135-Day Interventions of Yam Dioscorin and the Dipeptide Asn-Trp

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The 135-Day Interventions of Yam Dioscorin and the Dipeptide Asn-Trp (NW) Reduce Weight Gains and Improve Impaired Glucose Tolerances in High-Fat Diet-Induced C57BL/6 Mice Guang-Cheng Wu, Shyr-Yi Lin, Hong-Jen Liang, and Wen-Chi Hou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05564 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on January 8, 2018

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Journal of Agricultural and Food Chemistry

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The 135-Day Interventions of Yam Dioscorin and the Dipeptide Asn-Trp (NW)

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Reduce Weight Gains and Improve Impaired Glucose Tolerances in High-Fat

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Diet-Induced C57BL/6 Mice

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Guang-Cheng Wu,†,# Shyr-Yi Lin¶,▼,# Hong-Jen Liang,┴ and Wen-Chi Hou†,*

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†

Graduate Institute of Pharmacognosy, ¶Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan

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┴

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▼

Department of Primary Care Medicine, Taipei Medical University Hospital, Taipei 110, Taiwan

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These two authors contributed equally to this study

Department of Food Science, Yuanpei University of Medical Technology, Hsinchu 300, Taiwan

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Running title: Yam Dioscorin and Asn-Trp Interventions in High-Fat Diet-Induced C57BL/6 Mice

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ACS Paragon Plus Environment


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ABSTRACT

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The C57BL/6J mice were fed a 135-day normal diet or a high-fat diet (HFD) without, or

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concurrent with, a single yam dioscorin (80 mg/kg) or dipeptide NW (40 mg/kg) intervention

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every day. The final body weights (g) of mice were 26.1 ± 1.4, 34.97 ± 2.1, 31.75 ± 2.6, and

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31.66 ± 3.1, respectively, for normal diet-fed, HFD-fed, dioscorin-intervened, and

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NW-intervened group. The mice in both intervened groups showed similar less weight gains and

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had significant differences (P < 0.05) compared to those in the HFD group under the same

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cumulative HFD intakes. The blood biochemical index of mice with dioscorin interventions

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showed significantly lower contents in total cholesterol and low-density lipoprotein, and NW

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interventions showed significantly lower total triglyceride contents compared to those of the

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HFD group (P < 0.05). Both intervened mice exhibited similar reductions in total visceral lipid

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contents and have significant differences compared to those of the HFD group (P < 0.05). The

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dioscorin intervention was better than NW interventions in lowering blood glucose levels by oral

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glucose tolerance tests and both showed significant differences (P < 0.05) compared to those in

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the HFD group. Yam dioscorin or dipeptide NW will potentially use for preventive functional

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foods of less body weight gains and impaired glucose tolerance controls, which require further

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clinical trial investigations.

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KEYWORDS: Asn-Trp (NW), high-fat diet (HFD), obesity, oral glucose tolerance tests, yam

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dioscorin

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█ INTRODUCTION

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A sedentary lifestyle, alcohol consumption, a high-fat diet, stress, and urbanization are causing

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the increased prevalence of global obesity,1, 2 and direct healthcare costs for the overweight and

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the obese will reach US$957 billion by 2030.1 It is estimated that over 85% of adults in America

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will become overweight or obese, and the mean BMI will become 31.2 by the year 2030. Obesity

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generally entails excess fat accumulation in the central abdominals and around the organs. The

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characterized by the body mass index [BMI (kg/m2)], being overweight (25 to 29.9) and being

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obese (higher than 30) are positively correlated with several cancer risks, compared to those with

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normal BMI (18.5 to 24.9).4 It has been found that the parameters of waist circumference and

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BMI showed significant correlations in the frequencies of cardiovascular diseases and type 2

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diabetes mellitus.2,5 Research has proposed that excessive fat accumulations may result in

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adipocyte dysfunctions, which are closely associated with the increasing secretions of

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pro-diabetic and pro-inflammatory hormones/cytokines,6 and in turn elevate the risks of insulin

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resistance and blood pressure, accompanied by abnormal blood glucose, signifying a cluster of

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multiple metabolic risk criteria for cardiovascular diseases.7,8

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Many peptides derived from protein resources have physiological functions other than

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nutrient supply.9-13 The yam storage protein of dioscorin, which was different from dioscorine,

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an alkaloid found in tubers of Dioscorea hirsute, and the hydrolyzed peptides have been proved

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to have functional activities in enzymatic and animal models, including antioxidant activities,14,15

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anti-aging activities,16,17 anti-hypertensive activities,18,19 immunomodulatory activities,20 and

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dipeptidyl peptidase-IV inhibitory activities for glucose tolerance improvement.21 The yam

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(Dioscorea spp) tuber is one of five major tuberous crops in the world22 which contain an 85% to

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90% water-extractable protein, named dioscorin, in different yam species.19,22 3



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A high-fat diet (HFD) is generally used to study obesity-associated metabolic diseases, such

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as

dyslipidemia,

obesity,

non-enzymatic

glycation,

inflammation,

insulin

resistance,

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hypertension, and cardiovascular atherosclerosis in rodents.23,24 Previously, Wistar rats were fed

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with HFD for 5 weeks to induce obesity, and then a single yam dioscorin was orally

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administered once a day for another 5 weeks to these obese rats concurrent with HFD.25 The

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results revealed that yam dioscorin post-intervention toward pre-obese rats showed no reduction

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in body weight and fat tissues; however, the systolic blood pressure and impaired glucose

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tolerance of intervened rats significantly improved compared to the HFD group.25 Food peptides

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(such as HIRL and YPFVV) have been reported as improving dyslipidemia during lipid

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metabolisms in diet-induced animal models.26 The synthetic Asn-Trp (NW), from peptic

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hydrolysis in silico of yam dioscorin, has been reported to have antioxidant and anti-aging

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activities in vitro and in vivo.15,17 The HFD-induced obesity can promote inflammation and

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reactive oxygen species via the renin-angiotensin system of adipocyte tissues in obese animal

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models.6 Therefore, this study fed C57BL/6J mice with HFD concurrent with a single yam

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dioscorin (80 mg/kg) or NW (40 mg/kg) intervention once a day from day 1 to day 135 to

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investigate the anti-obesity and impaired glucose tolerance controls in obese mice.

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█ MATERIALS AND METHODS

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Chemicals. HFD (D12492) was from Research Diets, Inc. (NJ, U.S.A.). Laboratory Rodent

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Diet 5001 was the normal diet for rodents from LabDiet Co. (MO, U.S.A.). Other chemicals

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were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Dipeptide NW for animal experiments

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in vivo was from Cellmano Biotech Co. (Hefei, China) which the purity was above 95%

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(supplementary Figure S1).

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The Yam Dioscorin Purification. Following the reported methods,16,20 yam tuber (D. alata

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cv. Tainong No.1) was peeled and homogenized by a blender in Tris buffer (50 mM, pH 8.3).

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The dioscorin in crude extracts was purified by the DE-52 ion exchange chromatography. After

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being washed with Tris buffer (50 mM, pH 8.3) for four column volumes, the dioscorin was

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eluted by 150 mM NaCl in the same buffer, and collected by a fraction collector. The eluted

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fractions monitored by 280 nm were pooled and dialyzed against deionized water at 4 °C, the

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purified dioscorin was lyophilized and stored at 4 °C for animal experiments. The purity of

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dioscorin was higher than 99% as previous reported.20

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Effects of Dioscorin or Dipeptide NW on HFD-induced Obesity in C57BL/6 Mice.

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The C57BL/6J mice (male, N=40) were purchased from the National Laboratory Animal Center

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(Taipei, Taiwan) were used in diet-induced obesity; the design and protocol of the animal

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experiments were approved with No. LAC-2014-0349. The five-week-old C57BL/6J mice, free

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water access under temperature, humidity, and light/dark cycle controls, were acclimated for one

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week, then, the mice were randomly grouped with four similar body weights (N=10), including

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one normal diet group, one HFD group as the control, and two intervened groups: the dioscorin

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group (80 mg/kg) and the dipeptide NW group (40 mg/kg). The dioscorin or NW was prepared

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daily in isotonic normal saline. For the intervention experiments, the dioscorin or dipeptide NW

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was fed orally by oral gavage once a day with free access to HFD for 135 days; for the normal

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diet group or HFD group, aliquots of isotonic normal saline were fed orally by oral gavage once

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a day with free access to HFD for 135 days.

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The body weights and cumulative feed intakes of mice in each group were recorded during

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the 135-day experiments. To calculate the feed conversion, the total amounts of diet intake (g)

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was divided by mouse weight gain (g) in the final. After being sacrificed, mouse organs (brain, 5



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liver, heart, and kidney) and visceral adipose tissues (mesenteric fat, perirenal fat, and

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epididymal fat) were collected and weighed for comparisons. The mouse blood was collected

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and assayed for contents of total cholesterol (TC), low-density lipoprotein (LDL), and total

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triglyceride (TG) by the National Laboratory Animal Center (Taipei, Taiwan).

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Effects of Dioscorin or Dipeptide NW Interventions on Impaired Glucose Tolerances

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in HFD-induced Mice. Dysfunction glucose tolerances in HFD-induced mice were investigated

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by oral glucose tolerance tests (OGTT) with some modifications.27 Mice of each group fasted

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overnight, and glucose (2 g/kg) was orally administered by a gavage. Blood was withdrawn

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from mice by capillary tubes from the retro-orbital sinus at fixed times during 120 min after the

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glucose loads. The assay kit for glucose level determined was from Randox Laboratories-US.

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The areas under the curve (AUC0-120) of blood glucose levels during 0 min to 120 min were

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calculated by additions of each assay value at 0, 5, 30, 60, 90, and 120 min.

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Statistics. Data were determined and expressed as mean ± SD. One-way analysis of

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variance (one-way ANOVA) followed by the post hoc Tukey’s test were used for four group

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comparisons. The identical marked alphabet among groups showed no significantly different (P

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> 0.05). The blood glucose levels of the OGTT in the fixed sampling time were compared by

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Student’s t-test between the intervened group or the normal diet group and the HFD group (the

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control). The any difference in comparison with the control was considered significant

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difference when P < 0.05 (*), or P < 0.01 (**), or P < 0.001 (***) by the GraphPad Prism 5.0

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software (San Diego, USA).

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█ RESULTS 6


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Reduction of Weight Gains of Dioscorin or Dipeptide NW Intervention in

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HFD-Induced C57BL/6 Mice. Figure 1 shows the weight changes of mice in the normal diet

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group, HFD group, and intervened groups together with HFD (dioscorin group, 80 mg/kg;

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dipeptide NW group, 40 mg/kg) during the 135-day experiments. The average body weights (g)

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of mice after 135-day intervention are 26.1 ± 1.4, 34.97 ± 2.1, 31.75 ± 2.6, and 31.66 ± 3.1,

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respectively, for normal diet-fed, HFD-fed, dioscorin-intervened, and NW-intervened group

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(Figure 1). It is clear that the HFD-fed mice showed the highest average body weight, and those

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fed a normal diet showed the lightest average body weight among the four mice groups during

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the experiments; these two groups showed a significant difference (P < 0.05) from day 15 to the

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end of the experiments. The mice fed with dioscorin (80 mg/kg) concurrently with HFD

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showed less weight gains, had a significant difference (P < 0.05) compared to those fed with

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HFD (the control) from day 15 to the end, and also showed different significantly (P < 0.05)

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compared to those fed a normal diet from day 65 to the end. The mice fed with dipeptide NW

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(40 mg/kg) concurrent with HFD showed less weight gains, had a significant difference (P

135-Day Interventions of Yam Dioscorin and the Dipeptide Asn-Trp

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