Sea Buckthorn Fruit Oil Extract Alleviates Insulin Resistance through

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Sea Buckthorn Fruit Oil Extract Alleviates Insulin Resistance through the PI3K/Akt Signaling Pathway in Type 2 Diabetes Mellitus Cells and Rats Shan Gao, Qing Guo, Chengguang Qin, Rui Shang, and Zesheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04682 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on January 31, 2017

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Journal of Agricultural and Food Chemistry

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Title: Sea Buckthorn Fruit Oil Extract Alleviates Insulin Resistance through the

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PI3K/Akt Signaling Pathway in Type 2 Diabetes Mellitus Cells and Rats

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Short title: Sea Buckthorn Fruit Oil Extract Alleviates Insulin Resistance Shan Gaoa, Qing Guoa, Chengguang Qina, Rui Shanga, Zesheng Zhang ab*

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a

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Tianjin University of Science and Technology, Tianjin 300457, China.

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b

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Tianjin, 300457, China.

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

Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center,

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*Corresponding author:

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Tel: +86 022 6091 2431; Fax: +86 022 6091 2431

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Email address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Sea buckthorn fruit oil is rich in palmitoleic acid (POA), which has been reported

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to play roles in many metabolic processes. In this study, a sea buckthorn fruit oil

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(SBFO) extract was evaluated through in vitro experiments (the does were 50, 100,

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200 and 400 µM) and in vivo experiments (the does were 100, 200 and 300

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mg/kg·day) to explore its mechanism of action in the treatment of type 2 diabetes

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mellitus (T2DM). The results revealed that the SBFO extract effectively increased the

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glucose uptake from 12.23 ± 1.09 to 14.90 ± 1.48 mmol/l in IR HepG2 cells, lowered

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blood glucose (the reductions rates of blood glucose in groups treated with SBFO

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extract at 200 and 300 mg/kg·day were 10.47% and 13.79%, respectively) and

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improved insulin indices from -6.11 ± 0.10 to -5.45 ± 0.31 after 4 weeks treatment

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with SBFO extract at 300 mg/kg·day in T2DM SD rats. RT-PCR and western blotting

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analyses suggested that the SBFO extract could promote the expression of

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phosphatidylinositol-3-kinase (PI3K) and glycogen synthesis (GS) while inhibiting

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the expression of glycogen synthesis kinase-3β (GSK-3β). Thus, the SBFO extract

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played a positive role in alleviating T2DM through the PI3K/Akt signaling pathway

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inHepG2 cells and diabetic rats and could be used for the future development of

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functional food and dietary supplements.

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KEYWORDS: SBFO extract; POA; T2DM; Insulin Resistance; PI3K/Akt signaling

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pathway

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INTRODUCTION

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T2DM, characterized by elevated blood glucose secondary to IR and β-cell

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dysfunction with associated insulin deficiency 1, is a metabolic and endocrine disorder

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that is currently in the midst of a worldwide epidemic. At present, T2DM accounts for

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approximately 90% of diabetes cases 2, and its incidence has grown faster than

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expected 3, especially in youth 4. Furthermore, T2DM is a risk factor for many chronic

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complications, such as cardiovascular disease and kidney failure 3, and causes severe

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health and economic problems. Although commercial chemosynthetic T2DM

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medicines exhibit good antidiabetic activity, they also have some limitations and

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negative side effects which impair the health of patients5. For instance, commercially

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available T2DM medicine rosiglitazone may lead to the cardiovascular adverse

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events6, metformin may cause indigestion7, and most medications available failed to

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correct the underlying causes of insulin resistance. Natural nutraceutical was a

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preferable treatment method which has been accepted by consumers appeared to be

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generally safe8. There has been some investigation about the antidiabet natural

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nutraceutical, like chenpi extract and d-chiro-inositol which could alleviate IR, but the

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cost or efficacy need to improve

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targeting the origin of insulin resistance is preferable for the improvement of T2DM

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treatment.

9, 10

. Thus, developing new natural nutraceuticals

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POA, a monounsaturated fatty acid, displays unusual characteristics such as a low

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melting point (0.5–1 ºC) and good oxidative stability, which are beneficial for its use

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in food production11. Applications of POA in the production of industrial chemicals 3



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and biodiesel have also been reported11, and it has been found to regulate various

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physiological processes, such as blood glucose metabolism12, metabolic syndrome13

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and the inflammatory response14. Previous research has shown that POA can enhance

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Akt activation and increase plasma membrane GLUT1 and GLUT4 protein contents

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through AMPK or MAPK signaling pathway in skeletal muscle and adipocytes, in

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addition to improving glucose homeostasis and IR15-17. Furthermore, POA can reduce

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hepatic steatosis by inhibiting the expression of sterol regulatory element binding

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protein-1 (SREBP1)

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(ATGL) through the activation of nuclear receptor peroxisome proliferator activated

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receptor α (PPAR α) 15.

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high palmitoleate at baseline existed a higher chance to observe an increase in insulin

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sensitivity compared with subjects with low levels18, 19.

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and increase the amount of lipase adipose triglyceride lipase

And reports have been confirmed that human subjects with

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Sea buckthorn (Hippophae rhamnoides L.) is a widely cultivated herbaceous plant

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in China, with a high content of POA in its fruit oil. Sea buckthorn has long been used

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in traditional medicine

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dyspepsia12, 13. Previous report indicated that the aqueous extract of seabuckthorn seed

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residues has hypoglycemic, hypotriglyceridemic and antioxidant effects in

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streptozotocin-induced diabetic rats, and some other reports indicated the positive

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effects of POA on glucose homeostasis and insulin resistance18, 19. However, there

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have been no specific and systematical investigations addressing the function of

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SBFO extract in the treatment of T2DM.

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20, 21

for the treatment of sputum, cough, skin diseases and

Considering that the known effects on diabetes are of the POA and taking account 4


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that SBFO extract is rich in POA, the objective of this work was to study function of

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SBFO extract in the treatment of T2DM. Therefore, in this study, in vitro and in vivo

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models involving HepG2 cells and SD rats were established to evaluate the effects of

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the SBFO extract on investigated IR through the PI3K/Akt signaling pathway,

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systematically.

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MATERIALS AND METHODS

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Chemicals

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SBFO used in this study was provided by the Ordos Conseco Ecological Development

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Co, Ltd. (Ordos, China). HepG2 cells was obtained from the Tianjin University of

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Science and Technology (Tianjin, China). STZ was purchased from Sigma Chemical

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Co. (St. Louis, MO, USA). Antibodies PI3Kp85, PI3Kp110, Akt, phospho-Akt,

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GSK-3β, and GS were obtained from Cell Signaling Technology lnc. (Danvers, MA,

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USA). Secondary antibodies were purchased from ZSGB-BIO Technology Co., Ltd.

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(Beijing, China). The other laboratory chemicals were of analytical grade. Trizol,

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RIPA, AST and ALT kits were purchased from Solarbio Science and Technology Co.,

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Ltd. (Beijing, China). Antibodies were obtained from Cell Signaling Technology lnc.

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(Danvers, MA, USA). Secondary antibodies were purchased from ZSGB-BIO

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Technology Co., Ltd. (Beijing, China). The other laboratory chemicals were of

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analytical grade.

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Sample Preparation and Characterization. SBFO used in this study was extracted

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as previous reported by us (unpublished work). The obtained SBFO extract was

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analyzed via GC, and the fatty acid composition and structure are shown in Table 1. 5



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The main components of unprocessed SBFO were POA and palmitic acid, with

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contents of approximately 35% and 32%, respectively. Oleic acid, vaccenic acid, and

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linoleic acid (each 5-10%) and trace amounts of other fatty acids (nutmeg acid, stearic

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acid, linolenic acid, peanut acid, each at approximately 1% or less) were also found.

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After the extraction process, the POA concentration was increased to 81%, which

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turned the extract into a dark brown, oily liquid with a low melting point and good

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oxidative stability.

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Cell Culture.HepG2 cells was cultured in Dulbecco’s modified Eagle’s medium

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(DMEM) containing 4.5 mM glucose, 10% fetal bovine serum (FBS) and 1% mixed

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antibiotics under standard cell culture conditions (humidified atmosphere, 5% CO2

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and 37 ˚C). Cells were seeded into 96-well plates at a concentration of 1x105 cells/mL.

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When the cells reached confluence, the medium of group N (nomal control group)

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was replaced with DMEM, while group C and SBFO extract treated groups (group S1,

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S2, S3 and S4)was replaced with DMEM mixed with insulin at 10-6 mM for 36 h.

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Then the medium of group N and C was replaced by DMEM, while SBFO extract

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treated groups were subsequently replaced with DMEM containing different

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concentrations (50, 100, 200 and 400µMdesignated S1, S2, S3 and S4, respectively)

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of the SBFO extract for another 24 h. The cytotoxic or deleterious effects on cell

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viability was measured in an MTT assay. Finally, the medium was replaced with

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DMEM without phenol red for another 24 h, and glucose uptake was measured using

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a glucose assay kit (Nanjingjiancheng, Nanjing, China). At the same time, the cells

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were collected and washed with PBS for future use to analyze the activation of the 6


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PI3K/Akt signaling pathway 22.

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Animals and Treatments. Four-week old male SD rats (200 ± 20 g) were obtained

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from the animal house of the Beijing University Science Center (Beijing, China). All

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rats were kept in specific-pathogen-free animal rooms and raised in a controlled

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environment at 23±2 °C with a humidity of 55±10%, under a 12 h light/dark cycle,

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with unrestricted access to food and water. The animal protocols were approved by

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the Animal Care and Use Committee, and all of the related facilities and experimental

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procedures were executed according to the Technical Standards for the Testing &

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Assessment of Health Food (2003) 9.

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The rats were acclimatized to the animal room for one week and then divided into

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several groups. The normal group (N) was fed a normal chow diet throughout the

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study, whereas the other groups were fed a high-fat diet. After four weeks of feeding,

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the high-fat diet rats were injected intraperitoneally with STZ dissolved in buffer

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solution (citrate buffer at pH 4.2−4.5) at a dose of 30 mg/kg. After 3 days, the rats

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whose blood glucose levels exceeded 11.1 mmol/L were considered to be T2DM rats

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and were used in subsequent experiments. These T2DM rats were randomly divided

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into 6 groups. The low-dosage group (L) was treated with SBFO extract at a dose of

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50 mg/kg body weight daily via oral gavage; the intermediate-dosage group (I) was

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treated with POA at a dose of 100 mg/kg daily; the high-dosage group (H) was treated

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with POA at a dose of 150 mg/kg daily; the negative control group (NC) was treated

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with oleic acid at a dose of 150 mg/kg daily; the positive control group (PC) was

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treated with rosiglitazone at a dose of 0.36 mg/kg daily (calculated according to the 7



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manufacturer’s instructions); and the control group (C) received only distilled water.

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To determine the effect of the SBFO extract on glucose metabolism in rats, body

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weights and blood glucose (using the blood glucose meter and test strips) were

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measured weekly for four weeks in all groups. After an additional 4 weeks, the rats

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were anesthetized for venous blood collection, followed by tissue harvesting.

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Heparinized plasma samples were obtained through centrifugation of whole blood at

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4000 rpm for 15 min at 4 ºC, and the plasma was then stored at -80 ºC for further

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biochemical tests. Tissue samples were stored in liquid nitrogen for further analysis 10.

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Blood Glucose (BG) Measurements, Oral Glucose Tolerance Test (OGTT) and

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Plasma Insulin Measurements in Rats. The blood glucose levels of the rats in all of

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the groups were determined weekly with a glucometer via tail bleeding after overnight

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fasting.

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OGTT was performed in overnight-fasted rats from all groups after 4 weeks of

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treatment with the SBFO extract. All of the rats were orally administered glucose at a

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dose of 2 g/kg. Blood was withdrawn from the tip of the tail at 0, 30, 60, and 120 min

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to measure blood glucose levels.

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For plasma insulin measurements, the rats were fasted overnight, after which

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blood samples were collected and tested with an ultra-sensitive rat insulin ELISA kit

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(Nanjingjiangcheng). Insulin levels (INS) were detected in the rats after

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intraperitoneal injection of STZ and 4 weeks of treatment with the SBFO extract. The

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insulin sensitivity index (ISI) was calculated following the formula ISI = Ln (1/INS ×

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BG). The homeostasis model assessment-β (HOMA-β) was calculated following the 8


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formula HOMA-β = (20 × INS)/(BG− 3.5)9.

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Hepatic Glycogen Measurements in Rats. Rat liver tissue homogenates from all

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groups were collected and tested with a liver/muscle glycogen assay kit

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(Nanjingjiangcheng).

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Alanine Transaminase (ALT) and Aspartate Transaminase (AST) Measurements

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in Rats. Plasma samples collected from overnight-fasted rats from all groups were

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tested with alanine aminotransferase assay kits and aspartate aminotransferase assay

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kits (Nanjingjiancheng).

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Real-Time Polymerase Chain Reaction Analysis. The effects of the SBFO extract

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on the PI3K/Akt signaling pathway were analyzed via real-time polymerase chain

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reaction.HepG2HepG2 cells and ground livers were collected in TRIzol

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disrupted at room temperature. Total RNA was extracted according to the

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manufacturer’s instruction and re-suspended in nuclease-free water. The concentration

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of total RNA was determined with an ultraviolet spectrophotometer, and its integrity

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was confirmed through visualization of rRNA bands after agarose gel electrophoresis.

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The total RNA was then converted to cDNA using an RT-PCR kit (TaKaRa, Dalian,

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China). Real-time PCR analysis was performed with premixed SYBR green reagents

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(TaKaRa, Dalian, China) in a real-time detector (Bio-Rad Laboratories, Hemel

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Hempstead, UK).

185 186 187

and

Primer pairs （Beijing Dingguo Changsheng Biotechnology Co, Ltd. Beijing, China) were designed using the Primer Premier 5 program. The

sequences

were as follows: PI3K (sense: ACAAAGCTCTACTCTAGGCGTG, anti-sense: T 9



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TACCAGCATGGTCATGGGC), Akt (sense: AGAGAGCCGAGTCCTACAGAAT

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A, antisense: CCGAGAGAGGTGGAAAAACA), GSK-3β (sense: TCGTCCATC

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GATGTGTGGTC, antisense: TTGTCCAGGGGTGAGCTTTG), GS (sense: TTG

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CCAGAATGCACGCAGAA, antisense: TGCCTGCATCATCTGTTGAC), β-actin

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(sense: GATCGATGCCGGTGCTAAGA, antisense: TCCTATGGGAGAACGGCA

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GA).

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Western Blotting Analysis. Protein samples were extracted from rat liver tissue with

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RIPA bufferand then resolved via 10% SDS-PAGE for 0.5 h at 80 V, followed by 2.5

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h at 100 V. The protein samples were subsequently transferred to a nitrocellulose

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membrane and probed with commercially available primary antibodies against

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PI3Kp85 (85 kDa), PI3Kp110 (110 kDa), Akt (60 kDa), p-Akt (60 kDa), GSK-3β (46

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kDa), and GS (81−85 kDa), followed by incubation with the corresponding secondary

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antibodies. The presence of the target proteins on the membranes with determined

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based on the assessment of fluorescence using the ChemiDoc XRS reagent (Bio-Rad),

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and the band densities were quantified.

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Statistics. Data are presented as the mean ± SD. One-way ANOVA was used for all

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statistical comparisons among groups. And the t test was used to exhibit the

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significance of differences between independent samples. Statistical significance is

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denoted as * P < 0.05, ** P < 0.01 (SPSS version 20.0, Statistical Package for the

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Social Sciences Software, SPSS Inc., Chicago, IL, USA).

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RESULTS

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The SBFO Extract Improved Glucose Uptake inHepG2 Cells. The doses of fatty 10


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acids were higher than the standard dose encountered in human or rat plasma, they

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were confirmed to have no cytotoxic or deleterious effects on cell viability in an MTT

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assay (data not shown).As shown in Figure 1, glucose uptake exhibit a

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dose-dependent increase in the SBFO extract-treated groups including S1, S2, S3 and

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S4. And glucose uptake was remarkably improved in group S4 (400µM SBFO

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extract-treated group) compared with the control group (p < 0.01). These data

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suggested that the SBFO extract could improve insulin sensitivity in IRHepG2 cells.

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The SBFO Extract Affected the PI3K/Akt Pathway inHepG2 Cells. In this study,

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the effects of the SBFO extract on the expression of the PI3K (Figure 2A), Akt

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(Figure 2B), GS (Figure 2C) and GSK-3β (Figure 2D) genes were evaluated via

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RT-PCR. The results showed that the expression of PI3K and GS increased in all of

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the SBFO extract-treated groups. In contrast, the expression of GSK-3β decreased,

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especially in group S1, in which GSK-3β expression was significantly decreased (p
0.05). These

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results indicated that the SBFO extract could promote the synthesis of glycogen.

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The SBFO Extract Reduced Plasma AST and ALT in Rats. The ALT and AST

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indices were tested in the plasma of the rats from all experimental groups. The data in

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Table 6 show that the SBFO extract reduced ALT and AST levels significantly in the

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rats from group I, H and PC compared with the rats from group C (p < 0.05) . These 13



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results indicated that the SBFO extract could protect hepatic tissue from cellular

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damage caused by hyperglycemia.

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The SBFO Extract Ameliorated IR through the PI3K/Akt Pathway in Rats. In

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this investigation, the effects of the SBFO extract on PI3K, Akt, GSK-3β, and GS

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were evaluated at the gene expression level via RT-PCR (Figure 4). The results were

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consistent with the findings inHepG2 cells obtained in this study: the expression of

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PI3K and GS RNA was promoted, while the expression of GSK-3β was decreased in

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groups L, I, H and PC (p < 0.05).

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The effects of the SBFO extract on the expression of the main proteins in the

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PI3K/Akt pathway were investigated through western blotting (Figure 5). The results

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indicated that the SBFO extract increased the expression of PI3K (consisting of a

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catalytic subunit (p110) and a regulatory subunit (p85)) and GS but reduced the

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expression of GSK3β. Groups I, H and PC in particular showed significant differences

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compared with group C (p < 0.05). We also investigated the effect of the SBFO

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extract on Akt activity (Figure 5C and 5D) and found that treatment with the SBFO

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extract increased phosphorylated Akt levels.

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In summary, SBFO extract treatment significantly enhanced the expression of the

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main genes and proteins in the PI3K/Akt pathway. It was also demonstrated that the

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SBFO extract had a positive effect on ameliorating IR through the PI3K/Akt pathway

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in rats.

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DISCUSSION

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T2DM is a worldwide epidemic associated with hyperglycemia and 14


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hyperlipidemia, caused by an imbalance in the insulin sensitivity of the liver and

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endocrine system23. Traditional commercial T2DM medicines generate negative

300

effects, along with their antidiabetic activities, resulting in impaired health of the

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patients. For instance, rosiglitazone may lead to adverse cardiovascular events6, and

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metformin may cause indigestion7. Thus, developing nutraceuticals containing natural

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compounds with low side effect profiles is necessary. Recently, POA has attracted

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attention due to its positive effects on metabolic diseases. Evidences accumulated

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over the years have shown that POA could improve glucose uptake by affecting

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insulin responsivity, it could enhance Akt activation, increase the expression of

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glucose transporter type 4 (GLUT4) and effect the phosphorylation of Adenosine

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5'-monophosphate (AMP)-activated protein kinase (AMPK) and mitogen-activated

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protein kinase (MAPK).12, 15, 17, 24. Our GC analysis revealed that the main component

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of SBFO was POA, which has known positive effects on hyperglycemia. The POA

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content can enriched to an even higher level through our extraction process. Therefore,

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our SBFO extract is suitable for use in developing a T2DM nutraceutical.

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To evaluate the curative effect of the SBFO extract on T2DM, in vitro and in vivo

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T2DM models were explored in parallel. Insulin-treatedHepG2 cells were used as an

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in vitro model because the cells treated in this way exhibit low expression of IRS-1

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and some of its downstream genes, in addition to decreased glucose uptake 25. SD rats

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were selected as our in vivo model because they can easily develop pathological

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features resembling T2DM and are more sensitive to STZ and the SBFO extract 26.

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The physiological measurements conducted in the in vitro experiments showed 15



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that the standard dose encountered in human or rat plasma, they were confirmed to

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have no cytotoxic or deleterious effects on cell viability and glucose uptake inHepG2

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cells treated with the SBFO extract was increased, possibly because the SBFO extract

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enhances the glucose metabolism of liver cells and reduces IR. The physiological

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measurements performed in our in vivo model included the assessment of body

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weight, BG levels, OGTT, insulin levels, hepatic glycogen levels, and AST and ALT

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levels in the rats. The body weight of the SD rats showed recovery tendency, and BG

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and OGTT showed a dose-dependent reduction tendency in the SBFO extract-treated

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groups, indicating that the SBFO extract ameliorated the T2DM-induced symptoms

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regulated the glucose metabolism and weakly enhanced the body weight. Regarding

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insulin levels, during the initial establishment of the T2DM rat model, the insulin

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levels of the rats in the STZ-injected groups increased, which demonstrated that the

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established model was successful and that IR was occurring. After 4 weeks of

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treatment, insulin levels were measured again and were found to have decreased in the

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SBFO extract-treated groups compared with the control group. This result indicated

335

that SBFO extract treatment could alleviate IR in rats. The ISI and HOMA-β indices

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were calculated according to BG and insulin levels, to measure the efficiency of the

337

body in taking advantage of insulin and the degree of health of pancreatic β-cells,

338

respectively. The data showed that the ISI and HOMA-β indices of the rats in the

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SBFO-treated groups were improved compared with the control group. Thus, SBFO

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extract treatment could enhance insulin sensitivity and alleviate IR, resulting in

341

accelerated glucose metabolism. Furthermore, the analyses of hepatic glycogen levels 16


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and the degree of liver injury (via AST and ALT detection) showed improvements in

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all SBFO extract-treated groups. These results demonstrated the positive effects of the

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SBFO extract on relieving hyperglycemia and liver injury in T2DM rats.

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Next, we investigated the molecular mechanism underlying the effects of the

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SBFO extract on T2DM. Previous studies have demonstrated that the IR observed in

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T2DM is most likely attributable to a defect in the insulin receptor/IRS-1/PI3K/Akt

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cascade27. In this pathway, Ser/Thr phosphorylation of IRS-1 could inhibit

349

insulin-stimulated tyrosine phosphorylation of IRS-1 and has the ability to bind and

350

activate PI3K28. There were two subunits for PI3K, their important effects were that

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PI3K-p85 protein would give a negative feedback on insulin sensitivity in return and

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PI3K-p110 affect the activity of downstream molecules to regulate the continuous

353

conduction of signal. Akt is the major downstream target of PI3K, therefore the

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activation of Akt leads to inactivation of a specific isoform of GSK3β, which in turn

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enhances the expression of GS and (potentially) regulates glucose transport activity9,

356

29, 30

357

showed that the SBFO extract increased the expression of PI3K and GS, decreased the

358

expression of GSK-3β, and activated the phosphorylation of Akt, indicating that both

359

up- and downstream genes of the PI3K/Akt pathway were activated. The activated

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upstream gene PI3K could promote insulin sensitivity through the feedback on IRS-1,

361

and the activated downstream genes GS and GSK-3β (a GS inhibition protein)

362

regulate glucose metabolism through affecting glucose transport and the synthesis of

363

glycogen As a coclusion, the results of RT-PCR and western blotting analyses

. The results of RT-PCR and western blotting analyses obtained in our study

17



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complied with the action mechanisms described in the previous literature. Besides, the

365

results of RT-PCR and western blotting analyses were confirmed by the physiological

366

measurements conducted in theHepG2 cells and rat model. In summary, the SBFO

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extract had a positive effect on glucose metabolism through the PI3K/Akt signaling

368

pathway and could alleviate IR.

369

It is worth discussing the results obtained for the PC group, treated with

370

rosiglitazone, in our in vivo experiments. In these experiments, it was found that the

371

T2DM outcomes of the rats in the SBFO extract-treated groups presented similar

372

tendencies to those of the rats in the PC group, suggesting that the appropriate dosage

373

of the SBFO extract might have similar ameliorative effects to rosiglitazone. In

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addition, the SBFO extract is a natural product with fewer side effects

375

with artificially synthesized drugs and can be applied to develop nutraceuticals for

376

relieving T2DM symptoms. Furthermore, group NC (treated with oleic acid) was

377

investigated to determine whether oleic acid modulated T2DM symptoms, and the

378

results showed that oleic acid had no impact on T2DM. Thus, it can be inferred that

379

the main component of the SBFO extract, POA, plays the leading role in alleviating

380

T2DM.

11

compared

381

In conclusion, the metabolic parameters determined in HepG2 cells and rats

382

model of T2DM, including glucose homeostasis, insulin sensitivity and liver injury,

383

were improved by treatment with the SBFO extract tested in this study. While SBFO

384

extract show weak effects on the recovery of body weight of the rats and insulin

385

secreting of islet B cells. The results provide

evidence that the SBFO extract may be

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effective in alleviating T2DM through the PI3K/Akt pathway, as confirmed by the

387

results of PT-PCR and western blotting. The mechanism of action of the SBFO extract

388

was similar to that of rosiglitazone. The present study offers valuable information for

389

understanding the curative effects and mechanism of action of the SBFO extract in

390

ameliorating T2DM and provides key data for the future development of functional

391

food and dietary supplements, once sufficient further research on SBFO has been

392

performed.

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ACKNOWLEDGMENTS

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This research was supported by the National Science-Technology Pillar Program

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

396

Teams at the University of the Ministry of Education of the People’s Republic of

397

China (Grant IRT1166).

398

CONFLICTS OF INTEREST

399

The authors declare no competing financial interests.

400

19



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504

FIGURE CAPTIONS

505

Figure 1. Effects of the SBFO extract on glucose uptake in IRHepG2 cells. Glucose

506

uptake was calculated by subtracting the glucose content of the test wells from the

507

glucose content of the blank wells. The values are the mean ± SD and similar results

508

were obtained in three independent experiments.**p < 0.01 and *p < 0.05 vs. group C.

509

The normal group (N) was treated with DMEM without Insulin, others were treated

510

with 10-6 mM insulin and divided into groups followed: the control group (C)

511

received DMEM only , the dosage group treated with SBFO extract at different

512

concentrations of 50, 100, 200, 400 µM.

513

Figure 2. Effects of different concentrations of the SBFO extract on the expression of

514

genes in the PI3K/Akt pathway in IRHepG2 cells. RT-PCR was performed, and the

515

results are expressed as the mean ± SD and similar results were obtained in three

516

independent experiments. **p < 0.01 and *p < 0.05 vs. group C. The normal group (N)

517

was treated with DMEM without Insulin, others were treated with 10-6 mM insulin

518

and divided into groups followed: the control group (C) received DMEM only , the

519

dosage group treated with SBFO extract at different concentrations of 50, 100, 200,

520

400 µM.

521

Figure 3. OGTT was determined in all experimental rats after 4 weeks of treatment

522

with the SBFO extract. All of the rats were orally administered glucose at a dose of 2

523

g/kg. BG levels were measured at 0, 30, 60, and 120 min after the administration of

524

glucose. The area under the curve shown in panel A was calculated and is displayed in

525

panel B. The values are the mean ± SD and each group contained 9 rats. **p < 0.01 25



Page 26 of 40

526

and *p < 0.05 vs. group C

527

Figure 4. Effects of different concentrations of the SBFOextract on the expression of

528

genes in the PI3K/Akt pathway in T2DM rat livers. RT-PCR was performed, and the

529

results are expressed as the mean ± SD and similar results were obtained in three

530

independent experiments. **p < 0.01 and *p

531

(N) was fed a normal chow diet during the whole study and received distilled water

532

only, other rats were fed with a high fat diet and divided into groups followed: the

533

control group (C) received distilled water only, the dosage group (L, I and H) treated

534

with 100, 200, 300 mg/kg SBFO extract at daily by oral gavage, the negative control

535

group (NC) treated with oleic acid, the positive control group (PC) treated with

536

rosiglitazone.

537

Figure 5. Effects of different concentrations of the SBFO extract on key proteins in

538

PI3K/Akt pathway in T2DM rats determined via western blot analysis. Photos are

539

representative images. The values are the mean ± SD from densitometry analysis and

540

similar results were obtained in three independent experiments. **p < 0.01 and *p

541

0.05 vs. group C. The normal group (N) was fed a normal chow diet during the whole

542

study and received distilled water only, other rats were fed with a high fat diet and

543

divided into groups followed: the control group (C) received distilled water only, the

544

dosage group (L, I and H) treated with 100, 200, 300 mg/kg SBFO extract at daily by

545

oral gavage, the negative control group (NC) treated with oleic acid, the positive

546

control group (PC) treated with rosiglitazone.

< 0.05 vs. group C. The normal group

547 26


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