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Tormentic Acid, a Major Component of Suspension Cells of Eriobotrya japonica, Suppresses High-Fat Diet-Induced Diabetes and Hyperlipidemia by Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation Jin-Bin Wu, Yueh-Hsiung Kuo, Cheng-Hsiu Lin, Hui-Ya Ho, and Chun-Ching Shih J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503334d • Publication Date (Web): 15 Oct 2014 Downloaded from http://pubs.acs.org on October 17, 2014

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Journal of Agricultural and Food Chemistry

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Tormentic Acid, a Major Component of Suspension Cells of

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Eriobotrya japonica, Suppresses High-Fat Diet-Induced Diabetes and

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Hyperlipidemia by Glucose Transporter 4 and AMP-Activated

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Protein Kinase Phosphorylation

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Jin-Bin Wu,† Yueh-Hsiung Kuo,# Cheng-Hsiu Lin,‡ Hui-Ya Ho,§ and Chun-Ching

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Shih║,*

8 †

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Taichung City 40402, Taiwan #

Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources,

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China Medical University, Taichung City 40402, Taiwan ‡

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Department of Internal Medicine, Fong-Yuan Hospital, Department of Health,

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Executive Yuan, Fong-Yuan District, Taichung City 42055, Taiwan §

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Graduate Institute of Pharmaceutical Chemistry, China Medical University,

║,

Jen Li Biotech Co. Ltd., Yong-feng Road, Taiping City, Taichung 411, Taiwan

* Graduate Institute of Pharmaceutical Science and Technology, College of Health

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Science, Central Taiwan University of Science and Technology, No.666, Buzih Road,

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Beitun District, Taichung City 40601, Taiwan

19

1

ACS Paragon Plus Environment


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ABSTRACT:

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acid (PTA) on diabetes and dyslipidemia in high-fat (HF)-fed mice. Feeding

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C57BL/6J mice with a HF diet for 12 weeks induced type 2 diabetes and

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hyperlipidemia. During the last 4 weeks, the mice were given orally PTA (at two

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dosages) or rosiglitazone (Rosi) or water. In this study, HF-diet increases glucose,

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triglyceride, insulin and leptin levels, while PTA effectively prevented these

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phenomenons and ameliorated insulin resistance. PTA reduced visceral fat mass and

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hepatic triacylglycerol contents; moverover, PTA significantly decreased both the area

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of adipocytes and ballooning degeneration of hepatocytes. PTA caused increased

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skeletal muscular AMP-activated protein kinase (AMPK) phosphorylation and Akt

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phosphorylation and glucose transporter 4 (GLUT4) proteins, whereas reduced the

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hepatic expressions of phosphenolpyruvate carboxykinase (PEPCK) and

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glucose-6-phosphatase (G6Pase) genes. PTA enhanced skeletal muscular Akt

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phosphorylation and increased insulin sensitivity. PTA also enhanced phospho-AMPK

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in the liver. Therefore, it is possible that the activation of AMPK by PTA results in

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decreasing hepatic glucose production, while increasing skeletal muscular GLUT4

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contents, thus contributed to attenuating diabetic state. Moreover, PTA exhibits

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anti-hyperlipidemic effect by down-regulations of the hepatic sterol regulatory

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element binding protein-1c (SREBP-1c) and apolipoprotein C-III (apo C-III) while

It was designed to evaluate the effects and mechanism of tormentic

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increased peroxisome proliferator activated receptor (PPAR)-α expression, thus

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resulting in decreases in blood triglycerides. Our findings demonstrated that PTA was

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effective for the treatment of diabetes and hyperlipidemia in HF-fed mice.

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KEYWORDS: tormentic acid, anti-diabetes, anti-hyperlipidemia, AMP-activated

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protein kinase phosphorylation, glucose transporter 4

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Introduction

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Type 2 diabetes represents greater than 90% of all diabetes cases. Insulin resistance is

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the majority of type 2 diabetes caused by insensitivity to insulin in peripheral tissues.

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It is predicted that the world’s population of type 2 diabetes would be reached 6.1%

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by 2025.1 Therefore, to find the safer and less toxic substitute in the treatment of type

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2 diabetes mellitus becomes important. Type 2 diabetes mainly reduces glucose

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uptake.2 Type 2 diabetes is accompanied by several complications to cause a series of

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metabolic diseases including obesity and dyslipidemia. It is known that blood glucose

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and lipid constitutes fluctuating homeostasis. Thus, to find the good resolution of

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glucose uptake and hepatic gluconeogenesis is an important issue in Type 2 diabetes.

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Insulin is secreted after a meal, and followed by glucose transporter 4 (GLUT4)

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was increased and translocated to the plasma membrane, and thus lead to glucose

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uptake into cells, contributing to reduced blood glucose.3,4 Insulin resistance and

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hyperglycemia was caused by problems in GLUT4 translocation and uptake.5,6 Thus,

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it is an important issue to increase protein contents and/or translocation of GLUT4 in

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the management of diabetes.

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AMP-activated protein kinase (AMPK) has regulated various metabolic pathways,

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and it is considered as an important target for the management of metabolic diseases

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including type 2 diabetes and dyslipidemia.7,8,9 Type 2 diabetes is found dysfunctional

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in glucose and lipid metabolism, therefore AMPK modulators have been suggested to

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be promising therapies.10

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The plant Eriobotrya japonica Lindl. (Figure 1A) is an evergreen fruit tree and

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belongs to Rosacease. The most used part of this plant is the dried leaf to treat

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diabetes mellitus.11,12 It is composed of many pentacyclic triterpenes, which

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demonstrate various pharmaceutical effects including hepato-protection13 and

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anti-diabetes.14,15 Callus tissue culture of loquat is reported to produce large amounts

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of triterpenes.16 We have recently shown that loquat leaf extract as well as its cell

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suspension culture (which contains five main bioactive constituents including

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tormentic acid (PTA), see chemical structure in Figure 1B), could improve insulin

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sensitivity and hyperlipidemia,17,18 we think it is possible that the five constitutes act

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synergistically (a synergistic effect is one in which the combined effect of two

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chemicals is greater than the sum of the effect of each agent given alone) on diabetes

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and lipid. Nevertheless, the effect of single and pure PTA of anti-diabetes and

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anti-dyslipidemia is still not fully understood. The aerial part of Poterium ancistroides

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Desf. contained tormentic acid and exhibited the anti-diabetic activity.19 Tormentic

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acid has been shown to lower blood glucose in normoglycemic and glucose-induced

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hyperglycemic rats19 and initiates insulin secretion by isolated rat islets of

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Langerhans.20

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The mouse C57BL/6 model fed with a HF diet could induce insulin resistance,

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obesity, hyperlipidemia, hyperinsulinemia, hyperleptinemia, and excess circulating

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free fatty acid.21,22 Therefore, we conducted our animal study using HF diet-induced

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diabetic and hyperlipidemic states. AMPK activity is depended on phosphorylation of

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Thr 172 of α subunits.23 This study also examined the effect of PTA on the expression

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or protein of genes involved in antidiabetes and lipogenesis, including GLUT4, Akt,

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AMPK, phosphoenol pyruvate carboxykinase (PEPCK), and glucose-6-phosphatase

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(G6Pase), sterol regulatory element binding protein-1c (SREBP-1c), peroxisome

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proliferator-activated receptor α (PPARα) and apolipoprotein C-III (apo-CIII).

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MATERIALS AND METHODS

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Chemicals. GLUT4 was purchased from Santa Cruz Biotechnology (CA, USA),

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phospho-AMPK from Abcam Inc (Cambridge, MA, USA), phospho-Akt, total AMPK

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and total Akt from Cell signaling Technology, Inc. (Danver MA, USA) and BCA

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protein assay kit from Thermo Scientific (Rockford, IL, USA) and ECL reagent kit

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from GE Healthcare BioSciences (Buckinghamshire, UK). Secondary antibody

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anti-rabbit were from Jackson Immuno Research, Laboratories, Inc. (Pennsylvania,

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USA). Structural proteins GAPDH were from Santa Cruz Biotecnology (CA, USA)

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and β-actin from Santa Cruz Biotecnology (CA, USA).

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Isolation of tormentic acid

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PTA was obtained from Jen Li Biotech Co. The parts of Callus induction,

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suspension cultures, extraction and isolation of tormentic acid from suspension cells

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of E. japonica are shown as previously described.16,24 Briefly, sterilized seeds after

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callus induction, subculture were cultured in a bioreactor, and the cell suspension

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(ca.844.5g) was dried and extracted with ethanol, then concentrated to afford the

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white powder fraction (ca.6.1g).24 The white powder (0.5g) was chromatographed on

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a reverse silica gel column (LiChroprep RP-18, E.Merck, 40-63 µm) and then further

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purified by preparative high performance liquid chromatography (PHPLC) to yield

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tormentic acid.24 Tormentic acid (230.5 mg ) ： 1H- NMR(pyridine-d5): δ 1.00

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(H-25),1.07 (H-24),1.10 (H-26),1.11 (H-30),1.26 (H-23),1.42 (H-29),1.71 (H-27),

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3.04(H-18),3.36 (H-3α),4.09 (H-3β),5.58 (H-12). 1H- NMR(400MHz) spectra were

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measure by Bruker AMX-400 spectrometer as previously described.24

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Animals and experimental design.

Part 1: Oral glucose tolerance (OGTT)

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test was performed on 12 h fasted ICR mice (n=5) but were allowed access to 0.2,

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0.4, 0.8 g/kg tormentic acid (PTA) or an equivalent amount of vehicle (water) which

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were given orally 30 min before an oral glucose load (1 g/kg body weight). The

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control group was given with glucose, while the normal group was without glucose.

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Blood samples were collected from the retro-orbital sinus of fasted mice at the time of

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the glucose administration (0) and every 30 minutes until 120 minutes after glucose

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administration. Blood glucose level was monitored. Part 2: C57BL/6J mice (4 weeks

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old) were obtained from the National Laboratory Animal Breeding and Research

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Center. The mice were divided randomly into two groups after 7 days acclimation.

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The control (CON) group (n=9) was fed a low-fat diet (Diet 12450B, Research Diets,

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Inc., New Brunswick, NJ 08901 USA), whereas the experimental group was fed a

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45% high-fat diet (Diet 12451, Research Diets, Inc., New Brunswick, NJ 08901 USA)

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for 12 weeks.18,25 The compositions of the experimental diets are shown as previous

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study described.18,25 After 8 weeks, the high-fat treated mice were randomly

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subdivided into 4 groups (n=9) including PTA (0.06, 0.12 g/kg/day) or rosiglitazone

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(Rosi; 1% methylcellulose 10 mg/kg body weight) (GlaxoSmithKline) or vehicle were

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treated by oral gavage one time per day from the 9th to 12th week, while the mice

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were still on the high-fat diet, whereas the CON and high-fat control (HF) mice were

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treated with vehicle only. At the end, food is deprived from animal (from 10 p.m. to

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10 a.m.). The next day, the mice were sacrificed for blood and tissue collection and

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analysis. Livers, skeletal muscles and white adipose tissues (WATs) (including

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epididymal, mesenteric and retroperitoneal WAT) were weighed and excised, followed

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by immediately freezing and kept at -80 ℃ for target gene analysis. Heparin (30

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units/ml) (Sigma) were added into blood sample. Plasma samples were collected

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within 30 min by centrifugation at 1600 × g for 15 min at 4 ℃. Plasma was obtained

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for insulin and leptin assay.18,25

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Measurement of body weight, body weight gain and food intake.

Body

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weight was measured daily at the same time throughout the study. Body weight gain

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is defined as the differences between the day and the next day. The pellet food was

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weighed and followed by placing in food container. After 24 h, the remaining food

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was weighed, and the difference represented the daily food intake.

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Analysis of blood parameters.

Blood samples (0.8 mL) were collected from

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retro-orbital sinus of fasted mice and glucose level was analyzed by the glucose

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oxidase method (Model 1500; Sidekick Glucose Analyzer; YSI Incorporated, Yellow

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Springs, OH, USA).18,25 Plasma triglycerides (TG), total cholesterol (TC) and free

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fatty acids (FFA) were measured using commercial assay kits according to the

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manufacturer’s directions (Triglycerides-E test, Cholesterol-E test and FFA-C test,

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Wako Pure Chemical, Osaka, Japan).17,18 The levels of insulin and leptin in blood

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were analyzed by ELISA using a commercial assay kit according to manufacturer’s

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directions (mouse insulin ELISA kit, Sibayagi, Gunma, Japan and mouse leptin

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ELISA kit, Morinaga, Yokohama, Japan).18,25

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Analysis of histopathology.

Small pieces of epididymal WAT and liver tissue

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were fixed with formalin (200 g/kg) neutral buffered solution and embedded in

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paraffin. Sections (8 µm) were cut and stained with hematoxylin and eosin. For

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microscopic examination, a microscope (Leica, DM2500) was used, and the images

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were taken using a Leica Digital camera (DFC-425-C).18

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Analysis of hepatic lipids.

Hepatic lipids were extracted using a previously

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described protocol.17 For the hepatic lipid extraction, the 0.375 g liver samples were

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homogenized with 1 mL distill water for 5 min. Finally, the dried pellet was

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resuspended in 0.5 mL ethanol and analyzed using a triglycerides kit as used for

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serum lipids.18

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Isolation of RNA and relative quantization of mRNA indicating gene

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expression.

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(Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s

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directions. The integrity of the extracted total RNA was examined by 2% agarose gel

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electrophoresis, and the RNA concentration was determined by the ultraviolet (UV)

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light absorbency at 260 nm and 280 nm (Spectrophotometer U-2800A, Hitachi). Total

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RNA (1 µg) was reverse transcribed to cDNA with 5µL Moloney murine leukemia

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virus reverse transcriptase (Epicentre, Madison, WI, USA) as a previously described

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protocol.17,25 The polymerase chain reaction (PCR) was performed in a final 25µL

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containing 1U Blend Taq™ -Plus (TOYOBO, Japan), 1 µL of the RT first-strand

Total RNA from the liver tissue was isolated with a Trizol Reagent

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cDNA product, 10 µΜ of each forward (F) and reverse (R) primer, 75 mM Tris-HCl

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(pH 8.3) containing 1 mg/L Tween 20, 2.5 mM dNTP and 2 mM MgCl2.17,18,25 The

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primers are shown in Table 1. The products were run on 2% agarose gels and stained

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with ethidium bromide. The relative density of the band was evaluated using

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AlphaDigiDoc 1201 software (Alpha Innotech Co., San Leandro, CA, USA). All the

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measured PCR products were normalized to the amount of cDNA of GAPDH in each

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sample.17,18,25

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Western Immunoblotting analysis.

This part is according to a previous

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report,18,26,27 protein extractions and immunoblots for the determination of GLUT4,

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phospho-AMPK (Thr172) and phospho-Akt (Ser 473) proteins were carried out on

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frozen skeletal muscle and liver tissue from mice. Briefly, samples liver tissue (0.1g)

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was homogenized with lysis buffer (pH6.4) and protease inhibitors. 40 µg of each

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homogenate is used for SDS-PAGE and immunoblotting as a previously described

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protocol.18,26,27 Additionally, GLUT4 were carried out on frozen skeletal muscle from

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mice briefly described: Skeletal muscle (1 g) was powdered under liquid nitrogen and

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homogenized for 20 s in buffer (pH 7.4). The total membrane fractions were

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collected with buffer and centrifuged as a previously described protoco.28,29 The

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protein contents of GLUT4, phospho-AMPK, phospho-Akt, total AMPK and total Akt

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were detected by immunoblotting using a rabbit polyclonal antibody. The protein

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concentration in supernatant was determined with a BCA protein assay kit. The

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membranes were blocked with 5% slim milk in Tris-buffered saline (TBS) (Amershan

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BioSciences, Uppsala, Sweden) containing 0.05% Tween-20 (Bio Rad, CA, USA) and

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incubated overnight at 4℃ with anti-GLUT4, anti-phospho-AMPK and

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anti-phospho-Akt at 1:200 dilution and anti-total-AMPK and anti-total-Akt at 1:1000

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dilution. Subsequently, the membranes were washed three times with TBS containing

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0.05% Tween-20 and incubated with secondary antibody anti-rabbit (1:1000) for 1h.

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Immunoreactive bands were detected with ECL reagent kit. The density blotting was

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analyzed using Alpha Easy FCTM software (Alpha innotech corporation, Randburg,

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SA). Structural proteins GAPDH and β-actin were obtained by stripping the

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nitrocellulose membrane proteins of liver and skeletal muscle, respectively.18,26,27

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Statistical Analysis.

All results are presented as the mean and standard error.

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Statistical significance is calculated by Dunnett’s multiple range tests, using SPSS

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software (SPSS Inc., Chicago, IL, USA).

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RESULTS

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OGTT Assay.

214 215

Administration of 0.2, 0.4 and 0.8 g/kg tormentic acid decreased

the levels of blood glucose from 30 to 120 min following a glucose-loading. Body weight and food parameters.

It was measured body weight of mice at

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the end. Feeding with a high-fat diet increased body weight and body weight gain,

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while the 4-week average food intake of the HF group is decreased. Final body weight

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and food intake are unchanged between the PTA-treated groups and the HF group.

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The PTA1- and PTA2- treated groups decreased the body weight gain over 4-week

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treatment (Table 3).

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White Adipose Tissue Fat accumulation.

The final weights of epididymal,

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mesenteric and retroperitoneal WAT and visceral fat of the HF group were increased.

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Administration of PTA1, PTA2 and Rosi reduced epididymal WAT, mesenteric and

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retroperitoneal WAT and visceral fat weights (Table 3).

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Blood parameter, leptin, insulin and liver lipid. HF-diet increased blood

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glucose, and administration of PTA1, PTA2 and Rosi lowered blood glucose levels

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(Figure 2B). High-fat diets caused increases in circulating TG, FFA, leptin and insulin

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levels. The PTA1-, PTA2- and Rosi- treated mice displayed a decreased TG, FFA,

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leptin and insulin. The PTA2- and Rosi- treated mice decreased TC levels. HF-diet

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increased the total lipids of liver and concentrations of triacylglycerol whereas mice

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administrated PTA1, PTA2 and Rosi significantly decreased these phenomenon.

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Pathological Diagnosis.

HF-induced the adipocyte hypertrophy (the average

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area of adipocytes in the HF group and CON group is 6515.9 ± 495.1 and 2584.6 ±

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205.8 µm2, respectively), whereas mice administrated of PTA1 (2380.9 ± 108.6 µm2)

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and PTA2 (2243.2 ± 100.9 µm2) significantly lower their hypertrophy. The average

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area of the Rosi-treated mice is 4574.4 ± 162.7 µm2 (Figure 3A). According to a

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previous study,30 designation of histological hepatocellular ballooning findings

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included grade 0, none; grade 1, few cells; grade 2, many cells. The ballooning

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phenomenon in liver is visible on HF-diet (mean score: 1.7 ± 0.2). The ballooning

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phenomenon is lower in the PTA1 (1.0 ± 0.2), PTA2 (0.7 ± 0.2) and Rosi (0.9 ± 0.2)

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treated mice (Figure 3B).

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Hepatic Target gene expressions.

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The mRNA levels of PEPCK, G-6Pase,

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11beta hydroxysteroid dehydrogenase 1 (11β-HDS1), diacyl glycerol acyltransferase

244

2 (DGAT2), PPARα, SREBP1c, FAS (fatty acid synthase) and apo C-III were

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increased in the HF group. Following treatment, the PTA1-, PTA2- and Rosi- treated

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groups decreased the mRNA level of PEPCK, G-6Pase, DGAT2, 11β-HSD1,

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SREBP1c, FAS and apo-C-III. The PTA1- and PTA2- treated groups increased the

248

mRNA level of PPARα (Figure 4).

249

The protein contents of phospho-AMPK (Thr172), GLUT4 and phospho-Akt

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(Ser 473)/ total Akt in different tissues.

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protein were decreased in the HF group, while the PTA1-, PTA2- and Rosi- treated

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groups increased the protein contents of both hepatic and skeletal muscular

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phospho-AMPK. HF-induced the protein contents of GLUT4 and phospho-Akt

The protein contents of phospho-AMPK

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decreased, while PTA1-, PTA2- and Rosi- treated mice increased the protein contents

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of GLUT4 and phospho-Akt in skeletal muscule (Figure 5).

256 257

DISCUSSION

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The present results demonstrated that PTA is effective in lowering blood glucose and

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circulating triglyceride levels in HF-fed mice. We have previously shown that loquat

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leaf extract as well as its cell suspension culture (contained the total contents of five

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triterpenes were 85.35% including tormentic acid 44.30%, corosolic acid 19.50%,

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maslinic acid 14.65%, oleanolic acid 1.60% and ursolic acid 5.30%, respectively),18

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could improve insulin sensitivity and hyperlipidemia.17,18 Since cell suspension

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culture contained 44.3% tormetic acid, it is possible that the five constitutes act

265

synergistically and exert more potent effect on diabetes and lipid. Nevertheless, the

266

effect of single and pure tormentic acid (PTA) on diabetes and dyslipidemia is still not

267

fully understood; therefore, we hypothesized that the present study of pure PTA could

268

exhibit an effect in glucose control and lipid metabolism. Indeed, treatment with PTA

269

displayed hypoglycemic and hypolipidemic effect.

270

C57BL/6J mice fed with a high-fat diet is a mouse model to induce type 2

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diabetes.31 HF- induced model had also elevated levels of triglycerides.31 Therefore,

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this model was used in the present study for investigating PTA’s effects on diabetes

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and dyslipidemia. In the present study, feeding C57BL/6J mice with HF diet induced

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hyperglycemia, obesity, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and

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excess circulating free fatty acid, consistent with earlier observations.31 Following

276

treatment with PTA, blood glucose levels, circulating triglycerides and visceral fat

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mass were lowered as well as a reduction in free fatty acid and improved insulin

278

resistance.

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One of the difficulties in pursuing the mechanism of action of this compound is

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its limited availability, although at present PTA could be produced a great quantity.

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Therefore, PTA was firstly administered at the concentration of 0.2, 0.4, 08 g/kg in

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OGTT test by ICR mice. It was found PTA could lower blood glucose levels. Then,

283

PTA was administered at the concentration of 0.06, 0.12 g/kg in chronic HF-fed mice.

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Based on the few amounts of PTA and the duration of 4-week chronic treatment of

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experimental design and tormentic acid at dosage of 30 mg/kg exerting

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antihyperglycemic effect but did not act in rats with severe diabetes induced by an

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injection of streptozotocin [19,20], the concentration of PTA is lowered to 0.06, 0.12

288

g/kg (duplicated or four times) in HF-fed mice; the daily dosage is expected to an

289

accumulative effect and long-lasting in effectiveness.

290

The primary aim is to investigate the anti-diabetic mechanism of PTA, and we

291

evaluate the GLUT4 protein content in the skeletal muscle. The present study showed

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that PTA2 caused markedly increases GLUT4 protein and PTA1 displayed similar

293

GLUT4 levels as Rosi, which is an anti-diabetic agent and directly targets insulin

294

resistance and increases peripheral glucose uptake (GLUT4) lead to improved

295

glycemic control.32 These findings at the first time revealed that PTA caused increased

296

GLUT4 proteins and directly with relation to exhibit anti-diabetes. Thus, PTA

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displayed a very marked enhancement of GLUT4 accompanied by ameliorated insulin

298

resistance.

299

There are two pathways in regulated GLUT4 translocation including the insulin

300

signaling (those involving phosphatidyl inositol 3’ kinase (PI3K)/Akt) and the AMPK

301

pathway.33 To monitor the mechanism of enhanced GLUT4 by PTA, we evaluated its

302

effects on the phosphorylation of Akt in the skeletal muscle. The results demonstrated

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that PTA had a significant increased effect on skeletal muscular phosphorylation of

304

Akt, suggesting that PTA increased muscular GLUT4 contents is likely to be partly

305

mediated by Akt phosphorylation; moreover, PTA enhanced Akt phosphorylation and

306

increased insulin sensitivity.

307

Further, it was evaluated that PTA acts on phosphorylation of AMPK to the same

308

effect that people who exercise or take antidiabetic agents such as AICAR and

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metformin34,35 and rosiglitazone could activate AMPK. TZDs exhibit as an AMPK

310

activator but different from metformin, and reduces liver fat accumulation and

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ameliorated insulin resistance due to its effect on plasma adipocytokine (including

312

adiponectin and leptin) and more AMPK activation.36,37 Indeed, PTA increases

313

skeletal muscular phosphorylation of AMPK, suggesting that it is possibly partly

314

responsible for increased GLUT4 translocation. Further study on phosphorylation of

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AS160 is needed.

316

The liver is the organ responsible for the majority of hepatic gluconeogenesis.38 A

317

number of hormones regulate a set of genes (including PEPCK and G-6Pase) in the

318

liver that modulates the rate of glucose synthesis. PEPCK is a rate-controlling step of

319

gluconeogenesis in animals, and G-6Pase plays a vital role in glucose homeostasis.39

320

Over expression of PEPCK enzyme in mice results in symptoms of type 2 diabetes.

321

The hepatic G-6Pase activities of diabetic animals were increased.40 PTA treatment

322

reduced the expressions of PEPCK and G-6Pase. Therefore, antidiabetic effect of PTA

323

is due to down-regulation of PEPCK and G-6Pase. Furthermore, PEPCK is controlled

324

by hormonal mechanisms including 11β-HSD1. 11β-HSD1 knockout mice fed with a

325

HF diet are protected from developing insulin resistance.41,18 Therefore, compounds

326

that down-regulate 11β-HSD1 might contribute to antidiabetic activities.18 Besides

327

PEPCK and G-6Pase, PTA decreased hepatic 11β-HSD1 expressions also lead to

328

enhanced insulin sensitivity.

329

AMPK activation is beneficial in the management of type 2 diabetes.42 Metformin

18


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is a medication for the treatment of type 2 diabetes by the reason of increased skeletal

331

muscular glucose uptake whereas reduced gluconeogenesis in the liver.20 HF-induced

332

reduced phospho- AMPK in the liver, with increased in PTA- and Rosi- treated

333

groups, indicating improved hyperglycemia by AMPK activation. Besides increasing

334

muscular glucose uptake (GLUT4), PTA is likely to reduce hepatic glucose

335

production by down-regulations of PEPCK and G-6Pase via AMPK.

336

To monitor the mechanism of PTA on anti-hyperlipidemia, we found that PTA

337

increased PPARα expressions. PPARα ligands (such as fibrates) have been shown to

338

reduce the expression of the apo C-III gene,43 thus resulting in lowering fat values in

339

the blood and liver and exhibiting hypotriglyceridemic effect. We also measured

340

DGAT2 expressions. DGAT2 catalyzes the final step in the synthesis of

341

triglycerides.44,18 It was found that PTA decreased circulating TG levels and this may

342

be associated with decreased DGAT2 expressions. Previous study showed that leptin

343

activated AMPK.45 It is possible that PTA directly caused AMPK phosphorylation or

344

alter leptin secretion by PTA inducing AMPK activation. Blood leptin level is

345

markedly reduced in PTA-treated mice. It is consistent with its reduced fat mass.46

346

PTA significantly reduced SREBP1c, a key transcription factor controlling de novo

347

lipogenesis.47 Moreover, PTA suppressed the expression of FAS, which is a key

348

enzyme in fatty acid synthesis. The glucose-induced SREBP1c and FAS mRNA levels

19



349

were also down-regulated by AMPK.48 The AMPK activator metformin has been

350

shown to down-regulate the FAS expression via AMPK activation.7 Therefore, it is

351

possible that PTA down-regulated these genes through AMPK activation.

Page 20 of 46

352

The evidence for morphological analysis comes from the finding that treatment

353

with PTA1 and PTA2 decreased the hypertrophy of adipocytes. The liver is a major

354

organ to metabolize fat. The level of circulating TG is fluctuating,49 it is possible that

355

PTA caused fat moving from adipose to liver tissue by increasing hepatic lipid

356

catabolism thus resulted in decreased the size of adipocytes and liver lipid droplets is

357

almost absent.

358

In conclusion, this study demonstrated that PTA effectively lowers hyperglycemia

359

and hypertriglycemia in HF-fed mice. PTA improves glycemic control primarily via

360

increased skeletal muscular GLUT4 proteins to elevate glucose uptake, whereas

361

suppressed hepatic glucose production (down regulations of PEPCK and G-6Pase).

362

PTA enhanced AMPK phosphorylation both in skeletal muscle and liver. PTA

363

enhanced skeletal muscular Akt phosphorylation and increased insulin sensitivity.

364

PTA increased hepatic fatty acid oxidation (PPARα), while suppressed lipogenic

365

enzyme expression (including SREBP1c and FAS), thus contributing to lowering

366

triglyceride levels. Therefore, tormentic acid is effective on type 2 diabetes and

367

hyperlipidemia in HF-fed mice.

20


Page 21 of 46


368 AUTHOR INFORMATION

369

370

Corresponding Author

371

*Phone:+886-4-22391647 ext. 3978. Fax: +886-4-22394256. E-mail:

372

[email protected] Mail: Graduate Institute of Pharmaceutical Science and

373

Technology, College of Health Science, Central Taiwan University of Science and

374

Technology, No.666, Buzih Road, Beitun District, Taichung City 40601, Taiwan

375

Notes

376

The authors have declared that there is no conflict of interest.

377 378

ACKNOWLEDGEMENTS

379

Financial support for this study was partly provided by CTU103-A-1 form the Central

380

Taiwan University of Science and Technology.

381 382

ABBREVIATIONS USED

383

AMPK, AMP-activated protein kinase; apo C-III, apolipoprotein C-III; BAT, brown

384

adipose tissue; 11β-HSD1, 11beta hydroxysteroid dehydroxygenase; CON, control;

385

DGAT, acyl-coenzyme A: diacylglycerol acyltransferase; FAS, fatty acid synthase;

386

FFA, free fatty acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6Pase,

21



Page 22 of 46

387

glucose-6-phosphatase; GLUT4, glucose transporter 4; HF, high-fat control; OGTT,

388

oral glucose tolerance test; PEPCK, phosphenolpyruvate carboxykinase; PPARα,

389

peroxisome proliferator-activated receptor α; PTA, tormentic acid; Rosi, rosiglitazone;

390

RT-PCR, reverse transcription-polymerase chain reaction; SREBP-1c, sterol

391

regulatory element binding protein 1c; TC, total cholesterol; TG, triglyceride; WATs,

392

white adipose tissues

393 394 395 396 397

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546

Table 1. Primers used in this study Gene

Accession

Forward primer and reverse primer

numbers

PCR

Annealing

product temperature ( (bp)

℃)

330

52

350

50

300

50

149

50

352

55

219

50

240

50

Liver PEPCK

NM_011044. F: CTACAACTTCGGCAAATACC 2

G6Pase

R: TCCAGATACCTGTCGATCTC

NM_008061. F: GAACAACTAAAGCCTCTGAAAC R: TTGCTCGATACATAAAACACTC 3

11β-HSD1 NM_008288. F:AAGCAGAGCAATGGCAGCAT 2 DGAT2

NM_026384. F: AGTGGCAATGCTATCATCATCGT 3

PPARα

R: GAGCAATCATAGGCTGGGTCA

R: AAGGAATAAGTGGGAACCAGATCA

NM_011144 F: ACCTCTGTTCATGTCAGACC R: ATAACCACAGACCAACCAAG

SREBP1c NM_011480 F: GGCTGTTGTCTACCATAAGC R: AGGAAGAAACGTGTCAAGAA FAS

NM_007988 F: TGGAAAGATAACTGGGTGAC R: TGCTGTCGTCTGTAGTCTTG

31



apo C-III NM_023114. F: CAGTTTTATCCCTAGAAGCA 3 GAPDH

Page 32 of 46

349

47

99

55

R: TCTCACGACTCAATAGCTG

NM_031144 F: TGTGTCCGTCGTGGATCTGA R: CCTGCTTCACCACCTTCTTGA

547 548

32


Page 33 of 46


549

Table 2. Absolute tissue weight, food intake, liver lipids, and blood profiles Parameter

CON

HF

HF+PTA1

HF+PTA2

HF+Rosi

0.06a

0.12a

0.01a

1.074 ± 0.154*

Absolute tissue weight (g)

EWAT

0.598 ± 0.021

1.629 ± 0.169### 1.042 ± 0.089*

0.945 ± 0.079**

RWAT

0.168 ± 0.011

0.715 ± 0.063### 0.490 ± 0.022**

0.418 ± 0.046*** 0.472 ± 0.059**

MWAT

0.288 ± 0.016

0.518 ± 0.070### 0.414 ± 0.032

0.345 ±0.038*

0.233 ± 0.034**

Visceral fat

0.766 ± 0.028

2.344 ± 0.199### 1.532 ± 0.108*

1.363 ± 0.122**

1.546 ± 0.160*

BAT

0.146 ± 0.020

0.187 ± 0.023

0.145 ± 0.020

0.125 ± 0.009*

0.156 ± 0.034

Liver (g)

0.984 ± 0.057

1.177 ± 0.107

0.927 ± 0.038

0.913 ± 0.035

0.954 ± 0.037

Spleen

0.062 ± 0.004

0.077 ± 0.004

0.078 ± 0.004

0.072 ± 0.003

0.077 ± 0.053

Final body

24.04 ± 0.33

31.91 ± 1.95### 29.43 ± 1.05

29.17± 0.80

31.85 ± 1.75

0.21 ± 0.32

2.56 ± 0.39###

0.88 ± 0.29**

0.84 ± 0.38**

2.58 ± 0.31

2.21 ± 0.03

1.84 ± 0.06###

1.71 ± 0.04

1.69 ± 0.03

1.83 ± 0.04

weight (g)

weight (g)

Body weight

gain (g)

food intake

(g/day/mouse)

Liver lipids

33



Page 34 of 46

total lipid (mg/g) 55.2 ± 5.1

98.7 ± 6.4###

80.1 ± 4.6*

68.2 ± 5.9**

74.7 ± 5.7**

Triacylglycerol

31.5 ± 4.2

68.5 ± 7.1###

51.7 ± 5.1*

35.2 ± 4.9***

43.8 ± 4.7***

FFA (meq/L)

1.63 ± 0.14

2.75 ± 0.42#

2.15 ± 0.35*

1.88 ± 0.29 *

1.85 ± 0.28*

TC (mg/dL)

87.7 ± 5.7

188.5 ± 10.2### 167.0 ± 6.1

153.7 ± 6.9*

140.7 ± 14.1*

Leptin (μg/mL)

1.523 ± 0.072

3.124 ± 0.065### 2.345 ± 0.092*** 1.780 ± 0.094*** 2.151 ± 0.099***

(µmol/g)

Blood profiles

Insulin (μU/mL) 31.3 ± 5.9

162.6 ±16.5###

101.7 ± 15.2*

73.9 ± 11.8**

56.4 ± 13.8**

550

All values are means ± S.E. (n=9). # p < 0.05, ## p < 0.01, ### p< 0.001 compared with

551

the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the

552

high-fat + vehicle (distilled water) (HF) group. Tormentic acid (PTA) (PTA1: 0.06 and

553

PTA2: 0.12 g/kg bodyweight); Rosi: rosiglitazone (0.01 g/kg body weight). BAT,

554

brown adipose tissue; RWAT, retroperioneal white adipose tissue; MWAT, mesenteric

555

white adipose tissue; Visceral fat was defined as the sum of epididymal and

556

retroperioneal WAT. FFA, plasma free fatty acid; TC, total cholesterol; TG,

557

triglyceride. aDose (g/kg/day)

558

34


Page 35 of 46


559

1A

560 561

1B

562

563 564

Figure 1. (A). The plant Eriobotrya japonica Lindl. (B). structure of tormentic acid

565

(PTA).

566 35



567

Page 36 of 46

2A

###

200

Glucose (mg/dl)

Normal Control Tormentic acid (0.2 g/kg) Tormentic acid (0.4 g/kg) Tormentic acid (0.8 g/kg) ### ###

*** *** ***

160

###

* *** *** ***

120

* *** *** ***

* *** ***

80

0

30

60

90

120

min

568 569 570

2B

36


Page 37 of 46


###

Glucose levels (mg/dL)

150

125

**

100

***

***

75

50

Control

HF

HF+PTA1 HF+PTA2 0.06 0.12

HF+Rosi 0.01 (g/kg/day)

571 572

2C

Triglyceride levels (mg/dL)

160

140 ### 120

*

100

***

**

80

60

40

Control

HF

HF+PTA1 HF+PTA2 0.12 0.06

HF+Rosi 0.01 (g/kg/day)

573 37



Page 38 of 46

574 575

Figure 2. (A). Effects of tormentic acid on oral glucose tolerance test (OGTT) in

576

normal mice. Animals in all groups received oral glucose 30 minutes after tormentic

577

acid administration. Blood samples were collected and centrifuged at 3000 rpm for 10

578

minutes. Each point is the mean ± S.E. of 5 separate mice. ### p < 0.001 compared

579

with the control (CON) group; * p < 0.05, *** p < 0.001 was significantly different

580

compared with the control group in the same time by ANOVA. (B)~ (C) Effects of

581

tormentic acid (PTA) on (B). blood glucose levels and (C) circulating triglyceride

582

levels at week 12. Mice were fed with 45% high-fat diet (HF) or low-fat diet (CON)

583

for 12 weeks. After 8 weeks, the HF mice were treated with vehicle, or tormentic acid

584

(PTA), or rosiglitazone (Rosi) accompanied with HF diet for 4 weeks. All values are

585

means ± S.E. (n=9). # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the control

586

(CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the high-fat +

587

vehicle (distilled water) (HF) group by ANOVA. Tormentic acid (PTA1: 0.06 and

588

PTA2: 0.12 g/kg bodyweight); Rosi: rosiglitazone (0.01 g/kg body weight). WAT,

589

white adipose tissue; Epididymal WAT+ Retroperitoneal WAT, visceral fat.

590

38


Page 39 of 46


591

3A

592

CON

HF

HF+PTA1

HF+PTA2

593 594

595 596

HF+Rosi

597 598

39



599

3B

600

CON

Page 40 of 46

HF

601 602

HF+PTA1

HF+PTA2

603 604

HF+Rosi

605 606

Figure 3. Effects of tormentic acid (PTA) on epididymal WAT and liver tissue

607

morphology in the low-fat (CON), high-fat (HF), HF + PTA1, HF + PTA2, or HF +

608

Rosi groups. Pictures of hematoxylin and eosin-stained sections of (a) mean area of

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609

adipocytes (µm2) for epididymal WAT (Magnification: 10 (ocular) × 20 (object lens)

610

from mice fed with TA. White adipose tissue (named adipocytes) is polyhedral by

611

H&E stain, and the appearance displayed the string-like cytosol being surrounded by

612

a vacuole. Because the cytosol is embedded in paraffin as immersed in lipid solvents,

613

and all the fats were finally removed. It was carefully observed unobvious nucleus (N)

614

in the other side of cells; and (B). liver tissue (Magnification: 10 (ocular) × 20 (object

615

lens)) from mice fed with PTA. The high-fat diet induced the hepatic ballooning

616

degeneration in the HF group as compared with the CON group. The ballooning

617

degeneration is a form of liver parenchymal cell death and the nucleolus was

618

squeezed into the other side named balloon (as the arrow indicated). This may be due

619

to the heap of glycogen in the nucleus. High-fat diet induced obesity and insulin

620

resistance. Insulin levels affected the storage of hepatic glycogen. Treatment with

621

PTA1 and PTA2 significantly decreased the degree of ballooning degeneration. Each

622

presented is typical and representative of nine mice. Tormentic acid (PTA1: 0.06 and

623

PTA2: 0.12 g/kg bodyweight); Rosi: rosiglitazone (0.01 g/kg body weight).

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624

625

626

Figure 4. Semiquantative RT-PCR analysis on PEPCK, G-6Pase, 11β-HSD1, DGAT2,

627

PPARα, SREBP1c, FAS and apo C-III mRNA expression in liver tissue of the mice by

628

oral gavage extracts of tormentic acid (PTA) for 4 weeks. All values are means ± S.E.

629

(n=9). # p < 0.05, ### p < 0.001 compared with the control (CON) group; * p < 0.05,

630

** p < 0.01, *** p < 0.001 compared with the high-fat + vehicle (distilled water) (HF)

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631

group. Tormentic acid (PTA) (PTA1: 0.06 and PTA2: 0.12 g/kg bodyweight); Rosi:

632

rosiglitazone (0.01 g/kg body weight). Total RNA (1µg) isolated from tissue was

633

reverse transcripted by MMLV-RT, 10µL of RT products were used as templates for

634

PCR. Signals were quantitated by image analysis; each value was normalized by

635

GAPDH.

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636

637 638

Figure 5. The protein contents of GLUT4 in skeletal muscle, the ratio of

639

phospho-AMPK (Thr172) to total AMPK in liver tissue and skeletal muscle, and 44


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640

quantified results for the phosphorylation status of Akt (p-Akt normalized to total Akt

641

(pAkt / Akt)) in skeletal muscle of the mice by oral gavage tormentic acid (PTA) for 4

642

weeks. Protein was separated by 12% SDS-PAGE detected by Western blot. All

643

values are means ± S.E. (n=9). # p < 0.05, ## p < 0.01 compared with the control (CON)

644

group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the high-fat + vehicle

645

(HF) group. Tormentic acid (PTA) (PTA1: 0.06 and PTA2: 0.12 g/kg bodyweight);

646

Rosi: rosiglitazone (0.01 g/kg body weight).

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Tormentic acid Suppression of diabetes and hyperlipidemia

C57BL/6J+ HF diet

Activation of AMPK Decreases in gluconeogenesis (PEPCK, G6Pase) and fatty acid synthesis (SREBP1c, FAS) Regulation of leptin secretion and increased in fatty acid oxidation (PPARα) Activation of AMPK Increased in glucose uptake (GLUT4)


Tormentic Acid, a Major Component of Suspension Cells of

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