Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes

Journal Pre-proof Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes Qinghai Meng, Xu Qi, Yu Fu, Qi Chen, Peng Cheng, Xichao Yu, Xin Sun, Jingzhen Wu, Wenwen Li, Qichun Zhang, Yu Li, Huimin Bian PII:

S0378-8741(19)32444-4

DOI:

https://doi.org/10.1016/j.jep.2019.112326

Reference:

JEP 112326

To appear in:

Journal of Ethnopharmacology

Received Date: 18 June 2019 Revised Date:

6 October 2019

Accepted Date: 17 October 2019

Please cite this article as: Meng, Q., Qi, X., Fu, Y., Chen, Q., Cheng, P., Yu, X., Sun, X., Wu, J., Li, W., Zhang, Q., Li, Y., Bian, H., Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112326. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Flavonoids extracted from Mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes

Qinghai Meng1,✟, Xu Qi4,✟,Yu Fu1, Qi Chen1, Peng Cheng1, Xichao Yu1, Xin Sun1, Jingzhen Wu2, Wenwen Li2,Qichun Zhang1,3, Yu Li2, *, Huimin Bian1,3,* 1

School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023,

China 2

School of Medicine and Life Sciences, Nanjing University of Chinese Medicine,

Nanjing 210023, China 3

Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia

Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China 4

Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing

Medical University, Nanjing 210029, China ✟

Co-first author.

*Correspondence: Yu Li, E-mail: [email protected] and Huimin Bian, E-mail: [email protected]. Tel: +86 138 5149 5212. E-mail address: Qinghai Meng [email protected]; Xu Qi [email protected]; Yu Fu [email protected]; Qi Chen [email protected]; Peng Cheng [email protected]; Xichao Yu [email protected]; Xin Sun [email protected]; Jingzhen Wu [email protected]; Wenwen Li [email protected]; Qichun Zhang [email protected];

ABSTRACT Ethnopharmacological relevance: Mulberry (Morus alba L.) leaves have been widely applied to controlling blood glucose as a efficacious traditional Chinese medicine or salutary medical supplement. The extracts of mulberry leaf suppress inflammatory mediators and oxidative stress, protect the pancreatic β-cells and modulate glucose metabolism in diabetic rats. Our previous studies and others have shown that mulberry leaf extract has excellent therapeutic effects on type 2 diabetes mellitus (T2DM), however, the underlying mechanism remains to be studied. Aim of the study: Skeletal muscle insulin resistance (IR) plays an important role in the pathogenesis of T2DM. The aim of this study was to investigate the effects and mechanisms of Mulberry leaf flavonoids (MLF) in L6 skeletal muscle cells and db/db mice. Materials and methods: L6 skeletal muscle cells were cultured and treated with/without MLF for in vitro studies. For in vivo studies, the db/db mice with/without MLF therapy were used. Results: MLF and metformin significantly ameliorated muscle glucose uptake and mitochondrial function in L6 muscle cells. MLF also increased the phosphorylation of AMPK and the expression of PGC-1α, and upregulated the protein levels of m-GLUT4 and T-GLUT4. These effects were reversed by the AMPK inhibitor compound C. In db/db mice, MLF improve diabetes symptoms and insulin resistance. Moreover, MLF elevated the levels of p-AMPK and PGC-1α, raised m-GLUT4 and T-GLUT4 protein expression, and ameliorated mitochondrial function in skeletal muscle of db/db mice. Conclusions: MLF significantly improved skeletal muscle insulin resistance and mitochondrial function in db/db mice and L6 myocytes through AMPK-PGC-1α signaling pathway, and our findings support the therapeutic effects of MLF on type 2 diabetes. Key words: Mulberry leaf flavonoids; skeletal muscle insulin resistance; mitochondrial function; AMPK Abbreviations MLF, mulberry leaf extracted flavonoids; AMPK, adenosine monophosphate-activated protein kinase; PGC-1α, proliferator-activated receptor γ co-activator 1-α; T2DM, type 2 diabetes mellitus; IR, insulin resistance ; ROS, reactive oxygen species; PA, palmitic acid; CC, compound C; STZ, streptozotocin; FBS, fetal calf serum; DMEM, dulbecco's modified eagle medium; OGTT, oral glucose tolerance test; STT, starch glucose tolerance test; ITT , insulin tolerance test; CPT-1,

carnitine palmitoyl transferase I; NRF1, nuclear respiratory factor 1; COXIV, cytochrome c oxidase subunit 4; MMP, mitochondrial membrane potential; AUC, area under the curve.

1. Introduction Globally, approximately one in eleven adults have diabetes mellitus, 90% of whom have type 2 diabetes (T2DM) (Zheng et al., 2018). Asia is a major region of the rapidly emerging global epidemic of T2DM, especially China. T2DM leads to a variety of complications that cause serious psychological and financial burdens on patients and society (Hsieh et al., 2017). Although there are an abundant number of researches on the treatment of T2DM, effective therapeutic drugs are still needed. Insulin resistance (IR) is an important unwholesome peculiarity of the pathogenesis of T2DM (DeFronzo and Tripathy, 2009). IR is defined as a reduced responsiveness capability of target tissues (skeletal muscle, liver and adipocytes) to insulin. Skeletal muscle is responsible for 80% of insulin-stimulated glucose uptake, and thus performs a key role in the adjusting of the whole-body glucose homeostasis (Hesselink et al., 2016). Furthermore, it has been found that muscle mitochondrial dysfunction is closely related to T2DM and can result in IR of skeletal muscle (Di Meo et al., 2017). Skeletal muscle completes the uptake and use of glucose mainly through glucose transporter 4 (GLUT4). Insulin can stimulate the intracellular GLUT4 vesicles to the cell membrane transport, which can increase GLUT4 on the cell membrane through exocytosis, known as translocation (Klip et al., 2019). Adenosine monophosphate-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor γ co-activator 1-α (PGC-1α) signaling can enhance mitochondrial function and GLUT4 translocation (Leick et al., 2010). Therefore, regulating AMPK signaling to improve mitochondrial function may be an excellent strategy for the treatment of T2DM. Mulberry (Morus alba L.) leaves have been widely applied to controlling blood glucose as a efficacious traditional Chinese medicine or salutary medical supplement (Kim et al., 2015). The extracts of mulberry leaf suppress inflammatory mediators and oxidative stress, protect the pancreatic β-cells and modulate glucose metabolism in diabetic rats (Liu et al., 2017; Sheng et al., 2017). The mulberry 1-deoxynojirimycin, a kind of ethanol extract from mulberry leaf, was also displayed an ability to lower blood glucose levels in db/db mice (Hu et al., 2017). In our previous research, Mulberry leaf flavonoids (MLF) reduce the serums levels of glucose and lipids, and improve IR in mice with T2DM induced by combination of high fat diet with STZ (Kuai et al., 2016; Zhang et al., 2015), but the underlying mechanism remains to be studied. The objective of this experiment research was to ascertain the hypoglycemic ability and the relevant mechanism of MLF in L6 skeletal muscle cells and db/db mice. Our results suggested that MLF may be a promising therapeutic option for T2DM.

2. Material and Methods 2.1 Preparation and quality control of mulberry leaf extracts flavonoids The mulberry (Morus alba L.) leaves were presented by the Beijing Tong Ren Tang Co. Ltd (Beijing, China). The flavonoids extract of mulberry leaves was prepared with the following procedure. The mulberry leaves were putted in aluminum foil on ice. Then they were washed with distilled water. After the mulberry leaves were lyophilized, the dried mulberry leaves were ground into powder with a blender. The powder of the mulberry leaves was sieved by using a 100-mesh sieve. 10 g of lyophilized leaves were mixed in 100 ml 75/25 water/methanol. The mixture was bathed at 80°C in water for 120 min. After the liquid was filtered, the filtered fluid was concentrated under vacuum to obtain the extracts. The dilution of flavonoids extract of mulberry leaves was used in the following experiments. The main ingredient of rutin in mulberry leaf extracts flavonoids (1 g/ml) was confirmed by using an Agilent 1260 liquid chromatography system. In brief, 10 µL mulberry leaf extracts flavonoids was injected into the apparatus with an auto sampler. Chromatographic separation was proceeded by using an Agilent Zorbax SB-C18 column (4.6 ×250 mm, 5 µm) with a flow rate of 1 ml/min. The mobile phase was made up of solvent A (0.1% formic acid) and solvent B (acetonitrile). The linear gradient solution was performed from 2% to 8% solvent B for 0–5 min, 8–15% solvent B for 5–10min, 15–25% solvent B for 10–25 min, 25–45% solvent B for 25–40 min, 45–60% solvent B for 40–50 min, 60–70% solvent B for 50–60 min, 70% solvent B for 60– 70 min, and 70–2% solvent B for 70–71 min. The temperature of the separation experiment was 35℃, with a detection wave length of 256 nm. The contents of rutin was 320.5 µg/ml. The representative HPLC chromatograms of flavonoids that extracts from mulberry leaves was in figure 1. 2.2 L6 Cell culture Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China) provided the skeletal muscle L6 cells. They were nurtured in 10% FBS, 100 unit/ml penicillin, and 100 mg/ml streptomycin contained DMEM at 37°C. The medium was replaced with DMEM containing 2% FBS to let the myoblasts differentiate into myotubes for 7 days when the cells were filled with the culture flask. During the course of cell differentiation, the medium was replaced every two days. After the differentiated cells obtained, the cells were starved for 6 hours before the cells were incubated in DMEM medium with 100 nmol/L insulin and 0.75 mmol/L Palmitic acid (PA) for 24 hours. The cells were co-treated with MLF at different concentrations (5, 10, 20, 40 and 80 µg/ml) or other reagents in the 24 h. In other in vitro experiments, the concentration of flavonoids was 10 µg/ml and the concentrations of metformin and compound C were 10µM.

2.3 Animals All the male db/db mice and db/m mice at 7-week age were purchased from Changzhou Cavens Experimental Animal Co., Ltd. (Jiangsu, China). The mice were raised in a specific pathogen-free animal laboratory affiliated to the Experimental Animal Center of Nanjing University of Chinese Medicine. The using of animals and all operations on animals in this study got the accordance from the Animal Ethics Committee of Nanjing University of Chinese Medicine. All mice raised in circumstances that alternated between 12 hours of light and 12 hours of darkness at the temperature of 20-22 °C and relative humidity of 50% - 60%. They were fed with normal chow diet (Changzhou Cavens Experimental Animal Co., Ltd.) and sterile drinking water. All the male db/db mice and db/m mice were permitted to adapt to the growing environment for 7 days. After then, the random blood glucose (RBG) of the mice was detected. The db/db mice with RGB less than 11.1 mmol/L were excluded for the subsequent analyses. The other db/db mice and db/m mice were used in the following experiments. Fifty mice were randomly divided into four groups: the control group (Control), diabetes mellitus group (Model), metformin treatment group (Metformin), Mulberry leaf flavonoids treatment group (Flavonoids). The db/m mice were considered to be the control group, and the db/db mice were considered to be other groups. Each group consisted of ten mice (n=10). The db/db mice in metformin group were orally treated with 200 mg/kg metformin hydrochloride, the db/db mice in the MLF group were orally treated with 180 mg/kg MLF, and the mice in Model group and Control group were orally treated with 0.5% CMC-Na. All the treatments in mice were last for 7 weeks and applied by the dose volume: 0.1 ml/10g.

2.4 Measurement of RBG levels All mice were fasted and free for water for 12 hours every 7 days during the 49 days treatment period (including before treatment), and the blood glucose was detected by using One Touch Basic Blood Glucose Monitoring System (Accu-Chek) with one drop blood from the tip of the mouse tail. The value of blood glucose displayed on the instrument was recorded. 2.5 Oral glucose tolerance test (OGTT)， ，Starch glucose tolerance test (STT) and Insulin tolerance test (ITT) At the 4th week, all mice were fasted and free for water for 12 hours before the OGTT was carried out by intragastric giving the db/db mice and db/m mice a glucose solution (2 g/kg). At the 5th week, all mice were fasted and free for water for 4 hours before the ITT was carried out by intraperitoneally injected with insulin (1 U/kg, providing by Jiangsu Wanbang Biochemistry Medicine Co. Ltd) on the db/db mice and db/m mice. At the 6th week, all mice were fasted and

free for water for 12 hours before the STT carried out by intragastric giving the db/db mice and db/m mice a starch solution (6 g/kg). In OGTT, ITT and STT, the blood glucose levels were detected at the 0, 30, 60, 90,120, and 180 min time points after the glucose, insulin, and starch were given to the mice. The blood glucose curves of the mice in each group at different time of the three experiments were plotted, respectively. The area under the curve (AUC) was figured up by using the blood glucose levels.

2.6 Hematoxylin and eosin (H & E) staining All mice were fasted and free for water for 12 hours after 49 days treatment. After that, all mice were sacrificed, the pancreas and skeletal muscle were obtained and stored in 4% formaldehyde. The two kinds of tissues from the mice were dehydrated and buried into the embedding box by paraffin. The slices of pancreas and skeletal muscle of mice were obtained by using a microtome. After the paraffin was removed from the slices, the hematoxylin and eosin (H&E) staining was performed according to previous report (Luna, 1968) to evaluate the histopathological changes of the pancreas and skeletal muscle.

2.7 Serologic detection All mice were fasted and free for water for 12 hours after 49 days treatment. After that, the blood was taken from the mice eye socket. The blood samples were placed at 37 °C for 0.5 hours and then centrifuged (3000 rpm, 20 minutes) to obtain the serum. Insulin and glycated serum protein (GSP) contents in the serum of the mice were detected by using a mouse insulin ELISA kit and a mouse GSP ELISA kit refer to the respective manufacturer's directions. For the total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) in serum, they were detected by using a biochemical analyzer (Thermo 1510) and serum lipid detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The following formulas were used to evaluate the homeostasis model of assessment-insulin resistance (HOMA-IR) and insulin sensitive index (ISI). HOMA-IR = RBG (mmol / L) × Insulin (mIU / ml) / 22.5, ISI = 1 / [RBG (mmol / L) × Insulin (mIU / L)] (Hanley et al., 2002).

2.8 Liver and muscle glycogen measurement All mice were fasted and free for water for 12 hours after 49 days treatment. After that, fresh liver and muscle of the mice were obtained and rinsed with pre-cooling sterile saline solution at 4 °C. The muscle glycogen detection kit and liver glycogen detection kit, which purchased from

Nanjing Jiancheng Bioengineering Institute (Nanjing, China), were used to detect the liver and muscle glycogen contents refer to the instructions prepared by the manufacturer of the kits.

2.9 Immunohistochemistry The expression of GLUT4 (Abcam, UK), p-AMPK (bioworld, USA) and PGC-1α (Abcam, UK) in mouse skeletal muscles were determined by immunohistochemistry. The slices of skeletal muscle of the mice were obtained refer to 2.6, after the paraffin was removed from the slices, citric acid solution (0.01 M, pH=60) was used to incubate the slices for 25 minutes at 95℃ water bath. After incubated with 3% H2O2 for 10 min, the slices were incubated for 2 h at 37°C with the antibodies against GLUT4, p-AMPK and PGC-1α, respectively. After rinsing, the slices were incubated for 30 min at room temperature with HRP-conjugated goat anti-rabbit IgG antibody (1:1000). The DAB color development kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to turn the positive position of protein brown. The expression levels of GLUT4, p-AMPK and PGC-1α in skeletal muscle of the mice were quantified by using Image Pro Plus 6.0.

2.10 Western bolt assay The protein expressions of CPT-1 (Proteintech, USA), NRF1 (Proteintech, USA), COXIV (Proteintech, USA), AMPK (Bioworld, USA), p-AMPK (Bioworld, USA), PGC-1α (Abcam, UK) and GLUT4 (Abcam, UK) were detected by western blotting. 60 mg of fresh skeletal muscle was lysed in 500µL protease and phosphatase cocktails containing RIPA buffer. After 30 minutes at 4 ° C, the samples were then centrifuged (12000 rpm, 15 min) to get the supernatant within total protein. The separation of membrane proteins from the cells and tissue was performed refer to the directions prepared by the manufacturer of the membrane protein extraction kit (Beyotime Biotechnology, Shanghai, China). The content of protein was detected by using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). The samples (within total protein 30 µg) were added to SDS-PAGE gel and electrophoresis was performed to separate the proteins. Then, the proteins on the gel were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The PVDF membrane within proteins was incubated for 2 hours in a blocking solution. Subsequently, the PVDF membrane was incubated in the desired primary antibody, including GLUT4 and PGC-1α, CPT-1, NRF1, COXIV and p-AMPK antibody (dilute 1:1000). These were incubated with the PVDF membrane at 4 °C overnight. After incubated with appropriate secondary antibodies (dilute 1:10000) for 2 h, and washed with washing solution TBS-T, the PVDF membrane was treated with chemiluminescence reagent and exposure photographing procedure. The obtained blots were quantified by using the software supplied with the instrument (SYSTEM GelDoc XR+, Bio-Rad, USA). The standardization of

total protein on the membrane glut4 expression, Na-K-ATPase (Abcam, UK) was used as a control. The normalization of total protein on other proteins was performed by using β-actin (Bioworld, USA) as a control.

2.11 Identification with coomassie brilliant blue staining When the cells were grown to about 60% of the wall area of the culture dish, the cell culture medium was changed to differentiation medium (DMEM containing 2% FBS), which was changed once a day for 7 days. The cells were washed with 2.5% glutaraldehyde for 30 min and washed with PBS. Then, 1% Triton X-100 was added for 10 min and distilled water was rinsed three times. The solution was stained with distilled water for 30 min. Images were taken under a microscope. 2.12 Immunofluorescence staining of striated muscle actin Both differentiated and undifferentiated L6 cells were fixed with 4% paraformaldehyde. After placing at room temperature for 30 minutes, the cells were washed twice with PBS containing 0.2% Triton X-100. After incubating the cells with PBS containing 3% BSA for 30 minutes, the cells were incubated with α-SMA (1:500) antibody (Abcam, UK) for overnight at 4 °C. After washing with PBS, the cells were incubated with FITC-labeled secondary antibody for 1 hour. The nucleus was stained with DAPI, and after the cells were washed again, the fluorescence images of the cells were gained by using a fluorescence microscope (Leica, Germany). 2.13 Detection of mitochondrial membrane potential (MMP) L6 cells were added to 6-well plates with a density of 1 x 10 6 cells per well. The cells were differentiated and treated as described in 2.2. Then, the JC-1 mitochondrial membrane potential detection kit (Beyotime Biotechnology, Shanghai, China) was used to detect the changes of L6 cell MMP in different groups of cells according to the manufacturer's instruction. Fluorescence microscopy (Leica, Germany) was used to obtain the photos of the cells. The complexes with red fluorescent were formed in cells with normal mitochondria. In mitochondria-damaged cells, JC-1 maintained a monomeric form and showed green fluorescence. The obtained photos were quantified by using Image Pro Plus 6.0.

2.14 Detection of reactive oxygen species (ROS) L6 cells were added to 6-well plates with a density of 1 x 10 6 cells per well. The cells were differentiated and treated as described in 2.2. Then, the contents of ROS in different groups were measured by using the ROS detection kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instruction. The cells were treated with trypsin without EDTA and different

groups of L6 cells were harvested. Then, the cells were incubated with DCFH-DA from the kit in the dark for 30 min at 37°C. After washed, the fluorescence intensity of the cells was detected by using a flow cytometer (Biosciences Accuri C6, Becton-Dickinson). 2.15 Statistical analysis All statistical analysis in this study was performed by using the SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). The mode of presentation of the data is mean ± SD. Differences between the two groups were analyzed by using a two-tailed Student's t-test, and differences between multiple groups were analyzed by one-way ANOVA and post hoc Dunnett's test. Differences between groups were considered statistically significant when P < 0.05. 3. Results 3.1 MLF ameliorates the insulin-resistant phenotype in L6 muscle cells. First, we establish the IR model of L6 skeletal muscle cells. The undifferentiated morphology of L6 skeletal muscle cells showed that the cells had no myotube structure (Fig. 2A). On day 7 after induction of differentiation, cells were stained with Coomassie brilliant blue, indicating that L6 cells had differentiated into mature skeletal muscle cells, and immunofluorescence showed positive staining of α-sarcomeric actin (a-SMA), which is the hallmark protein of myotube formation (Fig. 2A). After co-culture with insulin (100 nM) and with or without palmitic acid (PA) (0.75 µM) for 24 hours in differentiated L6 skeletal muscle cells, the glucose content in the cell-cultured medium supernatant was determined by the glucose oxidase-peroxidase method and the glucose consumption and decrement of glucose consumption were calculated. The results showed that exposure of L6 myotubes to the PA resulted in significant reduction of insulin-stimulated glucose uptake, indicating insulin resistance (Fig. 2B). Next, we observed the effect of MLF on glucose consumption at different concentration. The results showed that MLF increased glucose consumption in a dose-dependent manner (Fig. 2D). The results of MTT assay showed that the different concentration of MLF did not affect cell viability (Fig. 2C). Moreover, treatment with MLF significantly increased the translocation of m-GLUT4 and upregulated T-GLUT4 protein expression level (Fig. 2E-F). In addition, the therapeutic effect of MLF was consistent with metformin, which is currently the commonly used drug for clinical treatment of type 2 diabetes. 3.2 MLF improves skeletal muscle mitochondrial function in L6 cells Since mitochondrial dysfunction has been associated with the development of IR and T2DM, we next examined the effect of MLF on mitochondrial function. As shown in Figure 3A and 3B, MLK significantly increased the protein expressions of CPT-1, NRF1 and COXIV. We then used JC-1 fluorescent staining to detect mitochondrial membrane potential. Compared with the control group, the mitochondrial membrane potential of the skeletal muscle of the model group was

significantly decreased, and the mitochondrial membrane potential was significantly increased after administration of MLF (Fig. 3C-D). Moreover, MLF significantly increased the level of ATP in L6 cells, which was consistent with the effect of metformin (Fig. 3E). Next, we used DCFH-DA to detect changes in active ROS in L6 cells and found that MLF significantly reduced ROS fluorescence intensity in L6 cells (Fig. 3F-G). Flow cytometry results also showed that MLF significantly reduced ROS fluorescence intensity in L6 cells, which were consistent with immunofluorescence data (Fig. 3H). These results suggest that MLF improves mitochondrial function in L6 cells. 3.3 AMPK is required for MLF to enhance glucose metabolism in L6 skeletal muscle cells Since MLF has the same effect as metformin, which is one of the AMPK activators, we evaluated the effect of MLF on the AMPK signaling pathway. Western blot results indicated that MLF increased the levels of p-AMPK and PGC-1α (Fig. 4A-B), and these effects were abolished by compound C (CC, 10µM), the inhibitor of AMPK (Fig. 4C-D). In addition, MLF significantly increased the translocation of m-GLUT4 and upregulated T-GLUT4 protein expression level, which was reversed by CC (Fig. 4E-F). These results indicate that AMPK is required for MLF to enhance glucose metabolism in L6 skeletal muscle cells. 3.4 MLF improves diabetes symptoms in db / db mice The db/db mice are spontaneously diabetic mice, characterized by IR, obesity, polydipsia, polyphagia, hyperglycemia and hyperlipidemia. Because of their similar pathogenesis to human T2DM, they are ideal animal models for studying human T2DM (Burke et al., 2017). In this study, db/db mice were used as diabetic model to investigate the effects and the mechanisms of MLF. As shown in Fig.5, a significant increase in body weight was observed in db/db mice compared to the control group, however, administration of MLF or metformin did not affect the body weight of db/db mice (Fig. 5A). The food intake of mice after metformin administration was significantly reduced compared with the model group at 36d, while there was no significant difference between the MLF group and the model group (Fig. 5B). Mice treated with metformin or MLF showed significantly lower blood glucose levels compared to the model group (Fig. 5C). Glycogen synthesis and glycogenolysis can affect blood glucose levels, and glycosylated serum protein (GSP) and glycated hemoglobin (GHB) reflect immediate average blood glucose levels. Our results show that MLF and metformin significantly reduced the levels of GSP and GHB (Fig. 5D-E) and increased hepatic glycogen and muscle glycogen levels (Fig. 5F-G). Lipid examination showed that MLF and metformin significantly reduced the levels of TG, TC and LDL-C and increased the level of HDL-C in serum (Fig. 5H). These results demonstrate the superior role of MLF in the treatment of type 2 diabetes, consistent with our previous results.

3.5 MLF attenuates insulin resistance in db/db mice At the 8th week of MLF treatment, glucose tolerance was examined using the OGTT method. The results showed that MLF and metformin had significantly stronger hyperglycemia responses to oral glucose administration (Fig. 6A), and the AUC of MLF-treated and metformin-treated mice was significantly lower than that of db/db mice (Fig. 6D). At the 10th week of MLF and metformin treatment, insulin resistance was examined using the ITT method. The results showed a significant reduction in blood glucose levels in the MLF group and metformin group compared to the model group (Fig. 6B), and MLF and metformin reduced the area under the time-glycemic curve compared to the model group (Fig. 6E). At week 11 of MLF and metformin treatment, STT results showed that the blood glucose levels of the MLF treatment group and the metformin treatment group continued to decrease after 30 minutes (Fig. 6C). Similarly, the AUC of the MLF and metformin treatment groups was significantly lower than the model group (Fig. 5F). At week 12 of MLF treatment, we examined the effect of MLF on serum insulin levels. Treatment with MLF and metformin reduced serum insulin levels in db/db mice compared to model mice (Fig. 6G). Subsequently, HOMA-IR and ISI were calculated to check the exact difference between these groups. The HOMA-IR was significantly reduced in the MLF and metformin treatment groups compared to the model group (Fig. 6H). As expected, treatment with MLF in db/db mice correspondingly enhanced ISI (Fig. 6I). H&E staining results showed that db/db mice rarely showed islets with many empty areas. However, in the control group, the islets were round and showed clear boundaries. Islet is larger and more common than the other groups. In the MLF group, there are some islets, but they are irregular in shape and contain many central particles (Fig. 6J-K). H&E staining also showed skeletal muscle cell disorders and muscle fiber deformation in model mice compared to control mice, but these pathological changes were partially improved by treatment with MLF or metformin (Fig. 6J-K). 3.6 MLF activates AMPK to improve skeletal muscle mitochondrial function and glucose uptake in db/db mice We validated the effects of MLF on skeletal muscle glucose uptake and mitochondrial function in vivo. Western blot results showed that protein levels of m-GLUT4 and t-GLUT4 were significantly lower in model mice compared with control mice, but MLF and metformin treatment significantly upregulated the expression levels of m-GLUT4 and t-GLUT4 (Fig. 7A-B). Similarly, western blot results showed that protein expression of CPT-1, NRF1 and COXIV was decreased in skeletal muscle of db/db mice, while MLF and metformin significantly increased the expression of these proteins (Fig. 7C-D). Next, we validated the effect of MLF on the AMPK signaling pathway in vivo. The results showed that the levels of p-AMPK and PGC-1α in skeletal muscle of db/db mice decreased compared with the control group, while metformin and MLF upregulated the expression of p-AMPK and PGC-1α in skeletal muscle (Fig. 7E-F). In addition,

immunohistochemistry results showed that MLK and metformin significantly increased membrane transport of GLUT4 and phosphorylation of AMPK, consistent with the results of western blot (Fig. 7G-H). 4. Discussion In the current study, we found that MLF and metformin significantly ameliorated muscle glucose uptake and mitochondrial function in L6 muscle cells via AMPK-PGC-1α signaling pathway. In db/db mice, MLF improve diabetes symptoms and insulin resistance. Moreover, MLF elevated the levels of p-AMPK and PGC-1α, and ameliorated mitochondrial function in skeletal muscle of db/db mice. Overall, MLF attenuated IR and mitochondrial dysfunction in skeletal muscle by activation of AMPK, and MLF could be developed as a novel therapeutic agent for the treatment of T2DM. Mitochondria dysfunction and fatty acids accumulation in skeletal muscles are closely related to the development of IR (Heo et al., 2017). Mitochondria are the organelles necessary for aerobic ATP production: a molecular currency that provides chemical energy to promote important cellular processes. After mitochondrial dysfunction, the ability of cells to oxidize fat is decreased, resulting in their accumulation in the muscle, which further undermines insulin signaling and leads to IR. Mitochondrial fatty acid oxidation is usually the main muscle energy source during rest or endurance exercise. CPT1 is a key rate-limiting enzyme fatty acid β oxidation in the skeletal muscle mitochondrial. MMP is a prerequisite for maintaining oxidative phosphorylation of mitochondria and producing ATP (Zorova et al., 2018). When certain causes lead to obstacles in the electron transport process of respiratory chain, MMP will be decreased. Further oxidative phosphorylation and ATP synthesis of cells are reduced, resulting in mitochondrial Ca2+ release, ROS production, and release of cytochrome C and other mitochondrial contents (Schwarz et al., 2014). Among them, COX is the terminal compound enzyme of mitochondrial respiratory chain electron transfer and the COXIV is a subunit of COX (Klinge, 2008). When the expression of respiratory chain protein changes, it will inevitably cause obstacles in the process of electron transfer of respiratory chain and eventually oxidative phosphorylation. Research has been reported that NRF-1 not only regulate mitochondrial oxidative genes but also controls myocyte-specific enhancer factor 2A (MEF2A), the main transcription factor for glucose transporter GLUT4 (Ramachandran et al., 2008). Our results showed that excessive fatty acids entered the mitochondria after PA treatment, resulting in the barrier of mitochondrial electron transport, reducing MMP, COXIV and NRF1, downregulating the synthesis of ATP, and overproducing ROS, thus blocking the insulin signal transduction pathway. The changes of these data were observed after the treatment with MLF in L6 skeletal muscle cells and db/db mice. PGC-1α is the main regulator of skeletal muscle mitochondrial function and insulin resistance. PGC-1α transgenic mice have increased expression of mRNA for mitochondrial enzymes and more muscle fiber compared to wild-type mice (Lin et al., 2002). In patients with

insulin resistance and diabetes, many of the PGC-1α-responsive genes involved in oxidative phosphorylation are down-regulated (Mootha et al., 2003; Patti et al., 2003). In addition, exercise training, one of the insulin sensitizating treatments, leads to parallel restoration of both PGC-1α and insulin sensitivity (Meex et al., 2010). Similarly, rosiglitazone, the insulin sensitizing drug, recovers PGC1α expression in patients with T2DM, and significantly increases insulin sensitivity and muscle oxidative capacity (Mensink et al., 2007). Phosphorylation of PGC-1α is mediated by AMP-activated protein kinase (AMPK), which is a key factor in diabetes and metabolic syndrome. Metformin, exercise and many natural exert have a multitude of health benefits by activation of AMPK. Of note, Metformin, commonly used in the first-line treatment of T2DM, affects mitochondrial function (Madiraju et al., 2014; Vial et al., 2019). These studies suggest that AMPK-PGC-1α is an important therapeutic signal for improving mitochondrial function and insulin sensitivity. There is evidence that IR is closely associated with reduced GLUT4 expression. Translocation of GLUT4 into the cell membrane was promoted by activation of AMPK. As a serine/threonine protein kinase, AMPK has been postulated to be an important target in diabetes treatment for its role in the adjusting of glucose metabolism (Coughlan et al., 2014). After phosphorylated, the activated AMPK has a capacity to increase the glucose utilization by raising the expression of PGC-1α and GLUT4 and accelerating the translocation of GLUT4 from intracellular storage vesicles to cell membrane in skeletal muscle and adipose tissues (Kurth-Kraczek et al., 1999; Yamaguchi et al., 2005). A depressing activity of p-AMPK in peripheral tissue has been noted in obesity T2DM rodent model(Carling, 2017). To explore the hyperglycemic mechanism of MLF, the p-AMPK1/2 levels in skeletal muscle was detected in the present study. Our current data revealed that in the groups treated with MLF, the levels of p-AMPK1/2, PGC-1α and GLUT4 were significantly increased compared with the model control group, which is consistent with in vitro data. These results indicate that AMPK-PGC-1α is involved in the therapeutic effect of MLF on diabetes. Traditional Chinese medicine is considered to be an excellent solution for type 2 diabetes. Our current study found that MLF has excellent therapeutic effects on T2DM. Plentiful number of researches have confirmed that mulberry leaves have antihyperglycemic, antioxidant and antiglycation

activities

in

streptozotocin-induced

chronic

diabetic

rats

(Andallu

and

Varadacharyulu, 2003; Hu and Wang, 2004; Naowaboot et al., 2009). Moreover, Mulberry leaf extract stimulates glucose uptake and GLUT4 translocation (Naowaboot et al., 2012) and inhibits pancreatic islet cell apoptosis in diabetic rats (Zhang et al., 2014). However, we cannot rule out that its effects may be caused by inhibition of oxidative stress. Superoxide and other reactive oxygen species (ROS) are produced as an inevitable by-product of mitochondrial respiration. In the absence of adequate antioxidant capacity, excessive ROS production may be due to dysfunctional mitochondria, which may lead to lipid peroxidation and other forms of oxidative stress, such as nuclear and mitochondrial DNA damage (Song et al., 2007), which in turn may directly promote insulin resistance (Anderson et al., 2009). Previous researchers have suggested

that mulberry leaf extract can significantly inhibit oxidative stress and thereby improve glucose metabolism in diabetic rats. In fact, our results also show that MLF can significantly inhibit the production of ROS. Since the composition of mulberry leaf extract is very complicated, here we only study the effect of flavonoids in mulberry leaves. Our results show that the total flavonoids of mulberry leaves can mimic the action of metformin. It is important that AMPK inhibitor CC can eliminate the effect of MLF. Therefore, at least the effect of flavonoids in mulberry leaves is mainly to reverse skeletal muscle insulin resistance by increasing glucose uptake and mitochondrial function through AMPK-PGC-1. In conclusion, we demonstrated that MLF activated the AMPK-PGC-1α signaling to improve glucose metabolism, enhance mitochondrial function, and correct IR in L6 skeletal muscle cells and db/db mice. These discoveries suggested that MLF might be a promising therapeutic option for the treatment of T2DM. Acknowledgements This work was sponsored by Qing Lan Project. This work was supported by the Open Project Program of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica [No. JKLPSE201809] and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) [No. JKLPSE201605]. Conflict of interest The authors declare that they have no competing financial interests. Authors’ contributions Huimin Bian, Yu Li, Xu Qi and Xin Sun conceived and designed the experiments; Xichao Yu, Qi Chen, Peng Cheng and Xin Sun performed the experiments; Qinghai Meng analyzed all the data and prepared the manuscript; Yu Fu helped to analyze the data and prepare the manuscript; Jingzhen Wu and Wenwen Li contributed reagents/materials/analysis tools; Qichun Zhang drafted the manuscript. All authors read and approved the final manuscript.

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Figure legends Fig. 1. Representative HPLC chromatograms of flavonoids that extracts from mulberry leaves. A: Rutin.

Fig. 2. The flavonoids regulate glucose metabolism in IR model of L6 muscle cells. (A) The inducing of L6 muscle cells and the identification of mature skeletal muscle cells by CBB staining and α-SMA immunofluorescence staining (200×). (B) The effects of insulin (100 nM) with or without PA (0.75 µM) on glucose consumption (up panel) and glucose consumption decrement between two groups (down panel) in L6 myotubes (n=10). ##p<0.01, compared with control group. (C) The effects of flavonoids (5, 10, 20, 40 and 80 µg/ml) or metformin (10µM) on cell viability of L6 myotubes by MTT. (D) The effects of flavonoids (5, 10, 20, 40 and 80 µg/ml) or metformin (10µM) on glucose consumption in L6 myotubes (n=10). ##p<0.01, compared with control group. **p<0.01, compared with model group. (E) The representative result of western blot analysis of the total GLUT4 and the membranes of GLUT4 in L6 myotubes. (F) Related to E, the quantitative of the total GLUT4 and the membranes of GLUT4 protein expressions in L6 myotubes. n=3. ##p<0.01, compared with control group; **p<0.01, compared with model group. Fig. 3. The flavonoids improve skeletal muscle mitochondrial function in L6 cells (A) The representative result of western blot analysis of GPT-1, NRF1, and COXIV in L6 myotubes. (B) Related to A, the quantitative of GPT-1, NRF1, and COXIV protein expressions in L6 myotubes. n=3. ##p<0.01, compared with control group; **p<0.01, compared with model group. (C) The effects of flavonoids (10 µg/ml) or metformin (10µM) on mitochondrial membrane potential of L6 myotubes by JC-1 fluorescent staining. (D) Numerical data were expressed as Red/Green fluorescence + cells. ##p<0.01, compared with control group; **p<0.01, compared with model group. (E) The effects of flavonoids (10 µg/ml) or metformin (10µM) on ATP level of L6 myotubes. ##p<0.01, compared with control group; **p<0.01, compared with model group. (F) The effects of flavonoids (10 µg/ml) or metformin (10µM) on ROS level of L6 myotubes by DCFH-DA. (G) Related to F, fluorescence intesity of ROS level in L6 myotubes. ##p<0.01, compared with control group; **p<0.01, compared with model group. (H) The effects of flavonoids (10 µg/ml) or metformin (10µM) on ROS level of L6 myotubes by flow cytometry.

Fig. 4. The flavonoids activate AMPK-PGC-1α signaling in IR model of L6 cells. (A) The representative result of western blot analysis of AMPK, p-AMPK, and PGC-1α in L6 myotubes. (B) Related to A, the quantitative of AMPK, p-AMPK, and PGC-1α protein expressions in L6 myotubes. (C) The representative result of western blot analysis of AMPK, p-AMPK, and

PGC-1α in L6 myotubes. (D) Related to C, the quantitative of AMPK, p-AMPK, and PGC-1α protein expressions in L6 myotubes. (E) The representative result of western blot analysis of the total GLUT4 and the membranes of GLUT4 in L6 myotubes. (F) Related to E, the quantitative of the total GLUT4 and the membranes of GLUT4 protein expressions in L6 myotubes. n=3. ##p<0.01, compared with control group; **p<0.01, compared with model group.

Fig. 5. The flavonoids improve diabetes symptoms in db / db mice (A) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on body weight of db / db mice. (B) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on food intake of db / db mice. (C) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on blood glucose of db / db mice. (D) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on glycated hemoglobin of db / db mice. (E) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on glycated serum protein of db / db mice. (F) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on liver glycogen of db / db mice. (G) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on muscle glycogen of db / db mice. (H) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on serum lipid level of db / db mice. ##p<0.01, compared with control group; *p<0.05, **p<0.01, compared with model group.

Fig. 6. The flavonoids attenuate insulin resistance in db/db mice (A) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on blood glucose of db / db mice after oral glucose. (B) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on blood glucose of db / db mice after oral starch. (C) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on blood glucose of db / db mice after oral insulin. (D) Related to A, the AUC of OGTT in db / db mice. (E) Related to B, the AUC of OGTT in db / db mice. (F) Related to C, the AUC of OGTT in db / db mice. (G) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on serum insulin of db / db mice. (H) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on HOMR-IR of db / db mice. (I) The effects of flavonoids (180mg/ml) or metformin (200 mg/kg) on insulin sensitive index of db / db mice. ##p<0.01, compared with control group; *p<0.05, **p<0.01, compared with model group. (J) HE staining of pancreas and skeletal muscle in db / db mice (100×). (K) Related to J, pathology score of pancreas and skeletal muscle in db / db mice. ##p<0.01, compared with control group; **p<0.01, compared with model group.

Fig. 7. The flavonoids activate AMPK to improve skeletal muscle mitochondrial function and glucose uptake in db/db mice (A) The representative result of western blot analysis of the total GLUT4 and the membranes of GLUT4 in L6 myotubes. (B) Related to A, the quantitative of the total GLUT4 and the membranes of GLUT4 protein expressions in L6 myotubes. (C) The representative result of western blot analysis of GPT-1, NRF1, and COXIV in L6 myotubes. (D) Related to C, the quantitative of GPT-1, NRF1, and COXIV protein expressions in L6 myotubes. (E) The representative result of western blot analysis of AMPK, p-AMPK, and PGC-1α in L6 myotubes. (F) Related to E, the quantitative of AMPK, p-AMPK, and PGC-1α protein expressions in L6 myotubes. n=3. ##p<0.01, compared with control group; **p<0.01, compared with model group. (G) Immunohistochemistry staining of GLUT4 and p-AMPK of skeletal muscle in db / db mice (100×). (H) Related to G, the quantitative of immunohistochemistry staining of GLUT4 and p-AMPK of skeletal muscle in db / db mice. n=6. ##p<0.01, compared with control group; **p<0.01, compared with model group.