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Swine Research Group, Purina Mills Research Center, Gray Summit, MO 63039
2To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • isoflavone • genistein • L8 cells • muscle • protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). Most soy products, especially fermented products,
contain appreciable amounts (1–3 mg/g) of genistein and daidzein
(Coward et al. 1993
), and soy-based infant formula
contains ~3–287 µg/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 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., insulin-like 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 formula–fed 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 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Rat L8 myoblast cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD). The base culture medium (Dulbecco's modified Eagle's medium, DMEM3 ) was supplemented with specific sera (fetal bovine serum or horse serum), antibiotics (100,000 U penicillin, 100 mg streptomycin, and 250 µg 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 µmol/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 µmol/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 200x 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 µmol/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 µmol/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
µmol/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 (
= 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.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). Myoblast proliferation.
Myoblast proliferation, as measured by the rate of incorporation of
methyl-3H-thymidine 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
genistein's inhibition was evident at a concentration as low as 1.0
µmol/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 µmol/L (P < 0.05, Fig. 1
B),
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.
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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.
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A;
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. 2
B). 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,
genistein's 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
x treatment P < 0.001; Fig. 2
A). 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.
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) and genistin (Fig. 4
C) both inhibited tritiated amino acid
incorporation into total cell protein pools after chronic exposure (24
h), and the most pronounced effect occurred at 100 µmol/L.
A 2-h acute exposure to genistein had a detrimental effect on total
protein synthesis only at 100 µmol/L (Fig. 2
A),
and there was no effect of genistin at 2 h (Fig. 4
C).
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. 4
B), whereas the inhibition due to genistin was acute (2 h,
Fig. 4
D). Additionally, at low concentrations (1.0
µmol/L) comparable to the serum genistein concentration
achievable in human or rats fed soy diets, genistein increased myosin
synthesis rate (Fig. 4
B) and genistin increased total
protein synthesis rate (Fig. 4
C). 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.
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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
).
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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 + genistin accounting for 50% of total isoflavones, 50%
absorption and 70% body water, this intake represents a tissue and
circulation concentration of 5–10 µmol/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 ~0.7–1.5 µmol/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, require protein tyrosine
kinase activities. Genistein actually blocks epidermal growth factor-,
insulin- and growth hormone–induced 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BCA, bicinchoninic acid;
DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum;
HS, horse serum.
Manuscript received January 7, 1999. Initial review completed February 22, 1999. Revision accepted April 13, 1999.
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