Biochemical and Molecular Action of Nutrients

Soybean Isoflavones, Genistein and Genistin, Inhibit Rat Myoblast

Proliferation, Fusion and Myotube Protein Synthesis1
Shaoquan Ji,2 Gawain M. Willis, G. Robert Frank, Steven G. Cornelius
and Michael E. Spurlock
Swine Research Group, Purina Mills Research Center, Gray Summit, MO 63039

KEY WORDS:

isoflavone

genistein

L8 cells

muscle

Plant isoflavones, including genistein (49,5,7-trihydroxyisoflavone), genistin (the glycosylated genistein), daidzein
(49,7-dihydroxyisoflavone) and daidzin (glycosylated derivative of daidzein), are the major phytoestrogens in some plants.
Genistein and genistin exist at ;4.6 18.2 and 200.6 960
mg/g, respectively, of soybean, soybean nuts and soy powder
(Fukutake et al. 1996). Most soy products, especially fermented products, contain appreciable amounts (13 mg/g) of
genistein and daidzein (Coward et al. 1993), and soy-based
infant formula contains ;3287 mg/g genistein (Irvine et al.
1998). Glycosylated genistin is believed to be readily hydrolyzed by the acidic environment in the stomach and/or converted to the more active genistein in the gastrointestinal tract
by bacterial galactosidase (Chang and Nair, 1995). It is not
known whether this conversion is necessary for genistin absorption and bioactivity.
In recent years, genistein has attracted considerable attention because epidemiologic studies showed that consumption
of soybean-containing diets was associated with a lower incidence of certain human cancers in Asian populations (Barnes

protein

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ABSTRACT The isoflavones, genistein and genistin, are cytotoxic in vitro (e.g., inhibition of cell proliferation), due
in part to inhibition of protein tyrosine kinase and DNA topoisomerase activities. Normal cell functions associated
with these enzymatic activities could potentially be impaired in animals through ingestion of soybean products. In
this study, cultured rat myogenic cells (L8) were used to determine whether genistein or genistin influences
myoblast proliferation and fusion, and myotube protein synthesis and degradation. Genistein or genistin was
dissolved in dimethylsulfoxide and included in the culture medium at 0, 1, 10 or 100 mmol/L. Myoblast proliferation
was measured by methyl-3H-thymidine incorporation over 48 h. Myoblast differentiation was evaluated by the
number of nuclei in multinucleated myotubes. Myotube protein synthesis was measured by 2-h 3H-amino acid
incorporation into the myosin and total protein pools after acute (2 h) or chronic (24 h) exposure to similar
treatments; protein degradation was measured by measuring radioactivity in protein pools following a time course
of protein breakdown after myotube proteins were prelabeled with 3H-amino acids. Genistein or genistin strongly
inhibited in vitro myoblast proliferation (P , 0.001) and fusion (P , 0.001) in a dose-dependent manner with
effective genistein concentration as low as 1 mmol/L. Genistein or genistin inhibited protein accretion in myotubes
(P , 0.001). Decreased protein accretion is largely a result of inhibition on cellular (myofibrillar) protein synthesis
rate. No adverse effect on protein degradation was observed. Results suggest that if sufficient circulating
concentrations are reached in tissues of animals consuming soy products, genistein/genistin can potentially affect
normal muscle growth and development. J. Nutr. 129: 12911297, 1999.

et al. 1990, Setchell et al. 1984). In vitro studies further

showed that such chemopreventive and antineoplastic effects
were associated with the antioxidant activity of genistein (Cai
and Wei 1996, Record et al. 1995, Ruiz-Larrea et al. 1997) and
multiple inhibitory activities on cell proliferation and angiogenisis (Fotsis et al. 1997). The inhibitory effect of genistein
was achieved largely through inhibition of tyrosine protein
kinase (Linassier et al. 1990) and/or of DNA topoisomerase II
activities (McCabe and Orrenius 1993). The antioxidation
and the chemopreventive activities associated with ingestion
of diet-derived genistein are beneficial. However, activities
associated with the inhibitory activities on protein tyrosine
kinases and DNA topoisomerase II could potentially be detrimental because DNA replication and receptor-mediated signal
transduction pathways for some important hormones (e.g.,
growth hormone or insulin) and growth factors (e.g., insulinlike growth factors or epidermal growth factors) are part of
normal cellular functions.
Soybean and soy products are an important part of the diet
for some human populations and have traditionally been consumed at a high percentage in some animal feeds. Soy formulafed infants had phytoestrogens circulating at concentrations 13,000 20,000 times higher than plasma estradiol
concentrations, suggesting some biological effect (Setchell et
al. 1997). Cellular functions that depend on those enzymatic

1
Published in abstract form [Ji, S., Willis, G. M., Frank, G. R., Cornelius, S. G.
& Spurlock, M. E. (1997) Soybean genistein inhibits myoblast proliferation, differentiation and myotube protein synthesis. J. Anim. Sci. 75 (suppl. 1): 56 (abs.)].
2
To whom correspondence and reprint requests should be addressed.

0022-3166/99 $3.00 1999 American Society for Nutritional Sciences.

Manuscript received 7 January 1999. Initial review completed 22 February 1999. Revision accepted 13 April 1999.
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activities inhibited by genistein will potentially be compromised if a sufficient quantity of genistein enters the circulation
and animals consuming it are unable to inactivate it quickly.
The objectives of this study were to evaluate whether soybean
genistein and glycosylated genistin have a direct effect on
cultured muscle cells and to determine their potential effect on
myoblast proliferation and differentiation, and on myotube
protein synthesis and degradation.
MATERIALS AND METHODS

3
Abbreviations used: BCA, bicinchoninic acid; DMEM, Dulbeccos modified
Eagles medium; FBS, fetal bovine serum; HS, horse serum.

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Muscle cell culture. Rat L8 myoblast cell line was purchased

from American Type Culture Collection (ATCC, Rockville, MD).
The base culture medium (Dulbeccos modified Eagles medium,
DMEM3) was supplemented with specific sera (fetal bovine serum or
horse serum), antibiotics (100,000 U penicillin, 100 mg streptomycin,
and 250 mg amphotericin B/L), 0.584 g L-glutamine/L and 4.0 g
glucose/L. The medium, sera, antibiotics, soybean genistein and
genistin were obtained from Sigma Chemical, St. Louis, MO. Media
in culture plates for all experiments were changed every 48 h unless
specifically indicated otherwise. Myoblast cultures were maintained
in a complete medium containing 10% fetal bovine serum (FBS) in
6-cm culture dishes. When myotubes were needed, myoblasts were
grown to 100% confluence and then kept for 24 h in a medium
containing 1% horse serum (HS) to induce myoblast fusion to form
myotubes. Myotube cultures were maintained thereafter in a complete medium containing 10% HS. Treatments using myotubes were
initiated when myotubes were in 10% HS-DMEM for 4 d, except in
protein degradation experiments in which myotubes were treated in
2% HS-DMEM. Fusion percentage (percentage of nuclei within the
myotube) was ;95% when treatments were initiated. Soybean
genistein and genistin were dissolved in dimethylsulfoxide at 100
mmol/L as a stock. All culture plates contained the same amount of
dimethylsulfoxide. All experiments were repeated two or three times
with between 5 and 10 replicating plates for each treatment in each
experiment.
Myoblast proliferation. Myoblasts were plated at 500 cells/cm2.
Twenty-four hours after plating, myoblasts were treated with 0, 1, 10
or 100 mmol/L of genistein or genistin in 10% FBS-DMEM containing 9.25 MBq/L of methyl-3H-thymidine (Amersham Life Science,
Arlington Heights, IL). At 48 h OF incubation, medium was removed
and plates washed three times with ice-cold PBS to remove unincorporated methyl-3H-thymidine. Cells were scraped in 1.0 mL of a
buffer containing 10 mmol/L Tris-acetate, 1 mmol/L NaCl and 0.1
mmol/L EDTA, pH 7.6, transferred into a scintillation vial containing ScintiVerse cocktail (Fisher Scientific, St. Louis, MO) and
counted.
Myoblast differentiation. Myoblasts were plated at 3000 cells/
cm2. At 100% confluence, cells were induced to differentiation in 1%
HS-DMEM for 24 h. Cells were treated with genistein or genistin at
specific concentrations (0, 1, 10 or 100 mmol/L) in the following
ways: 1) in the differentiation medium for 24 h and in the myotube
medium (10% HS-DMEM) for an additional 48 h (d 2); half of the
plates were treated the same but incubated for an extra 2 d with
genistein excluded in the myotube medium (d 4); 2) only in the
myotube medium for 48 (d 2) or 96 h (d 4) after initiation of fusion
without genistein or genistin. At the end of treatments, cells in
culture plates were then stained with a Giemsa solution, and nuclei
within myotubes (.3 nuclei) were numerically counted in 10 randomly selected fields (under 2003 magnification) on each replicate
plate. The average number of myotube nuclei per field was calculated
for that plate for statistical purposes.
Protein synthesis. Synthesis rates for both myosin and total
cellular protein were measured in myotubes after acute (2 h) or
chronic (24 h) exposure to genistein or genistin. In acute exposure
experiments, cultured myotubes (4 d in 10% HS-DMEM) were exposed to genistein or genistin (0, 1, 10 100 mmol/L) for 2 h in the
presence of 92.5 MBq/L of 3H-labeled amino acid mixture (leucine,

lysine, phenylalanine, proline and tyrosine) available commercially

(Cat. # TRK 550; Amersham Life Science). In chronic exposure
experiments, 4-d old myotubes were incubated in the treatment
medium containing genistein or genistin (0, 1, 10 or 100 mmol/L) for
24 h, followed by an additional 2-h incubation with fresh medium
containing genistein or genistin at the same concentrations and the
tritiated amino acid mixture (92.5 MBq/L).
At the end of the radiolabeling period, plates were washed three
times with ice-cold PBS. Tritiated amino acid incorporation into the
total and myosin protein pools was evaluated using a method described previously (Ji and Orcutt 1991, Orcutt and Young 1982).
Protein degradation. Myotube protein degradation was measured using pulse-chase methodology as described previously (Ji and
Orcutt 1991). Briefly, myotubes (4-d old in 10% HS-DMEM) were
first labeled with 9.25 MBq/L of 3H-amino acid mixture for 24 h and
then rinsed three times with prewarmed DMEM to remove unincorporated labeled amino acids. The myotubes were subsequently incubated in DMEM containing 2% HS and genistein or genistin (50
mmol/L). The incubation medium also contained excess concentrations of nonlabeled amino acids to minimize reincorporation of the
labeled amino acids released from cellular proteins by degradation
processes. The medium was changed at 12-h intervals and myotubes
were harvested at 0, 6, 12, 24, 36, 48 and 60 h of treatment. After
harvesting, cells were washed three times with cold PBS, and the
myotubes from each plate homogenized in a total of 1.5 mL of myosin
extraction buffer (10 mmol/L Tris, pH 7.5; 250 mmol/L KCl; 5
mmol/L MgCl2). Aliquots of each homogenate were identified for
protein determination, myosin extraction and trichloroacetic acid
(0.61 mol/L) precipitation of total cellular proteins. Radioactivity in
myosin and total cellular protein pools was measured by scintillation
counting in CytoScint cocktail (Fisher Scientific) after the protein
pellet was dissolved in a NCS-II tissue solubilizer (Amersham Life
Science).
Protein determination. All protein determinations were accomplished using the bicinchoninic acid (BCA) assay (Smith et al.
1985). The BCA reagents were purchased as a kit (Pierce, Rockford,
IL). Standard curves were constructed using known concentrations of
bovine serum albumin.
Statistical analysis. Data were analyzed using General Linear
Model protocols of SAS (SAS 1997) for a completely randomized
design. Mean comparisons for experiments to measure cell proliferation, fusion and protein synthesis were accomplished by t test using
the least significant differences procedure (a 5 0.05). Radioactivity
(disintegration per minute) in the protein pools for protein degradation experiments were log-transformed before analysis to ensure a
linear radioactivity-time relationship. In experiments to measure
protein accumulation and protein degradation rates, ANOVA was
used to evaluate the main effects and their interactions with time.
Least-square means and pooled SEM are reported.

RESULTS AND DISCUSSION

Genistein and/or some of the other structurally similar
isoflavones in soybean products have antiproliferative and
chemopreventive effects in cultured tumor cells and in some
animal cancer models through mechanisms that include their
inhibitory activities on protein tyrosine kinases and DNA
topoisomerase II. It is unclear what effect genistein has on
normal cell functions and whether genistin, the major isoflavone in soy products, has a direct effect. Perhaps it is of more
interest to understand the effect of genistein or genistin on
embryonic and fetal growth and development when cells are
rapidly proliferating. From the perspective of animal growth
and development, it is imperative to examine whether muscle
growth and development are influenced by dietary isoflavones.
Myogenic cell line L8 was naturally derived from rat skeletal
muscle primary cultures and maintains many characteristics for
myoblast proliferation and differentiation (Richler and Yaffe
1970).
Myoblast proliferation. Myoblast proliferation, as measured by the rate of incorporation of methyl-3H-thymidine

GENISTEIN, GENISTIN AND MUSCLE GROWTH

during DNA synthesis, is a direct indicator for muscular hyperplasia. Methyl-3H-thymidine incorporation during muscle
cell proliferation was decreased (P , 0.05) by inclusion of
genistein or genistin in the culture medium (Fig. 1). The
inhibition by both genistein and genistin was dose dependent,
and genisteins inhibition was evident at a concentration as
low as 1.0 mmol/L, a concentration probably achievable in
animal serum after diets containing soy products are consumed. It is important to note that glycosylated genistein
(genistin) also was effective in inhibiting myoblast proliferation when added to the culture medium at 10 mmol/L (P
, 0.05, Fig. 1B), suggesting a direct role for genistin. It
remains to be determined whether such a direct effect of
genistin is a result of direct uptake of genistin by myoblasts or
uptake of genistein after hydrolysis of genistin in the medium.
Although some galactosidase activities in the medium from
damaged cells may exist, genistin may enter cells directly
because it is small in size and practically insoluble in water.

Although the inhibitory effect of genistein on proliferation in

several cancer cell types has been reported (Peterson and
Barnes 1996), this study represents the first documentation of
a direct inhibitory action for both genistein and genistin on
muscle cell proliferation.
Myoblast differentiation. Myofiber formation is a result of
both proliferation and differentiation of myoblasts. Initiation
of myoblast fusion and the fusion process itself are important
aspects of myoblast differentiation during prenatal muscle development. When genistein or genistin was included in the
cell culture medium for induction of myoblast fusion throughout the test period (i.e., induction of fusion and the fusion
process), myotube formation (myoblast fusion) was strongly
inhibited because genistein or genistin decreased the number
of nuclei within myotubes (Fig. 2A and C, P , 0.05). The
inhibition of myoblast fusion was also dose dependent.
This direct inhibitory effect of genistein and genistin on
myoblast fusion appeared to be stage specific. Typically, when
confluent L8 myoblasts are induced to fuse by using a low
serum medium, there is very little myotube formation during
the first 24 h of initiation. When genistein was included in the
low serum differentiation medium for the first 24 h, but excluded from the subsequent postinduction medium, there were
fewer nuclei within myotubes per microscopic field after an
additional 2-d postinduction incubation (Fig. 2A; P , 0.05).
However, when genistein was included only in the postinduction medium (i.e., after confluent myoblasts were exposed to
low serum medium for 24 h with no genistein), there was no
difference in the number of myotube nuclei between the
genistein-treated and control plates (Fig. 2B). It appeared,
therefore, that genistein did not inhibit the myoblast fusing
process that occurred primarily after 24-h fusion initiation, but
rather inhibited cellular processes immediately after confluent
myoblasts were exposed to low serum differentiation medium.
The inhibitory effect of genistein at this specific stage of
muscle formation abolished the ability of the myoblast to fuse.
It is not yet known exactly how genistein exerts its control
over the initiation of the myoblast fusion/differentiation process. But such inhibition of the initiation of myoblast fusion is
not likely related to its inhibition of myoblast proliferation
because genistein was included after myoblasts were 100%
confluent on culture plates. Furthermore, genisteins inhibition of initiation of myoblast fusion appeared to be a partially
reversible process; inhibition of myoblast fusion by genistein
decreased after a 2-d withdrawal period (i.e., day 4, time
3 treatment P , 0.001; Fig. 2A). It is possible that inhibition
of myoblast fusion by genistein is related to expression or
activities of myogenic factors such as myogenin, MRF-4 or to
a recently identified muscle-specific growth differentiation factor 8 (myostatin, McPherron et al. 1997).
Protein accumulation and synthesis rate. Muscle growth
in adult animals is achieved primarily by muscular hypertrophy
(i.e., increased muscle fiber diameters) through myofibrillar
protein accretion, and perhaps, to a lesser extent, by muscular
hyperplasia through satellite cell (a dormant form of myoblast)
proliferation and differentiation. Although we did not measure
the effect on satellite cells, effects of genistein and genistin on
satellite cell proliferation and fusion events would presumably
be similar to that on myoblasts. In the terminally differentiated L8 myotubes (myofibers), genistein or genistin consistently decreased protein accumulation relative to control cells
(Fig. 3). Such inhibition of myotube protein accumulation
was evidently a result of decreased protein synthesis rate,
increased protein degradation rate, or both.
To determine how myotube protein accretion is altered, we
subsequently measured the rate of tritiated amino acid incor-

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FIGURE 1 Effect of soybean genistein (panel A) or genistin (panel

B) on rat L8 myoblast proliferation. The myoblasts were plated at 500
cells/cm2 in culture dishes (6 cm diameter) and incubated for 24 h at
37C. Cells were then incubated for 48 h in a medium containing 10%
fetal bovine serum, 9.25 MBq/L methyl-3H-thymidine, and 0, 1.0, 10.0
or 100 mmol/L of genistein or genistin. At the end of incubation, plates
were washed with PBS and cells were harvested for scintillation counting. All variables were tested in 10 independent cultures per treatment
for each experiment, and each experiment was conducted three times.
Least-square means and SEM (n 5 10) are reported. The data were
analyzed by t test using the least significant difference (LSD) procedure.
Values in a panel that do not share a letter are significantly different (P
, 0.05).

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FIGURE 3 Effect of soybean genistein (panel A) or genistin (panel

B) on rat L8 myotube protein accretion. Myoblasts were plated at 3000
cells/cm2 and allowed to grow to 100% confluence. After induced to
fuse in 1% horse serum medium for 24 h, myoblasts were grown in a
medium containing 10% horse serum for 4 d before initiation of treatment. Genistein or genistein at 50 mmol/L was included in the medium.
Cells were harvested every 12 h and homogenized. Total protein content was quantified by bicinchoninic acid (BCA) assay. All variables
were tested in six independent plates for each experiment and each
experiment was repeated once. Values reported herein represent leastsquare means and SEM (n 5 6). ANOVA was used to evaluate the main
effects and their interactions.

FIGURE 2 Effect of soybean genistein (panels A and B) or genistin

(panel C) on rat L8 myoblast fusion. Myoblasts were plated at 3000
cells/cm2. At 100% confluence, cells were induced to fuse in a medium
containing 1% horse serum (fusion initiation medium) for 24 h and then
cultured in a medium containing 10% horse serum (myotube medium).
Genistein was included in the medium at specific concentrations in the
fusion initiation medium and myotube medium (0, 10 or 100 mmol/L) for
48 h (d 2), and plates treated the same but followed by an additional
48-h withdrawal period (d 4, panel A) or in the myotube medium (0, 1,
10, 100 mmol/L) for 48 h (d 2) or 96 h (d 4), but not in fusion initiation
medium (panel B). Genistin was included in fusion initiation medium for
24 h and also in the myotube medium for an additional 48 h (0, 10, 100
mmol/L). Each experiment (8 plates/treatment) was repeated once. At

poration into the cultured myotubes as an indicator of protein

synthesis. Genistein (Fig. 4A) and genistin (Fig. 4C) both
inhibited tritiated amino acid incorporation into total cell
protein pools after chronic exposure (24 h), and the most
pronounced effect occurred at 100 mmol/L. A 2-h acute exposure to genistein had a detrimental effect on total protein
synthesis only at 100 mmol/L (Fig. 2A), and there was no effect
of genistin at 2 h (Fig. 4C). However, genistein and genistin
had different inhibitory effects on THE synthesis rate of the
myofibrillar protein myosin; inhibition by genistein was primarily chronic (24 h, Fig. 4B), whereas the inhibition due to

the end of treatments, cells were stained with Giemsa solution, and the
number of nuclei within myotubes (.3 nuclei) was counted in 10
randomly selected fields (under 200X magnification) on each replicate
plate. The average number of myotube nuclei per field was calculated
for that plate for statistical purposes. Least-square means and SEM (n
5 8) are reported. The data were analyzed by t test using the least
significant difference (LSD) procedure. Values in a panel that do not
share a letter are significantly different (P , 0.05).

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FIGURE 4 Effect of soybean genistein (panels A and B) or genistin (panels C and D) on rat L8 myotube protein synthesis. Myoblasts were plated
at 3000 cells/cm2, and allowed to grow to 100% confluence. After induced to fuse in 1% horse serum medium for 24 h, myoblasts were grown in
a medium containing 10% horse serum (HS) for 4 d before initiation of treatment. Synthesis rates for both total cellular protein (panels A and C) and
myosin (panels B and D) were measured in myotubes after acute (2 h) or chronic (24 h) exposure to genistein or genistin at 0, 1, 10 or 100 mmol/L.
In acute exposure experiments, cultured myotubes [4 d in 10% HS-Dulbeccos modified Eagles medium (DMEM)] were exposed to genistein or
genistin for 2 h in the presence of 92.5 MBq/L of 3H-labeled amino acid mixture (leucine, lysine, phenylalanine, proline and tyrosine). In chronic
exposure experiments, myotubes were incubated in the treatment medium containing genistein or genistin for 24 h, followed by an additional 2-h
incubation with fresh medium containing genistein or genistin and the tritiated amino acid mixture (92.5 MBq/L). Incorporation of tritiated amino acids
into the total and myosin pools was measured by scintillation counting. All variables were tested in six independent plates per treatment for each
experiment and each experiment was repeated once. Values reported herein represent least-square means and SEM The data were analyzed by t test
using the least significant difference (LSD) procedures. Values in a panel that do not share a letter are significantly different (P , 0.05).

genistin was acute (2 h, Fig. 4D). Additionally, at low concentrations (1.0 mmol/L) comparable to the serum genistein
concentration achievable in human or rats fed soy diets,
genistein increased myosin synthesis rate (Fig. 4B) and genistin increased total protein synthesis rate (Fig. 4C). The stimulatory effect on protein synthesis at low concentrations is
likely a result of the estrogenic effect of genistein or genistin.
Indeed, recent reports showed that maternal exposure to physiologic doses of genistein (compared with estrogen concentrations) mimics the effects of estrogen on mammary gland development in female mouse offspring, suggesting that genistein
can act as an estrogen in vivo (Hilakivi-Clarke et al. 1998,
McIntyre and Sylvester 1998). It may indeed be true as demonstrated by Wang et al. (1996) in a breast cancer cell line
that genistein (or genistin) acts as an estrogen at low concentrations, and at high serum concentrations, it acts as an estrogen receptor antagonist.
Protein degradation. On the other hand, genistein or
genistin did not appear to affect protein degradation rate in
myotubes; neither genistein nor genistin had any effect on the
calculated half-life of the total protein pool or the slopes of the
total protein degradation curves (data not shown). In the case

of myosin degradation curves, genistein tended to slow myosin

degradation, as shown by a significant time-by-treatment interaction (Fig. 5). Genistin exerted a similar effect on myosin
degradation (data not shown). If all isoflavones follow the
same mechanism, decreased myofibrillar protein degradation
and increased protein synthesis rate at low concentrations of
genistein and genistin may be partially responsible for the
purported increase in muscle growth associated with daidzein
injection of Wistar rats (Wang and Han 1998a), with quercetin treatment of cultured quail embryonic muscle cells (Wang
and Han 1998b) or with dietary intake of soybean extract
containing a mixture of isoflavones in gravid rats (Cook and
Stahly 1998).
Overall, genistein and genistin inhibited cultured muscle
cell growth through their strong inhibitory activities on proliferation and differentiation of myoblasts, and on protein
accumulation through inhibiting protein synthesis in myofibers. Genistein and genistin decreased myosin degradation
rate, although total cellular protein degradation rate was unaltered. Like genistein, glycosylated genistin exerted direct
effects on myoblasts and fused myotubes and demonstrated a
similar in vitro activity, suggesting a possibility that genistin

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does not have to be hydrolyzed to be biologically active.

However, genistin may be less potent than genistein because
of a smaller proportionate inhibition of myoblast proliferation
at the same concentration.
Some human diets, e.g., soy-based infant formulas, contain
32 47 mg/L isoflavones, equivalent to an intake of 4.0 8.0
mg/kg body weight for a 4-mo old infant (Setchell et al. 1997).
Assuming genistein 1 genistin accounting for 50% of total
isoflavones, 50% absorption and 70% body water, this intake
represents a tissue and circulation concentration of 510
mmol/L, not considering the turnover rate and conjugation
processes. Similarly, some feeds for pregnant dams and nursing
as well as growing animals (e.g., pigs) contain ;30% soybean
meal, and intake of isoflavones would be very high. Because
both genistein and genistin are small molecules and very
hydrophobic, they probably cross gastrointestinal epithelial
cell membranes and placental barriers easily. Indeed, King et
al. (1996) investigated absorption and excretion of genistein
in rats and showed that the extent of absorption is similar for
the glycone and aglycone forms of genistein. Our preliminary
data showed that isoflavones in pig sera are primarily in glycosylated forms with genistin concentration at ;0.71.5
mmol/L, thus supporting the notion of direct absorption of
genistin. It is therefore likely that high oral intake of both
genistein and genistin has a detrimental effect on prenatal and
postnatal muscle growth through inhibition of myoblast/satellite cell proliferation, differentiation and/or myofiber protein
synthesis.
Several lines of additional evidence also suggest a negative
role for genistein and genistin, and possibly other isoflavones,
on animal performance. First, signal transduction pathways for
many important hormones, growth factors such as epidermal
growth factors, insulin-like growth factors, or cytokines, re-

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FIGURE 5 Effect of soybean genistein on rat L8 myotube protein

degradation. Myotubes (4 d old in myotube medium) were incubated in
10% myotube medium containing 9.25 MBq/L of 3H-amino acid mixture for 24 h, then rinsed three times with prewarmed Dulbeccos
modified Eagles medium (DMEM) and incubated in DMEM containing
2% horse serum, genistein at 50 mmol/L and excess concentrations of
nonlabeled amino acids. The medium was changed at 12-h intervals
and myotubes were harvested at 0, 6, 12, 24, 36, 48 and 60 h of
treatment. The tritiated amino acid incorporation into the total protein
and myosin pools was quantified by scintillation counting. All variables
were tested in six independent plates at each time point in each
experiment and each experiment was repeated once. Values reported
herein are least-square means (predicted) of log-transformed DPM
(disintegration per minute, 1 DPM 5 60 Bq) and SEM. ANOVA was used
to evaluate the main effects and their interactions. Time by treatment
interaction represents the comparison of the two treatments in each
experiment.

quire protein tyrosine kinase activities. Genistein actually

blocks epidermal growth factor-, insulin- and growth hormoneinduced and tyrosine/MAP-kinase mediated proliferation
of several fibroblastic cell lines (Akiyama et al. 1987, Linassier
et al. 1990, Pertseva et al. 1996), and epidermal growth factor
receptor down-regulation was associated with genistein inhibition of normal mammary epithelial cell proliferation (McIntyre and Sylvester 1998). Second, genistein has been shown to
inhibit insulin-dependent or insulin-independent glucose
transport (Smith et al. 1993, Vera et al. 1996) and decrease
immunocytochemical labeling of the glucose transporter 4
(GLUT4) carboxyl terminus in isolated rat adipocytes (Smith
et al. 1993). Although such an effect in adipocytes may be
advantageous for animal production, a similar effect on muscle
cells would be disadvantageous. Third, isoflavones including
genistein have been detected in human and rat body fluids at
concentrations sufficient to cause biological effects (Franke
and Custer 1996 King et al. 1996); they are rapidly absorbed
and highly bioavailable in rats (Sfakianos et al. 1997), despite
the fact that the major portion of the intestinally absorbed
isoflavone undergoes conjugation with glucuronic acid in the
liver and finally is excreted in the urine or bile (Yasuda et al.
1996). Moreover, in vitro work demonstrated that genistein
could affect reproduction because it arrested cell cycle progression at G2-M phases (Matsukawa et al. 1993), inhibited cleavage of one-cell mouse embryos in a dose-dependent and reversible manner (Besterman and Schultz, 1990) and inhibited
in vitro maturation of cumulus enclosed and denuded pig
oocytes (Jung et al. 1993).
The results of this study in combination with the above
evidence suggest that diet-derived genistein (genistin) could
potentially have a dramatic effect on animal muscle growth.
More attention should be paid to the effect of isoflavones on
prenatal or neonatal muscle growth. However, any negative
effect on growth and development associated with feeding
soybean products would depend on how isoflavones are absorbed and metabolized, and what interactions with endocrine
systems of animals and feed components occur. Recently,
feeding rat dams a mixture of soybean-derived isoflavones
(genistin, daidzin and glycitin), primarily in glycosidic forms,
did not produce a negative effect on postnatal growth rate and
efficiency in pups (Cook and Stahly 1998). The negative and
direct effect of pure genistein and genistin on in vitro muscle
formation and protein metabolism necessitates further evaluation of the benefits and negative effect of isoflavones in
soybean-based infant formulas and animal diets.
ACKNOWLEDGMENTS
We thank Becky Godat, Joanne Kuske and Robyn Pelker for their
excellent technical assistance.

LITERATURE CITED
Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N.,
Shibuya, M. & Fukami, Y. (1987) Genistein, a specific inhibitor of tyrosinespecific protein kinases. J. Biol. Chem. 262: 55925595.
Barnes, S., Grubbs, C., Setchell, K. D. & Carlson, J. (1990) Soybeans inhibit
mammary tumors in models of breast cancer. Prog. Clin. Biol. Res. 347:
239 253.
Besterman, B. & Schultz, R. M. (1990) Regulation of mouse preimplantation
development: inhibitory effect of genistein, an inhibitor protein phosphorylation, on cleavage of one-cell embryos. J. Exp. Zool. 256: 44 53.
Cai, Q. & Wei, H. (1996) Effect of dietary genistein on antioxidant enzyme
activities in SENCAR mice. Nutr. Cancer 25: 17.
Chang, Y. C. & Nair, M. G. (1995) Metabolism of daidzein and genistein by
intestinal bacteria. J. Nat. Prod. 58: 18921896.
Cook, D. R. & Stahly, T. S. (1998) Effects of dietary soy isoflavone concentrations in gravid rats on rate and efficiency of growth and muscle content of
the offspring. J. Anim. Sci. 76 (suppl. 1): 163 (abs.).

GENISTEIN, GENISTIN AND MUSCLE GROWTH

K. V. (1996) On the tyrosine kinase mechanism of the novel effect of insulin

and insulin-like growth factor I. Stimulation of the adenyl cyclase system in
muscle tissues. Biochem. Pharmacol. 52: 18671874.
Peterson, G. & Barnes, S. (1996) Genistein inhibits both estrogen and growth
factor-stimulated proliferation of human breast cancer cells. Cell Growth
Differ. 7: 13451351.
Record, I. R., Dreosti, I. E. & McInerney, J. K. (1995) The antioxidant activity of
genistein in vitro. J. Nutr. Biochem. 6: 481 485.
Richler, C. & Yaffe, D. (1970) The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev. Biol. 23: 122.
Ruiz-Larrea, M. B., Mohan, A. R., Paganga, G., Miller, N. J., Bolwell, G. P. &
Rice-Evans, C. A. (1997) Antioxidant activity of phytoestrogenic isoflavones. Free Radic. Res. 26: 6370.
SAS Institute Inc. (1997) SAS Users Guide: Statistics. SAS Institute, Cary, NC.
Setchell, K.D.R, Borriello, S. P., Kirk, D. N. & Axelson, M. (1984) Nonsteroidal
estrogens of dietary origin: possible role in hormone-dependent disease.
Am. J. Clin. Nutr. 40: 569 578.
Setchell, K.D.R, Zimmer-Nechemias, L., Cai, J. & Heubi, J. E. (1997) Exposure
of infants to phytoestrogens from soy-based infant formula. The Lancet. 350:
2327.
Sfakianos, J., Coward, L., Kirk, M. & Barnes, S. (1997) Intestinal uptake and
biliary excretion of the isoflavone genistein in rats. J. Nutr. 127: 1260 1268.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Malia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C.
(1985) Measurement of protein using bicinchoninic acid. Anal. Biochem.
150: 76 81.
Smith, R. M., Tiesinga, J. J., Shah, N., Smith, J. A. & Jarett, L. (1993) Genistein
inhibits insulin-stimulated glucose transport and decreases immunocytochemical labeling of GLUT 4 carboxyl-terminus without affecting translocation
of GLUT 4 in isolated rat adipocytes: additional evidence of GLUT4 activation
by insulin. Arch. Biochem. Biophys. 300: 238 246.
Vera, J. C., Reyes, A. M., Carcamo, J. G., Velasquez, F. V., Rivas, C. I., Zhang,
R. H., Strobel, P., Iribarren, R., Scher, H. I. & Slebe, J. C. (1996) Genistein
is a natural inhibitor of hexose and dehydroascorbic acid transport through
the glucose transporter GLUT 1. J. Biol. Chem. 271: 8719 8724.
Wang, J. & Han, Z. K. (1998a) Effects of daidzein on muscle growth and some
endogenous hormone levels in rats. Am. J. Clin. Nutr. 68: 1539S (abs.).
Wang, J. & Han, Z. K. (1998b) Effects of isoflavone and flavonoids on quail
skeletal muscle cell growth in vitro. Am. J. Clin. Nutr. 68: 1539S1540S (abs.).
Wang, T. T., Sathyamoorthy, N. & Phang, J. M. (1996) Molecular effects of
genistein on estrogen receptor mediated pathways. Carcinogenesis 17: 271
275.
Yasuda, T., Mizunuma, S., Kano, S. Y., Saito, K.-I. & Ohsawa, K. (1996) Urinary
and biliary metabolites of genistein in rats. Biol. Pharm. Bull. 19: 413 417.

Downloaded from jn.nutrition.org by guest on April 7, 2015

Coward, L., Barnes, N. C., Setchell, K.D.R. & Barnes, S. (1993) Genistein and
daidzein, and their b-glycoside conjugates: anti-tumor isoflavones in soybean
foods from American and Asian diets. J. Agric. Food. Chem. 41: 19611967.
Fotsis, T., Pepper, M. S., Aktas, E., Breit, S., Rasku, S., Adlercreutz, H., Wahala,
K., Montesano, R. & Schweigerer, L. (1997) Flavonoids, dietary-derived
inhibitors of cell proliferation and in vitro angiogenesis. Cancer Res. 57:
2916 2921.
Franke, A. A. & Custer, L. J. (1996) Daidzein and genistein concentrations in
human milk after soy consumption. Clin. Chem. 42: 955964.
Fukutake, M., Takahashi, M., Ishida, K., Kawamura, H., Sugimura, T. & Wakabayashi, K. (1996) Quantification of genistein and genistin in soybeans and
soybean products. Food Chem. Toxicol. 34: 457 461.
Hilakivi-Clarke, L., Cho, E. & Clarke, R. (1998) Maternal genistein exposure
mimics the effects of estrogen on mammary gland development in female
mouse offspring. Oncol. Rep. 5: 609 616.
Irvine, C. H., Fitzpatrick, M. G. & Alexander, S. L. (1998) Phytoestrogens in
soy-based infant foods: concentrations, daily intake, and possible biological
effects. Proc. Soc. Exp. Biol. Med. 217: 247253.
Ji, S. Q. & Orcutt, M. W. (1991) Effect of the b-adrenergic agonist isoproterenol
on protein accretion, synthesis, and degradation in primary chicken muscle
cell cultures. J. Anim. Sci. 69: 28552864.
Jung, T., Fulka, J., Jr., Lee, C. & Moor, R. M. (1993) Effects of the protein
phosphorylation inhibitor genistein on maturation of pig oocytes in vitro. J.
Reprod. Fertil. 98: 529 535.
King, R. A., Broadbent, J. L. & Head, R. J. (1996) Absorption and excretion of
the soy isoflavone genistein in rats. J. Nutr. 126: 176 182.
Linassier, C., Pierre, M., Le Pecq, J. B. & Pierre, J. (1990) Mechanisms of
action in NIH-3T3 cells of genistein: an inhibitor of EGF receptor tyrosine
kinase activity. Biochem. Pharmacol. 39: 187193.
Matsukawa, Y., Marui, N., Sakai, T., Satomi, Y., Yoshida, M., Matsumoto, K.,
Nishino, H. & Aoike, A. (1993) Genistein arrests cell cycle progression at
G2-M. Cancer Res. 53: 1328 1331.
McCabe, M. J., Jr. & Orrenius, S. (1993) Genistein induces apoptosis in
immature human thymocytes by inhibiting topoisomerase-II. Biochem. Biophys. Res. Commun. 194: 944 950.
McIntyre, B. S. & Sylvester, P. W. (1998) Genistein and erbstatin inhibition of
normal mammary epithelial cell proliferation is associated with EGF-receptor
down-regulation. Cell Prolif. 31: 35 46.
McPherron, A. C., Lawler, A. M. & Lee, S. J. (1997) Regulation of skeletal
muscle mass by a new TGF-b superfamily member. Nature (Lond.) 387:
8390.
Orcutt, M. W. & Young, R. B. (1982) Cell differentiation, protein synthesis rate
and accumulation in muscle cell cultures isolated from embryos of layer and
broiler chickens. J. Anim. Sci. 54: 769 776.
Pertseva, M. N., Plesneva, S. A., Kuznetsova, L. A., Shpakov, A. O. & Derkach,

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