Chlorogenic acid modulates glucose and lipid metabolisms via AMPK activation in HepG2 cells and shows its anti-hyperglycemic effect on streptozocin-induced diabetic mice

Hong-Ji Yao, Zhao-Yi Xue, Rui-Qi Wang, Cheng-Zuo Jiang, Jing-Yan Xiong, Zhi-Xuan Xia, Qiang Liu, Qi-Bing Li , Yong Zhang?

1. Department of Pharmacology, Hainan Medical University, Haikou 571199, China

2. Research Center of Pharmaceutical Engineering Technology, Harbin University of Commerce, Harbin 150076, China

Keywords:Chlorogenic acid Glucose and lipid metabolism Anti-diabetes activity AMPK

ABSTRAC T Objective: In this study, we focus on the hypoglycemic effects of chlorogenic acid (CGA) in vitro and in vivo and its mechanism base on regulate glucose and lipid metabolism via AMPK activation. Methods: The cytotoxicity, glucose consumption and intracellular triglyceride assay were been detected by commercial kits. The western blots were used to detection the associated protein levels after CGA treatment, and the inhibiter blocking experiments were also be done. In vivo experiment, the fasting blood-glucose, lipid metabolism, liver function, insulin resistance, glucose tolerance, and pathological change were assessed on streptozocin induced diabetic mice. Results: We found that CGA exhibited no cytotoxicity at concentrations of 100μM, it caused a significant increasing of glucose consumption and reducing of the PA-BSA induced intracellular TG level on HepG2 cells at 50μM and 100μM treatment, CGA exhibited up-regulating the level of p-AMPK (Thr172) and p-ACC (Ser79) in dose-dependent manners in vitro and in vivo. The stimulating activities of CGA on AMPK were completely blocked by compound c (CC) on HepG2 cells. And the efficacies of CGA on glucose consumption and intracellular TG accumulation were also completely blocked by CC pretreatment. The CGA also exhibited potent anti-diabetic effects with hypoglycemic activity, improve insulin resistance and glucose tolerance, regulate glucose and lipid metabolism and protect the liver function in vivo. Conclusion: Our results suggested that CGA can regulate glucose and lipid metabolism by AMPK activation, and exhibit potent anti-hyperglycemic effect in streptozocin induced diabetes mice, and may be used as a potential effective anti-diabetes drug.

1. Introduction

Diabetes has become a worldwide epidemic chronic metabolism disease, and increase global health burden with a total of 463 million patients in 2019 (International Diabetes Federation, 2019)[1,2]. The diabetes complications usually associate and damage the liver, heart, eyes, kidneys and nerves[3]. The glucose and lipid metabolism disorder is the main phenotype for diabetic and one of main inducement for diabetes complication[4].

The liver is one of the main organs to regulate the glucose and lipid metabolism in human body. Glucose uptake and oxidative catabolism commonly occur in liver, and contribute to the blood glucose decreasing for diabetes[5]. The liver is important for glucose homeostasis maintaining, and it can transform glucose into glycogen, and provide glucose via glycogenolysis and gluconeogenesis in body[6]. At the same time, the liver is also the main organ of participating in lipid metabolism. AMP-activated protein kinase (AMPK) is one of the key enzymes associated with the glucose and lipid metabolism in liver. AMPK activation can promote glucose consumption, glucose uptake, fatty acid oxidation of liver, and inhibit lipid synthesis, and reduce glucose output, which beneficial for people with diabetes[6-8]. Many studies have shown that the liver AMPK activation usually is good for improving glucose and lipid metabolism disorder and insulin resistance[9], and many anti-diabetes drugs in clinical could activity the AMPK of liver, such as metformin[10], thiazolidinediones[11], berberine[12].

Chlorogenic acid (CGA), molecular formula as C16H18O9, is widely found in many famous traditional chinese herbal, such as Honeysuckle and Eucommia ulmoides Oli. Many in vitro and in vivo research reports have found that CGA own many important biological activity, such as antioxidant, anti-inflammatory, antibacterial, hypoglycemic, lipid-lowing, anti-cardiovascular, antimutagenic, anti-cancer, immune regulation [13-15], and suggested that it is a potential drug for the prevention or treatment of diabetes mellitus and related complications.

In this study, we focus on its anti-diabetes activity and mechanism.The anti-diabetes activity of CGA is definite, and has been certified in clinical trial and animal experiment in vivo [16]. The reported hypoglycemic mechanism of CGA associated with G-6-P enzyme in liver and glucose uptake in skeletal muscle. In our study, we are interested in its effects on glucose and lipid metabolism and its associate with AMPK activation in HepG2 hepatocyte in vitro, and its anti-diabetes activity on STZ induced diabetic mice.

2. Materials and Methods

2.1 Cell culture and treatment

HepG2 cells were cultured in DMEM with 10% fetal calf serum(FBG) and appropriate antibiotics at 37℃ in incubator with 5%CO2. Chlorogenic acid (CGA) (purity≥98%, lot number:20191120,purchased from Biofurify Phytochemicals Ltd.) and metformin(Met) (purity≥98%, lot number:20191008, purchased from Biofurify Phytochemicals Ltd.) were dissolved in DMEM medium,and Met as the positive drug. The cells were seeded into 96-well plates (2.0×104/well), 24-well plates (1.0×105/well), or 6-well plates (4.0×105/well) and cultured for about 24 h, then after the cell confluence reach about 75%-85%. The cells were culture with 0.5%serum medium for 12 h for serum starvation, CGA and Met were added to treat the cells at indicated concentrations or time intervals,DMEM medium as a negative control.

2.2 Cytotoxicity assay

Cell viability was evaluated by the MTT assay. In brief, HepG2 cells were seeded into 96-well plates, cultured as above, then treatment with CGA at series of concentration for 24 h. After treatments, the medium were removed and the cell were incubated with 50 μl MTT solution for 4 h, the level of formazan was tested by detection the absorbance at 490 nm under a microplate reader.

2.3 Glucose consumption assay

As our previous study, HepG2 cells were seeded into 96-well plates at 1.0×105cells per well concentration, then cells were treated with DMEM medium or CGA or Met at indicated concentration for 24 h with 5 replicates for each treatment. Then the glucose levels of culture supernatants were assayed by the randox glucose assay kit base on glucose oxidase method. The Glucose consumption of drug treatment was calculated by the initial glucose level minus the remaining glucose level of medium.

2.4 Intracellular triglyceride Assay

The method of palmitic acid coupled with bovine serum albumin(BSA) was as previous study17): First make the fatty free BSA solute in DMEM medium to get the 10% BSA solution, then palmitic acid powder was added to the 10% fatty acid free BSA solution and dissolved by shaking gently 1-2 h at 50 oC to yield an 7.5 mM solution of palmitic acid complexed to BSA (PA-BSA).

HepG2 cells were cultured and seeded as above. After starving in 0.5% FBS-containing medium overnight, the cells were treated with 300 μM PA-BSA for 24 h, and 10%BAS solution treatment as control at the same time. Then PA-BSA, CGA and Met at indicated concentration were added to PA-BSA pretreatment wells for treatment 24 h. The intracellular triglyceride (TG) levels were tested by the cell and tissue TG assay kit, and the TG levels were normalized to protein concentrations of samples.

2.5 Western blot

The HepG2 cells were treatment with drugs as above, the cell total proteins were extracted, quantified, and the samples of western blot were produced by cell total protein. The western blots were done as follow: the samples with 50μg total protein were subjected to 10%or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE); bands were electrically transferred to a PVDF membrane (Merck KGaA, Darmstadt, Germany). After blocking,the detection of p-AMPKα (Thr172) (p-AMPK), AMPKα (T-AMPK),p-ACC (Ser79) (p-ACC), ACC (T-ACC) and β-actin (ACTIN)proteins were be done with specific monoclonal antibodies and secondary antibodies. The protein bands were developed by using an ECL kit (Beyotime biotechnology, Nanjing, china). The blots were scanned and quantified, the level of p-AMPKα and p-ACC was normalized to that of T-AMPK and T-ACC. The level of target protein was showed as fold of control treatment.

2.6 Blocking experiments

HepG2 cells were cultured and seeded as above. Before drug treatment, the cells were pretreated with compound c (dissolved in DMSO) at 10 μM for 1 h, and the untreated wells were added DMSO as no compound c wells; After pre-treatment, the compounds of this study were added to corresponding wells, and co-cultured with the cells for 24 h. The total protein of cells were extracted after treatment, and the samples were produced by protein loading buffer for western blots; In parallel experiments, the supernatants of cell culture were taken by centrifugation at 500 g for 10 min, the glucose consumption of every treatment were tested by the glucose assay kit as above. In other parallel experiments, intracellular TG levels were determined by TG assay kit as above, and normalized to protein concentrations every treatment.

2.7 Animals

Sixty male Kunming mice, 6-week old, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).The animal experiments of this study were performed in accordance with the national institutes of health regulations for the care and use of animals in research at Animal Research Center of Hainan Medical University (SYSX-(qiong)-20160013). The experimental protocols were approved by the medical ethics committee of Hainan Medical University (HMU-IACUC/20200625). All the animals were kept in Animal Research Center of Hainan Medical College under typical environmental surroundings at 24 ± 0.5 °C and 12 h light-dark cycle with free standard laboratory water as well as to food freely.

2.8 Streptozocin induced diabetic mice

The animals were feed with basal food and water freely for three days first. Six mice were feed the basal diet as the normal group, and the others mice feed the high fat diet for modeling. The modeling mice were injected of freshly prepared STZ (70 mg/kg/d)dissolved in 0.1 mM citrate buffer (pH 4.5) after overnight fasting by intraperitoneal injection, and consecutive administrated three days. The fasting blood-glucose was detected at the fourth day after STZ injection with eight hours overnight fasting. The fasting bloodglucose level greater than 11.1 mM was served as diabetic mice and used in the present study.

2.9 Animals grouping and treatment

All successful modeling diabetes mice were randomly divided into 4 groups (n=6 in each group) as STZ induced diabetes model group, and two therapy groups as 50 mg/kg/d CGA and 200 mg/kg/d CGA, and one positive control group as 200 mg/kg/d Met. The normal group was no diabetes mice as above. The normal control and diabetes model group mice were given equal volume of saline solution. The fasting blood-glucose was detected every week. At the end of the 4-week treatment, all animals were fasted for 8 h, the blood samples were collected from the orbits, and the serum was separated by centrifugation at 3000 rpm for 15 min, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST),TG, low density lipoprotein cholesterol (LDL-c), and cholesterol(CHO) in serum samples were detected by appropriate kits. After blood samples taken, animals were euthanized, the liver was taken from each animal for further assay.

2.10 Oral glucose tolerance test and insulin tolerance test

The oral glucose tolerance test (OGTT) was done before experiment finish after CGA treatment on all animals. Blood samples were taken from tail vein after fasting overnight, and the blood glucose were detected by kits, and define it as the 0 min blood glucose. Then 2 g/kg of glucose was oral administrate to all animals, and blood samples were taken from tail vein for blood glucose detection at 0, 30, 60, 120, 180 min. The blood glucose levels were tested by a glucose meter (Roche, ACCU-CHEK Active).

The insulin tolerance test was also done before experiment finish after CGA treatment on all animals. Blood samples were taken from tail vein after 6 h fasting, and the blood glucose were detected by a glucose meter (Roche, ACCU-CHEK), and define it as the 0 min blood glucose. Then 0.5 U/kg of insulin was conduct to all animals,and blood samples were taken from tail vein for blood glucose detection at 0, 30, 60, 120, 180 min after administration. The blood glucose levels were tested by a glucose meter (Roche, ACCUCHEK), and define it as the 0 min blood glucose. Then 0.5 U/kg of insulin was intraperitoneal injected to all mice. Blood samples were collected from tail veil for blood glucose detection by a glucose meter (Roche, ACCU-CHEK Active) at 30, 60, 90 and 120 min after administration. The blood glucose levels were determined by a glucose meter.

2.11 Hematoxylin and eosin staining pathological detection

The liver tissue of each mouse was taken from the same site and fixed in 10% formalin. The tissues were subjected to paraffin embedding, histological section (4 μm), and hematoxylin and eosin(H&E) staining. Pathological changes of the liver were evaluated and scored by steatosis (0, ＜ 5% of parenchyma involved; 1, 5%to 25% of lobular parenchyma involved; 2, 25% to 50% of lobular parenchyma involved; 3, 50% to 75% of lobular parenchyma involved; 4, ＞75% of lobular parenchyma involved).

2.12 Statistical analysis

In vitro, all the values present as mean ± SD, and at least 3 separate experiments; In vivo, all animal experiment results shown with the value as mean ± SD of 6 mice in each group. The GraphPad Prism 5 software (GraphPad Software, Inc, La Jolla, CA, USA) was used for statistical analysis. After validation of the test for homogeneity of variance, differences among studied groups were examined by one-way ANOVA followed by multiple comparisons. p < 0.05 was considered to be statistically different.

3. Results

3.1 Cytotoxicities of CGA in HepG2 cells

As shown in Figure1, CGA has no cytotoxicities at 100 μM work concentration with 24 h treatment in HepG2 cells. But it can importantly reduce the viability of HepG2 cells when its concentration reached 200 μM (p＜0.05 vs CK).

Figure1 The Effect of CGA on cell viability.

3.2 CGA stimulates glucose consumption

Next, the activity of CGA on glucose metabolism was investigated by glucose consumption assay. The results as Figure2A, CGA can importantly increase the basal glucose consumption level in HepG2 cells (p ＜ 0.01 or p ＜ 0.001 vs CK), and this activity of increasing is in a dose-dependent manner (Figure2A). The CGA at 50 μM caused a significant increase of glucose consumption (Figure2A, p＜ 0.01 vs CK),and when the concentration of CGA reached 100 μM, its efficacy was comparable to that of 5mM metformin (Figure 2A).

Figure 2 The Effects of CGA on glucose consumption and intracellular TG.

3.3 CGA reduced the intracellular TG accumulation in HepG2 cell by PA-BSA induced

To investigate the effects of CGA on lipid metabolism, HepG2 cells were induced by PA-BSA to accumulate TG, and CGA were used to withstand the PA-BSA induced intracellular TG accumulation in HepG2 cell. As shown in Figure 2B, 0.3 mM of PA-BSA treatment for 24 h increased intracellular TG level (Figure2B, p ＜ 0.01 vs CK) dramatically in HepG2 cells. And CGA could reduce intracellular TG content by PA-BSA induced in dose-dependent manners (Figure2B, p ＜ 0.01 or p ＜ 0.001 vs PA-BSA). The CGA at 50 μM showed a significant efficacy on reduce intracellular TG accumulation (Figure2B, p ＜ 0.05 vs PA-BSA). At the same time,the efficacy of positive drug metformin was observably (Figure2B, p＜ 0.001 vs PA-BSA).

Figure 3 The Stimulating effects of CGA on the AMPK pathway.

3.4 CGA actived AMPK and could be block by AMPK specific inhibitor

As we know, AMPK is a highly conserved sensor of cellular energy and a master regulator of metabolic homeostasis. Our above results have suggested that CGA could modulate lipid and glucose metabolisms in HepG2 cells. we further explored that CGA could activate AMPK, and increased the level of p-AMPK(Thr172) and p-ACC (Ser79) in dose-dependent manners after 24 h of administration (Figure3A and B) in vitro. CGA at 50 μM could stimulate the cellular AMPK pathway significantly (Figure3A and B, p ＜ 0.05 vs CK). At the same time, the liver AMPK were detected by western blot and indicated that CGA could significantly activate the mice liver AMPK under 4-week treatment (Figure3C and D), exhibited up-regulating the level of p-AMPK (Thr172) and p-ACC (Ser79) in dose-dependent manners in vivo (Figure3D).This stimulating activitie of CGA on the p-AMPK on HepG2 cell were completely blocked by CC, a specific inhibitor of AMPK(Figure4A and B). At the same time, the efficacies of CGA on glucose consumption (Figure4C) and intracellular TG accumulation(Figure4D) were also completely blocked by CC pretreatment,indicating that AMPK was indispensable. The effects of Metformin on cellular AMPK activation, glucose consumption and intracellular TG accumulation were also abolished by compound C (Figure4),which was in agreement with previous reports [18].

Figure 4 AMPK inhibition abolished the stimulating effects of CGA on the AMPK activating and blocked its inhibitory effects on glucose and lipid metabolism.

3.5 Effects of CGA on blood glucose, plasma lipid, and liver function on STZ induced diabetic mice

For further exploration the anti-diabetes activity in vivo, CGA was given orally to the STZ induced diabetic mice. Before the drug treatment, the fasting blood glucose level of all STZ induced diabetes mice group were no significant difference (Figure5A).After 4-week experimental period, the STZ induced group (17.33± 0.47 mM) showed significantly increase in fasting blood glucose level (Figure5A, p ＜ 0.001 vs normal group) compare to the normal group (5.4 ± 0.35 mM) and before drug treatment (13.6 ±0.91 mM). But administration of CGA, the average fasting blood glucose level of the dosage groups at 200 mg/kg/d reached to 14.58± 0.63 mM, exhibited significantly hypoglycemic effect compare to the STZ diabetes groups (17.33 ± 0.47 mM) (Figure5A, p <0.05 vs STZ diabetes group), and have not significantly increased compare to before drug treatment (13.8 ± 1.03 mM). The positive drug metformin also showed potent effect (figure5A, p ＜ 0.05 vs STZ diabetes group). Those results showed hypoglycemic activity of CGA in vivo.

Diabetes patients usually present with dyslipidemia, especially hyperlipidemia. As shown of the serum samples detection,STZ induced diabetic mice showed more higher serum level of triglyceride (TG) (Figure5B, p < 0.01 vs normal group), low-density lipoprotein cholesterol (LDL-c) (Figure5C, p < 0.001 vs normal group) and cholesterol (CHO) (Figure5D, p < 0.05 vs normal group)compare to normal group. Compare to the STZ diabetic mice group,treatment with CGA at 200mg/kg substantially reduced the elevated levels of TG, LDL-c and CHO. (Figure5B, C and D, p < 0.05 vs STZ induced diabetes group). The positive drug metformin also showed potent effect (Figure5B, C and D, p ＜ 0.05 vs STZ induced diabetes group). Those results showed that CGA could regulate the lipid metabolism in vivo.

Figure 5 The effects of CGA on blood glucose, plasma lipid, and liver function on STZ induced diabetic mice. The STZ diabetes mice were orally administered with CGA or Met at indication dosage with 4-week.

The liver damage is common occur during the development of diabetic. Compared to the nomal group, serum ALT and AST levels increased significantly in STZ induced diabetes mice(Figure5E and F, p ＜ 0.001 vs normal groups), which indicated that the function of liver was impaired, and the liver was damage by the hyperglycemia and hyperlipidemia of diabetes mice. The liver treated with CGA and Metformin had a noticeable decreased with ALT and AST levels (Figure5E and F). Furthermore, in the examination of liver histopathology, serious fatty degeneration and accumulation were also observed in STZ induced diabetes mice (Figure6A-E), and the pathological score of steatosis was significantly higher than normal groups (Figure6F , p ＜ 0.001 vs normal group), but which were effectively improved by CGA dose-dependently (Figure6, p ＜ 0.05 vs STZ diabetes group). Those results suggested that CGA could improve the liver function and steatosis in vivo.

Figure 6 CGA improved the hepatic steatosis of STZ induced diabetes mice at pathological examination level.

3.6 Effects of CGA on glucose tolerance and insulin resistance

For further evaluate the effects of CGA on glucose metabolism and insulin sensitivity in diabetic animals, we evaluated OGTT and ITT respectively. OGTT results as shown in figure 7A, blood glucose in all mice reached peak concentration at 30min after intragastric administration of glucose solution, and then gradually decreased.Compared with normal control Kunming mice, the AUC value of OGTT in diabetic model group was significantly increased (p < 001),indicating that glucose tolerance was significantly decreased and impaired (Figure 7A). After CGA treatment, the glucose tolerance of diabetic mice was significantly improved, and the AUC value of OGTT decreased in a dose-dependent manner (Figure 7C, p < 0.01).ITT results are shown in figure 7B. The blood glucose of kunming mice in the normal group decreased gradually after intraperitoneal injection of insulin and reached the lowest value about 30 minutes later. In the untreated diabetic model mice, the blood glucose reached its lowest level at 90 minutes later (Figure 7B), showing obvious insulin resistance, and the AUC value of ITT was significantly higher than that of the normal control group (Figure 7D, P < 0.01).After insulin injection, blood glucose of mice in all CGA treatment groups decreased significantly, and the AUC value of ITT in 200mg/kg/d CGA treatment group reached the lowest value at 60min, and the AUC value of ITT was significantly lower than that in untreated STZ diabetes model group, which was similar to that of positive Met (Figure 7D, p < 0.05). These results showed that CGA could significantly improve glucose tolerance and insulin resistance in STZ induced diabetes mice.

Figure 7 CGA improve glucose tolerance and insulin resistance in STZ induced diabetes mice.

4. Discussion

Diabetes is an increasingly serious global health problem, and estimated prevalence of it may expected to increase to 592 million people by 2035 [19]. Glucose and lipid metabolism disorder is usually the main characterized by diabetes, include type 1 and type 2 [20,21].There is no doubt that regulating glucose and lipid metabolism to decrease the level of serum glucose and lipid is good for diabetic.As we know, liver is one of the main organs for glucose and lipid metabolism [22]. And people with diabetes have an increased risk of developing many serious health problems with diabetes complications, usually damage to liver, eyes, kidneys and nerves etc organs [3,4,23]. Many anti-diabetic drugs, such as metformin,rosiglitazone, acarbose are widely used for treatment diabetes with blood glucose control, but the adverse effects are not avoid, and still are not meet the clinical needs [5]. So new effective anti-diabetes drugs are still strongly necessary for diabetic own more benefits and treatment options. Here, we reported that nature product CGA has beneficial effects against glucose and lipid metabolic disorder, may be a new potential effective anti-diabetes drugs.

Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in many chinese herbal, such as Eucommia, Lonicera,and it is also the main polyphenol compounds in coffee. Previous studies of CGA showed that it could suppress the N-nitro-sating reaction [24], inhibit glucose 6-phosphatase [25], and exhibited antioxidation [26], anti-lipidperoxidation activity [27], include antidiabetes activity [28,29]. In this study, we further used STZ induced diabetes mice model to observe the impact of CGA on glucose and lipid metabolism disorder and its anti-diabetes activity. our results showed that CGA at 200 mg/kg/d dosage significantly reduced FBG in STZ induced diabetes mice, and also reduced the serum level of LDL-c, TG, CHO, ALT and AST, improved the lipid metabolism disorder and liver disfunction. We also demonstrated that CGA could regulate the glucose and lipid metabolism by the activation of AMPK signal pathway in HepG2 liver cells.

AMPK is a key highly conserved kinase that controls energy balance in organism, and considered as an important molecular target for the treatment of glucose and lipid metabolic disorders such as diabetes, dyslipidemia, fatty liver and obesity [8]. The effects of AMPK activation, such as increasing glucose consumption, glucose uptake and fatty acid oxidation, inhibiting lipid synthesis and hepatic glucose output, are known as the main causes of hypoglycemic[7,30]. Our results suggested that CGA activated AMPK significantly in HepG2 Cells and the hepatic tissue of STZ induced diabetic mice, which improve of glucose and lipid metabolic disorders in vivo. From the previous studies by other scientist suggested that CGA could inhibit the activity of G-6-P enzyme and reduce the produce endogenous glucose from gluconeogenesis and glycogen decomposition for contributing its anti-diabetes activity [31]. It was also found that CGA could up-regulate the glucose transporters4(Glut4) expression and transposition by AMPK activation to provoke the glucose uptake in skeletal muscle [32]. And many other studies have revel that CGA could promote insulin secretion through glucagon-like peptide1 pathway and regulate blood glucose [33]. All these research suggest that CGA produce anti-diabetes effect may through many different mechanisms at the same time.

Insulin resistance is one of the main and common risk factors for type 2 diabetes, and is closely relate to glucose and lipid metabolic[34,35]. When peripheral tissue produced insulin resistance, the insulin is failure to control blood glucose levels with damage glucose tolerance and glucose and lipid metabolic disorder [34,36].At the same time, glucose and lipid disorder could increase the risk of diabetic complication, especially diabetes-related cardiovascular and cerebrovascular diseases. ITT and OGTT are usually as two main tests for insulin sensitivity and glucose tolerance for diabetes.In this study, we demonstrated that the CGA can improve the insulin sensitivity and glucose tolerance of STZ induced diabetes mice, and regulate the glucose and lipid metabolic in vitro and in vivo.

In this study, we explored the anti-diabetic effects of CGA in regulating glucose and lipid metabolisms via AMPK activation in vitro and in vivo. Our results demonstrated that CGA could stimulate glucose consumption and reduce the PA-BSA induced intracellular TG level in HepG2 cell, and those effects is depend on AMPK activation. The CGA exhibited potent anti-diabetic effects with hypoglycemic activity, improve insulin resistance and glucose tolerance, regulate glucose and lipid metabolism and protect the liver function in vivo. Furthermore, the potential anti-diabetes mechanism of CGA may closely relate to activation of AMPK, which contribute the activity of CGA on regulation glucose and lipid metabolism.Collectively, these results suggested CGA could be utilized as an effective agent for diabetes treatment.

Conflict of interest

Authors report no conflict of interest.

Authors’ Contributions

Zhang Yong was responsible for experimental design, data processing and article writing; Xia Zhi-xuan, Liu Qiang, Liu Qibing were responsible for manuscript proofreading and experimental guidance; Yao Hong-ji, Wang Rui-qi, Jiang Cheng-zuo, Xiong Jingyan, Xue Zhao-yi were responsible for the implementation of the experiment and data collection.