Insulin Sensitivity-Enhancing Activity of Phlorizin Is Associated with

Sep 16, 2016 - College of Life Sciences, Sichuan Normal University, Longquan, Chengdu 610101, China. ‡ .... Journal of Agricultural and Food Chemist...
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Insulin Sensitivity-Enhancing Activity of Phlorizin is Associated with Lipopolysaccharides decrease and Gut Microbiota Changes in obese and type 2 diabetes (db/db) Mice Xueran Mei, Xiaoyu Zhang, Zhanguo Wang, Ziyang Gao, Gang Liu, Huiling Hu, Liang Zou, and Xueli Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03474 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

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Insulin

Sensitivity-Enhancing

Activity

of

Phlorizin

is

Associated

with

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Lipopolysaccharides decrease and Gut Microbiota Changes in obese and type 2

3

diabetes (db/db) Mice

4 5

Xueran Mei,†,§,& Xiaoyu Zhang,†,& Zhanguo Wang,*,§ Ziyang Gao,† Gang Liu,† Huiling

6

Hu,# Liang Zou,§ and Xueli Li,†

7 8



9

China

College of Life Sciences, Sichuan Normal University, Longquan, Chengdu 610101,

10

§

11

Chengdu University, Longquan, Chengdu 610106, China

12

#

13

Chengdu 610730, China

14

* To whom correspondence should be addressed: Dr. ZG Wang, School of Medicine and

15

Nursing, Chengdu University, No.1 Shiling Street, Chengdu 610106, China. Tel/Fax: +86

16

28 84617082, E-mail: [email protected]

17

&

Metabonomics Synergy Innovation Laboratory, School of Medicine and Nursing,

School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Wenjiang,

Both authors are identified as Co-First Author.

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Abstract

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Phlorizin exists in a number of fruits and foods and exhibits many bioactivities. The

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mechanism of its anti-diabetes has been known as it can competitively inhibit

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sodium–glucose symporters (SGLTs). However, phlorizin has a wide range of two-phase

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metabolism in systemic circulation and shows poor oral bioavailability. An alternative

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mechanism may involve gut microbiota in intestine. Sixteen obese mice with type 2

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diabetes (db/db) and eight age-matched control mice (db/+) were divided into three

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groups: diabetic group treated with phlorizin (DMT group), vehicle-treated diabetic group

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(DM group) and normal control group (CC group). Phlorizin was given in normal saline

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solution by intragastric administration for 10 weeks. After the last treatment course, body

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weight, energy intake, serum lipopolysaccharides (LPS), insulin resistance and fecal

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short-chain fatty acids (SCFAs) were compared. 16S rRNA gene denaturing gradient gel

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electrophoresis (DGGE) and quantitative PCR were used to determine the changes in

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microbiome composition. Co-administration of phlorizin significantly prevented metabolic

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syndrome by decreasing weight gain, energy intake, serum lipopolysaccharides, and

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insulin resistance. And the fecal level of total SCFAs were dramatically increased,

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especially butyric acid. DGGE and quantitative PCR demonstrated that phlorizin

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co-administration increased the gut microbial diversity, the growth of Akkermansia

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muciniphila and Prevotella. Meanwhile, the gut microbiota structure of db/db mice after

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phlorizin treated was improved and approached to normal group. The mechanism of the

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hypoglycemic action of phlorizin is associated with LPS decrease and gut microbiota

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changes, briefly, it acts in the intestine to modify gut microbial community structure,

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resulting in lower LPS load in the host and higher SCFAs producing beneficial bacteria.

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Key words: Phlorizin; Lipopolysaccharides; Gut microbiota; Short-chain fatty acids;

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Insulin resistance

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INTRODUCTION

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The dihydrochalcone phlorizin is a natural polyphenol and dietary constituent found in

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a number of foods and fruits including apple, strawberry, pomegranate, and some

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medicinal plants and tisane (Lippia graveolens, Malus toringoides leaves, Pyrus

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betulaefolia leaves, etc.). Phlorizin was isolated from the bark of apple trees 180 years

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ago, and has been used in human medicine for long because of its extensive

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bioactivities.1-5 This polyphenol was discovered as the first specific and competitive

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inhibitor of sodium–glucose symporters (SGLTs) located in the mucosa of small intestine

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(SGLT1) and proximal renal tubule of kidney (SGLT2), therefore the constituent could

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improve hyperglycemia through blocking renal glucose reabsorption and intestinal

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glucose absorption.6-9 However, they don’t have much attention to the structure changes

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of phlorizin, of which phlorizin has a wide range of one phase and two-phase metabolism,

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and shows a poor oral bioavailability.10, 11 On the one hand, most of phlorizin after oral

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administrated would be hydrolyzed to phloretin by β-glucosidase and lactase-phlorizin

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hydrolase (LPH) located in the membrane of small intestine epithelial cells.12, 13 Then, the

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phloretin is further metabolized into its phase Ⅱ

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remarkable enterohepatic circulation in systematical pharmacokinetics circulation. Briefly,

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less than 1% of phlorizin could be detected as the prototype in plasma, and majority of the

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phlorizin was in the form of its metabolite in systematical circulation.11 On the other hand,

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phlorizin, like other polyphenols, can be biotransformed by gut microbiota into simpler

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phenolic compounds, the levels and bioactivities of circulating metabolites may not be

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sufficient to explain the pharmacological effects of polyphenols.6 Therefore, the poor oral

conjugate, which experiences

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bioavailability of phlorizin indicated that its hypoglycemic effect may be attributed to a

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potential mechanism besides SGLT mechanism.

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Recently, increasing evidence strongly supports that the etiology or development of

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Type 2 diabetes (T2D) is closely associated with gut microbiota.14-17 Structure imbalance

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in gut microbiota may impair gut barrier function and increase the levels of endotoxin

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especially lipopolysaccharides (LPS) in circulating systems, which provokes metabolic

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endotoxemia and induces insulin resistance, obesity, and even diabetes.18-22 Literatures

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showed that the development of metabolic diseases is along with changes in gut

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microbiota structure, including beneficial bacterium decreased and pernicious bacteria

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increased.18, 23-25 In addition, the imbalance in gut microbiota community may promote gut

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permeability and release endotoxin such as LPS. LPS is the main endotoxin produced

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from the outer membrane of Gram-negative bacteria, and identified as a novel factor

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triggering the high fat diet-induced obesity and T2D.18, 26 Recent reports showed that the

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level of LPS in T2D or high-fat diet induced mice is two or three folds higher of normal

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ones.20, 27 And single administration of LPS induced a high plasma level of glucose and

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insulin resistance.21 As similar as the obesity, T2D shows a decreased expression of

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intestinal tight junction proteins along with greater intestinal epithelium permeability and

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increased the LPS across the gut enterocyte into the systemic circulation, which induces

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the intestinal barrier function change with the chronic low-grade inflammation and

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ultimately leads to dysfunction of insulin receptor, insulin resistance, and glucose

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intolerance.28

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In conclusion, the inducement or development of T2D is closely related to gut

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microbiota and LPS level. Therefore, phlorizin may express anti-diabetic effect by

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mediating gut microbiota structure and reducing LPS concentration in serum. The aim of

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the current study was to investigate whether phlorizin has beneficial effects on obesity and

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diabetes, by decreasing serum level of LPS, improving the gut barrier function and

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regulating the structural changes of gut microbiota, as well as the abundance in main

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microbial groups.

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MATERIALS AND METHODS Experimental

Design.

Six-week-old

male

type

2

diabetic

db/db

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(BKS.Cg-Dock7m+/+Lepdb/J) mice and db/+ (heterozygote; control) mice littermates

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(Modal Animal Research Center of Nanjing University; Jiangsu, China) were housed in a

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controlled environment (room temperature 20-22 °C, room humidity 40%-60%; inverted

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12-h daylight cycle, lights off at 8:00 A.M.) in groups of four mice per cage, with free

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access to food and water. The mice were kept under observation for one week prior to the

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start of the experiments. All of the following animal experimental procedures were

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approved by the animal ethics committee of Chengdu University. The sixteen db/db mice

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were randomly divided into two groups: the phlorizin-treated diabetic group (DMT group, n

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= 8) and vehicle-treated (sterile saline solution) diabetic group (DM group, n = 8).

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Age-matched db/+ mice were chosen to be the control group (CC group, n=8). Phlorizin

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(purity >98%, Zhongren Biotechnology, Inc., Hunan, China) was given daily in sterile

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saline solution (20 mg/kg body weight) by intragastric administration and the

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vehicle-control group received the corresponding volume of sterile saline solution.

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Animal treatments lasted for 10 weeks, during which the body weight and food intake

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of each animal were measured once a week. Fresh stool samples were collected in weeks

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0, 4 and 10 by using the cryogenic vials and immediately stored at -80 °C for subsequent

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analysis.

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Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). OGTT

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and ITT were performed after 10 weeks of treatment in mice according to previously

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described methods.29 Briefly, the OGTT was executed after fasting for 12 h, after which

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2.0 g/kg body weight glucose was orally administered to the mice. Blood glucose

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determined through a glucose meter (Omron Healthcare, Japan) using 1µl of blood

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collected from the tip of the tail vein before and at 30, 60, 90 and 120 min after glucose

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administration. The ITT was performed after 6 h of food deprivation, which was followed

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by intraperitoneal injection of insulin (1.5 U/kg body weight). Blood glucose was measured

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as described for the OGTT.

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Biochemical Analysis. At the end of the trial, after 12 h of food deprivation, blood

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was collected from the caudal vein, and serum was isolated by centrifugation at 4000 rpm

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at 4 °C for 10 min. ELISA kits (Yinggong, Inc., Shanghai, China) were used to measure

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fasting insulin (FINS) and LPS. The homeostasis model assessment of insulin resistance

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(HOMA-IR) index was calculated as previously described.30 All animals were sacrificed by

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cervical dislocation.

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Fecal Short-chain Fatty Acids (SCFAs) Quantification by GC-MS. Quantification 31

and performed using an

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analysis of fecal SCFAs are same as the described method

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Agilent 7890A gas chromatography coupled with an Agilent 5975C mass spectrometric

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detector (Agilent Technologies, USA). For feces samples, fecal water was prepared by

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homogenizing feces in 0.005 M aqueous NaOH followed by centrifuging at 13,200 g at

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4 °C for 20 min. The supernatant fecal water was derivatization with PrOH/Pyridine

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mixture solvent (3:2, v/v) and propyl chloroformate (PCF). After derivatization, the

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derivatives were extracted by a two-step extraction with hexane. The concentrations of

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the SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid and n-valeric acid)

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were performed with a polar DB-WAX capillary column (30 m × 0.25 mm i.d., 0.25 µm film

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thickness, Agilent, CA). Helium was used as a carrier gas at a constant flow rate of 1

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mL/min. The initial oven temperature was held at 60 °C for 5 min, ramped to 250 °C at a

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rate of 10 °C/min, and finally held at this temperature for 5min. The temperature of the

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front inlet, transfer line and electron impact (EI) ion source were set as 280, 250 and

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230 °C, respectively. Data handing was performed with an Agilent’s MSD ChemStation

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(E.02.00.493, Agilent Technologies, Inc., USA).

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16S rRNA Gene-based Analysis. Genomic DNA was extracted from fecal samples

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using a TIANamp Stool DNA Kit based on a spin-column technology (Beijing, China). The

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extracted DNA from each sample was then used as a template to amplify the V3 regions

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of 16S rRNA genes using the universal primers Forward (5'-CGC CCG GGG CGC GCC

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CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3')

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and Reverse (5'-GTA TTA CCG CGG CTG CTG GCA C -3').32 A Polymerase Chain

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Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was performed to

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determine the bacteria communities’ dynamics and carried out with a D-code mutation

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detection system (Bio-Rad, USA) and a gradient from 35–65%. The PCR-DGGE was

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measured as described previously. 33

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Real-time Quantitative PCR. PCR was performed using the Bio-Rad real-time PCR

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system and software (Bio-Rad CFX manager, 3.0, USA) and SsoFast™ EvaGreen®

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Supermix (Bio-Rad Inc., USA) for detection according to the manufacturers’ instructions.

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All samples were performed in triplicate. The identity and purity of the amplified product

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were assessed by melting curve analysis at the end of amplification. The primer

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sequences of the bacteria are presented in Table 1.34,

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Primers were chosen to

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represent members of the main phyla of the gut microflora, Akkermansia muciniphila and

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Bacteroides–Prevotella, associated with diabetes, similar in many respects to the gut

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microflora of obesity or diabetes patients,23,

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signal.

36, 37

to provide a typical of the microbial

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Statistical Analysis. The DGGE spectra were converted into digital data using

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Quantity One software (Version 4.6.2, Bio-Rad, USA). Similarities between microbial

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community profiles generated by DGGE analysis were assessed by UPGMA clustering

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algorithm using the NTSYSpc software (Version 2.10e). Principal component analysis

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(PCA) was employed to compare the gut microbiota composition between treatment

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groups in the software SIMCA-P (Version 11.5). Every band pattern was shown as one

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plot, and highly similar band patterns were plotted close together. According to the

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quantities and intensities of the bands, the Shannon’s diversity index (H) was used to

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evaluate the diversity of the gut microbial community, and the phylotype richness was

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used to evaluate the number of the gut microbial species.33

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Statistical analyses were performed in the SPSS 20.0 (SPSS Inc., CO., USA). The

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differences were assessed by one-way ANOVA followed by post hoc. Correlations

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between parameters where assessed by Pearson’s correlation test. A value of p