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 
. It has been shown that kuding tea lowers serum TG, TC and LDL-c levels in dyslipidemia patients 
, and improves blood pressure in phase I hypertension patients, as well as preventing the progression of atherosclerosis 
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 
, while weight loss was usually caused by a reduction of the adipocyte mass and number by suppression of energy intake or overburn of excess calories. The specific mechanisms by which EK protects against weight gain is yet to be defined. There are several likely possibilities based on evidence available from our studies. Because a reduction in food intake may significantly affect body weight, blood glucose and lipid levels, we considered the possibility that the effect of EK may result from reduction of food intake. However, we did not observe the difference of food intake amount between the HF group and EK treatment mice. Green tea has been reported to inhibit intestinal lipid absorption 
. However, our data showed that kuding tea does not have an inhibitory effect on the lipids absorption. Thus, it is probable that the decreased lipid levels and body weight may not be associated with the decreased absorption of lipids at an intestinal level. Taken together, our data indicate that the protective mechanisms of EK against body weight gain are independent of the reduction of energy intake and absorption of the lipids in the gut.
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 
. Of these, ursolic acid has been studied for its effects on metabolic disorders. For example, ursolic acid enhances the binding of PPAR-α to PPRE, regulates the expression of lipid metabolism genes and significantly reduces intracellular triglyceride and cholesterol concentrations in hepatocytes 
, and decreases body weights, visceral adiposity, levels of blood glucose and plasma lipids, as well as increasing plasma leptin in high-fat diet-fed mice 
. Lupeol has also been reported to lower blood glucose in experimental diabetic animals 
. However, in the extract used in current study, it only contains 6.48% of ursolic acid (about 1/20 used the previous studies), suggesting that ursolic acid is not the only effective component. The active compounds in kuding tea need to be investigated for their roles in metabolic diseases. Or an intestinal permeation system could be used to elucidate the mechanism of kuding tea on 3T3L1 adipocyte.
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 
. The potential of the LXRs as drug targets for hyperlipidemia, AS, diabetes, hypertension and inflammation have been previously shown 
. Further investigation of the effects of EK with LXRβ knockout mice will confirm the signaling pathway of EK. And probably this will identify the inhibition of LXRβ as a therapy for metabolic diseases in vivo.
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 
. Recently two naturally occurring compounds, rhein and naringenin, have been verified as LXRα/β and LXRα antagonists respectively and have also been shown to have hyperlipidemia lowering properties 
. Kanaya et al have reported that white button mushrooms have protective effects against liver steatosis through the inhibition of LXR signaling 
. We show that EK selectively inhibits LXRβ transactivity in the presence of the LXRβ agonist GW3965, suggesting that EK contains an antagonist of LXRβ which competitively binds to LXRβ. The metabolic effect of LXRβ inhibition by EK is also shown on gene expression. The mRNA expression of LXRβ targets genes that control fatty acid oxidation, regulates fatty acid and cholesterol synthesis, such as ABCA1, ABCG1, LPL, and ApoE, and was significantly inhibited in the liver and fat tissue of EK treated mice. Identification of this specific LXRβ ligand may result in a novel therapy for metabolic diseases.
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.