This study was to explore the protective effects of Deepure tea against insulin resistance and hepatic steatosis and elucidate the potential underlying molecular mechanisms. C57BL/6 mice were fed with a high fat diet (HFD) for 8 weeks to induce the metabolic syndrome. In the Deepure tea group, HFD mice were administrated with Deepure tea at 160 mg/kg/day by gavage for 14 days. The mice in HFD group received water in the same way over the same period. The age-matched C57BL/6 mice fed with standard chow were used as normal control. Compared to the mice in HFD group, mice that received Deepure tea showed significantly reduced plasma insulin and improved insulin sensitivity. Deepure tea increased the expression of insulin receptor substrate 2 (IRS-2), which plays an important role in hepatic insulin signaling pathway. Deepure tea also led to a decrease in hepatic fatty acid synthesis and lipid accumulation, which were mediated by the downregulation of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthesis (FAS), and acetyl-CoA carboxylase (ACC) proteins that are involved in liver lipogenesis. These results suggest that Deepure tea may be effective for protecting against insulin resistance and hepatic steatosis via modulating IRS-2 and downstream signaling SREBP-1c, FAS, and ACC.
Nonalcoholic fatty liver disease (NAFLD) and insulin resistance are the main pathophysiological characteristic of metabolic syndrome [
In the liver, insulin is involved in a number of actions responsible for glucose control and lipid metabolism. Insulin receptor substrate (IRS) proteins are a family of cytoplasmic adaptor proteins that transmit signal from insulin receptor to its final biological actions through a series of intermediate effectors. Hepatic insulin signaling for these effects is mediated mainly through insulin receptor substrate 2 (IRS-2) [
Tea is one of the most popular beverages worldwide and can be categorized into three types: nonfermented green, partially fermented oolong, and fully fermented black and Pu-erh tea [
The present study was conducted to investigate whether Deepure tea, a specific tea concentrated from Pu-erh tea, could ameliorate insulin resistance and NAFLD in high fat diet (HFD) raised mice and possible mechanisms of its action. We demonstrated that Deepure tea decreased HFD-induced hyperinsulinemia and improved diet-induced NAFLD in C57BL/6 mice. HFD markedly inhibited hepatic IRS-2 protein expression in mice, which was reversed by treatment with Deepure tea. The improved hepatic steatosis appears to be mediated through the downregulation of SREBP-1c protein level, subsequently decreasing the level of FAS and ACC, which are involved in de novo lipogenesis in the liver.
Deepure tea was supplied by Tasly Pharmaceutical Co. Ltd. (Tianjin, China). The raw materials were extracted from leaves of old Pu-erh tea trees, which were from Yunnan province of China. The batch number of the Deepure tea used in this experiment was 20110918. The processing of the product followed a strict quality control, and the ingredients were subjected to standardization. Deepure tea was manufactured as nanometer level powder after dynamic cycle extraction and concentrated by evaporating and spray drying.
Antibodies recognizing IRS-2 and GAPDH were from Cell Signaling Technology (Boston, MA, USA). Antibodies against SREBP-1c, FAS, and ACC were from Abcam (Cambridge, MA, USA). BCA protein assay kit was purchased from Applygen Technologies (Beijing, China). ELISA kit for LDLR of mice was purchased from Andygene (Richardson, USA). All other reagents used in our study were of analytical grade.
Four-week-old male C57BL/6 mice (the animal certificate number was SCXK (Jing) 2006–2009) were purchased from Weitonglihua Animal Center, Beijing, China. The animals were housed at 21–23°C and a humidity level of 40–60%. They were exposed to a 12 h lighting cycle and allowed
C57BL/6 mice were fasted for 2 h before experiment. Blood samples were collected from tail veins for determination of baseline values of blood glucose (
After C57BL/6 mice were fasted for 6 h, tail vein blood was collected. The samples were centrifuged at 3500 rpm for 10 min at 4°C to separate plasma. Plasma insulin levels were measured by enzyme-linked immunosorbent assay (ELISA) using an insulin ultrasensitive ELISA kit (ALPCO, Salem, NH, USA), according to the manufacturer’s instruction. The concentrations of plasma triglyceride (TG) and total cholesterol (TC) were determined according to the kit instruction (Jian Cheng Biotechnology Company, Nanjing, China). Plasma glucose levels were detected to calculate the homeostasis model assessment (HOMA). HOMA-IR index = fasting blood glucose (mmol/L) × fasting plasma insulin (pmol/L)/22.5 [
Liver tissues were lysed in sample buffer containing 62 mM Tris-HCl, pH 6.8, 0.1% SDS, 0.1 mM sodium orthovanadate, and 50 mM sodium fluoride. The protein content was determined by the BCA protein assay. Equal amounts of proteins were loaded and separated by SDS-PAGE. After electrophoresis, the proteins were transferred on membranes, after being blocked with 3% nonfat dry milk the membrane with target proteins was incubated with an antibody against IRS-2, SREBP-1c, ACC, FAS, or GAPDH overnight at 4°C. The blots were incubated with a respective HRP-conjugated second antibody, and then immunoreactive bands were revealed using an enhanced chemiluminescence system (Applygen Technologies Inc., Beijing, China). The protein signal was quantified by scanning densitometry in the X-film by Image-Pro Plus 6.0 software (Bio-Rad, Hercules, California, USA) [
Liver tissue samples from each mouse were fixed in formalin saline solution (10%) and then embedded in paraffin, sliced at five micrometer thickness, and stained with HE for histological analysis under a light microscope.
Data were expressed as means ± SEM. Student’s
The mice in HFD and Deepure tea groups did not show any difference in body weight but both possessed a higher body weight than those in the control group, as shown in Figure
Deepure tea restored plasma level of insulin and improved insulin resistance in high fat diet mice. Plasma parameters were measured in the fasting state of the mice. (a) The body weight in different groups. (b)-(c) Plasma TG and TC level in different groups. (d) Plasma glucose level in different groups at baseline. (e) Plasma insulin level in different groups at baseline. (f) Insulin resistance of mice evaluated by homeostasis model assessment (HOMA) index. (g) Intraperitoneal glucose tolerance test (IPGTT). (h) Plasma glucose concentration tested 120 min after glucose administration. The number of animals included was 10 (control group), 9 (HFD group), and 8 (Deepure tea group), respectively. All experiments were performed in triplicate. Data are means ± SEM.
At baseline, plasma glucose level in HFD group did not differ from that in control group but decreased by 15.3% in Deepure tea treated group with significance (Figure
Histological examination was conducted at the end of the experiment, and the result is illustrated in Figure
Deepure tea diminished nonalcoholic steatohepatitis caused by HFD in C57BL/6 mice. Representative images of liver tissues collected from the mice of control (a), HFD (b), and Deepure tea (c) group with low magnification displayed in 1 and high magnification in 2, 3, and 4 are shown. The number of mice examined in each group was 6. Bar = 100
Insulin resistance is known showing a reduced insulin sensitivity of peripheral tissue with aberrant IRS-2 and downstream members of the insulin signaling pathway [
Deepure tea modulated the expression of genes involved in hepatic insulin signaling and lipid metabolism. (a) Representative western blot of IRS-2 expression in liver tissue of mice in different group. (b) Quantification of the western blot results of IRS-2. (c) Representative western blot of SREBP-1c, FAS, and ACC expression in liver tissue of mice in different group. (d) Quantification of the western blot results of SREBP-1c. (e) Quantification of the western blot results of FAS. (f) Quantification of the western blot results of ACC,
To gain insight into the protective mechanisms of Deepure tea against insulin resistance and hepatic steatosis in HFD-fed mice, we further examined the protein levels in hepatic tissue that are involved in insulin signaling and lipogenesis. SREBP-1c is a key transcriptional factor regulating de novo lipogenesis in liver [
This study provides evidence that short-term intake of Deepure tea protects against the development of hyperinsulinemia and NAFLD in HFD mice. Histologic results clearly showed that 14-day feeding of Deepure tea at 160 mg/kg reduced dietary-induced hepatic steatosis, which was correlated with the downregulation of SREBP-1c, FAS, and ACC expression in the liver. Consistent with reduced plasma insulin, the hepatic IRS-2 expression was significantly reduced in HFD mice. Thus, the findings from our investigation suggest that Deepure tea may be a desirable food for preventing insulin resistance and ectopic lipid accumulation, especially in HFD-induced obesity.
High-energy diets are used widely in nutritional experiments as a strategy to induce obesity in animals [
Insulin resistance manifests reduced insulin sensitivity of peripheral tissue with an abnormality in the insulin signaling pathway, including IRS and other downstream molecules [
Excessive intake of fatty acid will lead to fatty liver. In the present study, C57BL/6 mice fed with high fat diet displayed NAFLD, whereas after administration of Deepure tea, the lipid content in liver was significantly reduced, suggesting that the Deepure tea could significantly improve hepatic lipid accumulation.
The increased fatty acid de novo synthesis is one of the major sources of lipid accumulation in liver. The protein SREBP-1c, as a transcriptional regulator of lipid synthesis, activates genes required for de novo lipogenesis [
In conclusion, this study demonstrates that short-term oral administration of Deepure tea has beneficial effects on diet-induced hyperinsulinemia and ectopic lipid accumulation. These effects are most likely mediated through modification of IRS-2 and its downstream signaling SREBP-1c, FAS, and ACC. Importantly, the dose of Deepure tea administrated in the present study was close to that which human intakes as routing. Thus, these results also suggest that regular Deepure tea consumption is conducive to maintain metabolism homeostasis.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Jing-Na Deng and Juan Li contributed equally to this work.
This work was supported by the Production of New Medicine Program of Ministry of Science and Technology of China [2013ZX09402202].