transcription factors are important regulators of lipid and glucose homeostasis. Based on the inhibition of obesity and hyperlipidemia, we tested whether EK acts on PPARγ, α, β/δ and LXRα and LXRβ, which are drug targets for metabolic syndromes
(A, B): LXRα and LXRβ trans-activities. The expression plasmids of pCMXGal-mouse LXRα and LXRβ-LBD were co-transfected with Gal4 reporter vector MH100 × 4-TK-Luc to 293T cell for 24 hours. Then the cell was treated with 10 µM of LXR agonist GW3965 and/or 5–20 µg/ml of EK for another 24 hours. DMSO was used as the vehicle control. The relative luciferase activities were measured by comparison to rellina luciferase activities. The results represent at least three independent experiments and data are presented as means ± SE. *P<0.05. (C) Gene expression levels of LXR target genes in livers of EK treated mice were compared to that of HF control mice from preventive treatment, and β-actin was used as an internal control. Data are presented as means ± SE for 5 mice per group. *P<0.05.
LXR regulates lipid and glucose metabolism through the activation of expression of a set of target genes, including fatty acid synthase (FAS), sterol regulatory element-binding protein-1c (SREBP-1c), lipoprotein lipase (LPL), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1) and LXR itself
In this study, we provide the evidence that kuding tea could prevent and alleviate metabolic disorders in high-fat diet-fed mice. Our results clearly showed that EK treatment blocks the body weight gain, hyperlipidemia, and insulin resistance in the mice induced with HF diet. Chemical and histologic evidence showed that EK treatment resulted in a significant reduction of lipid accumulation in hepatic issues of DIO mice, suggesting that EK has protective effects against the development of metabolic disorders such as obesity, dyslipidemia, diabetes and hepatic steatosis in mice. We also found that EK could improve metabolic disorders in obese mice. Furthermore, we determined that EK selectively suppresses the transactivity of the nuclear receptor transcription factor LXRβ. Thus, the findings from this investigation suggest that the protective effect of EK against metabolic disorders is likely due to its inhibitory effect on LXRβ.
Kuding tea is a popular beverage in China. Like green tea, kuding tea is used in health care formulae to ameliorate metabolic disorders such as obesity. In recent years, kuding tea has been reported to have various biological effects
C57BL/6 mice can develop metabolic syndromes when fed a high-fat diet. In the current study, the body weight, serum TC, TG, LDL-c and liver TG levels, glucose tolerance were significantly increased in mice fed a high-fat diet for 5 weeks. EK treatment resulted in a significantly lower weight gain and serum TC, and improved glucose tolerance and lipid accumulation in the liver, suggesting that EK could prevent the development of metabolic syndromes.
Weight gain is a consequence of the increase in adipocyte mass and numbers caused by excess calories stored as TG
In our therapeutic experiment, the EK-treated obese mice displayed lower serum TG and fasting glucose levels than obese control mice. However, there is no significant body weight loss, TC or LDL-c reduction in the mice. There are two possible reasons for this discrepancy. First, removing of excess fat has been shown to be much more difficult than prevention of fat gain. Second, in the preventive therapy, we treated the mice for 5 weeks, but the mice were only treated for two weeks for therapeutic treatment. Therefore, the results of the present study do not support the weight-reducing effects of kuding tea in clinical trials reported by previous investigators. The glucose tolerancewas improved in preventive treatment, but was less effective in therapeutic treatments. This may result from the reduction of fat tissue because the deposition of TG in cells is responsive for the development of insulin resistance
Compared to the water extract of kuding tea, the ethanol extract inhibited the adipocyte differentiation of 3T3-L1 adipocytes suggesting that liposoluble components of kuding tea may act on the adipocytes. The chemical analysis has shown that the ethanol extract of kuding tea contains 11 major compounds: lupeol, 11-keto-α-amyrin palmitate, α-amyrin palmitate, 12-ursene-3,28-diol, ursolic acid, 3β-hydroxylup-20(29)-en-30-al, 3β-hydroxy-20-oxo-30-norlupane, tanacetene, β-sitosterol, n-behenic acid and n-hexacosane
Published data regarding the mechanism of kuding tea are limited, but the studies suggest that kuding tea may improve metabolic disorders through multiple mechanisms. Our data suggest that kuding tea may prevent metabolic disorders by selectively targeting nuclear receptors of transcription factors LXRβ. The LXR family are ligand-activated transcription factors including both LXRα and LXRβ. LXRα is expressed primarily in the liver, adipose tissue, and macrophages, while LXRβ is ubiquitously expressed
Previous studies have shown that LXR agonists could lower serum TC levels, but increase liver and serum TG levels, which excludes the LXR agonists as a therapy for metabolic diseases. Development of selective agonists or antagonists of LXRs may avoid the off-target effects
In conclusion, we provide evidence that EK protects against the development of obesity, hyperlipidemia and insulin resistance in high-fat diet-fed mice. These findings suggest that EK may be used as a potential dietary strategy for preventing metabolic disorders such as obesity, hyperlipidemia, diabetes and atherosclerosis. The potential of using naturally-occurring dietary supplements to regulate body weight and lipid metabolism is attractive. Because this traditional beverage is safe and cheap, it should be considered as a dietary therapy for metabolic syndromes. This is particularly important because weight loss and the treatment for non-alcoholic fatty liver disease have a poor long-term success rate. Further investigations are needed to define the mechanisms by which this component protects against obesity and its associated symptoms.
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We thank Dr. E. Saez for providing us LXRs plasmids, Dr. R. Evans for the PPARs plasmids.