Attenuated mTOR Signaling and Enhanced Glucose Homeostasis by

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Attenuated mTOR signaling and enhanced glucose homeostasis by dietary supplementation with lotus seedpod oligomeric procyanidins in streptozotocin (STZ)-induced diabetic mice Xiaopeng Li, Yong Sui, Qian Wu, Bijun Xie, and Zhida Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00233 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Attenuated mTOR signaling and enhanced glucose homeostasis by

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dietary supplementation with lotus seedpod oligomeric procyanidins

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in streptozotocin (STZ)-induced diabetic mice Xiaopeng Lia, Yong Suib, Qian Wu c, Bijun Xiea, Zhid Sun a*

4 5

a

6

430070, China

7

b

8

Technology; Hubei

9

China

College of Food Science and Technology, Huazhong Agricultural University, Wuhan,

Institute

for

Farm

Products

Academy

of

Processing Agricultural

and Science;

Nuclear-Agricultural Wuhan,

430064,

10

c

11

of Technology, Wuhan,430068, China

12

Email: [email protected]; Tel:+86-27-87283201; Fax: +86-27-87282966

13

KEYWORDS: lotus seed oligomeric procyanidins; synbiotics; mTOR; glucose

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homeostasis; type 2 diabetes

Hubei Collaborative Innovation Center for Industrial Fermentation, Hubei University

15 16 17 18 19 20 21 22 1


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ABSTARCT: This study investigated the protective role of lotus seedpod oligomeric

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procyanidins

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xylo-oligosaccharide) against high fat and streptozotocin (STZ)-induced diabetes.

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Administration of LSOPC or synbiotics have no effect on blood glucose in normal

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mice. Treatments with LSOPC for 12 weeks markedly reduced blood glucose, FFA,

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endotoxin, GHbA1c and improved glucose homeostasis, lipid metabolism and insulin

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level. In addition, administration of LSOPC significantly reversed the increase of

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mTOR and p66Shc in liver, skeletal muscle and white adipose tissue (WAT). LSOPC

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significantly increased glucose uptake and glycolysis in liver, skeletal muscle and

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WAT, while improving heat generation in brown adipose tissue (BAT) and inhibiting

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gluconeogenesis and lipogenesis in liver. Furthermore, synbiotics strengthened the

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improving effect of LSOPC. These findings demonstrated that LSOPC and synbiotics

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may regulate glucose disposal in peripheral target tissues through p66Shc-mTOR

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signaling pathway.

(LSOPC)

and

synbiotics

(Bifidobacterium

37 38 39 40 41 42 43 44 2


Bb-12

and


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INTRODUCTION

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Diabetes mellitus (DM) is becoming a global health issue with growing significance.

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It is strongly associated with nutritional oversupply in modern society. In China, The

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number of people with diabetes is estimated to reach as much as 642 million by 2040

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according to the International Diabetes Federation (IDF). A growing body of evidence

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also proves that the western dietary pattern in favor of calorie-dense food is associated

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with more weight increment and increased risk of T2DM1-3. This situation is

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deteriorating in face of obesity prevalence and a rapid aging population around the

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world. Therefore, dietary therapies have attracted attentions of researchers expecting

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to contribute to the prevention of diabetes, especially at the onset of diabetes and in

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mild hyperglycemia4-6.

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Recent studies have found that the mammalian target of rapamycin (mTOR) and

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p66Shc can mediate glucose metabolism in liver, adipose tissue and skeletal muscle7-10.

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As a serine/threonine protein kinase, mTOR interacts with several proteins to form

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two complexes with different functions, mTOR complex 1 (mTORC1) and 2

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(mTORC2)7,

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factors SREBP1-c7. Chronic mTORC1 activation contributes to insulin resistance by

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mediating excess fat deposition in WAT, liver and muscle, as well as inhibiting the

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insulin signaling through S6 kinase 1(S6K1)-dependent negative feedback loop.

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However, there is little information about the regulation of mTORC2. It is usually

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considered that growth factors controls mTORC2. Alternatively, mTORC2 suppresses

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gluconeogenesis by regulating Akt and also promotes glycolysis through activation of

11

. mTORC1 controls lipid synthesis by regulating its transcription

3


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glucokinase and other transcriptional factors12. In insulin resistance or type 2 diabetes,

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disorders of glucose metabolism, including elevated gluconeogenesis and aggravated

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enzymes related to glycolysis, occur in liver. Two elements known to play significant

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physiological roles in gluconeogenesis are Akt and FoxO1 (forkhead box protein

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O1)13. Reduced Akt blocks FoxO1 activity by altering nuclear accumulation, thus

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inducing the activation of genes required for gluconeogenesis in insulin resistance

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subjects, most prominently PEPCK and G6Pase12.

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The adapter protein p66Shc belongs to Shc protein family (ShcA, ShcB, ShcC and

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RaLP). ShcA, now called Shc, is expressed as three isoforms of p66Shc, p52Shc and

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p46Shc. In recent years, p66Shc has been recognized as a putative longevity gene14.

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Researchers suggested that mice deficient of p66Shc adaptor protein had better glucose

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tolerance and an apparent resistance to the development of obesity and diabetes15, 16.

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Diabetes increased p66Shc gene expression and p66Shc activation in a variety of tissues

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both in animals and humans, indicating that p66Shc mediated insulin resistance and

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glucose homeostasis17-19. The adaptor protein p66Shc may block the insulin signaling

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pathway by inhibiting the phosphorylation of insulin transducer IRS-1 and may also

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increase S6K activity which in return deteriorates insulin signaling pathway19.

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Additionally, the adaptor protein p66Shc appears to be able to potentiate the damage

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effect of mTOR/S6K pathway20.

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Procyanidins are naturally existing polyphenolic compounds that are widely found in

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plants and daily foods. Our laboratory has established the proper extraction

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technology of oligomeric procyanidins of lotus seedpod (LSOPC) in recent years21. 4



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Lotus seedpod is rich in B-type procyanidins which are linked through a C4–C8 or

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C4–C6 bond. The mean degree polymerization of LSOPC was 3.21 with 74%

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catechin and 26% epicatechin in the terminal units and 26.0% catechin, 43.1%

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epicatechin and 30.9% epigallocatechin in the extensive units21. Recently, several

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reviews have illustrated the protective roles of polyphenols in insulin resistance or

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type 2 diabetes4, 22-24. In addition, reviews screened from 2736 reports summarized

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that dietary interventions of synbiotics (combining pre- and probiotics) appear to have

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a beneficial role in the prevention and management of type 2 diabetes25. Dietary

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supplements of Bb-12 and xylo-oligosaccharides (XOS) have been reported helpful in

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improving antioxidant status and gut homeostasis26, 27.

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Under this background, this study intended to probe the protective role of dietary

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supplements of LSOPC and synbiotics against HF and STZ-induced type 2 diabetes28.

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Further explored are the underlying mechanisms in liver, skeletal muscle and adipose

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tissue involved in mTOR and p66Shc-meadiating glucose disposal, gluconeogenesis

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and lipogenesis.

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MATERIALS AND METHODS

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Chemicals. Lotus seedpod (Wu Zhi No.2) were harvested from Honghu District in

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July 2015 (Hubei, China) and stored at -18 ℃ until use. Freeze-dried DVS

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Bifidobacterium animalis subsp. lactis Bb-12(>311 cfu/g, material no. 706146, batch

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no.3227312) was supplied by Chr. Hansen Company (Beijing, China) and stored at

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-18 ℃

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Xylo-oligosaccharide (XOS, 95P, a001; XOS2-7 ≧ 95% and XOS2-4 ≧ 65%) was

(date of manufacture: 03, 2015; best before date: 03, 2017).

5


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manufactured by Shandong Longlive Bio-Technology Co., Ltd., China. Accu-Chek

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Active meter and active test strips were purchased from Roche Diagnostics Asia

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Pacific Pte., Ltd (Beijing, China).

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Streptozotocin (CAS. #18883-66-4) and commercial standard procyanidins (CAS. #

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4852-22-6) was obtained from Shanghai Yuanye Bio-technology Co.,Ltd.(Shanghai,

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China). Rabbit FoxO1a antibody (Cat. #ab52857), rabbit phosphor-FoxO1a antibody

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(p-FoxO1a, S319, Cat. #ab47326) were bought from abcam (Shanghai, China). Rabbit

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Akt antibody (Cat. #4691) and Rabbit phospho-Akt antibody (Cat. #4060) were

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gained from (Cell Signaling Technology, Danvers, MA). Rabbit polyclonal antibody

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PKCβ (Cat. #12919-1-AP), p66Shc (Cat. #10054-1-AP), rabbit monoclonal antibodies

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against GLUT2 (Cat. #20436-1-AP) and GK (Cat. #19666-1-AP), UCP-1(Cat. #

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23673-1-AP) and rabbit polyclonal antibody mTOR (Cat. #20657-1-AP) were

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obtained from Protein Tech (Wuhan, China). β-actin (Cat. #BM0627) and rabbit

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monoclonal antibodies against GLUT4 (Cat. #PB0143) were purchased from Boster

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Biological Tecnology (Wuhan, China). GLUT 1 was obtained from Bioworld

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Technology (Shanghai, China). More detailed information of used antibodies in our

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experiment were listed in Supplementary Table S5. Assay kits used for determination

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of total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein (LDL),

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high-density

130

aminotransferase

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immunosorbent assay (ELISA) kit for measuring insulin, GHbA1c, endotoxin were

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purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

lipoprotein

(HDL),

(ALT),

free

aspartate

fatty

acids

aminotransferase (FFA),

and

6


the

(AST),

alanine

enzyme-linked


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Preparation of Lotus Seed Oligomeric Procyanidins(LSOPC). The frozen lotus

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seed of Nelumbo nucifera Gaertn. (Number 2 Wuhan plant) were extracted to obtain

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LSOPC according to our laboratory method29. Lotus seed procyanidins was extracted

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by ethyl acetate to obtain the LSOPC, including proanthocyanidin 10.9% (+)-catechin,

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9.1%(-)-epicatechin, 53.6% dimer, 19.5% trimer and 1.9% tetramer by LC-MS

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analysis21. Its purity was 99.35±0.79% compared to that of commercial grape seed

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procyanidins measured by Butanol-HCl assay. The LSOPC is stable during the

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storage of -18℃.

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Experimental Animals and Diet. The experimental animals study was performed in

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accordance with the international guide lines and internationally accepted ethical

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principles for the care and use of laboratory animals. Ninety male ICR mice (20±2g)

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were obtained from Experimental Animal Center of Disease Prevention and Control

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in Hubei Province (Wuhan, China). The permission number is SCXK2015-0018. The

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common diet contained the following (g/kg dry diet): 500g corn starch; 220g full fat

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soybean meal; 90g wheat flour; 90g wheat bran; 70g fish meal; 30g bone powder.

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High fat diet contained 69% common diet, 10% lard, 15% sucrose, 5% egg yolk

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power, 1% cholesterol and 0.2% sodium deoxycholate. The animal housing room was

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maintained in a room temperature 25±2℃and 50-70% relative humidity under a 12 h

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dark-light cycle. 150mg LSOPC was dissolved in 10ml distilled water for B150 group.

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150mg LSOPC, 50mg Bb-12 and 2.8g XOS were dissolved in to 10ml distilled water

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to prepare for B150+P group. 300mg LSOPC, 50mg Bb-12 and 2.8g XOS were added

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in to 10ml distilled water to prepare for B150+P group. These mixtures were prepared 7


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every morning between 8:30-9:30 am. The volume of administration is 1% of body

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weight.

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Experimental Design. After one-week adaptation, the ICR mice were randomly

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divided into seven groups summarized in Figure 1. Each group in Part A contains 10

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mice, while 15 mice in Part B. 5 animals were housed in each cage and allowed

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access to diet and boiled water. Each group were administrated with corresponding

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mixture every morning (9:30-10:30am) followed in Figure 1. In part A, NC,

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NC+B150 and NC+P group were treated with water, 150mg/kg LSOPC and

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synbiotics, respectively. In this part, mice were injected with citrate buffer twice. In

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part B, after four weeks of separate diets and drinks, mice fed with HF diet were

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continuously injected for two days with low-dose STZ (0.1 mol/L citric acid buffer,

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pH= 4.5) at 45 mg/kg body weight or vehicle and kept on the same diet for the next 8

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weeks. Exactly 2 weeks after STZ treatment(8:00-10:00 am), mice in each group with

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postprandial 30 min blood glucose concentrations between 8.5-15 mmol/L were

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selected for the further experiment and each group was given the same diet and drink.

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Food, water intake, body weight and postprandial blood glucose were recorded

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weekly. Postprandial blood glucose levels were measured from the tail vein using

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Accu-Chek Active meter. At the end of the study, all mice were fasted overnight (12h)

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and sacrificed. Blood samples were collected from the vein behind the eye sockets

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and stored at -80℃ until use. The liver, kidney, skeletal muscle, white adipose tissue

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(perirenal fat) and brown adipose tissue (interscapular) were immediately removed

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and stored at -80℃ until use as well. 8



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Histological Examination of the Liver.

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Small parts of liver tissue specimens were investigated as previously described29. The

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morphology of the liver was observed using a Nikon Eclipse 100 research microscope

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(Tokyo, Japan).

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Blood analysis. The content of plasma insulin, GHbA1c and endotoxin were

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determined by ELISA according to instructions provided by the manufacture

183

(Jiancheng, Nanjing, China). The AST and ALT activities in plasma were measured by

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microplate reader using the commercial enzymatic kits (Jiancheng, Nanjing, China).

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The plasma TC, TG, HDL, LDL and FFA concentrations were carried out by

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enzymatic colorimetric methods with reagent kits (Jiancheng, Nanjing, China). The

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homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using

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the formula: HOMA-IR = fasting glucose in mmol/L × fasting insulin in

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mU/L)/22.530.

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Oral Glucose Tolerance Test (OGTT). In the 13th experimental week, the 12 h

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fasted mice were subjected to OGTT. The 12h fasted mice (21:00pm-9:00am) were

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administered of glucose at a dose of 1 g/kg body weight. Blood samples were

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collected from the tail vein at 0(before glucose administration), 15, 30, 60 and 120

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min after administration, and blood glucose level were measured as mentioned above.

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Western Blot Analysis. The following experiment was carried out as previously

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described30. Liver tissue lysates were prepared using lysis buffer consisting of 50mM

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Tris-HCl, pH 7.4, 150mM NaCl, 1% Triton X-100, 0.1% SDS, 1mM EDTA, 2mM

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sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM EGTA, 1 mM 9


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β-glycerolphosphate, 2 mg mL-1 leupeptin, 2 mg mL-1 aprotinin, 2 mg mL-1 pepstatin,

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and 1 mM PMSF, and then homogenised and centrifuged at 12,000g for 10 min at

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4℃. Western blot was performed as previously described30. Briefly, the protein

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concentrations were determined by BCA protein assay kit (Jiancheng, Nanjing, China).

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Subsequently, protein extracts were diluted in sample buffer and heated for 10 min in

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boiling water bath. The protein samples were separated by SDS-polyacrylamide gel

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electrophoresis (SDS-PAGE), and then electro-transferred to a PVDF membrane

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(Millipore, Bedford, MA, USA). The membranes were then blocked with 5% nonfat

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powered milk in TBST for 2h at room temperature in a shaker and incubated

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overnight at 4℃ with primary antibodies in TBST(PKCβ,1:2000; p66shcA, 1:2000;

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mTOR, 1:800; FoxO1a,1:2000; p-FoxO1a,1:500; Akt, 1:1000; p-Akt,1:1500;

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GLUT2,1:600;

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β-actin,1:200), followed by incubation with horseradish peroxidase (HRP)-conjugated

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secondary antibodies(1:50000) for 2h at room temperature in a shaker.

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Immunoreactive bands were visualized by the enhanced chemiluminescent reagents

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(ECL). The intensity of bands was measured using BandScan 5.0 software.

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Quantitative Real-Time RT-PCR. Total RNA from livers were homogenized and

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extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to

217

manufactory’s instructions, and cDNA was synthesized using a first-strand cDNA

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synthesis kit (GeneCopoeia, Rockville, MD, USA). The reaction mixture was

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incubated as follows: 25℃5min，50℃ 15min，85℃5min，4℃ 10min. Diluted cDNA

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(1:10) was subjected to quantitative RT-PCR amplification using the SYBR Green

GLUT4,1:300;

GK,1:500;

UCP-1,1:1000;

10


GLUT1,1:1600;


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PCR Master Mix (Toyobo) according to manufacturer's protocol. The reaction

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mixtures were incubated at 95℃for 10 min, followed by 40 cycles of incubation at 95℃

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for 30s, then 60℃ for 30s. The sequences of the primers used in this study are shown

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in supplementary Table S2. The expression of mRNA values was calculated using the

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threshold cycle value (CT). For each sample, ∆CT

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analyzing the difference between CT value of the target gene and that of reference

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gene (NC group). The relative expression levels were estimated by analyzing the

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∆∆CT (∆CT sample-∆CT reference) and using the 2-△△Ct method31.

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Statistical Analysis. All results are expressed as mean ± standard errors and

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calculated using one-way ANOVE of SPSS 19.0 followed by Tukey's test. The

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significant level was set at p﹤0.05.

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RESULTS

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Postprandial Blood Glucose

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We have successfully developed a mild type 2 diabetic model following the previous

235

studies28, 32. The non-fasting blood glucose was around 24mmol/L after 4 weeks of

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modeling, which is similar to previous study28. Abnormal water intake, typical

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character of diabetes, was significantly elevated in DM group and this situation was

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ameliorated in B150, B150+P and B300+P group (Supplementary figure S2). The

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PPG of NC group did not show significant change during the experiment (Figure 2).

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In contrast, the continuous increase of PPG from 13.15±2.59 to 25.25±4.86 in DM

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group was observed, suggesting a deterioration situation. After 1 week of modeling,

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the PPG of B150 (9.58±1.77) and B150+P (9.97±1.28) group demonstrated no

sample

11


was calculated through

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marked difference compared with that of NC group(8.1±0.84) and DM group(13.15

244

± 2.58), while the significant reduction of PPG in B300+P group was found

245

compared to that of DM group(p

Attenuated mTOR Signaling and Enhanced Glucose Homeostasis by

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