Insulin Sensitivity-Enhancing Activity of Phlorizin Is Associated with

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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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Journal of Agricultural and Food Chemistry

1

Insulin

Sensitivity-Enhancing

Activity

of

Phlorizin

is

Associated

with

2

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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ACS Paragon Plus Environment


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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

28

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

30

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

33

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

107

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

108

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

109

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

110

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

111

analysis.

112

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

114

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

116

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

118

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

119

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

130

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

151

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

154

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

155

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

157

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

167

(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

169

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

Insulin Sensitivity-Enhancing Activity of Phlorizin Is Associated with

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