The Multivariate Functions of Adipose Tissue and the Metabolic Syndrome:
The Effects of Soybean Protein on Fat Distribution and Adipocytokines (Part III)
Yuji MATSUZAWA
Osaka University Graduate School of Medicine, Osaka 565-0871
ABSTRACT
The metabolic syndrome, a cluster of insulin resistance, hypertension and
dyslipidemia, is a common basis of type 2 diabetes and atherosclerotic vascular
diseases in industrial countries. The most common cause of the metabolic syndrome
is accumulation of intra-abdominal visceral fat. Adipose tissue secretes a variety of
bioactive substances called adipocytokines. Dysregulation of adipocytokines by
overnutrition may play an important role in the development of the metabolic
syndrome. The Japanese traditional diet may protect against the development of the
metabolic syndrome. This study was aimed to clarify 1) the role of adipocytokines in
the development of the metabolic syndrome, and 2) the significance of soybean protein,
one of the major components of Japanese traditional diet, on the regulation of
adipocytokines. In the previous reports, we demonstrated that adiponectin, which we
discovered in adipose cDNA project, possessed anti-atherosclerotic properties, but its
plasma concentration was decreased in visceral fat obesity. Calorie-restricted diet
containing soy protein could raise plasma adiponectin in diet-induced obesity. In this
final report, we describe a global function of this adipocyte-derived factor and the
effect of soy protein diet on the regulation of adipocytokines. In adiponectin knockout
mice, we found that CRP and TNFαmRNAs were overexpressed in the adipose tissue.
Highly sensitive Plasma CRP level was correlated negatively with plasma adiponectin
level in humans. Adiponectin-null mice also showed severe hepatic fibrosis induced by
carbon tetrachloride. Overexpression of adiponectin suppressed hepatic fibrosis via
inhibition of smad 2 signaling. High plasma adiponectin level was a negative risk for
breast cancer. In mouse tumor model, injection of adiponectin inhibited the tumor growth. These data indicate that adiponectin has multivalent functions including
anti-inflammatory, anti-fibrosis and anti-tumor activities, and that the reduction of
adiponectin may have a causative role in inflammation, hepatic fibrosis and some types
of cancer frequently associated with visceral obesity. Soy protein diet suppressed the
expression of type 1 plasminogen activator inhibitor mRNA and induced mRNA
expression and plasma concentration of adiponectin as compared to the casein diet
without affecting the body weight. However, we did not observe any significant
change in plasma adiponectin level after the ingestion of the formula containing 20 g
soy protein per day for 12 weeks. Our results indicate that adiponectin has
multivalent functions and that the reduction of adiponectin may play an important
role in the development of the disorders in visceral obesity. Soy protein diet has
potential beneficial effect on adiponectin. However, further clinical studies will be
necessary.
Soy Protein Research, Japan7, 1-12, 2004.
Molecular Biomedical Evaluation in Monkeys of Efficacy of Soy Products,
Soy Protein and Soy Isoflavone for Treatment of Hypercholesterolemia (Part I)
Shin NAKAMURA1, Fusako MITSUNAGA1, A-Ram JOENG1, Hidenori YAMAUCHI1,
Takashi HAYASHI2 and Joseph GABRIEL2
1Primate Research Institute, Kyoto University, Inuyama 484-8506
2Ina Research /Ina Research Philippines, Nagano 399-4501
ABSTRACT
Soy Products are known to protect against both hypercholesterolemia and
hypercholesterolemia-mediated atherosclerosis, which are typical life-style-related
diseases in the advanced countries. These diseases are also closely linked with agerelated
dysfunction in postmenopausal women. In the current study, we examined
protective and/or therapeutic effect of soy protein (β-Conglycinin; β-CG) and the
soy isoflavone (Soyaflavone HG; IF), which are expected to be the functional
components in soy products, on hypercholesterolemia that was developed in
ovariectomized (OVX) crab-eating monkeys with experimentally created
postmenopausal condition. The molecular mechanism of protective and/or
therapeutic action of the soy products against hypercholesterolemia was investigated
by genomic biomedical analyses of plasma, liver, adipose tissue, and stool samples.
Plasma levels of cholesterol and other related biochemical markers were monitored
weekly. Plasma levels of adipocytokines, leptin and adiponectin, were also assayed by
a sandwich ELISA established for monkey samples. Gene expression profiling was
performed on thrombotic genes (TF, TFPI), Th1/Th2 cytokine genes (IL-4, INF-γ),
cholesterol metabolic genes (LDL-R, VLDL-R, HMG-CoA, SR) and adipocytokine
genes (leptin, adiponectin, TF) by means of a Real-Time RT-PCR/SYBRY method
with ABI PRISM 7700. (Also, using bacterial DNA prepared from monkey stool,
intestinal microflora (Bacteroides, Bifidobacteria, E. coli, and Clostridia) was
analyzed by means of PCR. β-Conglycinin revealed significant protective and/or
therapeutic effect on hypercholesterolemia in OVX-monkeys, whereas Soyaflavone
did not show any effect. Little change was observed in the plasma levels of
adipocytokines. β-Conglycinin caused up-regulation of gene expression of TF, LDL-R,
and HMG-CoA reductase in both peripheral MNL (monocytes) and the liver
(hepatocytes), down-regulation of gene expression of TF and leptin, and up-regulation
of adiponectin in adipose tissue (adipocytes). Interestingly β-Conglycinin had
significant effect on intestinal microflora. It was effective in reducing the cell
numbers of harmful bacteria, E. coli and Clostridia, and increasing those of beneficial
bacteria, Bifidobacteria. From these results using a monkey model, it is suggested
that β-Conglycinin, but not soyaflavone, is the functionally effective component that
protects or cures postmenopausal hypercholesterolemia. These results also suggest
that β-Conglycinin- or soy protein-enriched-food is a possible candidate for
alternative-complementary medicine for these age-related disorders.
Soy Protein Research, Japan7, 13-19, 2004.
Key words : β-conglycinin, OVX-hypercholesterolemia, monkey model, adipocytokines,
intestinal microflora, alternative medicine
1Graduate School of Agriculture, Kyoto University, Uji 611-0011
2Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
Japan 7, 20-25, 2004.
ABSTRACT
Both soybean (Glycine max) and wild soybean (Glycine soja) storage proteins are
composed of two major components, β-conglycinin (7S) and glycinin (11S). We
detected electrophoretic variants of the α' subunit of β-conglycinin and the A3 and
A4 polypeptides of glycinin of wild soybean storage proteins on SDS-PAGE and
designated them as small α', small A3, and large A4. Their N-terminal sequences
were identical to normal ones. We isolated cDNAs of smallα', small A3, and large A4
by RT-PCR and sequenced them. Their sequences indicated a deletion of 24 amino
acids in smallα', a deletion of 4 amino acids in a small A3 and substitutions of 2 amino
acids in large A4 in comparison with the sequences of normal subunits of wild
soybean. This deletion and substitution probably causes the difference in the mobility
on SDS-PAGE. Furthermore, we analyzed the solubility, emulsifying ability, and
thermal stability, of 7S from lines containing small α', and the 11S from lines
containing small A3 or large A4. Solubility and emulsifying ability of all samples were
very similar to those of normal wild soybean, but the denaturation-starting
temperatures of 11S from lines containing small A3 or large A4 were about 4oC lower
than that of 11S of normal wild soybean. This characteristic is valuable for lowering
the cost of food production from soybean proteins by heating.
Soy Protein Research, Japan7, 20-25, 2004.
Key words : wild soybean, seed storage protein, β-conglycinin, glycinin
Analysis of the Regulatory Mechanisms ofβ-Conglycinin Gene Expression in
Modification of Seed Storage Protein Components in Soybean
Akira KANAZAWA
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
ABSTRACT
Theβ-conglycinin, a major component of seed storage proteins in soybean, comprises
three subunits, α, α' and β. Reporter gene constructs comprising upstream
sequences of the gene encodingαsubunit ofβ-conglycinin andβ-glucuronidase (GUS)
were made in order to identify regulatory elements of transcription in seeds. The
constructs were introduced into Arabidopsis thaliana plants via Agrobacteriummediated
gene transfer. GUS gene expression was detected in Arabidopsis seeds
when a sequence comprising 545 bp upstream of the transcription start site was fused
to the GUS gene, suggesting that regulatory elements for gene expression in seeds
are present in this region. Multiple sequence motifs including box I sequence, which
are highly conserved among various seed storage protein genes, are present in the
upstream region of the α subunit gene. Interactions between these sequences and
nuclear factors detected so far suggest that sequences located particularly in the
vicinity of the box I are co-operatively involved in the transcriptional control within
the DNA region that enabled the expression in Arabidopsis seeds.
Soy Protein Research, Japan7, 26-31, 2004.
Key words : soybean, β-conglycinin, α subunit, transcription, DNA-protein interaction
Studies on Biosynthetic Enzymes of Soybean Saponins for the Breeding of Low
Unpleasant taste Soybean Variety
Masakazu SHIRAIWA and Kazumi YASUDA
School of Agriculture, Ibaraki University, Ibaraki 300-0393
ABSTRACT
Group-A and -DDMP saponins exist in the soybean seed as intact saponins. While
group-A saponin is the main cause of the unpleasant taste of the soybean, the group-
DDMP saponin has various medicinal properties. Therefore, it is thought that the
elucidation of characteristics of the enzymes which catalyze biosynthetic reaction of
saponins will be useful toward the breeding of the value-added soybean variety in the
future. In this research, we have characterized the glucuronosyltransferase and
hydroxylase which is the key enzyme of group-A saponin biosynthesis. The substrate
specificity of glucuronosyltransferase for each aglycon of group-A and -DDMP
saponins changed with metal, reaction temperature and pH. The possibility that by
controlling the cultivation conditions of soybeans based on the characteristics of this
enzyme, the composition of group-A and -DDMP saponins in an soybean seed might
be controlled, is suggested. Furthermore, we searched for the catalytic enzyme in the
biosynthetic pathway which could stop only the biosynthesis of group-A saponin.
Instead, we found a new hydroxylase, and characterized it. The finding of this
hydrohylase has enabled us to depress biosynthesis of group-A saponin and raise
biosynthesis of group-DDMP saponin relatively. The result is considered to
contribute to development of low unpleasant taste and high functional soybean
variety.
Soy Protein Research, Japan7, 32-41, 2004.
Key words : soybean, saponin biosynthesis, glucuronosyltransferase, hydroxylase
The Structure of Tofu for Preventing the Change of Lipid
Tomotada ONO, Takahiro WADA and Atsuko IMAI
Faculty of Agriculture, Iwate University, Morioka 020-8550
ABSTRACT
The tofu (soybean curd), which is a traditional food in Japan & China, includes 38% lipid
and 52% protein in dry matter. The lipid in the curd is extremely stable against ooze and
oxidation by cooking and storing. Thus the tofu is an important food source for a stable
lipid supply. Soy milk was prepared from the soybean ground with water, heated, and
filtered. We have already proposed that the soy milk proteins consist of particulate and
soluble proteins. The particulate protein is made up of mainly basic peptide (B) of 11S
globulin andβsubunit of 7S globulin. The soluble protein is made up of acidic peptide (A)
of 11S, α and α' subunits of 7S. The tofu was made from soy milk by the addition of
CaSO4 or glucono-δ-lacton (GDL) as coagulants. The protein and lipids from tofu were
extracted by the addition of urea or guanidine hydrochloride (GuHCl) as a denaturant
with or without 2-mercaptoethanol. The proteins extracted from tofu were up to 40%
even by the addition of 8M urea or 5M GuHCl without 2-mercaptoethanol. The yield of
lipids was also low. In the presence of 2-mercaptoethanol, yield of both protein and lipid
increased to 100% with each denaturant. The composition of extracted proteins was
analyzed with SDS-PAGE. Proteins liberated from tofu, in turn, A,αandα', andβand B
with increasing denaturants. These results indicate that oil body particle covered with
oleosin in tofu is packed with three protein layers, that is, oleosin, protein particles
(mainly B and β), and soluble proteins (A, α and α'). Therefore, the lipid of tofu is
extremely stable against ooze and oxidation by cooking and storing.
Soy Protein Research, Japan7, 42-47, 2004.
Key words : tofu, oil-body, proteins of tofu, structure of tofu
A Novel and Convenient Assay Method for Proteinase Activity by Measuring
Turbidity Change in Proteolytic Digestion of Soy Protein Isolates
Kuniyo INOUYE and Kouhei NAGAI
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502
ABSTRACT
A novel, convenient and accurate assay method for proteinase activity was developed.
This method is based on measuring the turbidity change directly in the proteolytic
digestion of soy protein isolates (SPI). For all proteases examined, bromelain (Bro),β-
chymotrypsim (Chy), ficin (Fic), subtilisin BPN' (SB), subtilisin Carlsberg (SC),
thermolysin (TLN), and pronase (Pro), the initial velocity (vo) of the turbidity decrease
changed depending on the enzyme and substrate concentrations following the
Michaelis-Menten equation. Values of the catalytic constant (kcat) and Michaelis
constant (Km) were evaluated for the respective proteinases: The kcat values ((OD
units/min)/(g/L)) were 11 ± 0 for Bro, 3.9 ± 0.1 for Chy, 29 ± 0 for Fic, 77 ± 1 for
SB, 66 ± 1 for SC, 13 ± 0 for Pro and 277 ± 3 for TLN; the Km values (mg/mL) were
0.83 ± 0.07 for Bro, 0.97 ± 0.08 for Chy, 0.60 ± 0.06 for Fic, 0.58 ± 0.06 for SB, 0.93
± 0.05 for SC, 1.3 ± 0.1 for Pro and 1.3 ± 0.1 for TLN. The kcat values obtained with
turbidity measurement showed high correlation (R=0.995) with those evaluated in the
SPI digestion accompanied with acid treatment, which was the general assay method
for the proteinase activity. It was demonstrated that the turbidity measurement in
the proteolytic digestion of SPI could provide us with a convenient and accurate
method for evaluating proteolytic activity without pre- and post-treatment of the
samples and reaction solutions.
Soy
Protein Research, Japan7, 48-51, 2004.
Key words : soy protein isolates, proteinase, proteolytic activity, turbidity
Interactions of β-Conglycinin and Phosphatidylcholine with Dough Components
Reiko URADE
Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
Soy protein, β-conglycinin is known to decrease plasma triacylglycerol and
cholesterol levels in humans. Since these effects of β-conglycinin are transient, it is
necessary to take it daily to keep in good health. Bread is a convenient vehicle for
introducing β-conglycinin into the daily diet. However, β-conglycinin decreases the
loaf volume of bread. Therefore, strategies to overcome this adverse effect should be
developed. When soy phosphatidylcholine (PC) was added to the ingredients including
β-conglycinin, the adverse effect of this protein on the loaf volume was shown to be
counteracted. This study was carried out to elucidate the mechanism of this
improving effect of PC on bread-making. By confocal microscopy, PC was shown to
associate with gluten in dough. The effects of PC on the volume of dough prepared
with de-lipidated wheat flour were determined. The addition of PC showed
improving effect on the volume of dough with or without β-conglycinin. This effect
reached a plateau when soy PC was supplemented at 4% of wheat flour weight.
However, when PC was supplemented in combination with wheat lipids, a synergistic
increase in dough volume was obtained. These results suggest that PC acts in two
ways, dependent or independent, on wheat lipids in dough. PC showed different
effects depending on the PC molecular species.
Soy Protein Research, Japan7, 52-56, 2004.
Key words : β-conglycinin, bread, dough, phosphatidylcholine, molecular species
Masking of Soyprotein-derived Astringency and Its Astringency-sensitive
Neuron Profile in Chorda Tympani Nerve Fibers
Michio KOMAI1, Mayuko FUKUNARI1, Hitoshi SUZUKI2 and Tsutomu SAITO3
1Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, 2School of Science and Engineering, Ishinomaki-Senshu University, Ishinomaki 986-8580,
3Fuji Oil Co., Ltd., Izumi-Sano 598-8540
ABSTRACT
Astringency perceived in the mouth upon ingestion of tannic acid and other substances
is generally regarded as a dry, puckering sensation, which seems more closely allied to
the tactile than to the gustatory sense. On the other hand, according to some studies
perceptual assessments of astringency may be closely linked to bitterness in human
taste, and some electrophysiological studies showed that the chorda tympani and
glossopharyngeal nerves transmit the astringency signal. We attempted to find tactile
sensitive nerve bundles from the lingual trigeminal nerves by applying 0.6 M tannic
acid, but found instead 18 lingual trigeminal nerve bundles that did not respond to this
at all. This indicates that the lingual trigeminal nerves do not respond to astringent
stimuli. On the other hand, many chorda tympani nerve fibers responded to the tannic
acid solution. Therefore, we analyzed the response profiles of potential astringentsensitive
neurons in the chorda tympani nerves in adult female Wistar rats. Astringent
compounds, tannic acid, and acidified soy protein isolate [glycinin] were applied to the
tongue of the rat. According to the cluster analysis of 80 chorda tympani nerve fibers,
the responses to glycinin (3% soln, pH 3.5) that cause astringency were most closely
related to the response to sour taste (0.01 M HCl, r=0.721) and tannic acid (0.01 M,
r=0.693). They were less related to bitter taste (0.02 M quinine-HCl, r=0.559) and
umami taste (0.035 M MSG, r=0.483), and not correlated with other basic taste stimuli.
Several candidate substances that reduce astringency derived from acidified glycinin
are proposed.
Soy Protein Research, Japan7, 57-62, 2004.
Key words : astringency, glycinin, taste nerve, nerve fiber profile, rat
High Efficiency Dye-adsorbent Produced from Okara, a By-product of
Soy Foods Processing
-Adsorption Characteristics and the Elucidation of the Mechanism-
Norie NISHIKAWA, Akihiko MASUI, Yoshihiro YOBIKO and Nobuaki FUJIWARA
Technology Research Institute of Osaka Prefecture, Izumi 594-1157
ABSTRACT
Electrophoresis is a preparative and analytical technique broadly used in biological
sciences, particularly in protein research. Avoiding background raised by dyestuff, gel
staining and destaining are crucial steps in detection of protein bands in the gel
electrophoresis. Particularly important is fast and efficient gel destaining. Okara, a byproduct
obtained during the production of tofu (bean curd) and soymilk, quickly
adsorbed Coomassie Brilliant Blue (CBB) stain. Compared to activated carbon and/or
carbon fiber adsorption, Okara efficiently adsorbs significantly higher amount of dye.
In this research, the characteristics and mechanism of adsorption were examined with
the aim to develop an efficient dyestuff adsorption system using Okara. The amount of
the saturation dye adsorption, Qm (mg-dye/g-okara) obtained from the Langmuir
adsorption isotherm (25oC) after 24 h at pH 3 was 197. It was found that Okara's
adsorption ability is approximately 66 times higher than the ability of activated carbon.
There was no significant difference in the adsorption of the dyestuff between defatted
sample with n-hexane and intact sample. It was shown that the dyestuff was not
adsorbed on the oil body in Okara. The adsorption efficiency of dye increased
proportionally to the content of remaining protein in Okara and was influenced by pH.
These results show that dye was attached to the residual protein in Okara. The
decrease in adsorption capacity, which was influenced by the addition of salt (NaClO4),
was only 30%, however the addition of methanol decreased the adsorption capacity
approximately 70%. These results showed that dye adsorption was enhanced by
hydrophobic interactions.
Soy Protein Research, Japan7,63-68, 2004.
Key words : okara, dye, coomassie brilliant blue R-250, adsorption, ionic bond,
hydrophobic interactions
Effects of Soy Protein or Its Derived Peptides on Muscle Protein Degradation
Takeshi NIKAWA, Katsuya HIRASAKA, Harumi FUROUCHI, Yuko OHNISHI
and Kyoichi KISHI
Institute of Health Bioscience, The University of Tokushima Graduate School,
Tokushima 770-8503
ABSTRACT
We have previously demonstrated that muscle proteases and the related molecules
play important roles in disorders of muscle function. For example, rats subjected to
an acute running exercise have significantly high calpain activity in gastrocnemius
muscle. Rat gastrocnemius muscle atrophied by spaceflight or tail-suspension is
specifically sensitive to the ubiquitin-proteasome proteolytic pathway. In this study,
we examined the effects of soy protein isolate (SPI) or soy protein-derived peptides on
these proteolytic pathways. SPI diet prevented exercise-induced protein degradation
in skeletal muscle possibly through inhibition of calpain-mediated proteolysis, as
compared with the casein diet. Soy protein-derived peptides inhibited proteinubiquitination
caused by oxidative stress in mouse myoblastic cells. Our results
suggest that soy protein may contain bioactive factors for regulating proteolysis in
skeletal muscle.
Soy Protein Research, Japan7, 69-74, 2004.
Key words : soy protein isolate, soy protein-derived peptides, calpain, ubiquitin, skeletal muscle
Studies on Interaction between Soy Constituents and Central Nervous System
Masaaki YOSHIKAWA and Soushi SONODA
Graduate School of Agriculture, Kyoto University, Uji 611-0011
ABSTRACT
Effect of a pentapeptide Leu-Pro-Tyr-Pro-Arg (LPYPR), which corresponds to
residues No. 63-67 of soy bean glycinin A5A4B3 subunit, on central nervous system was
investigated in mice. LPYPR showed analgesic activity after intracerebroventricular
(icv) administration at a dose of 30 nmol/mouse. The analgesic activity was nonopiodic
since it was not blocked by naloxone. In passive avoidance test, LPYPR
improved memory retention at a dose of 20 nmol/mouse (icv) or 100 mg/kg (per os).
Soy Protein Research, Japan7, 75-78, 2004.
Key words : soy, glycinin, peptide, central nervous system, analgesia, memory, learning
Isolation and Application of Physiologically Active Peptides from
Soybean Whey and Okara Proteins
Masami YONEKURA and Ayako YAMAMOTO
School of Agriculture, Ibaraki University, Ibaraki 300-0393
ABSTRACT
In this study, the antihypertensive effect of a peptide, Leu-Ala-Pro (LAP), which has
angiotensin I-converting enzyme (ACE) inhibitory activity and was isolated from
thermolysin digests of soybean whey proteins, was investigated in spontaneously
hypertensive rat (SHR). Oral administration of soybean whey peptide preparation
containing 0.1% LAP at a dose of 1 g/kg of body weight/day resulted in a significant
reduction of systolic and diastolic blood pressures in SHR. Single oral administration
of synthesized LAP at a dose of 10 mg/kg had ability to lower blood pressure in 21-
week-old SHR. The ACE inhibitory peptides from whey protein digested with
protease S were also isolated by 60% ethanol fractionation, gel filtration on Sephadex
G-25, and reverse phase HPLC, and their amino acid sequences were determined.
Seven kinds of the ACE inhibitory peptides were identified as LN, LHP, LKP, VTY,
LYQA, YEAP, and YQAP. These results suggest that the soybean whey peptide
preparations containing LAP prepared by thermolysin digestion and the protease S
digests of soybean whey proteins may be promising materials for the development of
a functional food with antihypertensive activity.
Soy Protein Research, Japan7, 79-84, 2004.
Key words : soybean, peptide, angiotensin I-converting enzyme inhibitor, whey protein
Faculty of Science and Technology, Ritsumeikan University, Kusatsu 525-8577
ABSTRACT
The growth of plants was promoted by the degradation of soybean meal products
(DSP). The root hair number of Brassica campestris was increased when DSP was
added. The whey protein, SPI protein, and the insoluble protein were separated, and
their degraded products obtained by digestion with protease from Bacillus circulans
HA12, were assayed for analyzing the root hair promoting activity. Degradation
products from the whey protein had the root hair promoting activity. The whey
protein was further separated by chromatography, and the degradation products
from each peak were analyzed by the root hair assay. Degradation products from 20
kDa protein showed the root hair promoting activity and the protein was identified as
Kunitz trypsin inhibitor.
Soy Protein Research, Japan7, 85-89, 2004.
Studies on Liver Triglyceride-lowering Action of Soyapeptide Prepared by
Using Hydrophobic Absorbent Resin Column
Mayumi MINAMI, Masanobu SAKONO and Nobuhiro FUKUDA
Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192
ABSTRACT
In previous studies, we have demonstrated a potent hypolipidemic effect of both LD3
(hydrolyzate of soy protein isolate obtained by digestion with fungus protease) and F5
(prepared by using hydrophobic adsorbent resin column). In the present studies, we
have attempted to clarify the active components occurring in the LD3 and F5 and to
elucidate the mechanism responsible for the observed reduction, using primary rat
hepatocytes. When the hydrolyzates of casein, LD3 and F5 were incubated with
primary hepatocytes, followed by addition of [1-14C]oleate as an exogenous fatty acid
substrate (experiment 1), the incorporation of oleate into the medium lipids, but not
into cellular lipids, was significantly lowered in the groups of LD3-D (enzyme
hydrolyzate of LD3) and F5-D (enzyme hydrolyzate of F5) in a dose-dependent
manner; the extent of lowering activity being greater in F5-D than in LD3-D. This
lowering activity was not true for casein hydrolyzate. Although the effects on lipid
synthesis of addition of three types of di-peptides, which may be absorbable from the
intestine into the blood stream, were not consistent in experiment 2 and 4, it
nevertheless appeared to have some beneficial effects on lipid synthesis. In the third
experiment, we examined the effects of isoflavone and saponin on the incorporation of
radiolabelled oleate into lipids. The results showed that there was a dose-dependent
reduction in incorporation of oleate into the cell and/or medium lipids, especially the
triglycerides, in the cells incubated with isoflavone and saponin, respectively. In the
fourth experiment, we further examined the effects of combinations of di-peptide with
isoflavone or saponin on the incorporation of [14C]oleate into lipids, and found that
under certain conditions there was an additional effect of this combination of di-peptide with the isoflavone or saponin in terms of the reduced synthesis of lipids,
especially triglycerides, as compared to those of either di-peptide, isoflavone or
saponin alone. On the other hand, there was an increased incorporation of
radiolabelled oleate into water soluble fraction (ketone body). These results suggest
that serum and liver lipid-lowering effect of LD3 or F5 is in part due to the
combinational effect of di-peptide and/or other components such as isoflavone,
flavone and saponin the mechanism responsible for the observed lowering effect is
attributed to an altered metabolism of fatty acid between oxidation and esterification
in the liver.
Soy Protein Research, Japan7, 90-97, 2004.
Key words : liver triglyceride-lowering action, soypeptide, rat, hepatocyte
Soy Proteins Affect Activities of Small Intestinal Transporters and
Improve Lipid Metabolism
Ryuichiro SATO
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657
ABSTRACT
Cholesterol efflux from human cultured intestinal cells, Caco-2, was studied.
[3H]cholesterol efflux to the apical side of Caco-2 cells was measured after induction of
ABC transporter gene expression by the LXR agonist. Cholesterol efflux to the apical
side was enhanced by the agonist and/or the addition of bile acid, but not apo A-1. This
indicates that certain transporters including ABCG5 and G8 induced by LXR stimulate
cholesterol efflux to the apical side in a bile acid-dependent manner. Among several bile
acid molecules, chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) are potent
acceptors. Crude peptide molecules digested from soy proteins as well as bile acids,
stimulated cholesterol efflux from the intestinal epithelial cells. We also developed a
novel assay system for evaluating bile acid absorption through the intestinal bile acid
transporter (IBAT). Some soy peptides did not affect bile acid absorption, but appeared
to stimulate the farnesoid X receptor (FXR) activity in cells after being absorbed. This
stimulating effect of soy peptides on the FXR activity might partly explain the
molecular mechanism by which dietary soy proteins reduce serum triacylglycerol and
cholesterol levels.
Soy Protein Research, Japan7, 98-101, 2004.
Key words : cholesterol, bile acid, IBAT, FXR, ABCG5, ABCG8
Beneficial Effects of a Soy Protein Diet and a Soybean Trypsin Inhibitor in
Genetically Obese and Diabetic Rats Lacking Cholecystokinin-A Receptor
Kyoko MIYASAKA, Setsuko KANAI, Minoru OHTA and Ayako SEKIME
Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology,
Tokyo 173-0015
ABSTRACT
Otsuka Long-Evans Tokushima Fatty (OLETF) rats were developed as a model for
noninsulin-dependent diabetes mellitus (NIDDM). OLETF rats are characterized by
late onset of hyperglycemia at 18 weeks of age, followed by insulin-dependent diabetes
mellitus at 65 weeks of age. Soybeans and their products are classical Japanese food
and are generally believed to be good for health. In this study, we compared the
effect of a special diet, consisting of soy protein, on energy metabolism of normal
LETO rats and genetically diabetic OLETF rats. Twelve-week-old male rats of LETO
and OLETF strains were fed this experimental diet in parallel with CRF-1 (control)
and examined for respiratory quotient (RQ) and the blood or plasma parameters
associated with diabetes mellitus. We also examined whether abnormal intestinal
triglyceride metabolism in OLETF rats with NIDDM could be improved by soy
protein and/or soy peptides diet. A mesenteric lyphphnode fistula was prepared.
Administration of a soy protein diet increased daily food intake but did not change
daily energy expenditure in LETO rats, resulting in increased body weight. In
diabetic OLETF rats, administration of a soy protein diet increased daily energy
expenditure, but daily food intake did not change therefore, the body weight slightly
decreased. Moreover, glucose tolerance test was slightly improved by administration
of a soy protein diet. The intestinal fat absorption was not influenced by long-term
administration of a soy protein diet. However, simultaneous infusion of a soy peptide
with fat into the small intestinal lumen, decreased the intestinal fat absorption.
Thus, ingestion of soy protein and/or peptide might prevent fat absorption and hyperlipidemia as well as obesity.
Soy Protein Research, Japan7, 102-107, 2004.
Key words : soy-protein diet, energy expenditure, non-insulin dependent diabetes
mellitus, aging, health
Mechanism of Inducing Satiety Effects by Peptides Derived from
Soybean β-Conglycinin
Hiroshi HARA, Toshihiro MAEKAWA and Tohru HIRA
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
ABSTRACT
Soybean β-conglycinin peptone (β-con) enhances satiety via cholecystokinin (CCK)
secretion, and the β-conglycinin β-subunit 51-63 (β51-63, VRIRLLQRFNKRS) is
responsible for this effect. The aim of the present study is to identify signaling
pathway induced with β-con by using the enteroendocrine cell line, STC-1. We found
that β-conglycinin peptone induced Ca signaling in the STC-1 cells, which correlated
with the binding activity of peptides derived from β-conglycinin to the rat jejunal
brush-border membrane. The Ca signaling induced by β-con was dependent on Ca
outside of cells passing through L-type Ca channel. Adenylate cyclase was partly
involved in the induction of Ca signaling byβ-con in STC-1 cells.
Soy Protein Research, Japan7, 108-113, 2004
1School of Life Studies, Sugiyama Jogakuen University, Nagoya 464-8662
2Graduate School of Agriculture, Kyoto University, Kyoto 611-0011
ABSTRACT
The aim of this study was to investigate the effects of chronic feeding with dietary
protein, on insulin resistance during weight loss, in type II diabetic mice. Obesity
was induced in KK-Ay mice by high fat feeding, followed by weight loss induced
through restrictive isoenergetic and isonitrogenous diets (35% of energy as protein
and 5% as fat) for five weeks. Restrictive diets were based on casein, wheat gluten
hydrolysate (GLU-H), or soy protein isolate- hydrolysate (SPI-H). The postprandial
RQ and generation of 13CO2 from ingested 13C-glucose were higher in nondiabetic mice
than in diabetic mice. The postprandial RQ was higher in GLU-H-fed and SPI-H-fed
mice than in casein-fed mice after 3 weeks. We also observed the elevated
postprandial RQ in GLU-H and SPI-H groups after all the mice had single feedings of
the casein diet. In an oral glucose tolerance test, the average oxidation of 13C-glucose
derived from orally administered glucose solution was higher in GLU-H-fed than in
casein-fed mice. The average plasma insulin level after 30 min. of glucose
administration was lower in GLU-H-fed than in casein-fed mice. In fasted mice, the
RQ and levels of plasma glucose and insulin did not differ significantly between
dietary groups. These results indicate that chronic feeding of GLU-H increases
postprandial carbohydrate oxidation and prevents development of insulin resistance
in type II diabetic mice.
Soy Protein Research, Japan7, 114-120, 2004.
Key words : KK-Ay, 13C, glucose uptake, diabetes, soybean
A Novel Hybrid-type Artificial Bone Prepared from Plant Protein
Toshiki MIYAZAKI
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology,
Fukuoka 808-0196
ABSTRACT
The so-called bioactive ceramics such as Bioglass®, sintered hydroxyapatite, and
apatite- and wollastonite-containing glass-ceramics (glass-ceramics A-W), bond to
living bone when implanted in bony defect. They have been already subjected to
clinical applications as bone substitutes in orthopedic surgery, neurosurgery,
dentistry, and so on. However, their higher brittleness and lower flexibility than
natural bone, limits their clinical application to low loaded conditions. Materials
exhibiting both high bioactivity and high flexibility are required for novel bone
substitutes. Natural bone is a kind of organic-inorganic hybrid, which is composed of
organic collagen and inorganic apatite nanocrystals. Based on this we think that
apatite-polymer hybrid produces novel bone-repairing materials, which exhibit both
bioactivity and flexibility. In living body, the bone tissue is produced by site-selective
precipitation of apatite on collagen fibers abundant in carboxyl group. In the present
study, we attempted to synthesize organic-inorganic hybrids from various plant
proteins such as wheat-derived gluten and corn-derived zein. This kind of plant
protein is free from diseases of virus and prion origin. Apatite deposition was
observed on the surfaces of these proteins in aqueous solution supersaturated with
respect to the apatite, when their surfaces were previously modified with calcium
ions. This type of organic-inorganic hybrid is quite attractive as a novel bone
substitutes in orthopedic and dental fields.
Soy Protein Research, Japan7, 121-124, 2004.
Key words : artificial bone, organic-inorganic hybrid, gluten, zein, apatite
1Tohoku University School of Medicine, Sendai 980-8575
2Tohoku University Graduate School of Dentistry, Sendai 980-8575
ABSTRACT
Genistein and daidzein, soybean isoflavones, have been described to bind to
estrogen receptors and to exert estrogenic and/or anti-estrogenic activity. We
have shown that heme binds to Bach1, a transcriptional repressor, and that this
binding abrogates the DNA-binding activity of Bach1 to enhance the transcription
of genes carrying Bach1-binding sequences. This mechanism is involved in heme
oxygenase (HO)-1 induction by heme. HOs degrade heme, a pro-oxidant, to produce
bilirubin, an anti-oxidant, and HO-1 is an inducible isozyme of HOs. In this study,
we investigated the effects of the isoflavones on gene regulation of HO-1 and its
related transcription factors, including Bach1, in human lung carcinoma cells. Both
genistein and daidzein inhibited the induction of HO-1 by sodium nitroprusside
(SNP), a nitric oxide donor, while genistein but not daidzein inhibited the hememediated
HO-1 induction. Genistein and daidzein had little effect on the mRNA
levels of Bach1 and Nrf2, the transcription factors which regulate transcription of
the HO-1 gene. Further study is under way to elucidate the regulatory mechanism
of the HO-1 expression by the isoflavones. As the HO-1 induction is an important
protective response against excessive free heme and other oxidative stresses,
daidzein might be, in some cases, a useful phytoestrogen in that it does not inhibit
the heme-mediated HO-1 induction.
Soy Protein Research, Japan7, 125-129, 2004.
Key words : isoflavone, genistein, daidzein, heme, heme oxygenase-1, phytoestrogen
Effect of Genistein on Immune Responses in NC/Nga Mice Used as an Animal
Model for Atopic Dermatitis
Tohru SAKAI, Mari KOGISO, Kaori MITSUYA and Shigeru YAMAMOTO
1Department of Clinical Nutrition, Osaka Prefecture College of Nursing, Habikino 583-8555
2Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima 770-8503
ABSTRACT
The isoflavone genistein is a phytoestrogen found in high levels in soy products that
has been associated with decreased incidence of breast and prostate cancers. In
addition, a recent clinical trial suggests that supplementation with the dietary
genistein may be as effective as hormone replacement therapy in attenuating
menopause-related bone loss without causing the associated side effects. In this
study, we examined the effect of genistein on the immune responses in NC/Nga mice
that are used as an animal model for atopic dermatitis. Groups of mice were exposed
to vehicle or the genistein by gavage. The doses of genistein used were 4 and 20
mg/kg body weight/day. Treatment with picrylchroride in the ear of NC/Nga mice
developed dermatitis. Mice that received genistein showed mild dermatitis when
compared to the control mice. We also determined serum IgE level, but significant
difference was not observed between the groups. Although genistein did not affect
the in vitro lymphocyte proliferation, it suppressed cytokine production by spleen
cells. These results showed that dietary genistein modulates an inflammatory
response resulting in amelioration of dermatitis in the skin of NC/Nga mice.
Soy Protein Research, Japan7, 130-136, 2004.
Key words : genistein, NC/Nga mice, IgE, cytokine, dermatitis
A Soybean Kunitz Trypsin Inhibitor Suppresses Ovarian Cancer Cell Invasion by
Blocking Urokinase Upregulation
Hiroshi KOBAYASHI and Mika SUZUKI
Department of Obstetrics and Gynecology,
Hamamatsu University School of Medicine, Shizuoka 431-3192
ABSTRACT
We have previously reported in a series of papers that a Kunitz-type protease
inhibitor, bikunin, suppresses upregulation of urokinase-type plasminogen activator
(uPA) and its specific receptor (uPAR) expression, phosphorylation of ERK1/2 and
cancer cell invasion in vitro and the peritoneal disseminated metastasis in vivo. In
the present study, we investigated the effects of soy bean trypsin inhibitor (SBTI) on
the net enzymatic activity of secreted extracellular uPA, the signal transduction
involved in the expression of uPA and the invasion in HRA human ovarian cancer
cells. SBTI contains a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk inhibitor
(BBI). Here, we show 1) that KTI and BBI were purified separately from soybeans, 2)
that neither KTI nor BBI effectively inhibits enzymatic activity of uPA, 3) that uPA
upregulation observed in HRA cells was inhibited by preincubation of the cells with
KTI with an IC50 of ~2μM, whereas BBI failed to repress uPA upregulation, as
measured by enzyme-linked immunosorbent assay, 4) that the cell invasiveness was
inhibited by treatment of the cells with KTI with an IC50 of ~3μM, whereas BBI
failed to suppress cell invasion, as measured by an in vitro invasion assay, 5) KTI
suppresses HRA cell invasion by blocking uPA up-regulation which may be mediated
by a binding proteins other than a bikunin-binding protein and/or its receptor, and 6)
that the transforming growth factor-beta 1 (TGF-β1)-mediated activation of ERK1/2
was significantly reduced by preincubation of the cells with KTI. In conclusion, KTI,
but not BBI, could inhibit cell invasiveness at least through suppression of uPA
signaling cascade, although the mechanisms of KTI may be different from those of bikunin.
Soy Protein Research, Japan7, 137-144, 2004.
Key words : bikunin, urokinase, cancer metastasis, ovarian cancer, soybean protein
Anti-platelets Effects of Genistein, an Isoflavonoid from Soybean
Atsushi ODA
Department of Preventive Medicine,
Hokkaido University School of Medicine, Sapporo 060-8638
ABSTRACT
It has been reported that genistein inhibits platelet functions in vitro. The
mechanisms of the inhibitory effects are poorly understood. Genistein is a wellknown
inhibitor of protein tyrosine kinases. However, I show here that genistein
inhibits platelet activating factor (PAF)-induced elevation of ionized calcium
concentrations and IP3 production in platelets, induced by PAF, in the presence of
staurosporin, which abolishes increased protein tyrosine phosphorylation. Thus, it
appears that genistein inhibits activation of phospholipase C in stimulated platelets,
apparently independent of its effects on tyrosine kinases. I also show that catechins,
which might be consumed along with genistein in the Japanese diet, had markedly
different effects on platelets in comparison with genistein. As people take different
flavonoids simultaneously, it is highly desirable that the combined effects of
flavonoids are elucidated in different systems. In this sense, platelets may be useful
in these settings.
Soy Protein Research, Japan7, 145-148, 2004.
Soy Protein and Isoflavone Intake and the Risk of Colon Cancer
Chisato NAGATA
Gifu University Graduate School of Medicine, Gifu 501-1194
ABSTRACT
This study examined the relationship between soy protein and isoflavone intake and
the subsequent risk of colon cancer in a population-based cohort of Japanese men and
women. In 1992, the usual diet including soy intake was determined in 13,893 men
and 16,331 women in a city of Gifu Prefecture, with the use of a validated foodfrequency
questionnaire. The length of follow-up was about 8 y. In men, there were
no significant associations between risks of colon cancer and intakes of soy products,
soy protein, and soy isoflavones. In women, the highest compared to the lowest
tertile of soy product intake was significantly associated with a decreased risk of
colon cancer after controlling for covariates. However, the associations of colon
cancer risks with soy protein and soy isoflavone intakes were not statistically
significant.
Soy Protein Research, Japan7, 149-151, 2004.
Key words : soy protein, isoflavones, colon cancer, prospective study
Clinical Usefulness of β-Conglycinin Containing Snack for the Diet Therapy
of Hyperlipidemia and Obesity
Tomomi ADACHI1, Junko NABIKI1, Takeshi MIYAWAKI1, Nayuta MORI1,
Tetsuhiko OKAJIMA2, Motohiko HIROTSUKA2, Aki HARANO3 and Yutaka HARANO1
1Koshien University College of Nutrition, Takarazuka 665-0006
2Fuji Oil Co.,LTD., Izumisano 598-8540
3Harano hospital, Minoh 562-0005
ABSTRACT
Protein cookie comprising β-Conglycinin (β-CG), one of the major components of soy
bean protein that has been reported to reduce blood TG and cholesterol level in
animal study, was used as a supplement for the diet therapy of hyperlipidemia and
obesity. The protein cookie (9 g β-CG) was ingested by 5 healthy subjects, and their
glycemic and insulin responses were compared with those of control subjects who
ingested starch cookie in which soy bean protein was replaced with flour starch.
Thirty subjects with life- style related diseases, who were obese or having
dyslipidemia, were encouraged to take this snack (6 g/day), replacing or reducing
regular snack or carbohydrate source). Significant reduction in body weight (0.7 kg),
TG (19 mg/100mL) and chol (11 mg/100mL) was observed after 1-3 months.
Hamburger (500 kcal containing 37 g fat) was fed daily for a week to each of a total of
25 healthy subjects who were divided into those with or without intake of β-CG
cookie. Cookie test was performed before and after the burger-feeding. By PAGE
analyses of serum collected at 0, 1, 2 hr, presence or appearance of atherogenic
lipoprotein (VLDL, mid-band, decrease of HDL, small-dense LDL) was scored as 1, and
judged in total scores. Disappearance of dyslipidemia was counted as -1. In those
havingβ-CG no rise in score was noted, whereas in placebo the points increased from
0.5 to 2.5, suggesting an improving effect of β-CG cookie on VLDL metabolism.
Hepatic TG lipase when measured after heparin injection (30 U/kg body weight), tended to be activated, but no change was noted for LPL. β-CG cookie was effective
in weight reduction or dyslipidemia when used as a supplement in diet therapy.
Subjects were encouraged to replace or reduce regular snack, rich in fat or
carbohydrate or meal starch, with the newly developed soy bean cookie. The
hypolipidemic effect seemed to be attributable to the additive effect of improved
insulin sensitivity due to weight reduction and direct effect of β-CG. Antiatherogenic
effect on lipoprotein metabolism was mainly observed in TG rather than
cholesterol metabolism. Activation of hepatic TG lipase was suggested as one of the
mechanisms involved.
Soy Protein Research, Japan7, 152-156, 2004.
Key words : dyslipidemia, β-conglycinin, soy-bean protein, obesity, life-style related
diseases
Effect of Soy Isoflavones on Premenstrual Syndrome (Part 2)
Naoko ISHIWATA1, Sayo UESUGI2, Mariko UEHARA2 and Shaw WATANABE2
1Department of Domestic Science, Atomi Junior College, Tokyo 112-8687
2Faculty of Applied Bio-Science, Tokyo University of Agriculture, Tokyo 156-8502
ABSTRACT
Fifty-four young women with premenstrual syndrome participated in a randomized,
double-blind, placebo-controlled intervention trial. They were divided into two
groups: Twenty-eight women took a 20 mg/day isoflavone supplement (20 mg/day IF
group); 26 women took a 40 mg/day isoflavone supplement (40 mg/day IF group).
During the three periods of baseline, IF supplement, and placebo (PL), blood and urine
samples were obtained from women at times corresponding to peak estrogen and
progesterone in each woman's menstrual cycle. There was a trend toward a decrease
in the estradiol/progesterone ratio among women with moderate to serious
symptoms, and a decrease in prostaglandin E2 (PGE2) among women with serious
symptoms. Independent of symptom severity, the serotonin tended to increase
among individuals in the 40 mg/day IF group. Prolactin tended to increase as
symptom severity increased, and was significantly lower in the 40 mg/day IF group
compared to the placebo among those with severe symptoms. Sex-hormone binding
globulin levels tended to increase among the 40 mg/day IF group when symptoms
were severe. Only twenty percent of subjects were equol producers, similar to the
level observed among Westerners.
Soy Protein Research, Japan7, 157-161, 2004.
Key words : premenstrual syndrome, soy isoflavones, sex steroid hormone, prostaglandin, equol