Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain Takashi Ichinose, Kazuki Moriyasu, Akane Nakahata, Mitsuru Tanaka, Toshiro Matsui & Shigeki Furuya To cite this article: Takashi Ichinose, Kazuki Moriyasu, Akane Nakahata, Mitsuru Tanaka, Toshiro Matsui & Shigeki Furuya (2015) Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain, Bioscience, Biotechnology, and Biochemistry, 79:9, 1542-1547, DOI: 10.1080/09168451.2015.1044932 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1044932
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Date: 07 October 2015, At: 02:22
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 9, 1542–1547
Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain Takashi Ichinose1, Kazuki Moriyasu1, Akane Nakahata1, Mitsuru Tanaka2, Toshiro Matsui2 and Shigeki Furuya1,3,* 1
Laboratory of Functional Genomics and Metabolism, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; 2Laboratory of Food Analysis, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; 3Bio-Architecture Center, Kyushu University, Fukuoka, Japan Received February 3, 2015; accepted April 8, 2015
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http://dx.doi.org/10.1080/09168451.2015.1044932
In this study, we examined the effect of orally administrated dipeptides containing Tyr (Y) on the metabolism of catecholamines in mouse brains. We found that among eight synthetic dipeptides whose sequences are present frequently in soy proteins, Ser-Tyr (SY), Ile-Tyr, and Tyr-Pro had the highest apparent permeability coefficients in monolayers of human intestinal epithelial Caco-2 cells. When administrated orally, SY markedly increased tyrosine content in the cerebral cortex compared to the vehicle control, Ile-Tyr, Tyr-Pro, and Y alone. The oral administration of SY more effectively increased 3-methoxy-4-hydroxyphenylethyleneglycol, the principal metabolite of noradrenaline, in the cerebral cortex and hippocampus than did Ile-Tyr, Tyr-Pro, or Y alone. Central noradrenergic turnover was also markedly stimulated by SY administration. These in vivo observations strongly suggest that SY is more potent in boosting central catecholamine transmission, particularly the noradrenergic system, than Y alone or other dipeptides that include Y. Key words: soy; dipeptide; tyrosine; brain; noradrenaline
A wide range of biological activities and regulatory functions of peptides derived from food proteins of animal or plant origins has been described.1–3) Among a variety of sources of bioactive peptides, soybean proteins are considered to have advantages over other dietary vegetable proteins owing to their low cost, abundance, and higher nutritional value. Indeed, it is well documented that peptides derived from soybean proteins exert various beneficial effects on human health such as anti-cancer, anti-hypertensive, anti-obesity, anti-oxidative, hypocholesterolemic, and immunomodulatory activities.4) Despite these favorable
effects on systemic health, the effect of soybeanderived peptides on central nervous system functions is largely unknown. Recent studies have demonstrated that a soy peptide mixture composed mainly of di- and tripeptides has high absorbability, low immunogenicity, and sequencespecific physiological effects.5) We have previously shown that the intake of this soy peptide mixture increases neuroactive amino acids such as Glu in the cerebral cortex and hippocampus of mice.6) In addition to these neuroactive amino acids, a significant and marked increase in Tyr (Y) was observed in the serum. Our recent study also demonstrated that oral administration of a dipeptide Ser-Tyr (SY) efficiently increases the free Y content relative to the sole administration of Y alone at the same intake dose in several brain regions of adult mice.7) This suggests that Y derived from soy proteins/peptides may affect catecholamine metabolism in the brain, since Y can be transported efficiently at the blood–brain barrier and serves as a precursor of catecholamines. Therefore, the objective of the present study was to examine the effect of orally administered dipeptides with a Y residue, which are potentially generated by digestion of soy proteins, on the metabolism of catecholamines in the mouse brain. We show that SY efficiently increases the free Y content and stimulates the metabolism of noradrenaline (NA) in the brain compared to other dipeptides containing the Y residue or Y alone.
Materials and methods Materials. Y (L-enantiomer) was purchased from Wako (Osaka, Japan). Adrenaline (also known as epinephrine), dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleicetic acid,
*Corresponding author. Email: [email protected] Abbreviations: dopamine, DA; 3,4-dihydroxyphenylacetic acid, DOPAC; Hanks’ balanced salt solution, HBSS; 5-hydroxyindoleicetic acid, 5-HIAA; homovanillic acid, HVA; 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, HEPES; 3-methoxytyramine, 3-MT; noradrenaline, NA; 3-methoxy-4-hydroxyphenylethyleneglycol, MHPG, 2-(N-morpholino) ethanesulfonic acid, MES; normetanephrine, NM; vehicle, Veh. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Ser-Tyr stimulates central noradrenaline metabolism
5-hydroxytryptamine (5-HT), homovanillic acid (HVA), 3-methoxytyramine (3-MT), NA (also known as norepinephrine), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and normetanephrine were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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Peptide synthesis. The dipeptides SY, Tyr-Leu (YL), Tyr-Arg (YR), Tyr-Ser (YS), YP, Tyr-Asn (YN), Phe-Tyr (FY), and IY were synthesized using a 9-fluorenylmethoxycarbonyl (Fmoc)-solid phase synthesis method according to the manufacturer’s instructions (Kokusan Chemical Co., Osaka, Japan). Transport assay. Dipeptide transport experiments in Caco-2 cell monolayers were performed using an Ussing Chamber System (Model U-2500; Warner Instrument Co., Hamden, CT, USA) as described previously.8) In brief, Caco-2 cell monolayers used in this study were grown in BD Falcon™ cell culture Transwell inserts (PET membrane, 0.9 cm2, 1.0 μm pore size; BD Biosciences) coated with type I collagen (collagen gel culturing kit, Cellmatrix type I-A, Nitta Gelatin, Osaka, Japan) at a density of 4.0 × 105 cell/mL and were cultured in a seeding basal medium containing MITO+™ serum extender (BD Biosciences, Franklin Lakes, NJ, USA) for 48 h. The medium was replaced with an enterocyte differentiation medium containing MITO+™ serum extender every day for three days to allow the cells to form monolayers with >100 Ω cm2 of transepithelial electrical resistance. The monolayers grown in Transwell inserts were gently rinsed with Hanks’ balanced salt solution (HBSS) buffer (pH 6.0), and mounted in the Ussing Chamber containing 7.5 mL of HBSS buffer (pH 6.0) (adjusted with 10 mM 2-(N-morpholino) ethanesulfonic acid) on the apical side and the same volume of HBSS buffer (pH 7.4) (adjusted with 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) on the basolateral side. After equilibration for 15 min at 37 °C, the transport experiments were started by replacing the apical buffer with fresh HBSS buffer (pH 6.0) containing 2 mM dipeptides (Table 1). During the experiments, solutions on both sides were bubbled continuously with a mixture of O2:CO2 (95:5). Aliquots (0.2 mL each) were drawn
Table 1. The frequency of Y-containing dipeptide sequences and their transport properties across Caco-2 cell monolayers. Sequence SY YL YR YS YP YN FY IY a
Frequencya
Papp × 10−8 (cm/s)2b
16 8 8 5 5 5 5 5
72.0 ± 27.2 59.6 ± 19.8 5.0 ± 1.1 24.8 ± 8.8 88.5 ± 21.5 4.4 ± 0.9 0.6 ± 0.01 291.9 ± 26.5
Number of dipeptide sequences in major soy proteins with the Y residue determined from protein sequence data for glycinin, β-conglycinin α-subunit, β-conglycinin α-prime subunit, and β-conglycinin β-subunit are shown in Supplementary Fig. 1. b The apparent permeability coefficient (Papp) was measured using Caco-2 cell monolayers as described in the materials and methods section.
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from the basolateral side at time intervals of 15, 30, 45, and 60 min to determine the amount of transported dipeptide. The same volume of fresh buffer was supplied to the basolateral side at each time point. The amount of peptide transported to the basolateral side was determined simultaneously for all samples using a fluorescent NDA (naphthalene-2,3-dialdehyde)-derivatization method as previously reported.9) The apparent permeability coefficient (Papp) was calculated from the following equation: Papp ðcm/sÞ ¼
V dC AC0 dt
where dC/dt is the change in concentration on the basolateral side over time (mmol/s), V is the volume of solution in the basolateral compartment (7.5 mL), A is the surface area of the membrane (0.2826 cm2), and C0 is the initial concentration on the apical side (mmol). Animal experiments. In this study, the animal experiments followed the “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain” (Notice No. 88, Ministry of the Environment, Government of Japan). All experiments were reviewed and approved by the Animal Experiment Committee of Kyushu University (Permit Number: A25–121). C57BL/6 mice at 8 weeks of age were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). They were maintained in a pathogen-free animal facility (25 ± 1 °C) with a 12 h light/ 12 h dark cycle for two weeks. They were then transferred to the breeding room at the laboratory in a controlled environment (described above) and kept overnight with access to water and standard laboratory chow (CE-2; CLEA Jaoan Inc., Tokyo, Japan). Subsequently, mice were sorted into five groups and each group was administered saline (vehicle, Veh), Y, SY, IY, or YP solution (50 μmol/mL/50 g body weight) in saline by oral gavage. Then, each group was divided into two groups. In one group, mice were sacrificed at 30 min after oral ingestion. In the other, mice were sacrificed 60 min after oral ingestion. The brains, blood, and livers were sampled. The cerebral cortex and hippocampus were individually isolated on an icecooled plastic plate, weighed, and stored at −80 °C. The liver was also stored in the same conditions. The serum was prepared by centrifuging blood at 20,000 × g for 10 min after standing still. The serum samples were kept at −20 °C. Amino acid analysis. To determine the free amino acid content, the cerebral cortex and liver were individually isolated on an ice-cold glass plate, weighed, and stored at −80 °C until use. Serum samples were prepared as above and kept at −20 °C until use. Amino acids in the cerebral cortex, liver, and serum of mice were determined using high-performance liquid chromatography (HPLC) after derivatization of free amino acids using the ninhydrin method. Briefly, samples were homogenized individually in 5 volumes of water and centrifuged at 20,000 × g for 30 min at 4 °C.
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Proteins in the supernatant were removed by adding a 1/10 volume of 60% perchloric acid,10) and the amino acid composition of the supernatants was determined with an amino acid analyzer (Amino TacTM JLC500 V; JEOL Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Determination of monoamines and their metabolites. The total monoamines and their metabolites in the cerebral cortex and hippocampus were determined by an HPLC-electrochemical detector (ECD) system HTEC-500 (Eicom Co., Ltd., Kyoto, Japan) according to the manufacturer’s instructions. In brief, the cerebral cortex and hippocampus were quickly isolated, weighed, and frozen in liquid N2 until use. Frozen tissues were homogenized in 5 volumes of ice-cold 0.2 M perchloric acid containing 0.1 mM EDTA・2Na (Dojindo Lab., Kumamoto, Japan) and 10 μL of 10 μg/μL isoproterenol. The mixture was centrifuged at 20,000 × g for 15 min. Then, 200 μL of the supernatant was mixed with 35 μL of 0.2 M sodium acetate and filtered through a membrane filter (0.22 μm; Merck Millipore, Billerica, MA, USA). The HTEC-500 system (reverse-phase chromatographic analysis; Eicom Co., Ltd.) consisted of a reversed-phase column (Eicompack SC-5ODS, 3 mm φ × 150 mm; Eicom Co., Ltd.) and a graphite carbon working electrode (EICOM WE-3G; 12 mm φ) with an Ag/AgCl reference electrode. The ECD potential was set at + 750 mV for the working electrode. The acetate–citrate buffer (pH 3.5) used for the separation (flow rate, 0.5 mL/min at 25 °C) contained 0.053 M citric acid and 0.047 M sodium acetate, 5 mg/L EDTA, 193 mg/L sodium octyl sulfonate (Nacalai Tesque, Inc., Kyoto, Japan), and 17% methanol (v/v). It should be noted that levels of adrenaline in the cerebral cortex and hippocampus, and DOPAC, HVA, and 3-MT in the hippocampus were below the limit of quantitative detection in our HPLC-ECD system. Statistical analysis. Results are expressed as the means ± SEM. The statistical significance between groups was analyzed using one-way ANOVA followed by Tukey–Kramer’s test for post hoc analysis. p-values 0.05.
It is thought that catecholamines are tightly connected to key brain functions including cognition, emotion, executive function, mood, working memory, and so on. Dysregulation of central catecholaminergic systems therefore leads to psychiatric and neurological diseases that are associated with cognitive and emotional alterations. So far, dysfunctions of central NA and DA transmission have been implicated in the pathophysiology of major depressive disorder.14,15) It is well documented that a reduction in these catecholamine stores results in an exacerbation of depression symptoms.16) Hence, it will be interesting to confirm the present observations using depression models to further study the effect on NA metabolism. In conclusion, the present study provides the first in vivo evidence that SY administration elicits the efficient stimulation of central noradrenergic turnover. Furthermore, the observation that SY intake results in the maintenance of Y at a higher level in the cerebral cortex than does intake of other di-peptides or Y alone may be of particular significance. Although SY can be generated by in vitro enzymatic digestion of crude soy protein isolate with Bacillus-derived endoproteases,17) it remains unclear whether the ingestion of soy proteins/ peptides results in the generation of SY in the intestine, other organs, and body fluids. Thus, it should be emphasized that the results of present investigation illustrate the effect of SY administration, and these results need to be verified using an appropriate soy peptide/protein preparation in vivo for the future development of SY as a food material that is beneficial for brain function.
Authors contribution T.I., T.M., and S.F. designed research; T.I., K.M., A.N., and M.T. performed research; T.I., K.M., M.T., T.M., and S.F. analyzed data; and T.I., T.M., and S.F. wrote the paper.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2015.1044932.
Disclosure statement The authors also declare no conflict of interest.
Funding This study was supported by a fund from Fuji Oil Co. (Osaka, Japan).
References [1] Korhonen H, Pihlanto A. Bioactive peptides: production and functionality. Int. Dairy J. 2006;16:945–960. [2] Hartmann R, Meisel H. Food-derived peptides with biological activity: from research to food applications. Curr. Opin. Biotechnol. 2007;18:163–169. [3] Möller NP, Scholz-Ahrens KE, Roos N, Schrezenmeir J. Bioactive peptides and proteins from foods: indication for health effects. Eur. J. Nutr. 2008;47:171–182. [4] Singh BP, Vij S, Hati S. Functional significance of bioactive peptides derived from soybean. Peptides. 2014;54:171–179. [5] Nakamori T. Chapter 18 soy peptides as functional food materials. In: Mine Y, Li-Chan ECY, Jiang B, editors. Bioactive proteins and peptides as functional foods and nutraceuticals. Ames (IA): Wiley-Blackwell Publishing; 2010. p. 265–271. [6] Esaki K, Kokido T, Ohmori T, et al. Soy peptide ingestion increases neuroactive amino acids in the adult brain of wild-type and genetically engineered serine-deficient mice. J. Nutr. Food Sci. 2011;1:109. doi:10.4172/2155-9600.1000109. [7] Esaki K, Ohmori T, Maebuchi M, et al. Increased tyrosine in the brain and serum of mice by orally administering dipeptide SY. Biosci. Biotechnol. Biochem. 2013;77:847–849. [8] Takeda J, Park HY, Kunitake Y, et al. Theaflavins, dimeric catechins, inhibit peptide transport across Caco-2 cell monolayers via down-regulation of AMP-activated protein kinase-mediated peptide transporter PEPT1. Food Chem. 2013;138:2140–2145.
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[9] Ueno T, Tanaka M, Matsui T, et al. Determination of antihypertensive small peptides, Val-Tyr and Ile-Val-Tyr, by fluorometric high-performance liquid chromatography combined with a double heart-cut column-switching technique. Anal. Sci. 2005;21:997–1000. [10] Yang JH, Wada A, Yoshida K, et al. Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an NMDA receptor co-agonist, in adult brain. J. Biol. Chem. 2010;285:41380–41390. [11] Walter DS, Eccleston D. Increase of noradrenaline metabolism following electrical stimulation of the locus coeruleus in the rat. J. Neurochem. 1973;21:281–289. [12] Sved AF, Fernstrom JD, Wurtman RJ. Tyrosine administration reduces blood pressure and enhances brain norepinephrine release in spontaneously hypertensive rats. Proc. Nat. Acad. Sci. 1979;76:3511–3514.
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[13] Lieberman HR, Georgelis JH, Maher TJ, et al. Tyrosine prevents effects of hyperthermia on behavior and increases norepinephrine. Physiol. Behav. 2005;84:33–38. [14] Lambert G, Johansson M, Ågren H, et al. Reduced brain norepinephrine and dopamine release in treatment-refractory depressive illness. Arch. Gen. Psychiatry. 2000;57:787–793. [15] Hasler G, Drevets WC, Manji HK, et al. Discovering endophenotypes for major depression. Neuropsychopharmacology. 2004;29:1765–1781. [16] Mendels J, Frazer A. Brain biogenic amine depletion and mood. Arch. Gen. Psychiatry. 1974;30:447–451. [17] Inoue N, Nagao K, Sakata K, et al. Screening of soy proteinderived hypotriglyceridemic di-peptides in vitro and in vivo. Lipids Health Dis. 2011;10:85. doi:10.1186/1476-511X-10-85.
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain Takashi Ichinose, Kazuki Moriyasu, Akane Nakahata, Mitsuru Tanaka, Toshiro Matsui & Shigeki Furuya To cite this article: Takashi Ichinose, Kazuki Moriyasu, Akane Nakahata, Mitsuru Tanaka, Toshiro Matsui & Shigeki Furuya (2015) Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain, Bioscience, Biotechnology, and Biochemistry, 79:9, 1542-1547, DOI: 10.1080/09168451.2015.1044932 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1044932
View supplementary material
Published online: 21 May 2015.
Submit your article to this journal
Article views: 45
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Download by: [University of Lethbridge]
Date: 07 October 2015, At: 02:22
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 9, 1542–1547
Orally administrated dipeptide Ser-Tyr efficiently stimulates noradrenergic turnover in the mouse brain Takashi Ichinose1, Kazuki Moriyasu1, Akane Nakahata1, Mitsuru Tanaka2, Toshiro Matsui2 and Shigeki Furuya1,3,* 1
Laboratory of Functional Genomics and Metabolism, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; 2Laboratory of Food Analysis, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; 3Bio-Architecture Center, Kyushu University, Fukuoka, Japan Received February 3, 2015; accepted April 8, 2015
Downloaded by [University of Lethbridge] at 02:22 07 October 2015
http://dx.doi.org/10.1080/09168451.2015.1044932
In this study, we examined the effect of orally administrated dipeptides containing Tyr (Y) on the metabolism of catecholamines in mouse brains. We found that among eight synthetic dipeptides whose sequences are present frequently in soy proteins, Ser-Tyr (SY), Ile-Tyr, and Tyr-Pro had the highest apparent permeability coefficients in monolayers of human intestinal epithelial Caco-2 cells. When administrated orally, SY markedly increased tyrosine content in the cerebral cortex compared to the vehicle control, Ile-Tyr, Tyr-Pro, and Y alone. The oral administration of SY more effectively increased 3-methoxy-4-hydroxyphenylethyleneglycol, the principal metabolite of noradrenaline, in the cerebral cortex and hippocampus than did Ile-Tyr, Tyr-Pro, or Y alone. Central noradrenergic turnover was also markedly stimulated by SY administration. These in vivo observations strongly suggest that SY is more potent in boosting central catecholamine transmission, particularly the noradrenergic system, than Y alone or other dipeptides that include Y. Key words: soy; dipeptide; tyrosine; brain; noradrenaline
A wide range of biological activities and regulatory functions of peptides derived from food proteins of animal or plant origins has been described.1–3) Among a variety of sources of bioactive peptides, soybean proteins are considered to have advantages over other dietary vegetable proteins owing to their low cost, abundance, and higher nutritional value. Indeed, it is well documented that peptides derived from soybean proteins exert various beneficial effects on human health such as anti-cancer, anti-hypertensive, anti-obesity, anti-oxidative, hypocholesterolemic, and immunomodulatory activities.4) Despite these favorable
effects on systemic health, the effect of soybeanderived peptides on central nervous system functions is largely unknown. Recent studies have demonstrated that a soy peptide mixture composed mainly of di- and tripeptides has high absorbability, low immunogenicity, and sequencespecific physiological effects.5) We have previously shown that the intake of this soy peptide mixture increases neuroactive amino acids such as Glu in the cerebral cortex and hippocampus of mice.6) In addition to these neuroactive amino acids, a significant and marked increase in Tyr (Y) was observed in the serum. Our recent study also demonstrated that oral administration of a dipeptide Ser-Tyr (SY) efficiently increases the free Y content relative to the sole administration of Y alone at the same intake dose in several brain regions of adult mice.7) This suggests that Y derived from soy proteins/peptides may affect catecholamine metabolism in the brain, since Y can be transported efficiently at the blood–brain barrier and serves as a precursor of catecholamines. Therefore, the objective of the present study was to examine the effect of orally administered dipeptides with a Y residue, which are potentially generated by digestion of soy proteins, on the metabolism of catecholamines in the mouse brain. We show that SY efficiently increases the free Y content and stimulates the metabolism of noradrenaline (NA) in the brain compared to other dipeptides containing the Y residue or Y alone.
Materials and methods Materials. Y (L-enantiomer) was purchased from Wako (Osaka, Japan). Adrenaline (also known as epinephrine), dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleicetic acid,
*Corresponding author. Email: [email protected] Abbreviations: dopamine, DA; 3,4-dihydroxyphenylacetic acid, DOPAC; Hanks’ balanced salt solution, HBSS; 5-hydroxyindoleicetic acid, 5-HIAA; homovanillic acid, HVA; 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, HEPES; 3-methoxytyramine, 3-MT; noradrenaline, NA; 3-methoxy-4-hydroxyphenylethyleneglycol, MHPG, 2-(N-morpholino) ethanesulfonic acid, MES; normetanephrine, NM; vehicle, Veh. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Ser-Tyr stimulates central noradrenaline metabolism
5-hydroxytryptamine (5-HT), homovanillic acid (HVA), 3-methoxytyramine (3-MT), NA (also known as norepinephrine), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and normetanephrine were obtained from Sigma-Aldrich (St. Louis, MO, USA).
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Peptide synthesis. The dipeptides SY, Tyr-Leu (YL), Tyr-Arg (YR), Tyr-Ser (YS), YP, Tyr-Asn (YN), Phe-Tyr (FY), and IY were synthesized using a 9-fluorenylmethoxycarbonyl (Fmoc)-solid phase synthesis method according to the manufacturer’s instructions (Kokusan Chemical Co., Osaka, Japan). Transport assay. Dipeptide transport experiments in Caco-2 cell monolayers were performed using an Ussing Chamber System (Model U-2500; Warner Instrument Co., Hamden, CT, USA) as described previously.8) In brief, Caco-2 cell monolayers used in this study were grown in BD Falcon™ cell culture Transwell inserts (PET membrane, 0.9 cm2, 1.0 μm pore size; BD Biosciences) coated with type I collagen (collagen gel culturing kit, Cellmatrix type I-A, Nitta Gelatin, Osaka, Japan) at a density of 4.0 × 105 cell/mL and were cultured in a seeding basal medium containing MITO+™ serum extender (BD Biosciences, Franklin Lakes, NJ, USA) for 48 h. The medium was replaced with an enterocyte differentiation medium containing MITO+™ serum extender every day for three days to allow the cells to form monolayers with >100 Ω cm2 of transepithelial electrical resistance. The monolayers grown in Transwell inserts were gently rinsed with Hanks’ balanced salt solution (HBSS) buffer (pH 6.0), and mounted in the Ussing Chamber containing 7.5 mL of HBSS buffer (pH 6.0) (adjusted with 10 mM 2-(N-morpholino) ethanesulfonic acid) on the apical side and the same volume of HBSS buffer (pH 7.4) (adjusted with 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) on the basolateral side. After equilibration for 15 min at 37 °C, the transport experiments were started by replacing the apical buffer with fresh HBSS buffer (pH 6.0) containing 2 mM dipeptides (Table 1). During the experiments, solutions on both sides were bubbled continuously with a mixture of O2:CO2 (95:5). Aliquots (0.2 mL each) were drawn
Table 1. The frequency of Y-containing dipeptide sequences and their transport properties across Caco-2 cell monolayers. Sequence SY YL YR YS YP YN FY IY a
Frequencya
Papp × 10−8 (cm/s)2b
16 8 8 5 5 5 5 5
72.0 ± 27.2 59.6 ± 19.8 5.0 ± 1.1 24.8 ± 8.8 88.5 ± 21.5 4.4 ± 0.9 0.6 ± 0.01 291.9 ± 26.5
Number of dipeptide sequences in major soy proteins with the Y residue determined from protein sequence data for glycinin, β-conglycinin α-subunit, β-conglycinin α-prime subunit, and β-conglycinin β-subunit are shown in Supplementary Fig. 1. b The apparent permeability coefficient (Papp) was measured using Caco-2 cell monolayers as described in the materials and methods section.
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from the basolateral side at time intervals of 15, 30, 45, and 60 min to determine the amount of transported dipeptide. The same volume of fresh buffer was supplied to the basolateral side at each time point. The amount of peptide transported to the basolateral side was determined simultaneously for all samples using a fluorescent NDA (naphthalene-2,3-dialdehyde)-derivatization method as previously reported.9) The apparent permeability coefficient (Papp) was calculated from the following equation: Papp ðcm/sÞ ¼
V dC AC0 dt
where dC/dt is the change in concentration on the basolateral side over time (mmol/s), V is the volume of solution in the basolateral compartment (7.5 mL), A is the surface area of the membrane (0.2826 cm2), and C0 is the initial concentration on the apical side (mmol). Animal experiments. In this study, the animal experiments followed the “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain” (Notice No. 88, Ministry of the Environment, Government of Japan). All experiments were reviewed and approved by the Animal Experiment Committee of Kyushu University (Permit Number: A25–121). C57BL/6 mice at 8 weeks of age were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). They were maintained in a pathogen-free animal facility (25 ± 1 °C) with a 12 h light/ 12 h dark cycle for two weeks. They were then transferred to the breeding room at the laboratory in a controlled environment (described above) and kept overnight with access to water and standard laboratory chow (CE-2; CLEA Jaoan Inc., Tokyo, Japan). Subsequently, mice were sorted into five groups and each group was administered saline (vehicle, Veh), Y, SY, IY, or YP solution (50 μmol/mL/50 g body weight) in saline by oral gavage. Then, each group was divided into two groups. In one group, mice were sacrificed at 30 min after oral ingestion. In the other, mice were sacrificed 60 min after oral ingestion. The brains, blood, and livers were sampled. The cerebral cortex and hippocampus were individually isolated on an icecooled plastic plate, weighed, and stored at −80 °C. The liver was also stored in the same conditions. The serum was prepared by centrifuging blood at 20,000 × g for 10 min after standing still. The serum samples were kept at −20 °C. Amino acid analysis. To determine the free amino acid content, the cerebral cortex and liver were individually isolated on an ice-cold glass plate, weighed, and stored at −80 °C until use. Serum samples were prepared as above and kept at −20 °C until use. Amino acids in the cerebral cortex, liver, and serum of mice were determined using high-performance liquid chromatography (HPLC) after derivatization of free amino acids using the ninhydrin method. Briefly, samples were homogenized individually in 5 volumes of water and centrifuged at 20,000 × g for 30 min at 4 °C.
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Proteins in the supernatant were removed by adding a 1/10 volume of 60% perchloric acid,10) and the amino acid composition of the supernatants was determined with an amino acid analyzer (Amino TacTM JLC500 V; JEOL Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Determination of monoamines and their metabolites. The total monoamines and their metabolites in the cerebral cortex and hippocampus were determined by an HPLC-electrochemical detector (ECD) system HTEC-500 (Eicom Co., Ltd., Kyoto, Japan) according to the manufacturer’s instructions. In brief, the cerebral cortex and hippocampus were quickly isolated, weighed, and frozen in liquid N2 until use. Frozen tissues were homogenized in 5 volumes of ice-cold 0.2 M perchloric acid containing 0.1 mM EDTA・2Na (Dojindo Lab., Kumamoto, Japan) and 10 μL of 10 μg/μL isoproterenol. The mixture was centrifuged at 20,000 × g for 15 min. Then, 200 μL of the supernatant was mixed with 35 μL of 0.2 M sodium acetate and filtered through a membrane filter (0.22 μm; Merck Millipore, Billerica, MA, USA). The HTEC-500 system (reverse-phase chromatographic analysis; Eicom Co., Ltd.) consisted of a reversed-phase column (Eicompack SC-5ODS, 3 mm φ × 150 mm; Eicom Co., Ltd.) and a graphite carbon working electrode (EICOM WE-3G; 12 mm φ) with an Ag/AgCl reference electrode. The ECD potential was set at + 750 mV for the working electrode. The acetate–citrate buffer (pH 3.5) used for the separation (flow rate, 0.5 mL/min at 25 °C) contained 0.053 M citric acid and 0.047 M sodium acetate, 5 mg/L EDTA, 193 mg/L sodium octyl sulfonate (Nacalai Tesque, Inc., Kyoto, Japan), and 17% methanol (v/v). It should be noted that levels of adrenaline in the cerebral cortex and hippocampus, and DOPAC, HVA, and 3-MT in the hippocampus were below the limit of quantitative detection in our HPLC-ECD system. Statistical analysis. Results are expressed as the means ± SEM. The statistical significance between groups was analyzed using one-way ANOVA followed by Tukey–Kramer’s test for post hoc analysis. p-values 0.05.
It is thought that catecholamines are tightly connected to key brain functions including cognition, emotion, executive function, mood, working memory, and so on. Dysregulation of central catecholaminergic systems therefore leads to psychiatric and neurological diseases that are associated with cognitive and emotional alterations. So far, dysfunctions of central NA and DA transmission have been implicated in the pathophysiology of major depressive disorder.14,15) It is well documented that a reduction in these catecholamine stores results in an exacerbation of depression symptoms.16) Hence, it will be interesting to confirm the present observations using depression models to further study the effect on NA metabolism. In conclusion, the present study provides the first in vivo evidence that SY administration elicits the efficient stimulation of central noradrenergic turnover. Furthermore, the observation that SY intake results in the maintenance of Y at a higher level in the cerebral cortex than does intake of other di-peptides or Y alone may be of particular significance. Although SY can be generated by in vitro enzymatic digestion of crude soy protein isolate with Bacillus-derived endoproteases,17) it remains unclear whether the ingestion of soy proteins/ peptides results in the generation of SY in the intestine, other organs, and body fluids. Thus, it should be emphasized that the results of present investigation illustrate the effect of SY administration, and these results need to be verified using an appropriate soy peptide/protein preparation in vivo for the future development of SY as a food material that is beneficial for brain function.
Authors contribution T.I., T.M., and S.F. designed research; T.I., K.M., A.N., and M.T. performed research; T.I., K.M., M.T., T.M., and S.F. analyzed data; and T.I., T.M., and S.F. wrote the paper.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2015.1044932.
Disclosure statement The authors also declare no conflict of interest.
Funding This study was supported by a fund from Fuji Oil Co. (Osaka, Japan).
References [1] Korhonen H, Pihlanto A. Bioactive peptides: production and functionality. Int. Dairy J. 2006;16:945–960. [2] Hartmann R, Meisel H. Food-derived peptides with biological activity: from research to food applications. Curr. Opin. Biotechnol. 2007;18:163–169. [3] Möller NP, Scholz-Ahrens KE, Roos N, Schrezenmeir J. Bioactive peptides and proteins from foods: indication for health effects. Eur. J. Nutr. 2008;47:171–182. [4] Singh BP, Vij S, Hati S. Functional significance of bioactive peptides derived from soybean. Peptides. 2014;54:171–179. [5] Nakamori T. Chapter 18 soy peptides as functional food materials. In: Mine Y, Li-Chan ECY, Jiang B, editors. Bioactive proteins and peptides as functional foods and nutraceuticals. Ames (IA): Wiley-Blackwell Publishing; 2010. p. 265–271. [6] Esaki K, Kokido T, Ohmori T, et al. Soy peptide ingestion increases neuroactive amino acids in the adult brain of wild-type and genetically engineered serine-deficient mice. J. Nutr. Food Sci. 2011;1:109. doi:10.4172/2155-9600.1000109. [7] Esaki K, Ohmori T, Maebuchi M, et al. Increased tyrosine in the brain and serum of mice by orally administering dipeptide SY. Biosci. Biotechnol. Biochem. 2013;77:847–849. [8] Takeda J, Park HY, Kunitake Y, et al. Theaflavins, dimeric catechins, inhibit peptide transport across Caco-2 cell monolayers via down-regulation of AMP-activated protein kinase-mediated peptide transporter PEPT1. Food Chem. 2013;138:2140–2145.
Ser-Tyr stimulates central noradrenaline metabolism
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[9] Ueno T, Tanaka M, Matsui T, et al. Determination of antihypertensive small peptides, Val-Tyr and Ile-Val-Tyr, by fluorometric high-performance liquid chromatography combined with a double heart-cut column-switching technique. Anal. Sci. 2005;21:997–1000. [10] Yang JH, Wada A, Yoshida K, et al. Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an NMDA receptor co-agonist, in adult brain. J. Biol. Chem. 2010;285:41380–41390. [11] Walter DS, Eccleston D. Increase of noradrenaline metabolism following electrical stimulation of the locus coeruleus in the rat. J. Neurochem. 1973;21:281–289. [12] Sved AF, Fernstrom JD, Wurtman RJ. Tyrosine administration reduces blood pressure and enhances brain norepinephrine release in spontaneously hypertensive rats. Proc. Nat. Acad. Sci. 1979;76:3511–3514.
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[13] Lieberman HR, Georgelis JH, Maher TJ, et al. Tyrosine prevents effects of hyperthermia on behavior and increases norepinephrine. Physiol. Behav. 2005;84:33–38. [14] Lambert G, Johansson M, Ågren H, et al. Reduced brain norepinephrine and dopamine release in treatment-refractory depressive illness. Arch. Gen. Psychiatry. 2000;57:787–793. [15] Hasler G, Drevets WC, Manji HK, et al. Discovering endophenotypes for major depression. Neuropsychopharmacology. 2004;29:1765–1781. [16] Mendels J, Frazer A. Brain biogenic amine depletion and mood. Arch. Gen. Psychiatry. 1974;30:447–451. [17] Inoue N, Nagao K, Sakata K, et al. Screening of soy proteinderived hypotriglyceridemic di-peptides in vitro and in vivo. Lipids Health Dis. 2011;10:85. doi:10.1186/1476-511X-10-85.
