Pelargonidin-3-O-glucoside Derived from Wild ... - ACS Publications

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Bioactive Constituents, Metabolites, and Functions

Pelargonidin-3-O-glucoside derived from wild raspberry exerts antihyperglycemic effect by inducing autophagy and modulating gut microbiota Hongming Su, Lianghua Xie, Yang Xu, Huihui Ke, Tao Bao, Yuting Li, and Wei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03338 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 20, 2019

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Journal of Agricultural and Food Chemistry

Graphic abstract

Our study indicated that pelargonidin-3-O-glucoside (Pg3G) derived from wild raspberry (Rubus hirsutus Thunb.) improved glucose metabolism. In vitro study demonstrated that Pg3G improved glucose uptake through inducing TFEB-mediated autophagy pathway. In vivo study confirmed that Pg3G prevented hyperglycemia and insulin resistance and induced autophagy. In addition, this anti-diabetic effect was associated with enrichment of Prevotella, increased Bacteroidetes/Firmicutes ratio, and reinforcement of gut barrier integrity.

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derived

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Pelargonidin-3-O-glucoside

raspberry

exerts

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antihyperglycemic effect by inducing autophagy and modulating gut microbiota

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Hongming Sua, Lianghua Xiea, Yang Xua, Huihui Kea, Tao Baoa, Yuting Lia, Wei

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Chena,b*

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a

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Processing, Zhejiang University, Hangzhou 310058, China.

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b

Department of Food Science and Nutrition, Zhejiang Key Laboratory for Agro-Food

Ningbo Research Institute, Zhejiang University, Ningbo 315100, China.

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* Corresponding author:

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Prof. Wei Chen, Ph.D.

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Department of Food Science and Nutrition, Zhejiang University, No.866 Yuhangtang

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Road, Xihu District, Hangzhou 310058, China.

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Tel: +86 571 88982861

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E-mail: [email protected]

Fax: +86 571 88982191

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Keywords: Autophagy; Diabetes; Gut microbiota; Pelargonidin-3-O-glucoside;

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Raspberry

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ABSTRACT

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Increasing evidence indicates that anthocyanins exert beneficial effects on type 2

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diabetes (T2D), but the underlying mechanism remains unclear. Herein, the

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hyperglycemia-lowering effect of Pg3G derived from wild raspberry was investigated

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on high-glucose/high-fat (HG+HF)-induced hepatocytes and db/db diabetic mice. Our

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results indicated that Pg3G promoted glucose uptake in HG+HF-induced hepatocytes.

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Moreover, Pg3G induced autophagy, whereas autophagy inhibitors blocked the

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hypoglycemic effect of Pg3G. Transcriptional factor EB (TFEB) was found to be

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linked to Pg3G-induced autophagy. In vivo study showed that Pg3G treatment

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contributed to the improvement of glucose tolerance, insulin sensitivity and induction

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of autophagy. Furthermore, Pg3G not only modified the gut microbiota

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composition-as indicated by an increased abundance of Prevotella, and elevated

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Bacteroidetes/Firmicutes ratio, but also strengthened the intestinal barrier integrity.

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This study unveils a novel mechanism that Pg3G attenuates hyperglycemia through

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inducing autophagy and modulating gut microbiota, which implicates a potential

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nutritional intervention strategy for T2D.

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INTRODUCTION Type 2 diabetes mellitus (T2D) is a chronic metabolic disease that have affected

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millions of people worldwide 1. T2D is characterized by hyperglycemia and insulin

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resistance with a range of metabolic complications including diabetic nephrology,

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diabetic retinopathy as well as neuropathy 2. Although several anti-diabetic agents

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have been approved by the FDA for the treatment of T2D 3, their potential side-effects

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cannot be ignored. Recently, increasing attention has been paid to natural products

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from daily consumed foods. Several foods derived small molecules have been

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reported to regulate glucose homeostasis, such as resveratrol, curcumin,

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sulphurophane etc 2. Therefore, it is urgently needed to develop novel agents from

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natural products for the prevention and treatment of T2D.

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Accumulating evidence indicates that intestinal microbiota plays a critical role in

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whole-body glucose metabolism 4. A number of natural products have been found to

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regulate blood glucose level through the modulation of the gut microbiota

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composition. For example, metformin changed the microbiome of T2D individuals,

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which resulted in the antidiabetic action of this drug 5. A water extract of Ganoderma

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lucidum mycelium reduced insulin resistance in HFD-induced obese mice through

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increasing Bacteroidetes/Firmicutes ratio and decreasing endotoxin- bearing

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Proteobacteria levels 6. A recent study suggested that selective increase of short-chain

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fatty acids (SCFAs) producing strains by dietary fibers alleviated T2D 7. In addition, a

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series of berry fruits, such as artic berry extract 8, blueberry extract 9, cranberry

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extract 10, was reported to have beneficial effects against diabetes through regulating

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the gut microbiota.

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Raspberry is rich in polyphenolic compounds 11. This fruit is generally regarded as

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the third generation of “gold fruits” with special nutritional and edible values. To date,

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a variety of raspberry species have been identified and their nutritional ingredients are

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found to be distinct among disparate raspberry species 12. Our previous study

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indicated that raspberry extract afforded a neuroprotective effect against

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peroxynitrite-induced DNA damage and hydroxyl radical formation 13. We also found

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that wild raspberry (Rubus hirsutus Thunb.) extract provided protection against

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oxidative stress, and this protective action could be attributed to the anthocyanins

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fraction 14. In addition to those beneficial effects, raspberry hitherto exhibited

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versatile health benefits against oxidative stress 15, obesity16 and diabetes 17.

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Anthocyanins, which are naturally occurring phenolic compounds, are presented

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in raspberry fruits. The content and composition of anthocyanins presented in

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raspberry are different depending on the cultivars and cultivation regions. Emerging

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evidence indicated that anthocyanins possessed beneficial roles on T2D 18-19. For

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example, a previous meta-analysis suggested that anthocyanins consumption was

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positively associated with the reduction of T2D 20. Several cellular and animal models

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also demonstrated the anti-diabetic role of anthocyanins derived from daily consumed

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food 21-22. Our recent work revealed a novel α-glucosidase inhibitor derived from

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natural anthocyanins for improving postprandial hyperglycemia 23. However, to the

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best of our knowledge, information is still lacking regarding the anti-hyperglycemic

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effect of natural anthocyanins extracted from wild raspberry. Therefore, the objective

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of this study is to investigate the hyperglycemia-lowering effect of

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pelargonidin-3-O-glucoside (Pg3G), a major anthocyanin extracted from wild

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raspberry, and to examine its underlying mechanism of action by studying the

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possible involvement of autophagy and gut microbiota.

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MATERIALS AND METHODS Reagents. Lyso-Tracker DND 99 was obtained from Life Technologies (Carlsbad,

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CA, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). 2-NBDG

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was obtained from Cayman Chemical (MI, USA). SYBR Green PCR Master Mix was

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purchased from Roche (Basel, Switzerland). All other reagents used were of

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analytical grade.

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Preparation of Pg3G from wild raspberry. Fresh wild raspberry was extracted

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using 70% ethanol aqueous solution containing 0.1% hydrochloric acid. After

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extraction, the ethanol extract was centrifuged to remove pomace. The collected

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supernatant was evaporated at 45°C and then purified by AB-8 macroporous resin

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column to remove protein and sugar. The gradient elution was performed as follows:

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the column was first eluted by 4 bed volume (BV) of distilled water, and then 4 BV of

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ethanol aqueous solution (5:95, 10:90, and 20:80, v/v) containing 1.5% hydrochloric

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acid (v/v) in sequence. 10% and 20% of ethanol eluent were collected and then

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lyophilized to yield raspberry anthocyanins fraction. The raspberry anthocyanins

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fraction was further purified by high-speed counter-current chromatography (HSCCC)

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using a two-phase solvent system of tert-butyl methyl ether - n-butanol - acetonitrile -

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water (2:2:1:5, v/v/v/v, acidified with 0.1% trifluoroacetic acid). The upper-phase was

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set as the stationary phase, while the lower-phase was defined as the mobile phase.

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The flow rate was 3 mL/min. The absorbance was detected at 280 nm. During the

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elution, the eluent was collected every 3 min and then determined by HPLC (Dionex

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ultimate 3000, ThermoFisher Scientific, USA). The major anthocyanins in wild

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raspberry were identified by LC-MS/MS (AB SCIEX, Triple-TOF 5600plus, USA)

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and NMR (Bruker AVANCETM III spectrometer, 14.1 T) according to our previous

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report 23.

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Cell culture and high-glucose and high-fat culture medium preparation.

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HepG2 cells were cultured in low-glucose (5.5 mM) DMEM medium. The medium

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contained 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml

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streptomycin. The cells were cultured in a humidified cell incubator with an

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atmosphere of 5% CO2 at 37°C. A 50 mM palmitic acid (PA) stock solution was

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prepared as previously described 24. The high-glucose and high-fat (HG+HF) culture

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medium containing glucose (22.5 mM) and PA (0.1 mM). Cell viability was

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examined by the MTT method as previously described 25.

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Determination of glucose uptake. Intracellular glucose uptake was determined by

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both enzymatic method and fluorescence microscopy according to previous reports 26.

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Cells were seeded in 24-well plates at a density of 3.5×104 cells per well in the

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low-glucose medium for 24 h. The low-glucose culture medium was replaced with

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HG+HF culture medium to mimic the hyperglycemia milieu for 24 h and then treated

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with Pg3G at different concentrations (5, 10 and 20 μg/mL) for 24 h. After that, the

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medium was discarded and incubated with 10 mM fresh DMEM medium for 24 h.

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The medium was collected and the amount of glucose uptake was determined by

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calculating the consumption of glucose using GOD-POD glucose assay kit. The

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glucose concentration of the blank well was subtracted from the glucose concentration

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in each well to obtain the amount of glucose uptake. In addition, glucose uptake was

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detected using 2-NBDG. Briefly, the cells were first treated with Pg3G for 24 h. The

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cells were incubated with 2-NBDG (100 mM) for 1 h. Cells were washed with PBS

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and then immediately evaluated by fluorescence microscope (Nikon, Tokyo, Japan).

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Fluorescence microscopy. The HepG2 cells were grown on 12-well plates and

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treated with Pg3G for 24 h. Then the cells were incubated with 100 nM Lyso-Tracker

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Red DND-99 for 30 min, followed by 4% formaldehyde fixation for 20 min. Then,

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the images were captured by fluorescence microscope.

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Plasmids transfection assay. The HepG2 cells were transfected with

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mRFP-EGFP-LC3 or EGFP-TFEB plasmids using Roche X-tremeGENE HP DNA

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Transfection Reagent for 24 hours according to the manufacturer’s protocol and then

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treated with Pg3G for 24 h. The images were captured by fluorescence microscope

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(Nikon, Tokyo, Japan).

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Molecular docking. The full-length human TFEB structure model was built using

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the hierarchical and ab-initio approach as previously reported 27-28. The 3D structure

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of Pg3G was generated with ChemBio3D Ultra 12.0, and energetically minimized

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with MM2 force field. Molecular Docking studies were performed using Autodock v4

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package. The docking results were analyzed in clusters, and the model with the lowest

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binding energy was selected as the proposed position for Pg3G and then visualized by

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Pymol 2.0.6. The interaction between Pg3G and human TFEB model was analyzed

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using Discovery Studio 2017R2 (BIOVIA, v17.2.0, San Diego).

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Animal study. All animal experiments were conducted according to the guidelines

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and laws on the use and care of laboratory animals in China (GB/T 35892-2018 and

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GB/T 35823-2018). The animal protocol was approved by the laboratory animal

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management and ethics committee of Zhejiang Chinese Medical University

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(201610087). All the animal experimental procedures were performed in the Animal

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Experiment Center of Zhejiang Chinese Medical University (Hangzhou, China). Male

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db/db mice with C57BL/6J background, aged six weeks, were purchased from Model

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Animal Research Center of Nanjing University (Nanjing, China). The db/db diabetic

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mice model has been used for years. After one week of acclimatization, the mice were

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divided into two groups (n=24): 1) db/db mice were daily administered with vehicle

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(distilled water) by oral gavage (n-12); 2) db/db mice were daily administered with

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150 mg/kg per mice Pg3G (n=12) by oral gavage. All mice have ad libitum access to

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autoclaved water and diet. The temperature in the cage was maintained with constant

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temperature (23 °C) and humidity under a 12-h/12-h light/dark cycle. After eight

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weeks, the mice were sacrificed following 12-h fasting. Blood was collected by

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cardiac puncture and centrifuged at 5000 rpm for 10 min for serum collection. Caecal

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contents were collected immediately after euthanasia and preserved in sterilized tubes

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and immediately frozen using liquid nitrogen. The liver and intestine were collected

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and weighed. Serum and all of the tissues were snap-frozen and stored at –80 °C for

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further investigation. In the present study, 150 mg/kg per mice Pg3G were equivalent

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to 16.5 mg/kg Pg3G consumption by humans. As 500 g of wild raspberry contains

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407.1 mg of Pg3G, the dose of Pg3G is achievable through regular wild raspberry

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consumption.

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Biochemical analysis. Serum triglyceride, cholesterol, FFAs, AST, ALT and LDH

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were measured by Hitachi automatic biochemistry analyzer. SOD, CAT and T-AOC

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were determined by commercially antioxidant kits (Beyotime Biotechnology, Haimen,

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China). Serum insulin, LPS, LPS-binding protein (LBP) were determined using

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ELISA kits (Elabscience, Wuhan, China).

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Glucose tolerance test (GTT) and insulin tolerance tests (ITT). At week 8, mice

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fasted for 12 h, and GTT was performed after 0.5 g/kg glucose was administered

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intraperitoneally. Blood glucose levels were measured from the tail before glucose

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administration and 15, 30, 60, 90 and 120 min after administration. ITT was

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performed after mice fasted for 6 h. Insulin (2 IU/kg) was administered

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intraperitoneally. Blood glucose levels were measured from the tail before insulin

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administration and 15, 30, 60, 90 and 120 min after the administration.

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RT-PCR. Total RNA was isolated from caecal tissue using Trizol (Invitrogen,

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Carlsbad, CA, USA). cDNA was synthesized using the PrimeScript RT reagent Kit

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according to the manufacturer’s instruction (TaKaRa, Japan). Quantitative real-time

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PCR was carried out in the QuantStudio 3 Real-Time PCR System (Applied

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Biosystems, Foster City, CA, USA). The 20 μL reaction mixture consisted of 1×

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SYBR Green PCR Master Mix, 0.25 μM of cDNA and 0.2 μM of each primer. The

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PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for

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15 seconds and 60°C for 1 min. The gut barrier permeability related genes (Ocln,

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Cldn3, ZO-1, Mucin 2 and Tlr2) and antimicrobial peptides related genes (Pla2g2 and

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Lyz1) in caecal tissue were determined, which indicated the condition of gut barrier

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function. The primers were indicated in Supplementary Table 1. A relative

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gene-expression quantification method was used to calculate the fold change of

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mRNA expression according to the comparative CT method using GAPDH for

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normalization.

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Western-blot. Western-blot analysis was performed as previously described 29.

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Total protein fraction was extracted using RIPA Lysis Buffer (BOSTER Biological

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Technology co.ltd) with the addition of Roche cOmplete™ Protease Inhibitor

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Cocktail. Nuclear and cytoplasmic fractions were separated by a commercial kit

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according to the manufacturer’s protocol (Thermo Fisher Scientific, 78833, USA).

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Protein was separated by electrophoresis on SDS-polyacrylamide gels and transferred

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to polyvinylidene fluoride (PVDF) membranes (Millipore, ISEQ00010). After

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blocking with 10% nonfat dry milk in PBS buffer containing 0.1% Tween-20 (PBST),

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the membrane was incubated with the primary antibody overnight at 4 °C. After three

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washes, the membrane was incubated with horseradish peroxidase-conjugated

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secondary antibodies (Bio-Rad, 170-6515 and 170-6516) for 1 h. After three washes

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with PBST, the immunoreactive protein bands were visualized by Chemiluminescent

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HRP Substrate (Millipore, WBKLS0100). The following primary antibodies were

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used: LC3B antibody (Abcam, ab192890), TFEB antibody (Bethyl Laboratories, Inc.,

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A303-673A), Lamin B (Santa Cruz Biotechnology, sc-365214), GAPDH (Abcam,

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ab181602).

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Homeostasis model assessment of insulin resistance (HOMA-IR). The

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homeostasis model assessment (HOMA-IR) index was calculated using the fasting

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values of glucose and insulin with the following formula: HOMA-IR index = insulin

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(μU/mL) × glucose (mM) / 22.5.

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Transmission electron microscopy (TEM). The liver samples were first fixed

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with 2.5% glutaraldehyde for 24 h. After that, the sample was washed three times

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with PBS (0.1M, pH 7.0) and then fixed with 1% OsO4 for 2 h and washed with PBS

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three times again. After dehydrating in a graded series of ethanol (30%, 50%, 70%,

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80%, 90% and 95%), the sample was transferred to acetone and embedded in Spurr

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resin. The sample was sectioned in LEICA EM UC7 ultratome and sections were

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stained by uranyl acetate and alkaline lead citrate for 5 to 10 min, respectively, and

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then observed in Hitachi Model H-7650 TEM.

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Faecal microbiota identification. Ceacal contents were subjected to 16S rRNA

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sequencing. The genomic DNA from ceacal contents were extracted using the

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QIAamp DNA Stool Mini Kit (Qiagen, Germany) according to the manufacture's

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instruction. Amplification of the 16S rRNA V3-V4 region was carried out using 16S

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universal primers. Amplicons were separated and collected using the AxyPrep DNA

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Gel Extraction Kit (Axygen Biosciences, CA, USA). The purified amplicons were

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analyzed on the Illumina MiSeq platform. Raw sequences were analyzed and

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processed using the Quantitative Insights into Microbial Ecology (QIIME) software

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package. The predicted functional genes were analyzed by PICRUSt based on KEGG

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pathway 30. Faecal short-chain fatty acids (SCFAs) levels in faecal content were

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analyzed using gas chromatography as previously described 5.

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Short-chain fatty acids (SCFAs) determination. Faecal SCFAs levels were

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analyzed according to a previous report 5. Briefly, 100 mg faeces were homogenized

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with 800 mL of phosphate buffer (pH 7.3) and then centrifuged at 16,000× for 15 min

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to remove sediment. The supernatants were acidified by adding 300 μL 50% (v/v)

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sulfuric acid and vortexed for 30 s and standing for 2 min. The organic acids were

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extracted by diethyl ether twice. The extract was filtered through a 0.22 μm filter and

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measured by gas chromatography (GC) on Agilent 6890 (Agilent Technologies, CA,

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USA) in comparison with known standards.

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Statistical analysis. Data are expressed as the mean ± SEM. Significant differences

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were evaluated by two-tailed Student’s t-test between two groups or one-way analysis

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of variance (ANOVA) followed by the Bonferroni’s post hoc test between multiple

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groups. p

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