Rep. Nat'l. Food Res. Inst No.71, 7 13
Alteration by dietary soy isoflavone of SERBP-1-dependent genes
in the liver of rats fed a high saturated-fat diet
and Takashi Ide
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
The effect of soy isoflavone on mRNA expression of sterol regulatory element-binding protein (SREBP)-1 isoforms and the tar-
get genes of SREBP-1 involved in fatty acid metabolism was examined in rats. Animals were fed a 20% palm oil diet containing
0, 0.05, 0.1, 0.2, or 0.4% of soy isoflavone for 14 d. The diets containing 0.1, 0.2, and 0.4% of isoflavone significantly increased
the mRNA level of hepatic SREBP-1a to a similar level compared to a diet containing 0.05% of isoflavone or a diet free of
isoflavone. In contrast, diets containing 0.05-0.2% isoflavone, compared to a diet free of isoflavone, did not influence the mRNA
level of SREBP-1c. However, a diet containing 0.4% of isoflavone lowered this parameter to one half that observed with an
isoflavone-free diet. Dietary isoflavone dose-dependently increased the mRNA levels of genes targeted by SREBP-1 (fatty acid
synthase, stearoyl-CoA desaturase 1 and 6-desaturase), and they peaked at a dietary level of 0.1 or 0.2%. However, a diet con-
taining 0.4% of isoflavone decreased these parameters to the levels observed in rats fed an isoflavone-free diet. Thus, it is appar-
ent that varying amounts of dietary isoflavone modulate mRNA expressions of SREBP-1a and -1c differently. The contours of
the changes in the gene expressions of SREBP-1 isoforms may account for the biphasic modulation by different dietary levels of
isoflavone in the mRNA expression of the genes targeted by these transcriptional factors.
through the alterations in the expression of genes targeted by
these nuclear receptors.
Sterol regulatory element-binding protein (SREBP)-1 is a
Soybean contains considerable amounts of compounds col-
transcription factor that regulates the expression of genes
lectively called isoflavone, which mainly exist as glycoside.
involved in lipogenesis . Also, several genes involved in
Among soy isoflavones, the predominant compounds of agly-
fatty acid desaturation are controlled by this transcription fac-
con are daidzein, genistein and glycitein. Soy isoflavone has
tor . There are two isoforms of SREBP-1, termed SREBP-1a
various meritorious physiological activities to prevent post-
and -1c, that are derived from a single gene through the use of
menopausal symptoms, prostate and breast cancers, cardio-
an alternative transcriptional start site. It has been reported
vascular disease, and osteoporosis . Genistein, daidzein and
that isoflavone aglycons decreased serum and liver triglyc-
its intestinal bacterial metabolite equol are considered to be
eride levels in rats
and diabetic mice . In addition, dietary
ligands of estrogen receptor . Also, genistein but not daidzein
isoflavone lowered the activity of hepatic
was found to be an activator of peroxisome proliferator-acti-
which is known as one of the genes modulated by SREBP-1 .
vated receptor (PPAR)
. Therefore, it is possible that the
These studies raise the possibility that soy isoflavone not only
pleiotropic physiological effects of isoflavone are mediated
affects nuclear receptor-dependent metabolic pathways, but
Corresponding author: Tel.: +81-29-838-8083
also modulates the expression of the genes under the control
to 100. Soy isoflavone was added to experimental diets
of SREBP-1 and hence affects lipogenesis. The observation
instead of sucrose. Body weight at the start of experiment was
that genistein and daidzein did not affect SREBP-1 expres-
119-138 g. Animals had free access to the diets and water
sion in cultured hepatic cells may be contrary to this
during the experiment. At the end of the experiment, the ani-
. However, studies regarding the impact of
mals were sacrificed by bleeding from the abdominal aorta
isoflavone on mRNA expression of SREBP-1 in experimental
under diethyl ether anesthesia. Livers were then quickly
animals have been lacking. To examine the hypothesis that
excised and weighed. This study followed the institute s
soy isoflavone modulates lipogenesis through SREBP-1-
guidelines in the care and use of laboratory animals.
dependent mechanism, we analyzed the effect of a prepara-
tion rich in isoflavone glycosides made of soybean on gene
expressions of SREBP-1a and -1c, and some of their target
RNA in the liver was extracted
, and mRNA levels of
genes in rat liver. We used high saturated-fat diets for this
specific genes were analyzed by a quantitative real-time PCR
purpose, since a previous study showed that a high saturated-
using a PRISM 7000 sequence detection system (Applied
fat diet evoked more than 7-fold and approximately 2-fold
Biosystems, Foster City, CA) as detailed previously
increase in mRNA expression of SREBP-1c and -1a, respec-
mRNA abundance was calculated as its ratio to the 18S rRNA
tively, in mice .
level in each cDNA sample and the abundance was expressed
as a percentage assigning the value in rats fed a isoflavone-
free diet as 100. The nucleotide sequences of primers and
probes to detect each mRNA were designed using the Primer
Animals and diets
Express Software (Applied Biosystems) according to the
Male Sprague-Dawley rats obtained from Charles River
sequences available from the GenBank database. The
Japan (Kanagawa, Japan) at 4 weeks of age were housed indi-
nucleotide sequences of primers and probes for SREBP-1a,
vidually in a room with controlled temperature (20-22
SREBP-1c, fatty acid synthase, and 18S rRNA were the same
humidity (55-65%), and lighting (lights on from 07:00 to
as reported elsewhere
. Those for forward and reverse
19:00 h) and fed a commercial nonpurified diet (Type NMF;
primers and probes to detect mRNA of stearoly-CoA desat-
Oriental Yeast, Tokyo, Japan). After 7 d of acclimatization in
urase 1 were:
our housing condition, rats were randomly divided into 5
groups and fed for 14 d the purified experimental diets con-
taining varying amounts (0, 0.11, 0.21, 0.43 and 0.85%) of a
5-ACCGTGCGTGCTAATTCTTCTCTCAAGGT-5, and of
preparation rich in soy isoflavone glycosides (Soyaflavone
-desaturase were 5-ACCGCTGCTCATCCCTATGT-3,
HG) kindly donated from Fuji Oil Co. Ltd. Osaka, Japan. As
the soy isoflavone preparation contained 47.0% (w/w)
isoflavones, our experimental diets contained 0, 0.05, 0.1, 0.2
and 0.4% of soy isoflavone. The composition of the
Analysis of isoflavone in serum
isoflavones in the preparation were (in weight %): daidzin,
One hundred l of 0.58 M acetic acid and 50 l of a
33.8; glycitin, 16.4; genistin, 6.89; malonyl daidzin, 25.5;
glucuronidase prepatation (Type HP2, Sigma, St. Louis, MO)
malonyl glycitin, 9.68; malonyl genistin, 6.09; acetyl daidzin,
were added to 1 ml of serum, and incubated at 37
for 4 h.
0.54; acetyl glycitin, 0.80; acetyl genistin, 0.13; daidzein,
After the enzymatic hydrolysis, each serum sample was
0.07; glycitein, 0.06, and genistein, 0.02. Thus, our experi-
spiked with 6 g of fluorescein dissolved in 50 l of
mental diets contained isoflavone aglycons in the following
methanol as an internal standard, and 4 ml of 70 mM sodium
composition (in weight %): daidzein, 59.9; glycitein, 27.0,
dihydrogenphosphate was added. Isoflavones in the samples
and genistein, 13.1. The basal composition of the experimen-
were extracted using solid phase extraction cartridges (Sep-
tal diet was (in weight %): casein, 20; palm oil, 20; corn
Pak C18 cartridge, Waters, Milford, MA). The cartridges
starch, 15; cellulose, 2; mineral mixture , 3.5; vitamin mix-
were preconditioned with 6 ml each of methanol and 70 mM
ture , 1.0; L-cystine, 0.3; choline bitartrate, 0.25 and sucrose
sodium dihydrogenphosphate. The samples were loaded onto
the cartridges and allowed to flow freely. The cartridges were
were detected among the groups.
then consecutively washed with 10 ml of 70 mM sodium
dihydrogenphosphate and 2 ml of water. Isoflavones and an
Serum isoflavone concentrations
internal standard were eluted with 6 ml of methanol, then
Isoflavone was not detected in the serum of rats fed on an
dried under nitrogen and re-constituted with 200 l of
isoflavone-free diet. As expected, serum concentrations of
methanol for HPLC analysis. Isoflavones were analyzed by a
daidzein, glycitein and genistein progressively increased as
reversed-phase HPLC using a CAPCELL PAK AG120 C18
dietary levels of isoflavone increased (Table 1). Daidzein,
column (250 x 4.6 mm, Shiseido, Tokyo, Japan) with a
glycitein and genistein comprised 62.5, 25.2 and 12.3% of
mobile phase of 0.5% phospholic acid in water/acetonitrile
total isoflavone in the serum of rats fed a 0.05% isoflavone
(70:30, v/v), at a flow rate of 1.0 ml/min, monitoring at 259
diet. These values were comparable to the proportions con-
nm for isoflavones and 224 nm for fluorescein.
tained in experimental diets (58.5, 28.5 and 13.1%, respec-
tively). However, the proportions of daidzein in serum were
considerably higher in rats fed 0.1, 0.2 and 0.4% isoflavone
The data were analyzed by one-way ANOVA, and a
diets (75.3-77.6%) than in the animals fed a 0.05% isoflavone
Tukey-Kramer post hoc analysis was used to detect signifi-
diet. This was accompanied by the decreases in the propor-
cant differences between the means at a level of p<0.05.
tions of both glycistein (14.7-18.3%) and genistein (5.0-
mRNA levels of SREBP-1a and -1c and their tar-
Animal growth and liver weights
No significant differences were detected in food intake
We quantified the mRNA levels of SREBP-1a and -1c that
among the groups of rats fed diets containing varying
were involved in the regulation of gene expression of various
amounts of isoflavone (18.1-20.2 g/d). The growth was sig-
and some enzymes involved in fatty
nificantly lower in rats fed a diet containing 0.4% of
acid desaturation . mRNA level of SREBP-1a was compara-
5 g/14 d) than in the animals fed diets con-
ble between rats fed diets containing 0 and 0.05% of
taining 0.05 and 0.1% of isoflavone (145
8 and 147
isoflavone (Fig. 1). However, diets containing 0.1, 0.2 and
g/14 d, respectively). However, significant differences were
0.4% of isoflavone, compared to an isoflavone-free diet,
not detected among rats fed diets containing 0% (137
caused significant 68, 98, and 64% increases, respectively, in
g/14 d), 0.2% (143
6) and 0.4% of isoflavone. Liver
this parameter. Response to dietary isoflavone of mRNA
weight was significantly lower in rats fed a diet containing
expression of SREBP-1c was considerably different. Diets
0.4% of isoflavone (5.18
0.14 g/100 g body weight) than
containing 0.05-0.2% of isoflavone, compared to an
in the animals fed a diet containing 0.2% of isoflavone (5.77
isoflavone-free diet, did not affect mRNA expression of
0.18). No other significant differences in this parameter
SREBP-1c, but a diet containing 0.4% of isoflavone signifi-
The values were expressed as percentages, assigning the value in animals fed a diet free of isoflavone as 100. Values represent
SE of 7 or 8 rats. Values with different superscript differ significantly at p<0.05.
cantly reduced the mRNA level (Fig. 1).
1c, whereas isoflavone at dietary levels greater than 0.1%
The mRNA levels of SREBP-1-dependent genes, involved
increases the mRNA level of SREBP-1a. Shimomura et al.
in fatty acid synthesis (fatty acid synthase ), and fatty acid
reported that mRNA level of SREBP-1c exceeded that of
desaturation (stearoyl-CoA desaturase 1
SREBP-1a in tissues of mice, including liver (8.8-fold in
desaturase ) were also analyzed (Fig. 2). The responses of
liver) . Thus, it is generally considered that SREBP-1c but
mRNA expression of these enzymes to dietary isoflavone
not SREBP-1a is primarily involved in the regulation of lipo-
were biphasic. Increasing dietary levels of isoflavone up to
genesis and fatty acid desaturation in the liver. On the other
0.1% up-regulated the mRNA levels of these enzyme genes.
hand, it is reported that transcriptional activity of SREBP-1c
However, a diet containing 0.2% isoflavone failed to cause
is much weaker than that of SREBP-1a due to its shorter tran-
additional increases in the mRNA levels, and isoflavone at a
scriptional regulatory domain
. In fact, Amemiya-Kudo et
0.4% dietary level decreased the mRNA levels comparable to
al. demonstrated that over-expression of SREBP-1a in mice
those observed in rats fed an isoflavone-free diet.
profoundly increased mRNA levels of several genes involved
in lipogenesis in liver, whereas SREBP-1c over-expression
caused only moderate increase in these values . In the pres-
ent study, isoflavone at dietary levels of 0.1 and 0.2%
Previous studies have demonstrated that soy isoflavone
increased mRNA expression of SREBP-1-dependent genes
lowers serum triacylglycerol concentration in experimental
(Fig. 2). The contours of the changes paralleled that of
. We hypothesized in the present study that alter-
SREBP-1a but not of SREBP-1c (Fig. 1). This observation
ations in the gene expression of SREBP-1 and consequently
suggests that the former but not the latter is mainly involved
of its target genes may account for triacylglycerol-lowering
in the regulation of SREBP-1-dependent genes under these
effect of genistein and daidzein in experimental animals. Our
nutritional conditions. A diet containing 0.4% of isoflavone
study showed that dietary isoflavone modulates mRNA
decreased mRNA expression of SREBP-1-dependent genes
expression of SREBP-1a and -1c isoforms differently in the
even though SREBP-1a mRNA level was still sustained at a
liver according to its dietary levels. These changes were
high level (Fig. 2). Thus, the down-regulation of the expres-
accompanied by the alterations in the mRNA expression of
sion of SREBP-1c observed at this dietary level of isoflavone
some SREBP-1-dependent enzymes.
may counteract the physiological activity of SREBP-1a to
The present study demonstrated that a relatively high dose
increase the expression of genes involved in lipogenesis and
of dietary isoflavone down-regulates mRNA level of SREBP-
fatty acid desaturation.
The values were expressed as percentages, assigning the value in animals fed a diet free of isoflavone as 100. Values represent
SE of 7 or 8 rats. Values with different superscript differ significantly at p<0.05.
We showed that isoflavone at low dietary levels increased
dependent genes. However, the 0.4% isoflavone diet
mRNA expression of SREBP-1-dependent genes, whereas a
decreased mRNA expression of these genes to the level
diet high in isoflavone decreased these parameters. Genistein
observed in rats fed an isoflavone-free diet. Therefore, pres-
and daidzein have similar molecular weights and structural
ent study demonstrated that varying amounts of dietary
characteristics to that of 17 -estradiol and hence exerts estro-
isoflavone modulated the mRNA expressions of SREBP-1a
genic or anti-estrogenic action
. In addition, genistein and
and -1c differently. The observed changes in mRNA levels
daidzein affect adipogenesis through PPAR -dependent
may account for the biphasic modulations by different dietary
mechanism . Dang and his colleagues
levels of isoflavone in the expression of SREBP-1-dependent
genistein at a low concentration (1 M) stimulates osteogene-
sis and suppresses adipogenesis through the interaction with
estrogen receptors. However, at high concentrations (>1 M),
genistein acted as a ligand for PPAR
and hence up-regulat-
ed adipogenesis and down-regulated osteogenesis.
01) McCue, P., Shetty, K., Health benefits of soy
Furthermore, it has been demonstrated that the growth of
isoflavonoids and strategies for enhancement: a review,
estrogen-responsive breast cancer cells was stimulated by
Crit. Rev. Food Sci. Nutr., 44, 361-377 (2004).
genistein at the concentration of 10
M, was but was sup-
02) Kostelac, D., Rechkemmer, G., Briviba, K.,
pressed at the concentration of 10 M . Apparently, physio-
Phytoestrogens modulate binding response of estrogen
logical responses to varying doses of genistein were biphasic
to the estrogen response element. J.
in several cases. Therefore, the observed biphasic alterations
Agric. Food Chem., 51, 7632-7365 (2003).
by different dietary levels of isoflavone in mRNA expressions
03) Kim, S., Shin, H. J., Kim, S. Y., Kim, J. H., Lee, Y. S.,
of SREBP-1-dependent genes are not surprising.
Kim, D. H., Lee, M. O., Genistein enhances expression
In conclusion, our study showed that isoflavone at dietary
of genes involved in fatty acid catabolism through activa-
levels greater than 0.1% increased the mRNA level of
tion of PPAR , Mol. Cell. Endocrinol., 220, 51-58
SREBP-1a in liver; while a diet containing 0.4% of
isoflavone decreased the value of SREBP-1c. Diets contain-
04) Shen, P., Liu, M. H., Ng, T. Y., Chan, Y. H., Yong, E.
ing 0.1-0.2% of isoflavone compared to an isoflavone-free
L., Differential effects of isoflavones, from Astragalus
diet significantly increased mRNA expressions of SREBP-1-
membranaceus and Pueraria thomsonii, on the activation
of PPAR , PPAR , and adipocyte differentiation in
14) Ide, T., Interaction of fish oil and conjugated linoleic
vitro. J. Nutr., 136, 899-905 (2006).
acid in affecting hepatic activity of lipogenic enzymes
05) Shimano, H., Sterol regulatory element-binding proteins
and gene expression in liver and adipose tissue, Diabetes,
(SREBPs): transcriptional regulators of lipid synthetic
54, 412-423 (2005).
genes, Prog. Lipid Res., 40, 439-452 (2001).
15) Lim, J. S., Adachi, Y., Takahashi, Y., Ide, T.,
06) Kawakami, Y., Tsurugasaki, W., Nakamura, S., Osada,
Comparative analysis of sesame lignans (sesamin and
K., Comparison of regulative functions between dietary
sesamolin) in affecting hepatic fatty acid metabolism in
soy isoflavones aglycone and glucoside on lipid metabo-
rats, Br. J. Nutr., 97, 85-95 (2007).
lism in rats fed cholesterol. J. Nutr. Biochem., 16, 205-
16) Horton, J. D., Goldstein, J. L., Brown, M. S., SREBPs:
activators of the complete program of cholesterol and
07) Park, S. A., Choi, M. S., Cho, S. Y., Seo, J. S., Jung, U.
fatty acid synthesis in the liver, J. Clin. Invest., 109,
J., Kim, M. J., Sung, M. K., Park, Y. B., Lee, M. K.,
Genistein and daidzein modulate hepatic glucose and
17) Amemiya-Kudo, M., Shimano, H., Hasty, A. H., Yahagi,
lipid regulating enzyme activities in C57BL/KsJ-db/db
N., Yoshikawa, T., Matsuzaka, T., Okazaki, H., Tamura,
mice, Life Sci., 79, 1207-1213 (2006).
Y., Iizuka, Y., Ohashi, K., Osuga, J., Harada, K., Gotoda,
08) Matsuzaka, T., Shimano, H., Yahagi, N., Amemiya-
T., Sato, R., Kimura, S., Ishibashi, S., Yamada, N.,
Kudo, M., Yoshikawa, T., Hasty, A. H., Tamura, Y.,
Transcriptional activities of nuclear SREBP-1a, -1c, and
Osuga, J., Okazaki, H., Iizuka, Y., Takahashi, A., Sone,
-2 to different target promoters of lipogenic and choles-
H., Gotoda, T., Ishibashi, S., Yamada, N., Dual regula-
terogenic genes, J. Lipid Res., 43, 1220-1235 (2002).
tion of mouse
-desaturase gene expression by
18) Tabor, D. E., Kim, J. B., Spiegelman, B. M., Edwards, P.
SREBP-1 and PPAR , J. Lipid Res., 43, 107-114
A., Identification of conserved cis-elements and tran-
scription factors required for sterol-regulated transcrip-
09) Mullen, E., Brown, R. M., Osborne, T. F., Shay, N. F.,
tion of stearoyl-CoA desaturase 1 and 2, J. Biol. Chem.,
Soy isoflavones affect sterol regulatory element binding
74, 20603-20610 (1999).
proteins (SREBPs) and SREBP-regulated genes in
19) Yousef, M. I., Kamel, K. I., Esmail, A. M., Baghdadi, H.
HepG2 cells, J. Nutr., 134, 2942-2947 (2004).
H., Antioxidant activities and lipid lowering effects of
10) Tovar, A. R., Torre-Villalvazo, I., Ochoa, M., Elias, A.
isoflavone in male rabbits, Food Chem. Toxicol., 42,
L., Ortiz, V., Aguilar-Salinas, C. A., Torres, N., Soy pro-
tein reduces hepatic lipotoxicity in hyperinsulinemic
20) Kawakami, Y., Tsurugasaki, W., Yoshida, Y., Igarashi,
obese Zucker fa/fa rats, J. Lipid Res., 46, 1823-1832
Y., Nakamura, S., Osada, K., Regulative actions of
dietary soy isoflavone on biological antioxidative system
11) Lin, J., Yang, R., Tarr, P. T., Wu, P. H., Handschin, C.,
and lipid metabolism in rats, J. Agric. Food Chem., 52,
Li, S., Yang, W., Pei, L., Uldry, M., Tontonoz, P.,
Newgard, C. B., Spiegelman, B.M., Hyperlipidemic
21) Kojima, T., Uesugi, T., Toda, T., Miura, Y., Yagasaki,
effects of dietary saturated fats mediated through PGC-
K., Hypolipidemic action of the soybean isoflavones
1beta coactivation of SREBP, Cell, 120, 261-273 (2005).
genistein and genistin in glomerulonephritic rats, Lipids,
12) Reeves, P. G., Nielsen, F. H., Fahey, G. C. Jr., AIN-93
37, 261-265 (2002).
purified diets for laboratory rodents: final report of the
22) Shimomura, I., Shimano, H., Horton, J. D., Goldstein, J.
American Institute of Nutrition ad hoc writing committee
L., Brown, M. S., Differential expression of exons 1a and
on the reformulation of the AIN-76A rodent diet, J.
1c in mRNAs for sterol regulatory element binding pro-
Nutr., 123, 1939-1951 (1993).
tein-1 in human and mouse organs and cultured cells, J.
13) Chomczynski, P., Sacchi, N., Single-step method of
Clin. Invest., 99, 838-845 (1997).
RNA isolation by acid guanidinium thiocyanate-phenol-
23) Shimano, H., Horton, J. D., Shimomura, I., Hammer, R.
chloroform extraction. Anal. Biochem., 162, 156-159
E., Brown, M. S., Goldstein, J. L., Isoform 1c of sterol
regulatory element binding protein is less active than iso-
form 1a in livers of transgenic mice and in cultured cells,
(PPAR ) as a molecular target for the soy
J. Clin. Invest., 99, 846-854 (1997).
phytoestrogen genistein. J. Biol. Chem., 278, 962-967
24) Lephart, E. D., Setchell, K. D., Handa, R. J., Lund, T. D.,
Behavioral effects of endocrine-disrupting substances:
26) Wang, T. T., Sathyamoorthy, N., Phang, J. M.,
phytoestrogens. ILAR J., 45, 443-454 (2004).
Molecular effects of genistein on estrogen receptor medi-
25) Dang, Z. C., Audinot, V., Papapoulos, S. E., Boutin, J.
ated pathways. Carcinogenesis, 17, 271-275 (1996).
A., Lowik, C. W., Peroxisome proliferator-activated