Gymnemic acid alleviates type 2 diabetes mellitus and suppresses

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Bioactive Constituents, Metabolites, and Functions

Gymnemic acid alleviates type 2 diabetes mellitus and suppresses endoplasmic reticulum stress in vivo and in vitro Yumeng Li, Mingzhe Sun, Yaping Liu, Junjie Liang, Tianxin Wang, and Zesheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00431 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Journal of Agricultural and Food Chemistry

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Gymnemic acid alleviates type 2 diabetes mellitus and suppresses endoplasmic reticulum

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stress in vivo and in vitro

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Yumeng Li1, Mingzhe Sun1, Yaping Liu1, Junjie Liang1, Tianxin Wang1, Zesheng Zhang1,2,*

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Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food

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Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457,

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China

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2

Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center,

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300457, Tianjin, China.

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*

Address for correspondence:

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Name:

Zesheng Zhang

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Mailing address:

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Telephone number:

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Fax number:

+86 022 6091 2431

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E-mail address:

[email protected]

No. 29, 13th Avenue, Tianjin, China +86 022 6091 2431

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ACS Paragon Plus Environment


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ABSTRACT

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Gymnemic acid (GA) is an herbal ingredient that can improves glucose metabolism in patients

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with diabetes mellitus. In this study, we evaluated the ameliorative effects of GA on insulin

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resistance (IR) and identified the mechanisms in type 2 diabetes mellitus (T2DM) rats and IR

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HepG2 cells. GA effectively enhanced glucose uptake in IR HepG2 cells from 11.9 ± 1.09 to 14.7

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± 1.38 mmol/l, lowered fasting blood glucose (blood glucose levels in groups treated with GA at

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40 and 80 mg/kg/day were reduced by 15.2 % and 26.7 %, respectively) and oral glucose

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tolerance. Both in vivo and in vitro, GA downregulated the expression of endoplasmic reticulum

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(ER) stress indicator proteins such as ORP150, p-c-Jun, p-PERK, and p-eIF2α. In addition, the

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improvement of ER stress regulated the insulin signal transduction proteins, reducing p-IRS-1(ser)

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levels and increasing p-IRS-1(tyr) in GA-treated T2DM rats and IR HepG2 cells. In summary, the

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mechanism underlying the hypoglycemic effects of GA may be associated with alleviation of ER

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stress and facilitation of insulin signal transduction in T2DM rats and IR HepG2 cells.

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Keywords: Gymnemic acid; endoplasmic reticulum stress; insulin resistance; T2DM rats; HepG2

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cells.

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1. Introduction

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Type 2 diabetes mellitus (T2DM), characterized by persistent insulin resistance (IR) and

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hyperglycemia, affects approximately 370 million people worldwide, and is a common cause

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of mortality[1]. People with T2DM consistently have three cardinal abnormalities:

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overnutrition-associated obesity, leading to inflammation and oxidative stress in peripheral

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tissues; chronic hyperglycemia, leading to decreased insulin sensitivity and insulin resistance

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(IR) in muscle, adipose and liver tissues; excessive insulin secretion, leading to pancreatic

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beta (β) cell dysfunction, especially in regulating the consumption and generation of glucose

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in peripheral tissues[2]. The majority of the existing hypoglycemic drugs have been developed

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to regulate blood glucose levels, promote insulin secretion and repair β cell damage; however,

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some have undesirable effects, such as the induction of a sharp drop in blood glucose, which

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could lead to liver and kidney dysfunction and increase the risks of side-effects including

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chronic cardiovascular complications[3-5]. Therefore, the identification of novel therapies and

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a comprehensive understanding of the mechanisms responsible for their hypoglycemic effects

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are urgently required for the safe and effective treatment of patients with T2DM.

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Endoplasmic reticulum (ER) is an important site of protein folding, maturation, transportation,

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signal transduction and stress responses[6-7]. Chronic exposure to hyperglycemic and

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hyperlipidemic environments can severely impair the adaptive response of the ER, leading to

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a series of inflammatory and stress responses known as ER stress[8, 9]. ER stress in peripheral

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tissues can inhibit insulin signaling by activating signaling cascades, especially c-Jun

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N-terminal kinase (JNK)[9], protein kinase-like ER kinase (PERK)[10], oxygen-regulated

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protein 150 (ORP150) [11] and eukaryotic translation initiation factor 2 (elF2α). IR in T2DM



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is caused by many defects, such as downregulation of the cognate receptor and receptor

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substrate levels, and decrease insulin sensitivity. Insulin receptor substrate (IRS) proteins is an

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important cytoplasmic molecules mediating insulin activation cascade[12]. Enhanced serine

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phosphorylation and suppressed tyrosine phosphorylation of IRS-1 down regulates insulin

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receptor signaling and is considered to be a common cause of functional suppression of IRS-1

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protein[13]. The generation of ER stress can affect insulin transformation and the synthesis of

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glucose metabolism-related proteins, which ultimately leads to the chronic metabolic diseases:

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obesity, diabetes, and cardiovascular disease. Therefore, new strategies to reduce ER stress,

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facilitate insulin signal transduction and relieve hyperglycemia are urgently required.

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Gymnemic acid (GA)[14-15], a crude saponin fraction obtained from Gymnema sylvestre leaf

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extracts, is a natural product that has been used as a herbal remedy for thousands of years

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worldwide[16-17]. Reports have shown that GA mediates anti-hyperglycemic effects by

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reducing blood glucose levels in patients with T2DM[18]. Applications of GA derived from

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natural plant extracts have also revealed its ability to regulate various physiological processes,

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such as metabolic syndrome and inflammatory responses[19, 20]. Recent studies also

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indicated that GA stimulates insulin secretion, improves insulin action and lowers blood lipids

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in murine models[15, 21]. However, the potential mechanism underlying the hypoglycemic

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effects of GA in the treatment of T2DM remains unclear. It was especially important to verify

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the capacity of GA to reduce ER stress and elucidate the hypoglycemic signaling mechanisms

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both in vitro and in vivo. Therefore, the main purpose of this investigation was to study the

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ability of GA to lighten ER stress and alleviate insulin signal transduction in a rat model of

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T2DM and insulin-resistant HepG2 cells.


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2. Materials and methods

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2.1 Chemicals

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GA (consisting of GA I–IV; 95 % purity) was obtained from Solarbio Science and Technology

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Co., Ltd. (Beijing, China). The human hepatoblastoma cell line (HepG2) was purchased from

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China Center of Type Culture Collection (Wuhan, China). Streptozotocin (STZ) was obtained

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from Sigma Chemical Co. (St. Louis, MO, USA). Palmitic acid (PA > 99 % purity) was purchased

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from Tianjin Guang Fu Chemical Research Institute. Fatty acid free bovine serum albumin (BSA)

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and Fetal bovine serum (FBS) were purchased from Gibco Company (Grand Island, NY, USA).

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DMSO was purchased from Sigma Aldrich Corporation (St Louis, MO, USA). Antibodies for the

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detection of ORP150, phosphorylated-PERK, phosphorylated-eIF2α and phosphorylated-JNK

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(c-Jun), IRS, phosphorylated-IRS (ser307), phosphorylated-IRS-1 (tyr) and β-actin were

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purchased from Cell Signaling Technology lnc. (Danvers, MA, USA). Secondary detection

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antibodies were obtained from ZSGB-BIO Technology Co., Ltd. (Beijing, China). All other

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laboratory chemicals were of analytical grade.

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2.2 Cell culture

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HepG2 cells were cultured in DMEM added with 10 % FBS, 0.1 mg/ml streptomycin, and 100

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units/ml penicillin. Cell culture dishes were kept at 37 °C in a humidified atmosphere containing

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5 % CO2. After reaching 80 % - 95 % confluence, cells (1×105 cells/ml) were seeded in 96-well

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microtiter plates prior to experiments.

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2.3 Apoptosis index assay

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Following treatment with different concentrations of GA (0.25, 0.5, 0.1, 1.5, 2.0 mg/ml), HepG2

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cell apoptosis was measured using a cell apoptosis kit (Nanjingjiancheng, Nanjing, China)



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according to the manufacturer’s instructions. Apoptosis of the treated cells was then detected by

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flow cytometry using a BD FACSCaliber.

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2.4 Microculture tetrazolium (MTT) assay

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MTT assays were used to determine the range of non-toxic GA concentrations according to the

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method described by Gu et al.[21]. Briefly, HepG2 cells were seeded in 96-well plates (100 μl,

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1×105 cells/ml) in DMEM added with 10 % FBS, 0.1 mg/ml streptomycin, and 100 units/ml

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penicillin. The plates were incubated for 24 h at 37 °C in a humidified atmosphere containing 5 %

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CO2. After aspiration of the culture medium, the cells were incubated with different doses of GA

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(0, 0.25, 0.5, 1.0, 1.5, 2.0 mg/ml) for a further 24 h prior to the addition of 20 μl MTT labeling

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reagent. After incubation for 4 h, the culture medium was discarded and the metabolized product

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of MTT (blue-violet crystals) were dissolved in DMSO. The absorbance of the samples was

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measured at 490 nm using an ELISA scanner to speculate the proportion of viable calls.

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2.5 Glucose uptake analysis

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Glucose uptake was determined according to the method described by Jin et al.[22]. Briefly,

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HepG2 cells (1 × 105 cells/well) were seeded in 24 well plates and cultured in normal DMEM as a

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normal control group (NC), DMEM plus PA (0.25 mM) as an IR group. The IR group was then

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added with different concentrations of GA for 24 h. In this study, glucose uptake was detected by

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adding the fluorescent D-glucose analog 2-NBDG (Solarbio, Beijing, China) to the culture

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medium as a tracer. Before measurements were recorded, cells were incubated with insulin (100

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nM, Sigma - Aldrich, Shanghai, China) for 30 min before 2-NDBG was added at a final

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concentration of 50 µM. Fluorescence of cell monolayers was detected with a fluorescence

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microplate reader (Molecular Devices, USA) set at an emission wavelength of 535 nm and an


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excitation wavelength of 485 nm.

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2.6 Glucose production analysis

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Glucose production was determined according to the method described by Gu et al.[21]. Briefly,

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HepG2 cells (1 × 105 cells/well) were incubated in 6 well plates, after model establishment and

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GA treatment, the cells were cultured for 2 h in glucose free DMEM added with 5 mM glycine,

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valine, and alanine, lactate and pyruvate. The culture solution was then gathered for glucose

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determination using a glucose oxidase method kit (Nanjingjiancheng, Nanjing, China).

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2.7 Glycogen content determination

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Glycogen content was detected according to Gupta et al.[23]. Briefly, HepG2 cells (1 × 105

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cells/well) were incubated in 6 well plates, after the model establishment and GA treatment, the

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cells were flushed three times with PBS and collected by centrifugation (4000 rpm for 15 min) for

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estimation of the glycogen content using a commercial kit (Nanjingjiancheng, Nanjing, China).

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2.8 Reactive oxygen species (ROS) level estimation

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In this experiment, ROS levels were detected according to the method described by Tian et al.[24].

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Briefly, HepG2 cells (1 × 105 cells/well) were incubated in 6 well plates, after the model

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establishment and GA treatment, the culture solution was discarded and washed three times with

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PBS. The cells were then cultured for 30 min at 37 °C in DMEM containing

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2’,7’-dichlorofluorescein diacetate (10 µM, DCFH-DA, Beyotime, Shanghai, China). Images of

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ROS levels were obtained using a Nikon ELIPSE Ti-S fluorescence microscope (Nikon, Tokyo,

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Japan).

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2.9 Animals and experimental design

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Six-week old Male Sprague–Dawley (SD) rats (aged 6 weeks, 200  10 g) were obtained from



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Sibeifu Biotechnology Co. Ltd (Beijing, China). All rats were raised adaptively for one week in

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normative animal rooms, which were in a manipulative environment consisting of 23 ± 2 °C with

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a 12 h light/dark cycle, humidity of 55 ± 10 %, and unrestricted access to water and food. All the

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experimental procedures, animal care was handled in accordance with the guidelines provided by

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the Animal Care Committee and executed according to the Technical Standards for Testing &

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Assessment of Health Food (2003).

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2.10 Rat model of T2DM

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Rats model of T2DM were established as previously described by Zhang et al.[1]. Briefly, the rats

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were randomly divided into two groups: (1) the normal control group (NC) consisting of 20 rats

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fed a normal chow diet; (2) the T2DM group fed a high-fat diet (HFD). After one month, the

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T2DM rats were fasted for 12 h with free access to water, then intraperitoneally injected with 30

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mg/kg STZ (pH 4.5, dissolved in citrate buffer). The rats in the NC group were injected with the

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vehicle. Three days later, the rats in the T2DM group with blood glucose level ≥ 16.7 mM were

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considered as T2DM. These T2DM rats were continuously fed the HFD throughout the study.

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Subsequently, the T2DM rats were randomly divided into three groups (n = 10 per group). GA

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was administered to the T2DM rats at a doses 40 and 80 mg/kg/day for 6 weeks, respectively, as

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low dose (GA-L) and high dose (GA-H) groups, respectively; an equal volume of water was

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administered to the NC and T2DM groups. Finally, 10 rats in the normal group received GA at 80

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mg/kg/day, and were classed as the normal high-dosage control group (GA-NH). Oral glucose

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tolerance tests (OGTT) were handled as described by Zhang [1]. All rats were fasted for 12 h

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before receiving 2 g/kg glucose by gavage. The blood glucose levels were then determined at 0,

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30, 60, 90 and 120 min. At the end of the study, blood samples were drawn from the posterior


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vena cava. Liver, muscle and adipose tissues were gathered for further analyses.

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2.11 Metabolic parameters analyses

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The following metabolic parameters were measured in the serum using commercial kits

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(Nanjingjiancheng, Nanjing, China): insulin, non-esterified fatty acids (NEFA), total triglyceride

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(TG), cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein

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cholesterol (LDL-C). HbA1c assay kit hemoglobin assay kits (USCN Life Science, Wuhan,

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China) were used to detected HbA1c level, which was expressed as the ratio of HbA1c to total

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hemoglobin as reported by Mang et al.[25].

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2.12 Electron microscopy

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Transmission electron microscopy (TEM) was used to visualized the ultrastructural characteristics

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of the ER. HepG2 cells and T2DM rat livers were gathered and fixed with 2 % paraformaldehyde

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and 2.5 % glutaraldehyde at 4 °C for 1 h, and then fixed in 1 % osmium tetroxide for 1 h. The

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tissues were dehydrated in a series of ethanol before infiltration with epoxy resin. Ultrathin

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sections (50 nm) were cut and stained with 0.2 % lead citrate and 4 % uranyl acetate. Finally,

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sections were observed using an HT7700 electron microscope.

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2.13 RT-PCR analysis

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The expression levels of target genes were determined by RT-PCR. Total RNA was separated

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from HepG2 cells and T2DM rats using Trizol reagent. cDNA was synthesized from total RNA by

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reverse transcription using a RT-PCR kit (TaKaRa Biomedical Technology, Beijing, China).

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RT-PCR analysis was handled with premixed SYBR green reagents in a real time detector using

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the following thermal cycling profile: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, followed by 58 °C

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for 5 s, and a extension step at 72 °C for 30 min. The melting curves of the RT-PCR products were



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evaluated to analyze the amplification efficiency of the target gene (ORP150), which was found to

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be 94 %. The relative expression levels were determined using the 2-ΔΔCt method, with β-actin as

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an endogenous control. The PCR primer pairs used to amplify target cDNA fragments were

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designed

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TTGACTCAAACCTGTCCAAC; antisense, ACAAGAATGAACCTGGCTGT); β-actin (sense,

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AACTGGGACGACATGGAGAA; antisense, ATACCCCTCGTAGATGGGCA).

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2.14 Western blot analysis

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Protein expression was analyzed by Western blotting. In brief, total proteins were extracted form

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treated HepG2 cells and T2DM rat livers using RIPA reagent, the concentration was measured

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using the Bradford colorimetric method. Equal amounts of proteins from each sample were

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separated by 10 % sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and

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transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA).

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Membranes were then incubated in blocking reagent containing 5 % non-fat dried milk for 1.5 h at

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room temperature and incubated overnight at 4 °C with primary detection antibodies: TBST

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containing anti-β-actin (1:10,000), anti-ORP150 (1:1,000), anti-PERK (1:1,000), anti-p-PERK

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(1:1,000), anti-eIF2α (1:1,000), anti-p-eIF2α (1:1,000), anti-JNK (1:1,000), anti-p-c-Jun (1:1,000),

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anti-IRS (1:1,000), anti-p-IRS(Ser) (1:1,000), and anti-p-IRS(Tyr) (1:1,000). Membranes were

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then incubated with horseradish peroxidase secondary detection antibodies for 2 h at room

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temperature. Protein expression was determined using ECL, and bands were visualized through

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autoradiography. Band intensities were quantified using ImageJ software.

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2.15 Statistical analysis

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All data were presented as means ± standard deviation (SD). Statistical analysis was performed

with

the

Primer

Premier

5

program

as


follows:

ORP150

(sense,

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with SPSS version 20.0 (Statistical Package for the Social Sciences Software, SPSS Inc., Chicago,

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IL, USA). Differences were considered significant when P < 0.05, whereas P < 0.01 was regarded

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as very significant

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3. Results

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3.1 Cytotoxicity and apoptosis of HepG2 cells following GA treatment

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It is well-known that high concentrations of drugs can inhibit cell activities and induce

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apoptosis[26]. To determine the optimal concentration of GA for use in this study, we assessed the

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toxic and apoptotic effects of GA on HepG2 cells. When GA concentrations were lower than 1.5

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mg/ml, cells activities were not affected (Fig. 1). However, GA significantly increased HepG2 cell

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apoptosis at concentrations exceeding 1 mg/ml (Fig. 1B), indicating that GA at the concentrations

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below 1 mg/ml were suitable for use in bioactivity assays. Based on these results, GA was used at

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0.25, 0.5 and 1.0 mg/ml in the subsequent experiments.

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3.2 GA regulates glucose metabolism and oxidative stress in IR HepG2 cells

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As a high-fat component, PA is widely used to induce the IR in HepG2 cells, which can lead to

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ER stress and affect glucose metabolism when the effect is severe. In this study, we first

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demonstrated that GA markedly increased glucose uptake and suppressed glucose production in

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IR HepG2 cells. Additionally, the PA-induced decrease in glucose uptake was enhanced by GA at

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1.0 mg/ml (Fig. 2A), while glucose production was significantly inhibited (Fig 2B). Our data also

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indicated that GA increased the glycogen content of IR HepG2 cells in a dose-dependent manner

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(Fig. 2C). Moreover, GA significantly reduced ROS levels in IR HepG2 cells (Fig. 2D).

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According to these results, we concluded that GA regulates hyperglycemia and reduces ROS

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levels in IR HepG2 cells, which provides the experimental basis for the study of the mechanism



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responsible for hypoglycemia.

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3.3 Body weight, blood glucose and metabolic parameters

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Weekly measurements of fasting blood glucose (FBG) levels showed that T2DM rats had higher

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blood glucose levels than those in the NC group after being fed HFD for 4 weeks (Table 1).

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Compared to the T2DM group, oral administration of GA at 40 and 80 mg/kg/day prominently

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reduced the FBG level by 15.2 % and 26.7 %, respectively, by the end of the study. Furthermore,

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weekly measurement of the body weight showed that the weight of rats in the T2DM, GA-L and

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GA-H groups before GA treatment was prominently lower than that of rats in the NC and GA-NH

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groups (Table 2). After 6 weeks of GA treatment, the GA-L and GA-H groups showed a

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prominent increase in body weight. the FBG level was enhanced in T2DM group, and the area

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under curve (AUC) was approximately 3-fold higher than that in the NC group. Treatment with 40

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or 80 mg/kg/day GA alleviated the FBG level, and prominently decreased the AUC compared

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with that in the T2DM group.

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In addition to the decreased blood glucose level, diabetes induced increase in the HbA1c ratio and

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insulin level were decreased by GA administration. Moreover, serum concentrations of TG, TC,

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LDL-C, HDL-C and NEFA were analyzed to confirm the anti-hyperglycemic and

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anti-hyperlipidemic syndrome effects of GA. In the present study, the T2DM group exhibited an

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obviously increase in TG, TC, LDL-C and NEFA, while HDL-C was decreased. After GA

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treatment, the TC, TG, LDL-C and NEFA levels were dramatically decreased, while HDL-C

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levels were increased compared with those in the T2DM group (Table 2). Furthermore, the serum

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levels of AST and ALT were also prominently decreased by GA treatment (Table 2), indicating

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that GA treatment can significantly protect against liver damage and necrosis in T2DM rats. Taken


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together, these findings indicate that GA treatment mitigates glycaemia, promotes lipid

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metabolism and improves liver function in T2DM rats.

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3.4 Effect of GA on ER in T2DM rats and IR HepG2 cells

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To explore the molecular mechanism underlying the anti-diabetes properties of GA, we initially

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studied the microstructure of the ER for sign of stress in GA-treated HepG2 cells and T2DM rats.

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As shown in Figure 4, ultrastructural analysis revealed that GA treatment significantly improved

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the dilatation and degranulation of the smooth ER in IR HepG2 cells and T2DM rat livers. At the

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same time, we examined the expression of the ER stress indicator proteins and genes in HepG2

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cells. Compared with the NC group, ORP150 levels in HepG2 cells were remarkably increased in

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the Mod group, while treatment with GA strongly reduced the levels of ORP150 (Figure 5).

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Similarly, the levels of p-eIF2α, p-PERK and p-c-Jun in IR HepG2 cells were significantly

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increased, and these changes were reversed by treatment with GA. Furthermore, the effect of GA

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on the activation status of ER stress were validated in vivo. After 6 weeks of GA treatment, the

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levels of ORP150, p-PERK, p-eIF2α and p-c-Jun in the liver of T2DM rats were significantly

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reduced (Figure 6). Together, these results indicate that GA protects against ER stress both in vivo

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and in vitro.

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3.5 Effect of GA on insulin signal transduction in T2DM rats and IR HepG2 cells

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In this study, we explored whether the effects of GA on ER stress were related to the enhanced

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insulin signaling by examining the expression of the main molecules involved in insulin signal

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transduction. As shown in Figure 7, there were no changes in the expression of total IRS-1, while

272

p-IRS-1(ser307) was upregulated and p-IRS-1(tyr) was downregulated both in the IR HepG2 cells

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and T2DM rat livers. In contrast, treatment with GA greatly decreased the levels of



274

p-IRS-1(ser307) and also significantly increased the levels of p-IRS-1(tyr) in the IR HepG2 cells

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and T2DM rat livers. Therefore, these data indicated that GA enhanced insulin sensitivity by

276

inhibiting p-IRS-1(ser307) and facilitating p-IRS-1(tyr) without any change in the total level of

277

IRS-1both in vivo and in vitro.

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4. Discussion

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In this study, GA treatment maintained body weight, and alleviated blood glucose and lipid levels

280

in T2DM rats. In addition, GA increased glucose uptake and inhibited glucose production by

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hepatocytes in vitro. Moreover, ER stress and insulin signal transduction in high glucose

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consuming cells were ameliorated following exposure to GA in vivo and in vitro. Taken together,

283

our findings demonstrate an advantageous effect of GA in the regulate of T2DM symptoms, and

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this effect is likely to be associated with the modulation of ER stress and insulin signal

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transduction.

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T2DM is a chronic metabolic disease characterized by hyperglycemia accompanied with IR[27].

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IR is manifested mainly as the deterioration of insulin signaling and biological activity in response

288

to insulin stimulation in peripheral tissues[28]. T2DM occurs when insulin secretion can no longer

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compensate for IR[27]. Although increasing knowledge of T2DM, few natural hypoglycemic

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agents without side-effects are available to treat T2DM in the clinic. In addition, T2DM is the

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underlying cause of many chronic metabolic syndromes. Therefore, natural products with

292

anti-hyperglycemic activity and no side-effects are urgently required.

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In this study, the insulin and blood glucose levels, as well as the HbA1c ratio were prominently

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increased in STZ-treated rats fed with a HFD for 4 weeks, suggesting that T2DM was successfully

295

established[29]. Various studies have verified that GA plays an important role in the treatment of


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T2DM, although the mechanism responsible for its anti-hypoglycemic effects has not been

297

studied[30, 31]. Based on previous studies, we choose the doses of 40 and 80 mg/kg/day to

298

investigate the potential antidiabetic effects of GA in T2DM rats. The results showed that 6 weeks

299

of treatment with GA alleviated the diabetic symptoms as evidenced by reduced FBG and insulin

300

levels, and HbA1c ratio. Moreover, the OGTT is the most commonly used indicator of diabetes,

301

reflecting islet beta cell function, and generally representing the basal secretion of insulin. Our

302

data showed that the OGTT was greatly improved after GA treatment, indicating that GA

303

enhances insulin sensitivity and significantly improves the symptoms of IR. All these findings

304

indicate that GA increases the response to insulin to accelerate glucose metabolism.

305

The liver is the main organ that regulates glucose conversion and plays an important role in

306

glycemic control. Under the pathological status of T2DM, glucose uptake is disrupted and insulin

307

action is compromised, leading to hyperglycemia[32]. In this study, GA treatment increased

308

glucose uptake by HepG2 cells in a dose dependent manner without weakening the activity of

309

these cells, indicating that GA lowers blood glucose by enhancing glycol-metabolism in

310

hepatocytes.

311

ER is verified to play an important role in maintaining metabolic homeostasis[33], and more than

312

75% of proteins are synthesized in the ER[34]. Therefore, ER dysfunction can affect the folding

313

and maturation of the proteins, gradually leading to ER stress[35]. The important relationship

314

between the ER and metabolic regulation has increased the recognition of the ability of the ER to

315

regulate insulin transduction and glucose metabolism. In consideration of the special role of ER

316

stress in the pathogenesis status of T2DM, we explored the effect of GA on ER stress and insulin

317

transduction in vivo and in vitro. As an ER stress related chaperone, ORP150 increases glucose



318

assimilation and inhibits protein oxidation in T2DM[36]. Previous investigations demonstrated

319

that ORP150 expression was enhanced in cells under conditions of ER stress[37]. In this study, we

320

found that GA markedly inhibited ORP150 expression in IR HepG2 cells and T2DM rat livers, at

321

both the mRNA and protein levels. We further explored the effect of GA on the activity of several

322

other ER stress indicators (including JNK, PERK and eIF2α) in vivo and in vitro. We found that

323

GA lowered the expression of p-c-Jun while simultaneously inhibiting the expression of p-eIF2α

324

and p-PERK. All these results showed that GA protect hepatocytes from the damage associated

325

with ER stress.

326

Insulin signaling plays a pivotal role in maintaining the synthesis and degradation of glycogen,

327

lipids and proteins[38]. IRS proteins existing inside the cytoplasm exert important functions in the

328

insulin activation cascade. Impairment of IRS protein phosphorylation could impede the signaling

329

cascade, leading to T2DM[36]. Studies have demonstrated that p-IRS-1(ser) negatively regulates

330

insulin receptor signaling and is considered to be a common cause of IRS-1 protein dysfunction.

331

Moreover, decreased of p-IRS-1(tyr) is also a risk factor of IR[39]. In this investigation, we found

332

that GA effectively prevents damage to the insulin receptor, which is related to the suppression of

333

p-IRS-1 (ser) and promotion of p-IRS-1 (tyr). These findings suggest that the reparative function

334

of the ER and IRS in hepatocytes can effectively regulate glucose metabolism levels and relieve

335

the symptoms of hyperglycemia.

336

In conclusion, our study finds that GA reduce blood glucose levels and improve glucose and lipid

337

metabolism in T2DM rats, and also enhance glucose uptake in IR HepG2 cells. These functions

338

are likely to be regulated by amelioration of ER stress and insulin transduction. Furthermore, our

339

findings indicate the potential of GA as a hypoglycemic agent for the treatment of T2DM.


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340

Acknowledgments This research is supported by the National Science-Technology Pillar Program

341

(2012BAD33B05) and the Program for Changjiang Scholars and Innovative Research Team in

342

University of the Ministry of Education of the People’s Republic of China (Grant IRT1166).

343

Conflict of interest

344

The authors declare no conflict of interest.

345

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456

Figure 1. Effect of GA on cytotoxicity and apoptosis of HepG2 cells. (a, b) Means within a group

457

with different letters are significantly different (P < 0.05). Data represent the means ± SD (n = 10

458

per group).

459

Figure 2. Effect of GA on glucose uptake and production, glycogen content and levels of reactive

460

oxygen species in HepG2 cells. Glycogen content is represented as a percentage of the control

461

group. (a–c) Means within a group with different letters are significantly different (P < 0.05). Data

462

represent the means ± SD (n = 6 per condition). NC, normal control group; Mod, model control

463

Group; L, GA-0.25 mg/ml; M, GA-0.5 mg/ml; H, GA-1.0 mg/ml.

464

Figure 3. The effects of GA treatment on OGTT in experiment rats. The area under curve of panel

465

A is shown in panel B. (a–d) Means within a group with different letters are significantly different

466

(P < 0.05). Data represent the means ± SD (n = 8 per group).

467

Figure 4. The effect of GA treatment on histopathological features of experimental rat liver and

468

HepG2 cells.

469

Figure 5. Effect of GA on the expression of ER stress indicator protein in IR HepG2 cells. (a–c)

470

Means within a group with different letters are significantly different (P < 0.05). Data represent

471

the means ± SD of three independent experiments.

472

Figure 6. Effect of GA on the expression of ER stress indicator protein in T2DM rats. (a–c)

473


474


475

Figure 7. Effect of GA on insulin signaling transduction in T2DM rats and IR HepG2 cells. (a–d)

476


477




478

479 480

Page 24 of 33

Table 1. Effect of GA on fasting blood glucose in experimental rats group

0 weeks

1week

3 weeks

5 weeks

Reduction rate (%)

NC

4.65 ± 0.36

5.16 ± 0.14

5.05 ± 0.05

4.75 ± 0.22

T2DM

20.6 ± 4.17**

21.6 ± 5.17**

23.7 ± 3.09**

22.2 ± 3.12**

GA-L

21.4 ± 2.25

21.0 ± 4.25

18.6 ± 2.35

18.1 ± 1.90##

15.2 %

GA-H

20.7 ± 3.20

20.6 ± 2.20

17.3 ± 1.04##

15.2 ± 3.10##

26.7 %

GA-NH

5.15 ± 1.31

4.98 ± 0.87

4.98 ± 0.87

5.09 ± 1.69

Data are expressed as the means ± SD (n = 8 per group), **P < 0.01 vs. NC group; ## P < 0.01 vs. T2DM group;

481


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482

Table 2. Effect of GA on metabolic parameters of experimental rats. Parameters

NC

T2DM

GA-L

GA-H

GA-NH

Before GA treatment (g)

424 ± 8.10

346 ± 26.5**

346 ± 5.90**

339 ± 16.6**

419 ± 10.2

After GA treatment (g)

483 ± 16.0

330 ± 17.2**

369 ± 11.5##

377 ± 8.30##

476 ± 10.5

Body weight gain (g)

58.9 ± 10.4

-16.0 ± 8.97**

23.0 ± 5.75##

38.1 ± 7.55##

56.3 ± 14.1

FINS (mIU/l)

14.8 ± 1.51

21.1 ± 1.06**

18.3 ± 1.69

17.7 ± 1.35##

14.6 ± 1.98

HbA1c (%)

4.36 ± 0.86

12.5 ± 3.00**

10.1 ± 3.82

6.91 ± 1.78##

4.29 ± 0.37

NEFA(μmol/l)

184 ± 40.8

609 ± 142**

442 ± 102

372 ± 69.4##

195 ± 52.1

TC (mmol/l)

1.58 ± 0.05

4.32 ± 0.09**

3.98 ± 0.53##

2.24 ± 0.06##

1.60 ± 0.17

TG (mmol/l)

0.69 ± 0.06

2.31 ± 0.11**

1.90 ± 0.34##

1.06 ± 0.07##

0.73 ± 0.07

LDL-C (mmol/l)

1.07 ± 0.31

5.12 ± 0.71**

3.54 ± 0.52##

2.25 ± 0.34##

1.13 ± 0.14

HDL-C (mmol/l)

0.61 ± 0.03

0.24 ± 0.07**

0.43 ± 0.05##

0.52 ± 0.01##

0.65 ± 0.05

AST (mmol/l)

53.2 ± 10.1

142 ± 19.4**

108 ± 15.7##

72.0 ± 17.5##

56.2 ± 13.8

ALT (mmol/l)

29.3 ± 12.0

89.4 ± 14.3**

67.1 ± 9.2#

45.1 ± 6.04##

27.3 ± 11.9

483 484 485

Data are expressed as the means ± SD (n = 8 per group), **P < 0.01 vs. NC group; ## P < 0.01 vs. T2DM group;

486



487

Figure 1. Effect of GA on cytotoxicity and apoptosis of HepG2 cells. (a, b) Means within a group

488

with different letters are significantly different (P < 0.05). Data represent the means ± SD (n = 10

489

per group).

490 491


Page 26 of 33

Page 27 of 33


492

Figure 2. Effect of GA on glucose uptake and production, glycogen content and levels of reactive

493

oxygen species in HepG2 cells. Glycogen content is represented as a percentage of the control

494

group. (a–c) Means within a group with different letters are significantly different (P < 0.05). Data

495

represent the means ± SD (n = 6 per condition). NC, normal control group; Mod, model control

496

Group; L, GA-0.25 mg/ml; M, GA-0.5 mg/ml; H, GA-1.0 mg/ml.

497 498 499



500

Figure 3. The effects of GA treatment on OGTT in experiment rats. The area under curve of panel

501

A is shown in panel B. (a–d) Means within a group with different letters are significantly different

502

(P < 0.05). Data represent the means ± SD (n = 8 per group).

503 504 505


Page 28 of 33

Page 29 of 33


506

Figure 4. The effect of GA treatment on histopathological features of experimental rat liver and

507

HepG2 cells.

508 509



510

Figure 5. Effect of GA on the expression of ER stress indicator protein in IR HepG2 cells. (a–c)

511


512


513 514 515


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516

Figure 6. Effect of GA on the expression of ER stress indicator protein in T2DM rats. (a–c)

517


518


519 520



521

Figure 7. Effect of GA on insulin signaling transduction in T2DM rats and IR HepG2 cells. (a–d)

522


523


524 525 526


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527


Graphic for table of contents

528


Gymnemic acid alleviates type 2 diabetes mellitus and suppresses

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