Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes

Subscriber access provided by UNIV OF DURHAM

Bioactive Constituents, Metabolites, and Functions

Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes Neurotrophic Factor Expression in the Mouse Brain Ayano Shimizu, Takakazu Mitani, Sachi Tanaka, Hiroshi Fujii, Motohiro Maebuchi, Yusuke Amiya, Mitsuru Tanaka, Toshiro Matsui, Soichiro Nakamura, and Shigeru Katayama J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01581 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes Neurotrophic

2

Factor Expression in the Mouse Brain

3

4

Ayano Shimizu†, Takakazu Mitani‡, Sachi Tanaka†, Hiroshi Fujii‡, Motohiro Maebuchi§,

5

Yusuke Amiya∥, Mitsuru Tanaka∥, Toshiro Matsui∥, Soichiro Nakamura†, and Shigeru

6

Katayama*†‡

7

†

8

8304 Minamiminowa Kamiina, Nagano 399-4598, Japan

9

‡

Department of Agriculture, Graduate School of Science and Technology, Shinshu University,

Interdisciplinary Cluster for Cutting Edge Research (ICCER), Shinshu University, 8304

10

Minamiminowa Kamiina, Nagano 399-4598, Japan

11

§

12

Ibaraki 300-2497, Japan

13

14

∥

Research Institute for Creating the Future, Fuji Oil Holdings Inc., 4-3, Kinunodai, Tsukuba,

Division of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Graduate

School, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

15

16

Corresponding author:

17

Shigeru Katayama, Ph.D.

18

Telephone/FAX: +81-265-77-1603. E-mail: [email protected]

1 ACS Paragon Plus Environment


19

ABSTRACT

20

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays an

21

important role in cognitive abilities, including memory and learning. We have demonstrated that

22

soybean protein hydrolysate (SPH) diet suppresses age-related cognitive decline, via the

23

upregulation of BDNF in a mouse model of senescence. Our purpose was to identify novel

24

bioactive peptides in SPH, which enhance BDNF expression. We treated mouse primary

25

astrocytes with SPH, as well as with its positively-charged chromatographic fraction. Significant

26

increases in the expression of BDNF were observed in the treatment with positively-charged

27

fraction of SPH. Among the synthesized peptides, the dipeptide glycine-arginine (GR) increased

28

BDNF expression in vitro, and TNBS-LC-TOF-MS analysis showed the presence of GR in the

29

SPH. Furthermore, its administration in vivo increased the expression of BDNF in the cerebral

30

cortex, and the number of neurons in hippocampus and cerebral cortex. These data indicate that

31

GR might promote neurogenesis by upregulating BDNF levels.

32

33

Key words: astrocytes, BDNF, soybean protein hydrolysate, glycine-arginine dipeptide,

34

neurogenesis


Page 2 of 34

Page 3 of 34


35

INTRODUCTION

36

Aging is an inevitable biological process which constitutes the progressive decline of

37

physical and functional tissue capacities. Brain aging is defined as a progressive loss of

38

neurophysiological functions, which is often accompanied by age-related neurodegeneration.

39

Particularly, cognitive impairment and dementia, including Alzheimer's disease (AD), have

40

emerged as major debilitating illnesses associated with old age.1,2. In recent years, there has been

41

an increasing interest in natural dietary bioactive compounds with potential neuroprotective

42

properties, in order to prevent cognitive impairment and dementia.3 Omega-3 (ω-3)

43

polyunsaturated fatty acids (PUFAs), and especially docosahexaenoic acid (22:6,n-3), mainly

44

present in fish oil, play an important role in normal brain development and cognitive function.4

45

Some studies in animal models and humans suggest that dietary ω-3 PUFA intake can slow down

46

cognitive decline and attenuate the physiological brain disabilities.5 On the other hand, botanical

47

polyphenols have been reported to attenuate mood disorders and cognitive impairment,6,7 and

48

some flavan-3-ols, including anthocyanidins, such as catechin and epicatechin, have been shown

49

to selectively reach the brain and accumulate in the form of metabolites.8

50

Neurotrophins have emerged as a group of powerful molecular mediators in central

51

synaptic plasticity. Among these, brain-derived neurotrophic factor (BDNF) and neurotrophin-3

52

(NT-3) are considered as key players in the neurobiological mechanisms of learning and

53

memory.9 Thus, the therapeutic modulation of BDNF and NT-3 levels is a promising treatment

54

strategy for neurological and psychiatric disorders, in which BDNF levels are dysregulated. 3 ACS Paragon Plus Environment


55

Moderate exercise has been demonstrated to increase BDNF levels in the plasma.10 Maejima et

56

al. reported that moderate exercise contributes in the protection against cognitive function

57

decline in the elderly, through the up-regulation of BDNF expression in the hippocampus.11

58

Food-derived peptides have various functions in human health. Recently, functional

59

peptides with brain-health benefits have been reported. Yamada et al. demonstrated that the

60

enzymatic digestion products of β-lactoglobulin, a major component of bovine milk whey,

61

exhibit anxiolytic-like activity in mouse behavioral experiments.12 We have previously reported

62

that oral administration of soybean protein hydrolysates (SPH) for 6 months results in

63

suppression of the age-related cognitive decline in senescence-accelerated prone 8 (SAMP8)

64

mice, and in upregulation of BDNF and NT-3 expression in the brain of SPH-fed mice.13

65

However, the bioactive peptides in SPH remain unknown. In this study, we identified the

66

bioactive peptides in SPH, which exhibited a BDNF-enhancing effect. We further investigated

67

the in vivo effects of the oral and intraperitoneal administration of SPH, as well as of a specific

68

bioactive peptide, in healthy mice.

69

70

MATERIALS AND METHODS

71

Chemicals

72

SPH composed mainly of di- and tripeptides was obtained from Fuji Oil Co. Ltd. (Osaka,

73

Japan). The synthesized peptides KGRKG, KGRK, GRKG, KRG, KGR, GRK, RKG, KG, GR,

74

and RK were obtained from GenScript Japan (Tokyo, Japan). 4 ACS Paragon Plus Environment

Page 4 of 34

Page 5 of 34


75

76

Isolation of aromatic and positively-charged peptide fraction from SPH

77

For the preparation of aromatic peptides, 20 % (w/w) SPH solution (pH 7.0) was prepared,

78

by adding 19 volumes of 95 % ethanol, and kept at -20 °C for 30 min. After centrifugation at

79

5,000 rpm for 10 min, the supernatant, containing the aromatic peptides, was collected. For the

80

preparation of positively-charged peptides, SPH was dissolved in 10 mM ammonium formate

81

(pH 8.0) and loaded onto a DEAE-Sepharose Fast Flow column (GE Healthcare, Wauwatosa, WI,

82

USA). The flow-through fraction, containing the positively-charged peptides, was collected .

83

84

Animals

85

Eight-week-old male and ten-week-old female C57BL/6J mice were purchased from

86

Charles River Laboratories Japan, Inc., (Kanagawa, Japan) and mated to obtain embryos.

87

Pregnant females were sacrificed and embryos were collected at embryonic day 18 for primary

88

cultures. For in vivo experiments, six-week-old male C57BL/6L mice were also obtained from

89

Charles River Laboratories Japan, Inc. The mice were provided with free access to a

90

commercially-available pellet diet (MF; Oriental Yeast Co., Tokyo, Japan) and water. They were

91

acclimatized for one week before the start of the study. The animal room was maintained at

92

constant temperature (22 ± 2°C) and humidity (55 ± 10%), with a 12-h light/dark cycle. All

93

experiments were performed according to the institutional guidelines for animal experimentation

94

at Shinshu University (Animal ethics, No. 270076 and 270077). 5 ACS Paragon Plus Environment


95

96

Primary astrocyte cultures

97

Astrocyte cultures were prepared from brains of mouse embryos. After removing the

98

uterus, brains were immediately dissected from each embryo, immersed in Dulbecco's modified

99

Eagle's medium/nutrient mixture F12 (DMEM/F12; Gibco/Invitrogen, Carlsbad, CA, USA), and

100

treated with 0.25 % trypsin (Wako, Tokyo, Japan) in phosphate buffered saline (PBS), containing

101

0.01 % DNase I (Worthington Biochem, Freehold, NJ, USA), at 37 °C for 10 min. After

102

treatment, the cell suspension was centrifuged at 1500 rpm for 5 min and the supernatant was

103

discarded. The cells were resuspended in DMEM/F12, containing 10 ng/mL G-5 supplement

104

(Invitrogen, Carlsbad, CA, USA) and 10 % fetal bovine serum (Biowest, Nouaille, France), by

105

gentle trituration with a pipette and the cell suspension was filtered through a cell strainer to

106

remove undigested tissue. The cells were seeded at 2.0×106 cells in 75-cm2 flasks and incubated

107

in a humidified atmosphere of 95% air and 5% CO2, at 37°C. Seven days later, astrocytes were

108

harvested with trypsin and seeded at 2.0 × 105 cells in 60-mm dishes for real-time PCR, ELISA,

109

and western blotting analyses. After four days of incubation, cells were treated with 1 mg/mL

110

SPH or with 1 or 10 µM synthesized peptides for 6 or 24 h, and collected for qPCR or western

111

blotting. The cultured cells were confirmed to be astrocytes by immunostaining with an anti-glial

112

fibrillary acidic protein (GFAP) antibody.

113

114

Luciferase reporter assay for CREB phosphorylation 6 ACS Paragon Plus Environment

Page 6 of 34

Page 7 of 34


115

Primary astrocytes were cultured at a density of 2.5 × 106 cells/well in a 48-well plate and

116

transiently transfected with the reporter vectors pCRE-Luc14 and pRL-SV40 (control reporter

117

vector; Promega, Madison, WI, USA), by using Lipofectamine 2000, for 24 h. After the

118

transfection medium was replaced with fresh medium, the cells were incubated with SPH at a

119

concentration of 0.5 or 1 mg/mL for 4 h. The transfection efficiency was normalized to that of

120

pRL-SV40. Firefly and Renilla luciferase activities were measured using the Dual Luciferase

121

reporter assay kit and the GloMax 20/20 Luminometer (Promega). The relative light unit units

122

were normalized to the constitutively active Renilla luminescence of the same sample.

123

124

Real-time quantitative PCR (qPCR)

125

Total RNA was prepared using RNAiso Plus (TaKaRa Bio, Shiga, Japan). RNA was used

126

to reverse transcription using the ReverTra Ace real-time qPCR kit (Toyobo, Osaka, Japan).

127

Real-time qPCR was carried out using a Kapa SYBR Fast qPCR kit (Kapa Biosystems, Woburn,

128

MA, USA). Quantification of relative gene expression was performed by using the Thermal

129

Cycler Dice real time system software (Takara Bio). β-actin was used as an internal standard to

130

normalize the amount of total RNA present in each reaction mix, and the relative expression in

131

each experimental group was expressed as the fold change compared to a control sample, using

132

the

133

5'-GCGGCAGATAAAAAGACTGC-3' (forward) and 5'-CTTATGAATCGCCAGCCAAT-3'

134

(reverse), and for β-actin, 5’-CACTATTGGCAACGACAAGCGGTTC-3’ (forward) and

comparative

∆∆Cq

method.

The

primer


sequences

for

BDNF

were


135

5’-ACTTGCGGTGCACGATGGAG-3’ (reverse).

136

137

Western blot analysis

138

Astrocytes were collected using a cell scraper and homogenized in RIPA lysis buffer

139

(Santa Cruz Biotechnology, Santa Cruz, CA, USA) on ice. Cell lysates were centrifuged at

140

12,000 g for 10 min at 4°C, and the supernatants were collected. The total protein concentration

141

was determined using the BCA assay with bovine serum albumin (BSA) as the standard. Proteins

142

(15 µg) were separated by electrophoresis, on 15 % polyacrylamide gels, and transferred to

143

polyvinylidene difluoride membranes (Clear Blot Membrane-P; ATTO, Tokyo, Japan). After

144

blocking with 3 % BSA in Tris-buffered saline buffer, containing 0.1 % Tween 20, for 60 min at

145

room temperature, the membranes were incubated with primary antibodies against BDNF

146

(1:1,000, Abcam, Cambridge, MA, USA) and β-actin (1:5,000, Santa Cruz Biotechnology)

147

overnight at 4°C or for 1 h at room temperature, respectively. The membranes were then

148

incubated with the secondary anti-rabbit HRP-conjugated (1:5,000, Santa Cruz Biotechnology)

149

and anti-mouse HRP-conjugated antibodies (1:10,000, Santa Cruz Biotechnology) for 60 min at

150

room temperature, for the detection of BDNF and β-actin, respectively. Chemiluminescence

151

detection was performed using the Pierce Western blotting substrate (Thermo Scientific,

152

Rockford, IL, USA) and AE-9300 Ez-Capture (ATTO). proBDNF and mature BDNF (mBDNF)

153

were detected as a band of 35 kDa and 28 kDa, respectively.

154


Page 8 of 34

Page 9 of 34


155

Quantitative assay of peptides in SPH by LC-TOF-MS

156

Target peptides in SPH were assayed by a liquid chromatography time-of-flight mass

157

spectrometry (LC-TOF-MS) in combination with 2,4,6-trinitrobenzen sulfonate (TNBS)

158

derivatization technique.15 The quantification of peptides was performed according to our

159

reported standard additon method16, because the method could compensate unexpected

160

reduction of MS detection by interferring matrix without any use of appropriate internal

161

standard or expensive stable isotope labelling technique. Briefly, target peptides were spiked

162

into SPH solution (1 mg/mL in 0.1 M borate buffer, pH 8.0) with the final cncentration of 0, 4,

163

8, and 16 µg/mL. The spiked solutions (40 µL) were subjected to TNBS derivatization reaction

164

by adding 10 µL of 150 mM TNBS solution in 0.1 M borate buffer (pH 8.0) at 30 °C for 30 min.

165

Fifty µL of 0.2 % formic acid were, then, added to 50 µL of the resultant mixture, and an

166

aliquot (10 µL) was injected to LC-TOF-MS.

167

Chromatographic separation was carried on a Waters Biosuite C18 column (2.1 × 150 mm,

168

3-µm pore size) (Waters, Milford, MA, USA). A linear gradient elution of methanol (0–100%),

169

containing 0.1% formic acid, at a flow rate of 0.2 mL/min, was performed at 40°C. Electrospray

170

ionization (ESI)-TOF-MS analysis was carried out in positive ESI mode, and the mass-detection

171

range was set at 100-1,000 m/z. The conditions of the ESI source were as follows: drying gas

172

(N2) flow rate = 8.0 L/min; drying gas temperature = 200 °C; nebulizing gas pressure = 1.6 bar;

173

capillary voltage = 3,800 V. All data acquisition and analyses were performed by using a Bruker

174

data analysis 3.2 software. To ensure optimal conditions for the analyses, we calibrated the 9 ACS Paragon Plus Environment


175

detector using sodium formate clusters (10 mM sodium formate in water:acetonitrile (1:1, v/v)).

176

The calibration solution was injected at the beginning of the run and all spectra were calibrated

177

prior to characterization. The width was set at 0.05 m/z for the mono-isotopic isolation of the

178

target ions: trinitrophenyl (TNP)-KGR and TNP-GRK = 571.222 m/z; TNP-GR = 443.127 m/z;

179

TNP-RK = 514.201 m/z.

180

181

In vivo experiments

182

For oral administration, 7-week-old male C57BL/6J mice were treated with saline, GR

183

dipeptide (15 or 100 mg/kg, once a day), or SPH (100 mg/kg, once a day) for 14 days. For

184

intraperitoneal administration, mice were treated with saline, GR dipeptide (15 mg/kg, once a

185

day), or SPH (100 mg/kg, once a day) for five days. Twenty-four hours after administration, the

186

mice were sacrificed, and brains were collected, and processed for western blot analysis.

187

188

Immunostaining

189

The collected brain tissues were fixed with 30 % paraformaldehyde and then embedded in

190

paraffin. The sections (6 µm) were blocked in PBS containing 10 % goat serum and 0.1 % Tween

191

20 and were incubated with a rabbit anti-NeuN antibody (Abcam) diluted in 1:750. After

192

washing six times with 0.1 % BSA in PBS, the sections were incubated with secondary antibody

193

(Alexa Fluor 488 goat anti-rabbit IgG, Abcam) diluted 1:1,000, washed six times with 0.1 %

194

BSA in PBS, and then were mounted in a mounting medium with 4',6-diamidino-2-phenylindole 10 ACS Paragon Plus Environment

Page 10 of 34

Page 11 of 34


195

(DAPI) (ImmunoSelect Antifading Mounting Medium; Dianova, Hamburg, Germany). Images

196

were taken using an EVOS fl fluorescence microscope (Advanced Microscopy Group, Bothell,

197

WA, USA).

198

199

Statistical analysis

200

Data are presented as mean ± standard deviation (SD). Data on BDNF mRNA expression

201

in primary astrocytes were analyzed using Student’s t-test. For all other comparisons, one- or

202

two-way ANOVA followed by Dunnett’s post hoc test was used. Differences with p 0.988. Moreover, we found that GR dipeptide was

243

successfully detected and quantified in the SPH used in this study (2.02 mg/g of SPH, Figure S1),

244

whereas other peptides (KGR, GRK, and RK) failed to be detected within the present MS

245

conditions, suggesting that SPH contains at least GR dipeptide at >0.2 wt% level.

246

247

Effects of GR and SPH administration on BDNF expression in the mouse brain

248

To examine the effects of GR administration in vivo, we measured BDNF expression

249

levels in the brain after oral administration for 14 days. In the hippocampus, there was a trend for

250

increased proBDNF levels after treatment with 15 mg/kg/day of GR, but it was not statistically

251

significant (Figure 4A). Neither GR nor SPH treatment changed mBDNF expression in the

252

hippocampus. On the other hand, a significant increase in proBDNF expression was observed in

253

the cerebral cortex of mice treated with GR at 100 mg/kg/day, whereas mBDNF expression

254

remained unaffected (Figure 4B). 13 ACS Paragon Plus Environment


255

We next investigated the effects of the intraperitoneal administration of GR (15

256

mg/kg/day) and SPH (100 mg/kg/day). In the hippocampus, GR treatment increased proBDNF

257

expression, whereas no changes in mBDNF expression level were observed by either treatment

258

(Figures 5A). In contrast, in the cerebral cortex, significant increases in the expression of both

259

proBDNF and mBDNF were observed in the GR- and SPH-treated mice (Figure 5B). These

260

results suggest that GR administration enhances BDNF levels in the brain, and especially in the

261

cerebral cortex; this effect was stronger after intraperitoneal than oral GR administration.

262

We further examined whether intraperitoneally-administered SPH can promote

263

neurogenesis, a process that includes the proliferation, survival, and differentiation of neurons.

264

For this purpose, we used the neuron-specific nuclear protein NeuN, as a mature neuronal marker.

265

The number of NeuN positive cells was higher in GR- than saline-treated mice, in both the

266

dentate gyrus of the hippocampus (Figure 6A) and the cerebral cortex (Figure 6B), thus

267

suggesting that GR treatment might positively affect neurogenesis.

268

269

DISUCSSION

270

In this work, we demonstrated that the positively-charged peptides GRK and GR, induce

271

proBDNF expression in primary astrocytes, while GR administration in vivo enhances BDNF

272

expression in the brain. According to the LC-TOF-MS results, only the GR, but not the GRK

273

peptide, was detected in the SPH. It is possible that during SPH preparation, the enzymatic

274

treatment might have resulted in the cleavage of GRK to GR. To clarify the in vivo effect and 14 ACS Paragon Plus Environment

Page 14 of 34

Page 15 of 34


275

metabolism of the GR dipeptide, we compared the effects of the oral to those of the

276

intraperitoneal administration. The most significant increase was observed in proBDNF

277

expression in the cerebral cortex of mice intraperitoneally-treated with GR for five consecutive

278

days. However, a significant, but lower, increase was also observed in the cortex of mice

279

orally-administered with GR. This discrepancy between the oral and intraperitoneal

280

administration might be due to the potential cleavage of GR by enzymes of the digestive tract.

281

No significant changes were observed after oral administration of SPH, suggesting that GR is a

282

more potent agent than SPH. Our previous work has demonstrated that long-term administration

283

of SPH enhances BDNF and NT-3 expression in the brain and suppresses the cognitive decline

284

observed in SAMP8 mice.13 However, the effects of oral GR administration in SAMP8 mice still

285

remain unclear and require further studies.

286

BDNF is known to bind to TrkB and thus activate the phosphatidylinositol-3 kinase

287

signaling pathway and the release of neurotransmitters for presynaptic sites.18,19 Previous studies

288

have reported that the cyclic pentapeptide cyclo(-D-Pro-Ala-Lys-Arg-) was designed to act as an

289

effective BDNF-like agonist. Massa et al.20 also demonstrated that subregion b of the second

290

loop (Ser-Lys-Gly-Gln-Leu) of BDNF is involved in TrkB activation. Furthermore,

291

Cardenas-Aguayo et al.17 demonstrated that tetrapeptides, such as Ac-Ile-Lys-Arg-Gly-CONH2

292

and Ac-Ser-Lys-Lys-Arg-CONH2, which mimic different active regions of BDNF, activate the

293

TrkB receptor and induce BDNF expression. These reports suggest that positively-charged amino

294

acids in BDNF, such as lysine or arginine, might play important roles in its binding to TrkB. In 15 ACS Paragon Plus Environment


295

our study, we show that the positively-charged fraction of SPH increases BDNF expression in

296

primary astrocytes. GR was identified as one of bioactive peptides promoting BDNF expression

297

from the SPH; however, the GR content in SPH was estimated at around 0.2 wt% level,

298

suggesting that there must be still potential peptides in the positively-charged fraction of SPH.

299

Here we focused on the the amino acid sequence of KGRKG and did not investigated the

300

sequence combination except that. Therefore, there might be a possibility that oligopeptides

301

containing GR sequence exert a potent enhancing effect of BDNF expression.

302

In general, it is considered difficult for orally-administered peptides to reach the brain, as

303

they are easily digested to single amino acids; however, some bioactive peptides found in SPH

304

appear to directly contribute to BDNF regulation in the brain. Indeed, we found that both oral

305

and intraperitoneal administration of SPH and of its bioactive peptide, GR, increase BDNF levels

306

in the brain. However, it is still unknown whether these bioactive peptides cross the blood brain

307

barrier and reach different brain areas. Further investigation of the peptide distribution in the

308

brain will be necessary for the clarification of the underlying molecular mechanisms.

309

BDNF exerts an important role in the formation of appropriate synaptic connections, both

310

during development and during learning and memory in adults.21 It is well known that BDNF

311

protein is secreted as proBDNF, which is then cleaved extracellularly via the tissue plasminogen

312

activator/plasmin proteolytic cascade.22 Similarly, using western blotting, we detected BDNF in

313

the cell lysate mainly as proBDNF. We measured the secreted BDNF in the cell culture medium

314

by ELISA; however, BDNF levels were lower in the medium than the cell lysate (data not 16 ACS Paragon Plus Environment

Page 16 of 34

Page 17 of 34


315

shown). Since astrocytes are known to closely interact with neurons, a co-culture assay of

316

astrocytes and neurons will be necessary to examine the effects of SPH and its bioactive peptides

317

on BDNF secretion.

318

Several studies have demonstrated that orally administrated phytochemicals exhibit

319

positive effects on brain functions. For example, the oral administration of quercetin, a natural

320

flavonoid, increases the proliferation of mouse neural stem cells in the dentate gyrus, while

321

quercetin-3-o-glucuronide, one of the major quercetin metabolites, activates the Akt/cyclin D1

322

and BDNF signaling pathways.23 Further, oral administration of 7,8-dihydroxyflavone, a potent

323

TrkB agonist, prevents the deposition of amyloid beta (Aβ) and inhibits the synaptic loss seen in

324

the hippocampus of a transgenic mouse model of AD, by activating TrkB signaling.24

325

Concerning the beneficial effects of soybean-derived bioactive compounds on brain function,

326

isoflavone is considered as a potent neuromodulating agent. Oral administration of soybean

327

isoflavone in rats suppresses the Aβ1-42-induced neuronal damage, via activation of the

328

N-methyl-D-aspartate receptor signaling pathway.25 However, Chatterjee et al. reported that

329

genistein, an isoflavone abundantly found in soybean, enhances the accumulation of the Aβ

330

peptide, by increasing the expression of the amyloid precursor protein.26 Therefore, the

331

beneficial effect of soybean isoflavone against AD is still questionable. In this study, we

332

demonstrated that bioactive peptides of soybean ingredients are good candidates as

333

neuromodulating agents, which might contribute to the suppression of Aβ1-42-induced neuronal

334

damage. Hence, further studies on the potential beneficial effects of soybean peptides against AD 17 ACS Paragon Plus Environment


335

are required, with a special focus on the effects of the positively-charged fraction or the GR

336

dipeptide, identified in this study.

337

In conclusion, we demonstrated that the GR dipeptide contained in SPH enhances BDNF

338

expression both in vitro, in primary astrocytes, and in vivo, in the brain, and especially in the

339

cerebral cortex. These findings provide useful insight on the biochemical processes by which soy

340

proteins and peptides may promote good health in the elderly.

341

342

ACKNOWLEDGMENTS

343

This study was supported by the Ministry of Education, Culture, Sports, Science, and

344

Technology of Japan KAKENHI (Grant No. 15K07426); the Ministry of Agriculture, Forestry,

345

and Fisheries of Japan (Integration research for agriculture and interdisciplinary fields); and Fuji

346

Oil Holdings Inc. (Osaka, Japan).

347

348

Notes

349

The authors declare no competing financial interest.

350

351

References

352

(1)

353

supplementation on the aging process. Clinical Interventions in Aging. 2007, 2, 377.

354

(2)

Fusco, D.; Colloca, G.; Monaco, M. R. L.; Cesari, M. Effects of antioxidant

Ferrari, C. K. Functional foods and physical activities in health promotion of aging people. 18 ACS Paragon Plus Environment

Page 18 of 34

Page 19 of 34


355

Maturitas. 2007, 58, 327-39.

356

(3)

357

cognitive function. Nutr. Clin. Care. 2001, 4, 156-67.

358

(4)

359

and disease. Nature Reviews Neuroscience. 2014, 15, 771-85.

360

(5)

361

Bernoud-Hubac, N. The pleiotropic effects of omega-3 docosahexaenoic acid on the hallmarks of

362

Alzheimer's disease. The Journal of nutritional biochemistry. 2016, 38, 1-11.

363

(6)

364

“natural drugs” for promotion of resilience against stress-induced depression and cognitive

365

impairment. Neuromolecular Med. 2016, 18, 487-95.

366

(7)

367

supplementation up-regulates HSP70 and suppresses cognitive decline in a mouse model of

368

accelerated senescence. Journal of Functional Foods. 2018, 44, 292-8.

369

(8)

370

N.; Raftery, D.; Arrieta-Cruz, I. Brain-targeted proanthocyanidin metabolites for Alzheimer's

371

disease treatment. Journal of Neuroscience. 2012, 32, 5144-50.

372

(9)

373

Neurogenesis and neural plasticity, Springer: 2013; pp 117-36.

374

(10) Szuhany, K. L.; Bugatti, M.; Otto, M. W. A meta-analytic review of the effects of exercise

Nicolas, A. S.; Nourhashemi, L. F.; Lanzmann‐Petithory, D.; Vellas, B. Nutrition and

Bazinet, R. P.; Layé, S. Polyunsaturated fatty acids and their metabolites in brain function

Belkouch, M.; Hachem, M.; Elgot, A.; Van, A. L.; Picq, M.; Guichardant, M.; Lagarde, M.;

Ward, L.; Pasinetti, G. M. Recommendations for development of botanical polyphenols as

Kushimoto, S. U., Y.; Yanai, S.; Makabe, H.; Nakamura, S.; Katayama, S. Kale

Wang, J.; Ferruzzi, M. G.; Ho, L.; Blount, J.; Janle, E. M.; Gong, B.; Pan, Y.; Gowda, G.

Gómez-Palacio-Schjetnan, A.; Escobar, M. L., Neurotrophins and synaptic plasticity. In



375

on brain-derived neurotrophic factor. J. Psychiatr. Res. 2015, 60, 56-64.

376

(11) Maejima, H.; Kanemura, N.; Kokubun, T.; Murata, K.; Takayanagi, K. Effects of aging

377

and treadmill exercise on cognitive function and the expression of BDNF in the hippocampus.

378

The FASEB Journal. 2017, 31, 1020.5-.5.

379

(12) Yamada, A.; Mizushige, T.; Kanamoto, R.; Ohinata, K. Identification of novel β‐

380

lactoglobulin‐derived peptides, wheylin‐1 and‐2, having anxiolytic‐like activity in mice.

381

Mol. Nutr. Food Res. 2014, 58, 353-8.

382

(13) Katayama, S.; Imai, R.; Sugiyama, H.; Nakamura, S. Oral Administration of Soy Peptides

383

Suppresses Cognitive Decline by Induction of Neurotrophic Factors in SAMP8 Mice. J. Agric.

384

Food Chem. 2014, 62, 3563-9.

385

(14) Mitani, T.; Watanabe, S.; Yoshioka, Y.; Katayama, S.; Nakamura, S.; Ashida, H.

386

Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding

387

protein β degradation by adenosine receptor A1. Biochimica et Biophysica Acta

388

(BBA)-Molecular Cell Research. 2017, 1864, 2438-48.

389

(15) Nakashima, E. M.; Qing, H.-Q.; Tanaka, M.; Matsui, T. Improved detection of di-peptides

390

by liquid chromatography-tandem mass spectrometry with 2, 4, 6-trinitrobenzene sulfonate

391

conversion. Biosci., Biotechnol., Biochem. 2013, 77, 2094-9.

392

(16) Hanh, V. T.; Kobayashi, Y.; Maebuchi, M.; Nakamori, T.; Tanaka, M.; Matsui, T.

393

Quantitative mass spectrometric analysis of dipeptides in protein hydrolysate by a TNBS

394

derivatization-aided standard addition method. Food Chem. 2016, 190, 345-50. 20 ACS Paragon Plus Environment

Page 20 of 34

Page 21 of 34


395

(17) MdC, C.-A.; Kazim, S.; Grundke-Iqbal, I.; Iqbal, K. Neurogenic and neurotrophic effects

396

of BDNF peptides in mouse hippocampal primary neuronal cell cultures. PLoS One. 2013, 8,

397

e53596.

398

(18) Yamada, K.; Nabeshima, T. Brain-derived neurotrophic factor/TrkB signaling in memory

399

processes. J. Pharmacol. Sci. 2003, 91, 267-70.

400

(19) Schratt, G. M.; Nigh, E. A.; Chen, W. G.; Hu, L.; Greenberg, M. E. BDNF regulates the

401

translation

402

rapamycin-phosphatidylinositol 3-kinase-dependent pathway during neuronal development. J.

403

Neurosci. 2004, 24, 7366-77.

404

(20) Massa, S. M.; Yang, T.; Xie, Y.; Shi, J.; Bilgen, M.; Joyce, J. N.; Nehama, D.; Rajadas, J.;

405

Longo, F. M. Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal

406

degeneration in rodents. J Clin Invest. 2010, 120, 1774.

407

(21) Yamada, K.; Mizuno, M.; Nabeshima, T. Role for brain-derived neurotrophic factor in

408

learning and memory. Life Sci. 2002, 70, 735-44.

409

(22) Nagappan, G.; Zaitsev, E.; Senatorov, V. V.; Yang, J.; Hempstead, B. L.; Lu, B. Control of

410

extracellular cleavage of ProBDNF by high frequency neuronal activity. Proceedings of the

411

National Academy of Sciences. 2009, 106, 1267-72.

412

(23) Baral, S.; Pariyar, R.; Kim, J.; Lee, H.-S.; Seo, J. Quercetin-3-O-glucuronide promotes the

413

proliferation and migration of neural stem cells. Neurobiol. Aging. 2017, 52, 39-52.

414

(24) Zhang, Z.; Liu, X.; Schroeder, J. P.; Chan, C.-B.; Song, M.; Yu, S. P.; Weinshenker, D.; Ye,

of

a

select

group

of

mRNAs

by


a

mammalian

target

of


415

K. 7, 8-dihydroxyflavone prevents synaptic loss and memory deficits in a mouse model of

416

Alzheimer’s disease. Neuropsychopharmacology. 2014, 39, 638-50.

417

(25) Xi, Y.-D.; Ding, J.; Han, J.; Zhang, D.-D.; Liu, J.-M.; Feng, L.-l.; Xiao, R. The effect of

418

soybean isoflavone on the dysregulation of NMDA receptor signaling pathway induced by

419

β-amyloid peptides 1-42 in rats. Cell. Mol. Neurobiol. 2015, 35, 555-62.

420

(26) Chatterjee, G.; Roy, D.; Khemka, V. K.; Chattopadhyay, M.; Chakrabarti, S. Genistein, the

421

isoflavone in soybean, causes amyloid beta peptide accumulation in human neuroblastoma cell

422

line: implications in Alzheimer’s disease. Aging Dis. 2015, 6, 456.


Page 22 of 34

Page 23 of 34


423

FIGURE CAPTIONS

424

Figure 1. Effect of treatment with soybean protein hydrolysate (SPH) on brain-derived

425

neurotrophic factor (BDNF) expression in primary cultured astrocytes. (A) Western blot

426

analysis and quantification of proBDNF and mBDNF expression levels in the astrocyte lysates

427

treated with SPH (0.5 or 1 mg/mL). (B) Relative luciferase activity, reflecting CREB

428

phosphorylation levels, in primary astrocytes treated with different concentrations of SPH.

429

Values are expressed as mean ± SD, n = 3. Data were analyzed by one-way ANOVA followed by

430

Dunnett’s post hoc tests. *p

Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes

Recommend Documents