Hypoglycemic effect of pyrodextrins with different molecular weights

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Article

Hypoglycemic effect of pyrodextrins with different molecular weights and digestibilities in diet-induced obese mice Yan Cao, Xiaoli Chen, Ying Sun, Jialiang Shi, Xiaojuan Xu, and Yong-Cheng Shi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00404 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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Journal of Agricultural and Food Chemistry

Hypoglycemic effect of pyrodextrins with different molecular weights and digestibilities in diet-induced obese mice Yan Cao,†,# Xiaoli Chen,†,‡,# Ying Sun,† Jialiang Shi,§ Xiaojuan Xu,*,† and Yong-Cheng Shi*,§ †

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,

China ‡

College of Food Science and Technology, Modern Biochemistry Experimental Center,

Guangdong Ocean University, Zhanjiang 524088, China §

Department of Grain Science and Industry, Kansas State University, Manhattan,

Kansas 66506, United States

*

Corresponding authors:

Xiaojuan Xu, Tel/Fax: +86 27 68754188, E-mail: [email protected]. Yong-Cheng Shi, Tel/Fax: +1 7855326771, E-mail:[email protected].

1

ACS Paragon Plus Environment


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ABSTRACT ： Pyrodextrin shares some properties of resistant starch which is

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metabolically beneficial, and has potential applications as a functional food. In this

3

study, we report that oral administration of pyrodextrin (50 mg/kg/d for 7 weeks)

4

decreased the blood glucose (from 9.18±1.47 to 7.67±0.42 mmol/L), serum HbA1c,

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triglycerides, adipocyte size, and body weights (from 24.4±1.2 to 22.5±1.2 g) in

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high-fat diet-induced obese mice. The Western blotting analysis suggested that

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pyrodextrins decreased the intestinal SGLT-1 and GLUT-2 expression to ∼70% and

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∼60% of the obese control to slow down glucose transportation from gut into blood,

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and improved the hepatic metabolism tentatively. Moreover, the pyrodextrin with

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lower molecular weight of 44 kDa, more branched structure and increased

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non-digestible linkages of 46.2±0.3% showed stronger hypoglycemic activity. This

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work provides important information for developing pyrodextrins as functional food

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and dietary supplement for management of obesity and diabetes.

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KEYWORDS: pyrodextrin, molecular weight, digestibilities, obesity, hypoglycemic

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activity

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2


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INTRODUCTION

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Obesity and type 2 diabetes (T2D) have become a global epidemic. The prevalence of

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obesity is increasing globally at an epidemic rate with more than 1 billion adults

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overweight and at least 300 million clinically obese.1 These numbers are expected to

21

rise further in the next 20 to 30 years. Obesity is known to be an independent risk

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factor for serious health conditions, including hypertension, T2D, and cardiovascular

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diseases.1 According to statistics from International Diabetes Federation, diabetes has

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become one of the largest global health emergencies of the 21st century.2 Obesity and

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T2D can result in life-changing complications such as cardiovascular disease,

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blindness, kidney failure, and lower-limb amputation, being the major cause of death

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in most countries. It has been well recognized that more healthy food and improved

28

diet are an economical and effective way to prevent obesity and T2D.2 Therefore, a

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great deal of efforts have been focused on developing functional food and dietary

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supplement for management of obesity and diabetes.3

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According to FAO/WHO recommendations,4 the optimal diet to maintain health

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comprises at least 55% total energy from a variety of carbohydrate sources. Interest in

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the health benefits of carbohydrates, dietary fiber, and resistant starches (RS) among

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consumers, researchers, nutritionists, and food manufacturers has increased

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dramatically in the past decade. RS have gained wide attention because diets with an

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increased level of RS have been reported to result in a number of beneficial health

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effects, such as the improved animal gastrointestinal tract traits and human colonic 3



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health, as well as a reduced risk of diabetes, heart disease, and obesity.5-13 Pyrodextrin

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is the degraded starch product usually prepared by a process involving acidification

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and heat treatment of starches,14 and the resulted product is resistant to α-amylolysis

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and considered as a soluble dietary fiber.15, 16 Traditionally, pyrodextrins have wide

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application in industry as adhesives, coatings, binders, and encapsulating agents.17, 18

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However, the application of pyrodextrin in functional food for management of obesity

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and diabetes is seldom studied. As reported, pyrodextrins have reduced in vitro

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digestibility,19 and indigestible dextrins give lower plasma glucose and insulin

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response compared with glucose and maltodextrin in healthy human subjects.20

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However, supplemental indigestible dextrin did not affect the postprandial glycemic

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response to a rapidly digested starch.21 Due to the complex chemical reactions during

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dextrinization of starch involving hydrolysis, transglycosidation, repolymerization,

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and oxidation, the different structural changes at the molecular and granular levels

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have occurred.14, 22, 23 At present, the relationship between the subtle structural changes

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of pyrodextrin and its biological functionality is not completely understood.

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In the previously reported work, the structural changes occurring during the

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thermal

conversion

of

insoluble

native

waxy

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cold-water-soluble pyrodextrin under acidic conditions have been investigated by

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multiple techniques, and new glycosidic bonds formed during detrinization have been

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identified by NMR.14, 23 It is proposed that the starch backbone is hydrolyzed by acid

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in the amorphous region and the crystalline region with starch macromolecules being

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hydrolyzed into small molecular fractions but persisting in a radial arrangement.14 In 4


maize

starch

granules

to

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this paper, therefore, we explored the hypoglycemic effects and possible mechanism of

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pyrodextrins with different structures prepared from native waxy maize starch

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granules in obese mice induced by high-fat diet (HFD) for better understanding the

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correlation of structure to the functionality of pyrodextrin.

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MATERIALS AND METHODS

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Chemicals. Dulbecco’s modified eagle medium (DMEM, high glucose) and

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trypsin were purchased from Hyclone Laboratories (South Logan, UT). Fetal bovine

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serum (FBS) was purchased from Gibco (Grand Island, NY). Antibodies of β-actin,

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sodium-glucose transporter 1 (SGLT-1), glucose transporter 2 (GLUT-2) and

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phosphorylated insulin receptor substrate (IRS) 1/2 at Tyr612 (p-IRS) were purchased

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from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphorylated adenosine

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5-monophosphate-activated protein kinase (p-AMPK) antibody was purchased from

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Cell Signaling Technology (Boston, MA). All other reagents were analytical reagents

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from chemical companies in China. Normal diet and HFD consisting of 60% fat, 20%

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carbohydrates, and 20% protein were purchased from Beijing HFK Bioscience Co.,

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LTD.

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Sample Preparation. Pyrodextrin samples were prepared as described by Bai et

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al.14 Pyrodextrins were prepared at 170°C for 0.5, 1, 2, 3, and 4 h to obtain the final

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samples, and were coded as A1, A2, A3, A4, and A5 in this study, respectively, in the

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order of increasing time.

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Molecular Weight Determination. Molecular weights of the pyrodextrins were 5



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determined by size-exclusion chromatography (SEC) with laser light scattering (LLS)

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detection. Pyrodextrin samples were dissolved in 0.1 M NaNO3 solution to form 1

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mg/mL test solution. After filtering through 0.45 µm Millipore filters, the test solutions

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were subjected to the equipment combined with SEC column (Shodex-OHpak SB-806

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M HQ, 8.0 mm × 300 mm), Multi-angle LLS spectrometer (DAWN HELLEOS-II,

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He−Ne laser, λ = 663.4 nm), and Refractive Index Detector (Opitilab T-rEX). The

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flowing rate was 0.6 mL/min, and Astra software (Wyatt Technology Corporation,

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Santa Barbara, CA) was used to collect and analyze the data.

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Dietary Fiber Contents of Pyrodextrins. The dietary fiber contents of the

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pyrodetrxins were determined by the AOAC Method 2011.25 using an Integrated Total

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Dietary Fibre assay kit from Megazyme (Co. Wicklow, Ireland) with some

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modifications. The scheme of the modified method is depicted in Figure 1. Insoluble

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dietary fiber (IDF) and soluble dietary fiber precipitated in ethanol (denoted as SDF

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P) were determined by weighing the dried residues. The glucose content in the

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soluble dietary fiber in ethanol (SDFS) was determined by glucose oxidase/peroxidase

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(GOPOD) assay. The digestible fraction in SDFS filtrate was calculated to be

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0.9×glucose%. The enzyme resistant fraction, which was previously termed low

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molecular weight soluble dietary fiber (LMWSDF) and referred to as non-digestible

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oligosaccharides (NDO), in the SDFS was calculated as: LMWSDF% = 100% −

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IDF% − SDFP% − glucose% in the SDFS fraction. The total non-digestible starch

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content was calculated as 100 − digestible fraction (%).

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The

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium 6


bromide

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(MTT) assay. The intestinal epithelial Caco-2 cell line was obtained from the China

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Center for Type Culture Collection (Wuhan, China), which was used to evaluate the

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cytotoxicity of pyrodextrins. The cells were grown and kept in the medium of high

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glucose DMEM supplemented with glutamine, 10% FBS, 100 units/mL penicillin, and

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100 µg/mL streptomycin at 37 °C under a humidified atmosphere of 95% air and 5%

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CO2.

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The Caco-2 cells (5×103 cells in 0.2 mL) were seeded on 96-well plates and were

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treated with pyrodextrin samples (50, 100, 200, and 400 µg/mL) for 24 h after the

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confluence of ∼80%. Cells were incubated with MTT at 37°C for 4 h, and the

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supernatant was discarded. Dimethyl sulfoxide (DMSO) (150

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dissolve the blue precipitates and the optical density (OD) value was measured at 492

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nm on a microplate reader (BMG LABTECH, FLUOstar OPTIMA, Germany). The

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data were presented as cell viability and calculated as cell viability = experimental

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group / negative control group.

µL)

was added to

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HFD-induced Obese Mice Experiment. Female C57BL/6 mice (8-weeks old,

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19.2 ± 0.5 g) were purchased from the Animal Experiment Center of Wuhan

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University (Wuhan, China), and all animal protocols were approved by the Wuhan

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University Center for Animal Experiment/Animal Biosafety Level-III laboratory

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(Hubei Province, China). The mice were housed under controlled room temperature

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(22 ± 2 °C) and humidity (55 ± 5%) with 12:12 h light and dark cycle and free access

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to water and food. The mice were randomly divided into 5 groups (10 mice/group)

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including ND group (on normal diet, orally administered with an equal volume 7



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purified water), HF group (on HFD, orally administered with an equal volume of

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purified water), and A1, A3, and A5 groups (on HFD, orally administered with 50

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mg/kg/day A1, A3, and A5, respectively). After treatment for one week, fasting blood

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glucose (FBG) levels were measured. Before measurement, all mice were starved for 6

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h, and blood were collected from the mice tail vein for the blood glucose measuring

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using a medical blood glucose meter (On Call Plus REF G113-232, Acon Biotech,

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Hangzhou, CHN). Body weight, food consumption, and water intake in each group

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were monitored at the desired time interval.

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At the end of week 7, blood samples were collected from the mice orbital cavity

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after overnight starvation. The mice were then sacrificed, and jejunum (5−10 cm from

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the pylori, with intestinal contents, mesenteric border and lipid carefully removed off)

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and liver tissue samples were collected and frozen immediately by liquid nitrogen

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followed by storing at −86°C before use. The blood samples were centrifuged at 3000

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rpm for 15 min, and the supernatant serum was collected. The content of triglycerides

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

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high-density lipoprotein cholesterol (HDL-C) in serum were assayed with the

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respective Assay Kit following the manufacturer’s instructions (Nanjing Jiancheng

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Bioengineering institute, China). Serum HbA1c and insulin contents were assayed

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with ELISA Kits following the manufacturer’s instructions (Jianglaibio, Shanghai,

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China). The adipose tissues were stained with hematoxylin-eosin (H&E), and the

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histology morphology was observed under a microscope (NIKON ECLIPSE TI-SR,

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Japan). 8


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Oral Glucose Tolerance Test (OGTT). Healthy female mice (C57BL/6, 9-weeks

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old, 19.6 ± 0.6 g) on normal diet were used for OGTT. Before treatment, mice were

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fasted for 6 h (from 8 a.m. to 2 p.m.). Mice were then orally administered with 50

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mg/kg A1, A3, A5, and equal volume of water, respectively (denoted as A1, A3, A5,

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and CT group, 6 mice/group). 30 min later, all the mice were orally administered with

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1.5 g/kg glucose. The glucose levels in blood samples from the tail vein before (as 0

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min) and after glucose administration for 15, 30, 60, 120 min, were measured with a

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medical blood glucose meter (On Call Plus REF G113-232, Acon Biotech, Hangzhou,

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CHN).

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Western Blotting Analysis. The jejunum region of intestines and liver tissues of

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mice were homogenized in RIPA lysis buffer (ice bath), and centrifuged (13000 rpm)

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at 4°C for 5 min. The supernatant was obtained as the protein samples and kept at

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−86 °C before use. The protein concentration was measured by a BCA Protein Assay

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Kit (Beyotime Institute of Biotechnology). Aliquots of the protein solutions were

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mixed with the loading buffer of 4× sodium dodecyl sulfate (SDS) solution, and

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denatured in boiling water for 5 min. From intestine and liver tissues, 60 µg of

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proteins were subjected to 8%−10% SDS polyacrylamide gel electrophoresis

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(SDS-PAGE), and the resulting bands were electrically transferred onto a

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polyvinylidene fluoride (PVDF) membrane (0.45 µm, Millipore). After blocking with

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5% (w/v) bovine serum albumin (BSA) in the Tris buffered saline (TBS containing 10

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mM Tris-HCl, and 150 mM NaCl, pH 8.0) with 0.1% Tween 20 at room temperature

168

for 1 h, the membranes were probed with appropriate specific primary antibodies 9



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against β-actin, SGLT-1, GLUT-2, p-AMPK, and p-IRS-1/2 (Tyr612) overnight at

170

4°C. The reactive bands were incubated with

171

(HRP)-conjugated secondary antibody (Biosharp) for 50 min and visualized by

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enhanced chemiluminescence (ECL) Western blotting detection reagent on a

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ChemiDoc-ItTM imaging system (UVP, Upland, CA) according to the manufacturer’s

174

instructions. The images were quantified by densitometric analysis using the

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UVP-visionworks LS software. β-Actin was used as the internal control.

a horseradish peroxidase

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Biodistribution of Pyrodextrins in Mice. Two pyrodextrin samples of A1 and

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A5 were selected to react with fluorescein isothiocyanate isomer I (FITC) to obtain

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FITC-labeled pyrodextrins.24 Briefly, pyrodextrin (250 mg), FITC (35 mg), pyridine

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(100 µL), and dibutyltin dilaurate (20 µL) were dissolved in 25 mL DMSO and then

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heated at 100°C for 4 h. The mixture was precipitated with 4 volumes of ethanol and

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centrifuged at 6000 rpm for 10 min to discard the supernatant. The precipitates were

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re-dispersed in ethanol and precipitated following the same procedures, which were

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repeated four times for completely removing the unbound FITC. The FITC-labeled

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pyrodextrins, coded as A1-F and A5-F, were dried under vacuum at 60°C and then

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dispersed in water for use in the following experiment.

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Healthy female C57BL/6 mice (8-weeks old, 19.2 ± 0.5 g) were starved for 24 h,

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and were divided into three groups (6 mice/groups) of A1-F and A5-F groups

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intragastrically administered with 50 mg/kg body weight A1-F and A5-F respectively,

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and the blank control group given an equal volume of water. Blood samples from the

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mouse orbital cavity were collected at 24 h after A1-F and A5-F administration and 10


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centrifuged (3000 rpm, 15 min) to get the serum. The feces samples during 0−24 h

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were collected. Then A1-F and A5-F in feces were extracted with DMSO, followed by

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precipitating with ethanol and re-dissolving in water. The fluorescence intensity of

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serum and feces samples were measured at excitation wavelength of 484 nm and

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emission wavelength of 525 nm.

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Statistical Analysis. All data are expressed as means ± standard deviation (SD).

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The statistical analysis was performed using one-way ANOVA with Duncan’ multiple

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comparison among groups. Statistical significance was considered at p < 0.05.

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RESULTS

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Molecular Weights and In Vitro Dietary Fiber Contents of Pyrodextrins. The

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weight-average (Mw) and number-average (Mn) molecular weights of five pyrodextrin

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samples A1-A5 were measured by SEC-LLS, and the molecular weight values as well

203

as the polydisperse indexes (d) were summarized in Table 1. The Mw and Mn decreased

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with increasing heating time, leading to an increase in water-solubility. The d values of

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A1-A5 determined by Mw/Mn were in the range of 2.0−2.5, showing relatively narrow

206

molecular weight distribution. Pyrodextrins with different molecular weights were

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successfully obtained.

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Table 2 shows the IDF content, SDFP content, and the enzyme digestible and

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non-digestible fraction in the SDFS filtrate of the pyrodextrins. The total

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non-digestible starch content increased from sample A1 (28.4%) to sample A5 (46.3%)

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as the processing time increased from 0.5 to 4 h. Furthermore, sample A5 had the 11



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highest levels of IDF and SDFP. When the SDFS filtrate from each pyrodextrin

213

sample was analyzed by high performance anion exchange chromatography (HPAEC),

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no molecules larger than glucose were observed.

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The cytotoxicity of Pyrodextrins in vitro. To evaluate the possible cytotoxicity

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of pyrodextrins, the MTT experiments were carried out in intestinal epithelial Caco-2

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cells. As a result, all the five pyrodextrin samples showed no cytotoxicity in Caco-2

218

cells (Figure 2) at concentrations of 50, 100, 200, and 400 µg/mL, and they can be

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used in animal experiments.

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Reduction of Hyperglycemia in Mice. As reported, mice can become obese,

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hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant when

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exposed to a HFD.25 Therefore, A1, A3, and A5 with the highest, middle and lowest

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molecular weight were selected to be orally administered into HFD-induced obese

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mice to evaluate the possible hypoglycemic effect in vivo. After five weeks treatment,

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the FBG level in HF group was significantly higher than that in the ND group,

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indicative of successful model construction of hyperglycemia. Interestingly, the three

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pyrodextrin samples A1, A3, and A5 suppressed the fasting hyperglycemia induced by

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HFD. In particular, A3 and A5 decreased FBG levels more significantly when

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compared with A1 after 6 weeks (Figure 3A). It is well known that the HbA1c is more

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sensitive to the accurate diagnosis of diabetes because of its high stability, accuracy,

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and precise reflection of chronic glycemic levels.26, 27 Therefore, the HbA1c level in

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serum of mice fasted overnight was measured. As a result, A1, A3, and A5 all

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effectively reduced the concentration of HbA1c in contrast to HF group after 7 weeks 12


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(Table 3), showing hypoglycemic effect.

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In view of the hypoglycemic effect on FBG, the OGTT was also performed to

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evaluate the postprandial hyperglycemia regulation by pyrodextrins. As shown in

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Figure 3B−C, the postprandial hyperglycemia was tentatively decreased by A1 and A3,

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and was significantly decreased by A5. Taken together, pyrodextrins not only

239

decreased FBG and serum HbA1c levels in HFD-induced obese mice, but also

240

decreased postprandial blood glucose levels, further confirming that pyrodextrins can

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really reduce the hyperglycemia in obese mice.

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The food intake and levels of LDL-C, HDL-C, TG, TC and insulin in serum are

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also summarized in Table 3. It was found that the pyrodextrin samples decreased the

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TC level and had no obvious effects on other indexes.

245

Reduction of the Body Weight and Adipocyte Size in Obese Mice. Besides the

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hypoglycemic activity, the effect of pyrodextrins on body weights of DIO mice was

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also evaluated. As shown in Figure 4A, samples A1, A3, and A5 all decreased the body

248

weight after 3 weeks of treatment when compared with HF group. And group A3 and

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A5 showed a more remarkable reduction in body weight in contrast to group A1.

250

Moreover, histological analysis revealed that the size of adipocytes was decreased by

251

A1, A3, and A5 (Figure 4B). In particular, A5 significantly reduced the size of

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adipocytes, providing strong evidence for the significant decrease of body weight by

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A5.

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Suppression of SGLT-1 and GLUT-2 in Intestines by Pyrodextrins. Since

255

pyrodextrins, particularly sample A5, showed notable hypoglycemic activity in vivo, 13



256

the underlying hypoglycemic mechanism was thus investigated. In viewing that

257

pyrodextrins share some properties of resistant starches, we postulate that the orally

258

administered pyrodextrins may slow down glucose absorption from gastrointestinal

259

tract to blood circulation. So proteins in the intestine were extracted and subjected to

260

Western blotting analysis of SGLT-1 and GLUT-2, which are the major glucose

261

transporters in the intestine responsible for the glucose transportation from intestine

262

into blood.28 As shown in Figure 5, A3 and A5 suppressed both SGLT-1 and GLUT-2

263

expression, and A1 only suppressed the expression of GLUT-2 in contrast to HF

264

groups.

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Enhancing of p-IRS and p-AMPK Expression in Livers of Obese Mice. Liver

266

is a major metabolism organ responsible for glucose homeostasis in the body, and the

267

expression of p-IRS and p-AMPK in the liver were then analyzed by Western blotting.

268

It was found that, after 7 weeks treatment, A1, A3 and A5 enhanced the expression of

269

p-IRS and p-AMPK in the liver of obese mice compared with HF group. In particular,

270

A3 and A5 showed stronger up-regulation of p-IRS and p-AMPK (Figure 6).

271

Metabolic Distribution of Pyrodextrins after Oral Administration. As stated

272

in the introduction, pyrodextrins are resistant to α-amylolysis and considered as

273

soluble dietary fiber, which can escape the digestion in the gastrointestinal tract. In

274

this study, whether pyrodextrins were adsorbed in the gastrointestinal tract or not was

275

thus studied. Pyrodextrins of A1 (least converted) and A5 (most converted) were

276

labeled with FITC, the commonly used green fluorescent probe. The fluorescence

277

signals of FITC-labeled pyrodextrins (A1-F and A5-F) were found in both blood 14


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(Figure 7A) and feces (Figure 7B), indicating that pyrodextrins partially entered the

279

bloodstream and were partially excreted in the feces directly.

280

DISCUSSION

281

In this study, pyrodextrins exhibited hypoglycemic bioactivity in HFD-induced obese

282

mice. The pyrodextrin samples not only decreased the levels of FBG and HbA1c in

283

HFD-induced obese mice, but also decreased the TC level in serum (Figure 3 and

284

Table 3). In addition, the lower body weights and smaller adipocyte sizes were

285

observed in pyrodextrin-treated mice (Figure 4).

286

From the obtained data, we propose the following possible mechanisms for the

287

hypoglycemic activity of the pyrodextrins. Firstly, in obese mice, the orally

288

administered pyrodextrin decreased the intestinal expression of GLUT-2 and SGLT-1

289

(Figure 5), the major glucose transporters in the intestines. It has been reported that

290

SGLT-1 mediates glucose transport in intestinal mucosa and the depressed SGLT-1

291

partly contributes to the hypoglycemic effects of indigestible carbohydrates such as

292

oat β-glucan and yeast β-glucan.29,

293

glucose levels and body weights in obese mice by A3 and A5, we inferred that the

294

suppressed blood glucose was possibly through inhibition of GLUT-2 and SGLT-1 to

295

slow down the glucose transportation from gut into blood circulation system, similar

296

to our recently reported hypoglycemic effect of yeast β-glucan.31 Secondly, the

297

increased expression of p-IRS and p-AMPK was observed in the liver of obese mice

298

treated with pyrodextrins (Figure 6). Tyrosine phosphorylation of IRS can up-regulate

30

Considering the notable reduction of blood

15



299

the interaction between insulin receptors and insulin, playing important roles in the

300

insulin-dependent regulation of glucose and lipid metabolism. It has been suggested

301

that the decreased tyrosine phosphorylation of IRS is involved in the insulin resistance

302

of diabetes and obesity disease.32-35 Our recently reported work also demonstrates that

303

the yeast β-glucan ameliorated glucose and lipid metabolism through promoting p-IRS

304

in the liver.31 Therefore, in this study, the enhancement of p(Tyr612)-IRS observed in

305

the liver of obese mice suggested that the hypoglycemic effect of pyrodextrins was

306

associated with improved hepatic insulin signaling pathway. AMPK is a major cellular

307

energy sensor and a metabolic master switch, playing an important role in glucose

308

homeostasis.36, 37 The phosphorylation of AMPK decreases the level of blood glucose

309

through two pathways of increasing glucose uptake and decreasing hepatic

310

gluconeogenic gene expression in the liver. 37, 38 Some polysaccharides are reported to

311

protect hepatic cells against high glucose-induced damage by activation of AMPK,

312

which is a target for diabetes therapy.39, 40 It is thus deduced that p-AMPK played an

313

important role in decreasing the hyperglycemia by A1, A3, and A5.

314

As for the relationship between the structure and hypoglycemic bioactivity, we

315

found A5 and A3 were more effective than A1 to decrease the blood glucose level

316

(Figure 3) and body weights (Figure 4) of obese mice. Corresponding to these findings,

317

A5 and A3 exhibited stronger up-regulation of hepatic p-IRS and p-AMPK expression

318

(Figure 6) than A1. Moreover, comparing to sample A3, sample A5 with the highest

319

level of non-digestible starch (Table 2) and lowest molecular weight (Table 1) showed

320

stronger inhibition of body weight and adipocyte size. Sample A1 was more digestible 16


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(Table 2) because relatively fewer new bonds were formed at the early

322

dextrinization.41 In contrast, more new glyosidic linkages were formed in samples A3

323

and A5, and those new glyosidic linkages could not be digested by α-amylase.

324

Furthermore, transglycosidation occurred during dextrinization23 and samples A3 and

325

A5 became more branched. Therefore, it can be concluded that pyrodextrins with

326

lower molecular weight, more highly branched structure and increased level of

327

non-digestible linkages possessed higher hypoglycemic activity. Shi and his

328

co-workers have demonstrated that new glyosidic linkages and the hydrolyzation of

329

starch backbone in both amorphous and crystalline regions occur during the

330

preparation of pyrodextrin.14, 23 The structure and properties of pyrodextrins can be

331

controlled by pH, temperature, and heating time, through which biological

332

pyrodextrins can be screened. The findings obtained in this study will provide

333

guidelines for pyrodextrins as ingredients for down-regulation of hyperglycemia

334

resulted from obesity and diabetes.

335

In conclusion, oral administration of pyrodextrins suppressed body weight and

336

blood glucose level in HFD-induced obese mice, depending on pyrodextrins’

337

molecular weight, branched structure and non-digestible linkages. The hypoglycemic

338

effect of pyrodextrins can be partly ascribed to the inhibited expression of GLUT-2

339

and SGLT-1 in the intestine and the up-regulated expression of p-IRS and p-AMPK in

340

the liver of obese mice. This work provides the direction for developing functional

341

pyrodextrins with hypoglycemic effect which has potential applications in food and

342

medical fields. However, due to the complex structure of pyrodextrins, it is still very 17



Page 18 of 33

343

difficult to evaluate the detailed structure changes and the interactions with other

344

active substances in vivo. The reason why pyrodextrins with lower molecular weight,

345

more branched structure and increased non-digestible linkages showed stronger

346

hypoglycemic activity merits further investigation.

347

AUTHOR INFORMATION

348

Corresponding Authors

349

*E-mail: [email protected]. Phone: Phone: 86-27-68754188. Fax: 86-27-68754188.

350

*E-mail: [email protected]. Phone: +1 785-532-6771. Fax: +1 785-532-7010.

351

Notes

352

#

The authors contributed equally to this work. The authors declared no competing

353

financial interest.

354

ABBREVIATIONS USED

355

AMPK, adenosine 5-monophosphate-activated protein kinase; DIO, diet-induced

356

obese; FITC, fluorescein isothiocyanate isomer I; HDL-C, high-density lipoprotein

357

cholesterol; HFD, high-fat diet; HPAEC, high-performance anion exchange

358

chromatography; IDF, insoluble dietary fiber; IRS, insulin receptor substrate; LDL-C,

359

low-density lipoprotein cholesterol; OD, optical density; OGTT, oral glucose tolerance

360

test; p-, phospho-; RS, resistant starches; SDFP, soluble dietary fiber precipitated by

361

ethanol; SDFS, soluble dietary fiber that was still soluble after ethanol precipitation;

362

SEC-LLS,

size

exclusion

chromatography-laser 18


light

scattering;

SGLT-1,

Page 19 of 33


363

sodium-glucose transporter 1; TC, total cholesterol; TG, triglycerides; T2D, type 2

364

diabetes.

365

ACKNOWLEDGEMENTS

366

We gratefully acknowledge the financial supports from Special Key Research and

367

Development Program of China (2016YFD0400202), National Natural Science

368

Foundation of China (21574102, 21274114, 20874078 and 21334005), and New

369

Century Excellent Talents Program of Education Ministry (NCET-13-0442). We thank

370

Quan Li for the preparation of the pyrodextrin samples. This is contribution number

371

17-350-J of the Kansas Agricultural Experiment Station.

372

REFERENCES

373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391

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465

FIGURE CAPTIONS

466

Figure 1. The scheme of the modified AOAC Method 2011.25 used to determine

467

insoluble dietary fiber (IDF), soluble dietary fiber precipitated by ethanol (SDFP) and

468

soluble dietary fiber dissolved in ethanol (SDFS) of the pyrodextrins. The

469

modifications in this method were that glucose content in SDFS filtrate was

470

determined by glucose oxidase/peroxidase (GOPOD) reagent and the SDFS filtrate

471

was analyzed by high-performance anion exchange chromatography (HPAEC).

472

Glucose solutions (0.5 and 1.0 mg/mL) were used as standards.

473

Figure 2. Cytotoxicity of pyrodextrins (50, 100, 200, and 400 µg/mL) in Caco-2 cells

474

for 24 h. The cell viability was assessed by MTT test and normalized to that of

475

PBS-treated cells. Data represent mean ± SD of 3 independent experiments, and

476

groups bearing different small letters differ significantly at p < 0.05.

477

Figure 3.

478

diet (ND group), HFD (HF group) and HFD supplemented with 50 mg/kg/d

479

pyrodextrins (A1, A3, and A5 groups). Data represent mean ± SD of 10 mice, and

480

groups within the same week bearing different small letters differ significantly at p

Hypoglycemic effect of pyrodextrins with different molecular weights

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