Comparison of Regulation Mechanisms of Five Mulberry Ingredients

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Comparison of regulation mechanisms of five mulberry ingredients on insulin secretion under oxidative stress Yun-chong Zheng, Hao He, Xing Wei, Sheng Ge, and Yan-Hua Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03845 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 3, 2016

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Journal of Agricultural and Food Chemistry

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Comparison of regulation mechanisms of five mulberry ingredients on insulin

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secretion under oxidative stress

3

Yun-chong Zheng,†, ‡ Hao He,†, ‡ Xing Wei, †, ‡Sheng Ge §,*and Yan-Hua Lu †, ‡,*

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†

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and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China

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‡

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Meilong Road, Shanghai 200237, People’s Republic of China

8

§

9

People's Hospital, Shanghai 200233, People’s Republic of China

State Key Laboratory of Bioreactor Engineering, East China University of Science

Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130

Clinical Nutrition Department, Shanghai Jiaotong University Affiliated Sixth

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*Correspondence author

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Dr. Yan-hua Lu

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State Key Laboratory of Bioreactor Engineering

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East China University of Science and Technology

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Box 283#, 130 Meilong Road, Shanghai 200237, People’s Republic of China

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E-mail: [email protected]

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Phone: +86-21-64251185

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Dr. Sheng Ge

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Clinical Nutrition Department

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Shanghai Jiaotong University Affiliated Sixth People's Hospital

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600 Yishan Road, Shanghai 200233, People’s Republic of China

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E-mail: [email protected]

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Phone：+86-21-24058352

Fax: +86-21-64251185

ACS Paragon Plus Environment


23 24

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Abstract：： The effects of mulberry ingredients including 1-deoxynojrimycin (DNJ),

25

resveratrol

(RES),

oxyresveratrol

26

cyanidin-3-rutinoside (C3R) on insulin secretion under oxidative stress were

27

investigated. The results revealed that they had distinct effects on insulin secretion in

28

H2O2-induced MIN 6 cells, especially DNJ, C3G and C3R, While RES and OXY

29

showed modest effects in low dose (12.5 µM). The mechanisms were demonstrated in

30

signal pathway that after treatment with DNJ, C3G and C3R, the expressions of

31

glucokinase (GK) were up-regulated, leading to intracellular ATP accumulation and

32

insulin secretion. They also bound to glucagon-like peptide-1 receptor (GLP-1R),

33

improved GLP-1R, duodenal homeobox factor-1 (PDX-1) expression and stimulated

34

insulin secretion. Moreover, ROS production was inhibited, followed by decreasing

35

apoptosis rate, while RES and OXY accelerated the apoptosis at high dose (50 µM).

36

This work expounded the potential mechanisms of mulberry ingredients on insulin

37

secretion,

38

hyperglycemia.

39

Keyword：insulin secretion, mulberry, β-cells apoptosis, oxidative stress

indicating the

(OXY),

cyanidin-3-glucoside

potential application

(C3G),

in the intervention

40


and

against

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INTRODUCTION

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Hyperglycemia, insulin resistance and β-cell function loss are the main features of

43

type 2 diabetes. Pancreatic islets are crucial for the development of type 2 diabetes,1

44

as β-cells are the most fragile ones with less antioxidant ability compared with other

45

tissue cells.2 Oxidative stress induced by hyperglycemia is characterized by the

46

overproduction of reactive oxygen species (ROS) which further damages pancreatic

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islets and reduces insulin secretion.3 These functional defects are often accompanied

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by reduction of mRNA expression of glucokinase (GK) and glucose transporter-2

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(GLUT2), further characterized by increased apoptosis with improved caspases

50

activities.4,5 Therefore, therapies for improving the β-cells function6 and expanding its

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mass become the potential new strategies to control hyperglycemia, with mostly

52

untested mechanisms.

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Postprandial hyperglycemia is always accompanied by increasing insulin

54

secretion, however, two main signaling pathways are crucial for glucose induced

55

insulin secretion.7 On the one hand, glucose is transported into pancreatic β-cells

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through GLUT2 and hydrolyzed by GK, resulting in ATP accumulation which in turn

57

stimulates adequate Ca2+ influx, followed by the release of insulin.8 On the other hand,

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glucagon-like peptide-1 (GLP-1), a gut hormone secreted by enteroendocrine L cells

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after meals, can bind to GLP-1 receptor (GLP-1R).9 Then adenylate cyclase (AC) is

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activated, leading to cAMP accumulation which acts as a secondary messenger to

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activate protein kinase A (PKA), Ca2+ influx and translocation of PDX-1 to the

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nucleus. Moreover, PDX-1 is recognized as a key factor in the cascade regulating

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insulin secretion and β-cells proliferation.10,11 Nowadays, in addition to some new



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antidiabetic agents namely GLP-1R agonists12,13 and GK activators (GKAs)

14

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under exploiting, recent researches on supplementation with natural products have

66

demonstrated the effects on alleviation of hyperglycemic status by protecting the

67

function of pancreatic β-cells.15–18

are

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Mulberry (Moraceae, Morus alba L.) is a traditional medicinal plant, which is

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widely distributed in China. Its fruits and leaves have been used as medicinal herbs to

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treat hyperglycemia19 and dietary consumption of mulberry leaves can help improve

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the regeneration of β-cells20, 21. The anti-diabetes effect may be due to the active

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ingredients,

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cyanidin-3-rutinoside (C3R), resveratrol (RES), and oxyresveratrol (OXY) (Figure 1).

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It is reported that DNJ, a main alkaloid abundant in mulberry leaf, the content reached

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1.389-3.483 mg/g, can up-regulate PDX-1 and insulin expression22 which further

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improves the postprandial glycemic control.23 RES can increase glucose induced

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GLP-1 secretion24, followed by activation of Ca2+ channel and increase of insulin

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secretion in normal status.25 Although there were only a few researches focused on the

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anti-diabetic effect of OXY, a structural analogue of RES, it had shown similar effects

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with RES on α-glucosidase and hepatic glucose metabolism in our previous

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works.26,27 Interestingly, RES and OXY were synthesized and secreted in mulberry

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skin, which up to 0.0068 and 0.0373 mg/g, to defense microbial infection and

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mechanical damage. Moreover, mulberry fruits potentially exhibit anti-diabetic effect

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and contain many active ingredients including C3G and C3R which have strong

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antioxidant activity.28 Song et al. found that mulberry fruits contain higher amounts of

namely

1-deoxynojrimycin

(DNJ),

cyanidin-3-glucoside


(C3G),

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anthocyanins than other well-known fruits such as elderberry and black berry. Sun et

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al. and Jayaprakasam et al. reported that C3G-rich bayberry extract could decrease the

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ROS production. In turn C3G could increase cellular viability and insulin

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production.29, 30

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Recent studies show that these ingredients (DNJ, C3G, C3R, RES, and OXY)

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might improve β-cells insulin secretion. However, they mainly focused on the

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promoting effects on insulin secretion. Few studies had discussed the mechanisms on

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insulin secretion and anti β-cells apoptotic under oxidative stress, and so far no report

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focused on the differences among mechanisms. Which one is the most effective

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insulin secretagogues? Can they promote the survival of β-cells under oxidative stress?

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It is of broad interest to clarify these issues for developing more effective antidiabetic

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drugs. In this study, the effects of mulberry ingredients including DNJ, RES, OXY,

98

C3G, and C3R on insulin secretion under oxidative stress and its corresponding

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mechanism were investigated.

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MATERIALS AND METHODS

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Materials. DNJ, C3G, and C3R were purchased from Shanghai Yaji Biotechnology

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Co., Ltd. (Shanghai, China). Resveratrol was purchased from Jianfeng-Natural

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Research and Development Co., Ltd. (Tianjin, China). The purity (≥ 98%) of these

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ingredients was identified by HPLC. Oxyresveratrol (98%) was isolated and purified

105

in our laboratory. All of these five ingredients were naturally present in mulberry, and

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isolated from the plant. Polyclonal antibodies against GLP-1R (sc-66911),

107

monoclonal antibodies against PDX-1 (sc-390808) and GK (sc-17819) were



108

purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Gliclazide was purchased

109

from National Institute for the Control of Pharmaceutical and Biological Products

110

(Beijing, China).

111

Cell Culture and viability assay. The MIN 6 cell line was purchased from MeiXuan

112

biology science and technology (Shanghai, China). The cells were cultured in

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Dulbeccos modified eagle medium (DMEM) containing 10% fetal bovine serum at

114

37 °C in a humidified atmosphere incubator with 5% CO2. The cells were seeded at a

115

density of 2×105 cells/mL in 24-well or 96-well plates for 24 h. Then, the cells were

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pre-incubated with five ingredients. Induction of oxidative stress was achieved by

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treating cells with H2O2.31 The effects of mulberry ingredients on cell viabilities of

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both normal and H2O2 induced MIN 6 cells were measured via MTT assay.

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Detection of Insulin Secretion. The insulin secretion assay was performed according

120

to a modified method of Hu et al.8 After 24 h pre-incubation with ingredients, the cells

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were incubated in fresh DMEM medium at either a normal condition or a H2O2

122

induced condition for 1 h. The cells were firstly washed with Krebs-Ringer

123

Bicarbonate buffer (KRB buffer, pH 7.4). Subsequently, KRB containing 5 mM and

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25 mM glucose (basal and glucose-stimulated) were used to incubate cells for 2 h at

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37 °C to stimulate insulin secretion. The supernatant was tested for insulin by ELISA

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kit (Genetimes Technology Inc, shanghai, China).

127

Molecular Docking. The structure of GLP-1R (3C5T) was obtained from the RCSB

128

protein Data Bank (http://www.pdb.org/pdb/home/home.do.). All compounds (DNJ,

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RES, OXY, C3G, C3R) structure were obtained from the National Centre for


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Biotechnology Information (NCBI) PubChem compound database and converted to

131

Protein Data Bank (PDB) coordinates. The interactions between ligand and target

132

proteins were analyzed by using the Autodock v4.2 program (Autodock, Autogrid).

133

All groups were carried out with 250 individuals according to previous work29,30 and

134

the most populated cluster with the lowest energy was chosen as the most reliable

135

conformation. The details of interactions were displayed by using Pymol 0.99 and

136

ligplus v1.4.5.

137

Evaluation of ROS and ATP production in MIN6 cells. Intracellular ROS and ATP

138

were monitored by using Reactive oxygen species Assay Kit and ATP assay kit

139

(Jiancheng, Jiangsu, China) respectively. In brief, after treatment with ingredients and

140

H2O2, the cells were incubated with 10 µM DCFH-DA at 37 °C for 30 min and

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dispersed with trypsin. The fluorescence intensity (λex = 485nm, λem = 525 nm) of

142

endogenous ROS was measured with microplate reader (SpectraMaxi3, Wals, Austria).

143

For ATP evaluation, treated cells were lysed to measure ATP production according to

144

the manufacturer’s instructions. The total protein content was quantified by BCA

145

assay kit. To consider cell proliferation, ROS and ATP levels were confirmed by

146

protein contents and results were calculated as relative fold change compare to control

147

group.

148

Detection and quantification of apoptosis of MIN6. The apoptosis rate of MIN 6

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cells was monitored by using a modified method.32 After treated as mentioned above,

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5×105 cells were resuspension in 500 uL. 5 µL Annexin V-FITC and 5 uL propidium

151

iodide (PI) was added to cells at room temperature for 10 min in darkness. Apoptosis



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cells rate was analyzed with a flow cytometer (FACSAria, Becton Dickinson, NJ,

153

USA). Early apoptotic cells were located in the lower right quadrant (LR), which

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indicated Annexin-positive/PI-negative. The late apoptotic cells were in the upper

155

right quadrant (UR) which means Annexin-positive/PI-positive.

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Western Blot Analysis. After the ingredients and H2O2 treatment, the cells were lysed

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to collect the total protein for western blot analysis. Protein samples (20 µg) were

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separated by SDS-PAGE before transferred to PVDF membranes (Millipore, Bedford,

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MA, USA). After blocking with 5% BSA in Tris-buffered saline overnight, GK,

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GLP-1R and PDX-1 were discriminated by 1:1000 dilution primary antibodies and

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HRP-conjugated antibody. The immune complexes of antibody were visualized with

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an ECL system according to the manufacturer’s instructions (Biotech well Co., Ltd.

163

Shanghai, China). Protein contents were normalized by GAPDH and band intensity

164

was quantified by Smart View Bio-electrophoresis Image Analysis System (Furi Co.,

165

Ltd, SH, China).

166

Statistical Analysis. All data were expressed as the mean ± SD. All the grouped data

167

were statistically performed with IBM SPSS V22.0. One-way analysis of variance

168

(ANOVA) and LSD was employed to determine the significant differences between

169

groups and P < 0.05 were considered statistically significant.

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RESULTS

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Oxidative damage reduced insulin secretion. The cell viability was reduced

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significantly by treating with different concentrations of H2O2 (100-2000 µM). It was

173

less than 75% in the presence of 250 µM H2O2 for 1 h and did not rapidly decrease


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with the continuously increased concentration of H2O2 (Figure 2A). Meanwhile, the

175

basal and glucose-stimulated insulin secretion (BIS and GSIS) was reduced to 58%

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and 62% comparing with control respectively (Figure 2B). So, for establishing

177

diabetes model of pancreatic β-cells, treatment with 250 µM H2O2 for 1 h was chosen

178

to induce oxidative damage. Based on the results of MTT assay, it was suggested that

179

all compounds at 12.5-50 µM had no significant cytotoxicity in normal MIN 6 cells

180

(Figure S1) and two anthocyanins (C3G, C3R) could even improve normal MIN 6 cell

181

proliferation. RES and OXY showed a moderate cytotoxicity to normal MIN 6 cells at

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50 µM, but it did not reach a significant level.

183

Insulin secretion and cell morphology were changed by mulberry ingredients

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treatment in H2O2 induced MIN 6 cells. In order to investigate the effects of

185

mulberry ingredients on insulin secretion, MIN 6 cells were incubated with mulberry

186

ingredients for a sustained period of 24 h and then exposed to 250 µM H2O2 for 1 h.

187

As shown in Figure 3AB, H2O2-exposed MIN 6 cells had a reductive insulin secretory

188

response when glucose was raised from 5 to 25 mM, with a 38% and 42% decrease in

189

insulin secretion compared with control group respectively. 50 µM gliclazide, an

190

effective insulin secretagogue, almost completely restored the impaired insulin

191

secretion

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concentration-related manner, but it only showed a moderate effect on BIS. Only

193

when the concentration of DNJ reached 50 µM, it could increase BIS 22% compared

194

with H2O2 treating group (P < 0.05). C3G and C3R significantly promoted both GSIS

195

and BIS, and the most effective dose was 50 µM (P < 0.01). Compared with gliclazide,

in

oxidation

state.

DNJ

significantly


improve

GSIS

in

a


196

C3G and C3R showed stronger effects on GSIS at 50 µM. RES and OXY at low dose

197

(12.5 µM) could improve the GSIS, but less promoting effects were observed under 5

198

mM glucose. Unfortunately, RES and OXY failed to improve insulin secretion when

199

their concentrations were raised from 25 to 50 µM. In order to investigate the

200

relationship between insulin secretion and cell morphology, the morphology of the

201

tested cells was shown in Figure S3. Light cell contraction happened by treating with

202

H2O2, some of the mulberry ingredients could alleviate this effect, especially DNJ and

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C3G.

204

Effects of mulberry ingredients on ATP production and GK expression of MIN 6

205

cells. In order to clarify the mechanisms of mulberry ingredients on insulin secretion,

206

ATP production was first determined. As shown in Figure 4A, exposure to H2O2 led to

207

more than 25% (P < 0.01) ATP lose while 50 µM gliclazide could improve ATP

208

production under oxidative stress (P < 0.05). DNJ could significantly increase ATP

209

production when its concentration reached 50 µM (P < 0.05) while C3G and C3R

210

have even stronger effects. In RES and OXY groups, ATP production was reduced

211

modestly and only 50 µM RES showed a significant effect (P < 0.05).

212

In the pancreatic cells, GK was the rate limiting enzyme of glycolytic pathway

213

which was crucial for ATP production. As shown in Figure 4B, exposure to H2O2 led

214

to more than 50% GK lost while 50 µM gliclazide could up-regulate intracellular GK

215

level (P < 0.01). Treatment with DNJ, C3G and C3R could completely reverse the

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effects of H2O2 on GK protein (P < 0.01). RES and OXY at 12.5 µM up-regulated GK

217

expressions (P < 0.01). Unfortunately, GK expressions were significantly inhibited


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when they reached 25 µM. Their effects on GK expression were also observed in the

219

previous reports. Based on these results, it was indicated that mulberry ingredients

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could improve insulin secretion by up-regulating GK expression and ATP production.

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Mulberry ingredients bound to GLP-1R to mimicked GLP-1. To determine the

222

possible mechanism of mulberry ingredients on GLP-1R activation and subsequent

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insulin secretion, a 3D structure of GLP-1R (3C5T) extracellular domain33 was used

224

to analyze the interactions between GLP-1R and ligand. As shown in the Figure 5,

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DNJ could bind to GLP-1R and hydrogen bonds were formed with GLU24, GLU127

226

and GLU128. For C3G, RES and OXY, it seemed that they could bind to GLP-1R at

227

the same site which contain ASP67, GLN112, ARG121 and formed hydrogen bonds

228

with these residues respectively. The conformations of C3R were scattered, and no

229

populated cluster was observed. According to previous reports33,34, ASP 67, ARG121

230

and GLU128 belonged to an area located in extracellular N-terminal domain of

231

GLP-1R which was important for ligand binding and selectivity. This area contained

232

LEU32, TRP39, ASP67, ARG121, LEU123, GLU127 and GLU128. The conserved

233

ASP67 of GLP-1R was the center and interacted directly with the side chain of

234

TRP72 and ARH121, which was very important to ligand binding. During the binding

235

process of GLP-1, the backbone carbonyl of residues (LYS26* and VAL33*) of

236

GLP-1 could interact through hydrogen bonds with GLU128 and ARG121 of GLP-1R

237

respectively. Based on the results above, it was indicated that mulberry ingredients

238

might mimic the effects of GLP-1 to activate GLP-1R and subsequent insulin

239

secretion.



240

Effects of mulberry ingredients on H2O2 induced MIN 6 cells apoptosis. In order

241

to clarify mechanisms of mulberry ingredients on pancreatic cell biomass, the

242

viability of MIN 6 cells under oxidative stress was first investigated. As shown in

243

Figure 6A, DNJ, C3G and C3R prevented H2O2-induced MIN 6 cell death in a dose

244

related manner which showed significant effects until 50 µM (P < 0.05).

245

Unfortunately, RES and OXY accelerated the H2O2-induced cell death. It is suggested

246

that their modulatory effects on GSIS and BIS may be attributed to their effects on

247

pancreatic cell biomass.

248

As shown in Figure 6B, exposure to H2O2 led to about 60% cells apoptosis while

249

50 µM gliclazide could significantly inhibit cells apoptosis and the apoptosis rate was

250

lower than 40%.( P < 0.01). The apoptosis rates of MIN 6 cells were significantly

251

decreased after being incubated with DNJ, C3G, C3R (P < 0.01). The inhibitory

252

effects on cell apoptosis were much stronger than positive control even at low dose.

253

Unfortunately, although RES and OXY at low dose could inhibit MIN 6 cell apoptosis,

254

the processes of apoptosis were accelerated in the presence of 50 µM RES and OXY.

255

Based on above results, it is indicated that the regulatory effects of mulberry

256

ingredients on insulin secretion were partially attributed to their moderate effects on

257

MIN 6 cells apoptosis induced by H2O2.

258

Effects of mulberry ingredients on ROS production. During the apoptotic process,

259

H2O2 induced ROS production was an important factor causing pancreatic cell death.

260

As shown in Figure 7, the level of ROS in H2O2 treated group was much higher than

261

normal group which was almost ten times of normal group. Compared with gliclazide,


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DNJ, C3G and C3R at all concentrations showed much stronger inhibitory effects on

263

ROS production. By incubating with DNJ, C3G and C3R, the ROS level was

264

gradually reduced. DNJ was the most effective ingredient among the three and the

265

ROS level was reduced to 30% compared with H2O2 treated group (P < 0.01). For the

266

RES group, 12.5-25 µM RES could delay the production of ROS, but the level of

267

ROS was raised sharply in the presence of 50 µM RES. OXY showed a similar but

268

moderate effect on ROS, which gradually increased ROS level with the continuously

269

improved concentration of OXY. These results suggested that mulberry ingredients

270

could improve H2O2 induced MIN 6 cell apoptosis by changing ROS level.

271

Mulberry ingredients regulated β-Cell function via up-regulating GLP-1R and

272

PDX-1 in expressions level. In order to determine which step was impaired in

273

oxidative stress status in H2O2-induced MIN 6 cells, the expressions of a series of

274

regulators involving insulin secretion were compared. As shown in the Fig 8A and B,

275

H2O2 significantly down-regulated the protein expressions of both GLP-1R and

276

PDX-1, while gliclazide significantly improved their expressions. According to Fig

277

8A and B, mulberry ingredients showed similar effects on GLP-1R and PDX-1

278

expressions. DNJ, C3G and C3R at low dose (12.5 µM) up-regulated their expressions,

279

but did not reach a significant level. When the concentration of DNJ, C3G and C3R

280

continued to increase, the levels of GLP-1R and PDX-1 were higher than that of

281

control group. Interestingly, low dose of RES and OXY improved GLP-1R and

282

PDX-1 expressions under oxidative stress induced by H2O2. However, high dose of

283

RES and OXY gradually suppressed their expressions. Based on the results above, it



284

is indicated that mulberry ingredients could manage insulin secretion by regulating the

285

expressions of insulin secretion regulators including GLP-1R, PDX-1 and GK.

286

DISSCUSSION

287

In initial stage of type 2 diabetes, the target cells of insulin do not respond

288

properly to the insulin (insulin resistance). This result in a growing demand of insulin

289

to keep the blood glucose levels normal. At later stages of the disease, the

290

compensatory increase in insulin production apparently cannot keep up with the

291

increased insulin demand. This leads to a relative insulin shortage and β-cell

292

apoptosis.35 According to the development of diabetes, pancreatic islet β cells are the

293

key in regulation of blood glucose and more important for treatment of diabetes than

294

insulin resistance. Damage of β cell leads to reduction in insulin production, resulting

295

in hyperglycemia and continuous generation of additional oxidative stress, forming a

296

vicious cycle. It has been reported that pretreatment of mulberry ingredients including

297

DNJ, RES, OXY, C3G and C3R could improve insulin secretion. However, previous

298

studies mainly focused on their promoting effects on insulin secretion and few had

299

discussed their insulin secretion and anti β-cells apoptotic mechanisms under

300

oxidative stress.

301

In the present study, the effects and underlying mechanisms of mulberry

302

ingredients on MIN 6 cells were investigated. The basal insulin secretion (BIS) and

303

glucose-stimulated insulin secretion (GSIS) were reduced under oxidative stress.

304

According to the results, treatment with DNJ, C3G and C3R (12.5-50 µM)

305

significantly stimulated the GSIS compared with H2O2 induced group, meanwhile


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306

C3G and C3R could also gradually improve BIS. Unfortunately, only low dose of

307

RES and OXY could improve GSIS, and the insulin secretion was gradually

308

decreased with the continuously increased concentrations of RES and OXY. As

309

different impacts on cell viability were observed, such effects could be partly

310

attributed to not only the enhanced insulin secretion function, but also the improved

311

apoptosis status under oxidative stress.

312

According to previous reports,

7, 9

two main signaling pathways are crucial for

313

insulin secretion function. In the first pathway, glucose, which is transported into

314

β-cells by glucose transporter 2 (GLUT2), is metabolized by GK into

315

glucose-6-phosphate and ATP resulting in Ca2+ influx which in turn induces the

316

release of insulin. In the present study, DNJ, C3G and C3R showed similar effects.

317

They were observed to reverse the reduction of GSIS in MIN 6 cells under oxidative.

318

Meanwhile, the levels of GK and intracellular ATP were elevated by DNJ, C3G and

319

C3R, suggesting that the glucose stimulated insulin release pathway was affected. For

320

RES and OXY, two mechanisms may be involved in GK regulation. On the one hand,

321

low dose of RES and OXY could bind to GK at its allosteric site to stimulate GK,

322

which was confirmed in our previous paper (Figure S2). On the other hand, high dose

323

of RES could down-regulate the expression of GK, resulting in ATP loss and

324

subsequent reduction of insulin secretion. These phenomena were also observed in

325

hepatic cells.27

326

In the second pathway, insulin secretion is mediated by GLP-1 which is produced

327

in the intestine to stimulate its specific receptor (GLP-1R) located on the membrane of



328

β-cells.10,36 Adenylate cyclase (AC) is then activated to produce cAMP as a secondary

329

messenger, which in turn accelerates PDX-1 translocation and subsequent PRE-INS,

330

GK, GLUT2 expressions.10 Under simulated oxidative stress induced by H2O2, the

331

expression of GLP-1R in MIN 6 cells was decreased, accompanied with reduction of

332

both BIS and GSIS. Based on the regulatory mechanisms of GLP-1R, there were

333

mainly three ways to explain the reasons. First, it was reported that all mulberry

334

ingredients were effective α-glucosidase inhibitors in our previous study,26 in which

335

food was delayed to digest and could reach the terminal of intestinal, stimulating

336

GLP-1 expression.37 This indicated the in vivo promoting effects of mulberry

337

ingredients on insulin secretion might partially be attributed to the enhanced GLP-1

338

secretion. Second, they might bind to GLP-1R and mimic the effect of GLP-1,

339

promoting insulin secretary signal transduction. Third, the enhanced expression of

340

GLP-1R could increase the possibility of activation of downstream signals，finally

341

improved GK and GLUT 2 expressions, which aimed to confer glucose sensitivity to

342

β-cells.

343

level of GLP-1R, meanwhile bind to and stimulate GLP-1R to increase expression of

344

GLUT2 and GK. For RES and OXY, although they could also bind to GLP-1R like

345

C3G, the GLP-1R expression was gradually suppressed.

38

In the present study, DNJ, C3G and C3R could up-regulate intercellular

346

β-cells apoptosis is a main reason for the decreased insulin secretion in the late

347

stage of diabetes. Here, we further addressed the potential protective effects of

348

mulberry ingredients against H2O2 induced oxidative damage and cell apoptosis. In

349

the present study, DNJ, C3G and C3R could mitigate oxidative stress after treating


Page 16 of 42

Page 17 of 42


350

with H2O2 and reduce ROS production, which partially explained the anti-apoptotic

351

effect observed in MIN 6 cells. Moreover, PDX-1, a key factor required for β-cells

352

outgrowth and differentiation was also up-regulated by DNJ, C3G and C3R,

353

indicating that they could increase Pancreatic biomass. Unfortunately, high dose of

354

RES and OXY failed in inhibiting β-cells apoptosis. Instead, 50 µM RES increased

355

ROS production followed with accelerated β-cells apoptosis. The reason may be

356

attributed to their down-regulating effects on GK or PDX-1 expression, which results

357

in cell apoptosis and continuous generation of additional oxidative stress. Thus forms

358

a vicious cycle. However, this hypothesis need to be further confirmed in future study.

359

In this paper, the regulatory effects and mechanisms of five mulberry ingredients

360

(DNJ, RES, OXY, C3G, and C3R) on insulin secretion were investigated. Variant

361

effects on insulin secretion between different groups could be attributed to their

362

effects on β-cells secretary function and apoptosis under oxidative stress (Figure 9).

363

After treatment with mulberry ingredients, the intracellular GK, GLP-1R, PDX-1

364

levels in MIN 6 cells were significantly changed, leading to ATP accumulation and

365

subsequent insulin secretion. They could also reduce ROS production of MIN 6 cells,

366

followed by a decreased β-cells apoptosis rate. We speculate that GK is the connection

367

point between insulin secretion and apoptosis. However, the potential mechanisms on

368

cell proliferation and apoptosis were still not clear. In the apoptotic cells,

369

pro-apoptotic genes including Bad, Bid are up-regulated and anti-apoptotic genes

370

including Bcl-2, Bcl-xl are down-regulated, accompanied by activation of caspases.39

371

Moreover, the cell death is affected by IL-1β, NFκB.PDX-1 is a key regulatory factor



372

in the cascade stimulating insulin secretion and cell proliferation. The effects of

373

mulberry ingredients on PDX-1 protein expression were quantified via western

374

blotting, with the results showing increased PDX-1 level. Since PDX-1 is a key factor

375

in the cascade regulating insulin secretion and β-cells proliferation and also a

376

transcription factor which need to translocate into the nucleus, we hypothesized that

377

PDX-1 is another connection point between insulin secretion and apoptosis, and

378

increased PDX-1 protein level would enhance downstream gene expression activation,

379

and might be caused by activation of upstream signaling pathway such as PI3K-PKC

380

pathway.10,11 Effects of mulberry ingredients on these factors and subsequent PDX-1

381

activation will be systematically studied in our future research.

382

In summary, we have compared the regulatory effects of mulberry ingredients on

383

insulin secretion. Their impacts on β-cells secretory function and apoptosis are further

384

elaborated. It is suggested that they, especially C3G, might be used to improve

385

impaired insulin secretion in the late stage of diabetes. Dietary consumption of

386

mulberry leaves and fruits that are full of mulberry ingredients can significantly

387

improve impaired insulin secretion and subsequent hyperglycemia. Furthermore, the

388

bioavailability data reported by other studies supported our finding in vivo experiment,

389

such as DNJ concentration was 5908 ng/mL in plasma at 0.83 h after a single oral

390

dosing of SZ-A at 40 mg/kg.40 Additionally, the potential mechanisms of mulberry

391

ingredients on β-cells apoptosis, proliferation and GLP-1 secretion from intestine will

392

be investigated in the future.

393

ASSOCIATED CONTENT


Page 18 of 42

Page 19 of 42


394

Corresponding Authors

395

*(Y.-H. L.) Mail: State Key Laboratory of Bioreactor Engineering, East China

396

University of Science and Technology, Box 283,

397

130 Meilong Road, Shanghai 200237, People’s Republic of

398

China. E-mail: [email protected].

399

Phone: +86-21-64251185.

400

Fax: +86-21-64251185.

401

*(S. G.) Mail: Clinical Nutrition Department, Shanghai

402

Jiaotong University Affiliated Sixth People’s Hospital, 600

403

Yishan Road, Shanghai 200233, People’s Republic of China.

404

Email: [email protected].

405

Phone: +86-21-24058352.

406

Funding

407

This work was supported by the National Natural Science Foundation of China (No.

408

31600273) as well as the National Special Fund for State Key Laboratory of

409

Bioreactor Engineering (2060204)

410

Notes

411

The authors declare no competing financial interest.

412

ABBREVIATIONS USED

413

GK, glucokinase; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1

414

receptor， PDX-1, duodenal homeobox factor-1; ROS, reactive oxygen species; DNJ,

415

1-deoxynojrimycin; C3G, cyanidin-3-glucoside; C3R, cyanidin-3-rutinoside; RES,



Page 20 of 42

416

resveratrol; OXY ,oxyresveratrol; GKAs; glucokinase activators; DMEM, Dulbecco

417

minimum essential medium.GULT2, glucose transporter 2, AC, adenylate cyclase,

418

PKA,

419

SDS-Polyacrylamide gel electrophoresis, ELISA, enzyme linked immunosorbent

420

assay,

421

stimulated

422

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

protein

GAPDH,

kinase

A,

KRB,

Krebs-Ringer

glyceraldehyde-3-phosphate

insulin

secretion,

BIS,

Bicarbonate,

dehydrogenase,

basal

insulin


SDS-PAGE,

GSIS,

glucose

secretion,

MTT,

Page 21 of 42


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556

Figure captions

557

Figure

558

oxyresveratrol (OXY), Cyanidin-3-glucoside (C3G) and cyanidin-3-rutinoside

559

(C3R).

560

Figure 2 The impacts of H2O2 on the cell viability and insulin secretion. After

561

culturing in DMEM for 24 h, the cells were incubated with H2O2 for additional 1 h:

562

(A) cell viability was monitored by MTT, (B) glucose stimulated insulin secretion

563

(GSIS) and basal insulin secretion (BIS) were measured by ELISA. Each value

564

represents the mean ± SD of triplicate experiments.

565

Figure 3 The effect of mulberry ingredients on basal (5 mM glucose) and glucose

566

stimulated (25 mM glucose) insulin secretion under oxidative stress. (A) glucose

567

stimulated insulin secretion (GSIS), (B) basal insulin secretion (BIS). MIN 6 cells

568

were incubated with mulberry ingredients (12.5-50 µM) for 24 and with H2O2 for

569

additional 1h. Each value represents the mean ± SD of triplicate experiments. # vs

570

control; * vs ctrl+H2O2 (*) P < 0.05 and (**) P < 0.01 as compared with control.

571

Figure 4 The effects of mulberry ingredients on ATP production and GK

572

expression. After pretreatment, the cells were lysed and (A) ATP production and (B)

573

GK protein level were analyzed by ATP assay kit and western blotting respectively.

574

Each value represents the mean ± SD of triplicate experiments. # vs control; * vs

575

ctrl+H2O2 (*) P < 0.05 and (**) P < 0.01 as compared with control.

576

Figure 5 Docking model predicted interaction details between GLP-1R and

577

mulberry ingredients. (A, B) RES; (C, D) OXY; (E, F) DNJ; (G, H) C3G. The

1

Structures

of

1-deoxynojirmycin

(DNJ),


resveratrol

(RES),


578

docking study was carried out by using Autodock v4.2 program. The details of

579

interactions were displayed by using Pymol 0.99 and ligplus v1.4.5.

580

Figure 6 The effects of mulberry ingredients on H2O2 induced ROS changes.

581

After pretreatment, the ROS changes were monitored by using DCFH-DA kit and the

582

result was confirmed by protein contents. Each value represents the mean ± SD of

583

triplicate experiments. # vs control; * vs ctrl+H2O2 (*) P < 0.05 and (**) P < 0.01 as

584

compared with control.

585

Figure 7 The effects of mulberry ingredients on H2O2 induced cell viability and

586

apoptosis. After pretreatment, (A) the cell viability was measured by MTT, (B)

587

apoptosis rate determined by flow cytometry. The percentage of total apoptotic cells

588

represent the average (mean ± SD, n = 3) of the early and late apoptosis rates. # vs

589

control; * vs ctrl+H2O2 (∗) P < 0.05 and (∗∗) P < 0.01 as compared with control.

590

Figure 8 The effects of mulberry ingredients on H2O2 induced GLP-1R and

591

PDX-1 expressions. After pretreatment, the cells were lysed.（A）GLP-1R, （B）

592

PDX-1and GAPDH were analyzed by Western blotting. The relative band intensity

593

was analyzed by Smart View Bio-electrophoresis Image Analysis System, and protein

594

contents were normalized by GAPDH. Each value represents the mean ± SD of

595

triplicate experiments. # vs control; * vs ctrl+H2O2 (*) P < 0.05 and (**) P < 0.01 as

596

compared with control.

597

Figure 9 Schematic representation of the major regulatory steps. The signal

598

pathways include glucose-induced and GLP-1-induced insulin secretion. The potential

599

mechanisms of mulberry ingredients on β-cells apoptosis were written in gray color.


Page 28 of 42

Page 29 of 42


600

Figure 1

601

602



603

Figure2 (A)

604 605

(B)

606


Page 30 of 42

Page 31 of 42


607

Figure 3

(A)

608

(B)

609 610



611 612

Figure 4

(A)

613

(B)

614


Page 32 of 42

Page 33 of 42


615 616



617

Page 34 of 42

Figure 5 (A)

(B)

RES

RES 618

(D)

(C)

OXY

619 620

(F)

(E)

621 622

OXY

DNJ


DNJ

Page 35 of 42


(G)

623

(H)

C3G


C3G


624

Figure 6

(A)

625

(B)

626


Page 36 of 42

Page 37 of 42


627



628

Figure7

629


Page 38 of 42

Page 39 of 42


630

Figure8

(A)

631

632 633



(B)

634

635 636


Page 40 of 42

Page 41 of 42


637

Figure 9

638



639

Graphic for Table of Contents

640 641

Brief Summary

642

Five bioactive ingredients namely 1-deoxynojirimycin (DNJ), resveratrol (RES),

643

oxyresveratrol (OXY), cyanidin-3-glucoside (C3G) and cyanidin-3-rutinoside (C3R)

644

had been investigated for their regulatory effects on insulin secretion under oxidative

645

stress. The results indicated that the potential mechanisms of mulberry ingredients on

646

insulin secretion could be attributed to their effects on β-cells function and apoptosis

647

under oxidative stress, indicating the potential application of DNJ, C3G and C3R in

648

the intervention against hyperglycemia.


Page 42 of 42

Comparison of Regulation Mechanisms of Five Mulberry Ingredients

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