Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Flavonoids, polyphenolic compounds that exist widely
in plants, inhibit cell proliferation and increase cell differentiation in many cancerous and noncancerous cell lines. Because terminal differentiation of preadipocytes to adipocytes depends on proliferation of both pre- and postconfluent preadipocytes, we predicted that flavonoids would inhibit adipogenesis in the 3T3-L1 preadipocyte cell
line. The flavonoids genistein and naringenin inhibited proliferation of preconfluent preadipocytes in a time- and dose-dependent manner. When added to 2-day postconfluent preadipocytes at the induction of
differentiation, genistein inhibited mitotic clonal expansion, triglyceride accumulation, and peroxisome proliferator-activated receptor- expression, but naringenin had no effect. The
antiadipogenic effect of genistein was not due to inhibition of insulin
receptor subtrate-1 tyrosine phosphorylation. When added 3 days after
induction of differentiation, neither flavonoid inhibited
differentiation. In fully differentiated adipocytes, genistein
increased basal and epinephrine-induced lipolysis, but naringenin had
no significant effects. These data demonstrate that genistein and
naringenin, despite structural similarity, have differential effects on
adipogenesis and adipocyte lipid metabolism.
genistein; naringenin; preadipocytes; adipocytes; peroxisome
proliferator-activated receptor-; insulin receptor substrate-1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FLAVONOIDS, polyphenolic compounds that exist widely in plants, inhibit the proliferation of tumor and nontumor cells in cell culture, induce apoptosis (13), have estrogenic (21) and antiestrogenic (17, 22) effects, and function as antioxidants (24). Flavonoids also alter the activity of a number of intracellular enzymes, including tyrosine kinases (10), phosphodiesterases (6), and DNA topoisomerases (34). Primarily because of their antiproliferative effects, flavonoids have been the subject of active recent research as anticancer agents. Genistein, a soy isoflavone, inhibits proliferation of a number of cancer cell lines (reviewed in Ref. 3) and commonly induces differentiation. Naringenin, a grapefruit flavanone, alters cell growth in vitro, much as genistein does (26).
Although relatively little research exists on the effects of flavonoids on adipose cells, Kuppusamy and Das (14, 15) have found that several flavonoids potentiate epinephrine-induced lipolysis in primary rat adipocytes. Shisheva and Shechter (30) found that quercetin, a flavanol structurally similar to both genistein and naringenin, blocks insulin-mediated lipogenesis by preventing the insulin receptor tyrosine kinase from phosphorylating substrate. These potentially lipolytic and antilipogenic effects in rat adipocytes, coupled with antiproliferative activity in a number of cell lines, suggest that flavonoids may decrease adipose tissue mass or inhibit the signals that promote adipogenesis.
Given that flavonoids inhibit proliferation not only of cancer cells but also of normal cells, such as mammary (20) and intestinal (7) epithelial cells, we hypothesized that flavonoids would inhibit proliferation of preadipocytes. Because terminal differentiation to adipocytes depends on proliferation of both pre- and postconfluent preadipocytes (18), we predicted that flavonoids would also inhibit the differentiation process.
To test the effects of flavonoids on preadipocytes, we used murine 3T3-L1 cells. This cell line allows investigation of stimuli and mechanisms that regulate preadipocyte replication separately from those that regulate differentiation to adipocytes. As preadipocytes, these cells have a fibroblastic appearance. They replicate in culture until they form a confluent monolayer, after which cell-cell contact triggers G0/G1 growth arrest (9). Subsequent stimulation with 3-isobutyl-1-methylxanthine, dexamethasone, and high doses of insulin (MDI) for 2 days prompts these cells to undergo postconfluent mitotic clonal expansion, exit the cell cycle, and begin to express adipocyte-specific genes (25). Approximately five days after induction of differentiation, >90% of the cells display the characteristic lipid-filled adipocyte phenotype.
In this report, we showed that in 3T3-L1 cells, genistein and naringenin inhibited proliferation of preconfluent preadipocytes. Although genistein inhibited MDI-induced proliferation of postconfluent preadipocytes and their subsequent differentiation into adipocytes, naringenin had no effect on these processes. In mature adipocytes, genistein strongly induced lipolysis, both alone and in combination with epinephrine. Naringenin did not produce a statistically significant increase in lipolysis. These findings in cultured adipose cells suggest that dietary flavonoids, particularly genistein, may have inhibitory effects on adipose tissue enlargement in vivo.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Genistein (4',5,7-trihydroxyisoflavone) and all tissue culture
materials were from GIBCO (Grand Island, NY). Naringenin
(4',5,7-trihydroxyflavanone), epinephrine, insulin, dexamethasone, and
3-isobutyl-1-methylxanthine were from Sigma (St. Louis, MO). Rabbit
polyclonal anti-signal transducers and activators of transcription
(STAT) 3 antibody was from New England Biolabs (Beverly, MA). Mouse
monoclonal anti-peroxisome proliferator-activated receptor (PPAR)-
(E-8), rabbit polyclonal anti-insulin receptor substrate (IRS)-1 (C-20)
antibodies, and protein A/G plus agarose beads were from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody (PY20)
was from Pharmingen-Transduction Laboratories (Lexington, KY). The
enhanced chemiluminescence (ECL) detection kit and horseradish
peroxidase-conjugated secondary antibodies were from Amersham Pharmacia
Biotech (Piscataway, NJ).
Cell culture.
3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD)
were cultured in DMEM with 10% (vol/vol) fetal bovine serum (FBS), 1%
(vol/vol) penicillin-streptomycin (10,000 U/ml penicillin and 10,000 µg/ml streptomycin in 0.85% saline), and 1% (vol/vol) 100 µM
pyruvate at 37°C in 95% air-5% CO2. To induce differentiation, 2-day postconfluent 3T3-L1 preadipocytes (day 0) were stimulated for 72 h with 0.5 mM
3-isobutyl-1-methylxanthine, 0.5 µM dexamethasone, and 10 µg/ml
insulin (MDI) added to DMEM/10% FBS culture medium. On day
3, the MDI medium was replaced with DMEM/10% FBS, which was
changed every 2 days thereafter until analysis on days
7-10. Flavonoids were reconstituted as 100 mM stock solutions
in DMSO, filter sterilized, and stored at 20°C. For each
experiment, cells received flavonoids premixed with culture medium.
Unless otherwise noted, "vehicle" refers to 0.1% DMSO in culture
medium or MDI differentiation medium.
Proliferation assay. Preconfluent 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 10,000 cells/100 µl/well. Vehicle or flavonoids, in doses ranging from 1 to 100 µM, were added to culture medium with or without cells at the time of plating. At 0, 12, 24, and 48 h after plating, a colorimetric proliferation assay (CellTiter 96 AQueous nonradioactive cell proliferation assay; Promega, Madison, WI) was performed as directed by the manufacturer. To calculate the absorbance values at each dose and time point, the mean absorbance of two blank wells (containing flavonoids in culture medium but no cells) was subtracted from the mean absorbance of six wells containing cells.
Cytotoxicity assay. Preconfluent 3T3-L1 preadipocytes were seeded in 24-well plates at a density of 50,000 cells/ml/well or in six-well plates at a density of 130,000 cells/2 ml/well. Vehicle and 100 µM flavonoids were added to phenol red-free culture medium with or without cells at the time of plating. At 24 and 48 h after plating, all media were removed from the wells and stored at 4°C. Each remaining cell monolayer was washed three times with 37°C PBS, cells were scraped on ice in 1 ml lysis medium (0.05% Triton X-100 in PBS) per well, and the lysate was stored at 4°C until a colorimetric cytotoxicity assay (CytoTox 96 nonradioactive cytotoxicity assay; Promega) was performed as directed by the manufacturer. The percent lactate dehydrogenase (LDH) release was calculated as follows: % LDH release = (LDH in culture supernatant/LDH in culture supernatant + LDH in cell lysate) × 100.
Triglyceride assay. On days 7-10 after differentiation, 3T3-L1 cells were washed with PBS, scraped on ice in 100 µl of saline solution (2 M NaCl, 2 mM EDTA, 50 mM sodium phosphate, pH 7.4), sonicated to homogenize the cell suspension, and assayed for total triglyceride (GPO-Trinder; Sigma) according to the method of Shimabukuro et al. (29). Results were expressed as total triglyceride per cellular protein (DC protein assay; Bio-Rad, Hercules, CA). For lipolysis experiments, the conditioned medium was assayed for glycerol content 24 h after treatment (GPO-Trinder; Sigma).
Oil Red O staining. 3T3-L1 adipocytes were washed with PBS, fixed with 10% glutaraldehyde in PBS (pH 7.4), then stained with 0.5% Oil Red O.
Immunoblot analysis. 3T3-L1 cells were washed twice in ice-cold PBS with 1 mM orthovanadate, then placed immediately in sample buffer [1% Nonidet P-40, 20 mM Tris · HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% NaN3, 10 mg/ml aprotinin, 1 mM pepstatin, 16.4 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM Na3VO4, 2% SDS, and 10% glycerol] without dithiothreitol (DTT) or tracking dye. Lysates were heated and protein concentrations determined before adding 100 mM DTT and tracking dye. Protein concentrations were determined in cell lysates using the Bio-Rad DC protein determination kit. Bovine serum albumin (BSA) was used as a standard. Samples were heated for 5 min at 95°C, separated by 10% SDS-PAGE, and analyzed by immunoblotting as previously described (16, 19). Immunoblots were developed with the ECL kit.
Immunoprecipitation. Cells were placed in ice-cold RIPA lysis buffer [50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM Na3VO4, 1 mM PMSF, and 10 µg/ml aprotinin] and rocked on ice for 30 min. The lysed cells were harvested, centrifuged at 7,000 g for 20 min at 4°C, supernatants were removed, and protein concentrations determined using the Bio-Rad DC protein determination kit, with BSA used as a standard. For immunoprecipitations, 2.5-10 µg/ml of PY20 antibodies were added to lysates, which contained equal amounts of protein (200-300 µg); incubation occurred overnight at 4°C. Protein A/G plus agarose beads were added and agitated for 1 h at 4°C. Immunoprecipitates were recovered by centrifugation at 2,500 g and washed three times with ice-cold RIPA lysis buffer. Immunoprecipitated proteins were dissolved in 2× sample buffer and analyzed as described above.
Statistical analysis. Data are expressed as means ± SE. The significance of differences between means, set at P < 0.05, was assessed by one-way analysis of variance and Bonferroni post hoc testing (NCSS Statistical Software, Kaysville, UT).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Flavonoids inhibit proliferation of preconfluent
preadipocytes.
To determine whether flavonoids inhibit the proliferation of
preadipocytes, we cultured preconfluent 3T3-L1 preadipocytes in the
presence and absence of varying doses of genistein or naringenin, then
measured cell proliferation at several points during the course of
growth. As shown in Fig. 1, the
addition of genistein or naringenin at the time of plating inhibited
proliferation of 3T3-L1 preadipocytes in a dose- and time-dependent
manner. At 48 h, 100 µM genistein inhibited proliferation by
nearly 60%; similarly, 100 µM of naringenin reduced proliferation by
~40%.
|
|
Genistein inhibits postconfluent mitotic clonal expansion.
After stimulation with MDI differentiation medium, growth-arrested
postconfluent 3T3-L1 cells undergo two rounds of mitotic clonal
expansion before exiting the cell cycle and expressing adipocyte-specific genes (18). We treated 2-day
postconfluent preadipocytes with MDI plus 100 µM of flavonoids or
vehicle and measured proliferation 72 h later. Although genistein
inhibited postconfluent mitotic clonal expansion by roughly 30% (Fig.
3), naringenin had no effect on this
process. As shown in Fig. 4, neither
genistein nor naringenin triggered significantly more LDH release than
did control cells during postconfluent mitotic clonal expansion,
indicating that neither flavonoid produced significant cytotoxic
effects at this stage.
|
|
Genistein inhibits adipogenesis.
We next examined the effects of flavonoids on preadipocyte
differentiation. Given the effects of flavonoids on postconfluent mitotic clonal expansion, we hypothesized that genistein
would inhibit differentiation but that naringenin would not. We treated 2-day postconfluent preadipocytes with MDI plus 100 µM of flavonoids or vehicle for 72 h. From day 3 to days
7-9 after induction of differentiation, we maintained cells
in culture medium without flavonoids. Cells treated with MDI plus 100 µM of genistein accumulated roughly 40% of the intracellular
triglyceride contained in controls, as shown by both Oil Red O staining
(Fig. 5A) and triglyceride accumulation (Fig. 5B). Naringenin did not inhibit
triglyceride accumulation. We also determined the effects of flavonoids
or vehicle on the expression of PPAR-, a nuclear hormone receptor that regulates adipogenesis and is expressed during differentiation (32). Western blot analysis showed that vehicle- and
naringenin-treated cells expressed PPAR-
strongly and equally (Fig.
5C), whereas genistein-treated cells expressed considerably
less PPAR-
. The expression of STAT3, which does not change during
proliferation and differentiation of 3T3-L1 cells (8), was
unchanged by flavonoid treatment.
|
|
|
Genistein induces adipocyte lipolysis.
Genistein and naringenin both potentiate epinephrine-induced
lipolysis in primary rat adipocytes (14). To
determine whether flavonoids exert similar lipolytic effects in 3T3-L1
cells, we treated 3T3-L1 adipocytes (days 8-10 after
MDI stimulation) with 100 µM of flavonoids or vehicle with or without
0.1 µM or 1 µM epinephrine. As shown in Table
1, genistein alone induced a sixfold greater release of glycerol into the culture medium than did control (vehicle). The combination of genistein and epinephrine produced an
additive effect: a two- to fourfold increase over epinephrine, depending on the epinephrine concentration. Naringenin had no significant effects on lipolysis.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we found that genistein inhibited the proliferation of both preconfluent and postconfluent preadipocytes. In contrast, naringenin exerted significant dose-dependent antiproliferative effects only at the preconfluent stage. Naringenin did not inhibit mitotic clonal expansion of 2-day postconfluent 3T3-L1 preadipocytes, nor did it inhibit their differentiation to adipocytes. Genistein, on the other hand, blocked differentiation but only when administered at the onset of differentiation. Exposing 3T3-L1 cells to genistein from days 3-5 after induction of differentiation did not alter adipogenesis. These findings demonstrate that genistein's antiadipogenic effects occurred during the first 72 h after induction of differentiation. These data are consistent with studies showing the occurrence of two critical events during this 72-h period: mitotic clonal expansion and an irreversible commitment to differentiation (11, 28).
Genistein did not prevent insulin-stimulated tyrosine phosphorylation of IRS-1. This finding suggests that genistein exerts its antidifferentiation effects independently of insulin-stimulated activation of IRS-1. Genistein may block adipogenesis by inhibiting other receptor or nonreceptor tyrosine kinases activated by MDI during differentiation. Alternatively, genistein may inhibit the effects of insulin or other inducers of differentiation independently of tyrosine kinase activity. In isolated rat adipocytes, genistein suppresses the antilipolytic effects of insulin and inhibits insulin-stimulated glucose oxidation with 50% inhibitory dose values of 25 and 15 µg/ml, respectively. Genistein exerts these inhibitory effects without preventing autophosphorylation of the insulin receptor or its subsequent tyrosine phosphorylation of substrate (1).
When investigating the effects of the tyrosine kinase Syk on 3T3-L1 adipogenesis, Wang and Malbon (33) found that genistein exerts a dose-responsive antiadipogenic effect. Consistent with our results, they found that 100 µM genistein potently blocks differentiation. They also demonstrated that a 10-µM dose reduces 3T3-L1 adipogenesis. However, in that report, they did not determine whether genistein inhibits Syk activity.
In addition to its antiadipogenic effects, we also found that 100 µM of genistein, alone and in combination with epinephrine, strongly promoted lipolysis when administered to mature 3T3-L1 adipocytes. Naringenin did not have a statistically significant lipolytic effect. These findings differ from those of Kuppusamy and Das (14), who observed that in isolated rat adipocytes, 250 µM of naringenin promotes epinephrine-induced lipolysis to a greater degree than does a comparable dose of genistein (68 vs. 31% increase over control, respectively). Although the use of different cell systems may contribute to the varying results between studies, the differences in flavonoid doses may account for much of the discrepancy. Kandulska et al. (12) found that in isolated rat adipocytes, 1 mM of genistein enhances basal lipolysis but inhibits epinephrine-stimulated lipolysis.
Given that genistein promotes lipolysis and inhibits adipogenesis in cell culture, we anticipate that genistein will act similarly in vivo and potentially promote loss of body fat. According to Barnes et al. (4), the average Asian's intake of genistein ranges from 20-80 mg/day, yet the average American consumes only 1-3 mg/day. With high consumption of soyfoods, tissue concentrations of genistein typically do not exceed the 1-5 µM range (5). However, a nontoxic pharmacological dose of genistein, 8 mg/kg body wt, elevates serum genistein to the 10-µM range (personal communication from S. Zeisel, Univ. of North Carolina at Chapel Hill), a level sufficient to inhibit adipogenesis in 3T3-L1 cells (33).
The few existing animal studies that have evaluated the effects of genistein treatment on lipid metabolism or body weight support our findings in 3T3-L1 cells. Nogowski et al. (23) examined the effects of genistein on the lipid metabolism of ovariectomized rats and found that dietary genistein significantly decreases triglyceride levels in serum and muscle but increases the free fatty acid concentration in serum. After incubating isolated adipocytes with genistein, they found inhibited basal and insulin-induced lipogenesis as well as enhanced epinephrine-induced lipolysis. They concluded that dietary genistein may reduce the fattening process in ovariectomized rats. When Schleicher et al. (27) studied rats with accessory sex gland carcinoma, those treated with genistein had a decreased body weight compared with controls. Further animal studies will be required to verify that genistein promotes loss of body fat and to determine the optimal dose for such an effect.
Genistein holds great promise for nutrient-mediated regulation of body fat through its effects on preadipocyte replication, differentiation, and lipolysis. Naringenin, on the other hand, failed to inhibit differentiation and may, therefore, be ineffective as an antiadipogenic compound.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53398, the University Research Council, and the Institute of Nutrition at the University of North Carolina.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: J. B. Harp, Univ. of North Carolina at Chapel Hill, Dept. of Nutrition, CB# 7400 McGavran-Greenberg Hall, Chapel Hill, NC 27599 (E-mail: jharp{at}sph.unc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 May 2000; accepted in final form 30 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abler, A,
Smith JA,
Randazzo PA,
Rothenberg PL,
and
Jarett L.
Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.
J Biol Chem
267:
3946-3951,
1992
2.
Akiyama, T,
Ishida J,
Nakagawa S,
Ogawara H,
Watanabe S,
Itoh N,
Shibuya M,
and
Fukami Y.
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
J Biol Chem
262:
5592-5595,
1987
3.
Barnes, S.
Effect of genistein on in vitro and in vivo models of cancer.
J Nutr
125:
777S-783S,
1995[Medline].
4.
Barnes, S,
Peterson TG,
and
Coward L.
Rationale for the use of genistein-containing soy matrices in chemoprevention trials for breast and prostate cancer.
J Cell Biochem
22, Suppl:
181-187,
1995.
5.
Barnes, S,
Sfakianos J,
Coward L,
and
Kirk M.
Soy isoflavonoids and cancer prevention: underlying biochemical and pharmacological issues.
Adv Exp Med Biol
401:
87-100,
1996[Medline].
6.
Beretz, A,
Anton R,
and
Stoclet JC.
Flavonoid compounds are potent inhibitors of cyclic AMP phosphodiesterase.
Experientia
34:
1054-1055,
1978[ISI][Medline].
7.
Booth, C,
Hargreaves DF,
Hadfield JA,
McGown AT,
and
Potten CS.
Isoflavones inhibit intestinal epithelial cell proliferation and induce apoptosis in vitro.
Br J Cancer
80:
1550-1557,
1999[ISI][Medline].
8.
Deng, J,
Hua K,
Lesser SS,
and
Harp JB.
Activation of STAT3 during proliferative phases of 3T3-L1 adipogenesis.
Endocrinology
141:
2370-2376,
2000
9.
Green, H,
and
Kehinde O.
Sublines of mouse 3T3 cells that accumulate lipid.
Cell
1:
113-116,
1974[ISI].
10.
Hagiwara, M,
Inoue S,
Tanaka T,
Nunoki I,
Ito M,
and
Hidaka H.
Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonine protein kinases.
Biochem Pharmacol
37:
2987-2992,
1988[ISI][Medline].
11.
Ignotz, RA,
and
Massague J.
Type transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.
Proc Natl Acad Sci USA
82:
8530-8534,
1985[Abstract].
12.
Kandulska, K,
Nogowski L,
and
Szkudelski T.
Effect of some phytoestrogens on metabolism of rat adipocytes.
Reprod Nutr Dev
39:
497-501,
1999[ISI][Medline].
13.
Kuo, SM.
Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells.
Cancer Lett
110:
41-48,
1996[ISI][Medline].
14.
Kuppusamy, UR,
and
Das NP.
Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes.
Biochem Pharmacol
44:
1307-1315,
1992[ISI][Medline].
15.
Kuppusamy, UR,
and
Das NP.
Potentiation of -adrenoreceptor agonist-mediated lipolysis by quercetin and fisetin in isolated rat adipocytes.
Biochem Pharmacol
47:
521-529,
1994[ISI][Medline].
16.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
17.
Le Bail, JC,
Varnat F,
Nicolas JC,
and
Habrioux G.
Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids.
Cancer Lett
130:
209-216,
1998[ISI][Medline].
18.
MacDougald, OA,
and
Lane MD.
Transcriptional regulation of gene expression during adipocyte differentiation.
Annu Rev Biochem
64:
345-373,
1995[ISI][Medline].
19.
Marrero, MB,
Schieffer B,
Li B,
Sun J,
Harp JB,
and
Ling BN.
Role of Janus kinase/signal transducer and activator of transcription and mitogen-activated protein kinase cascades in angiotensin II and platelet-derived growth factor-induced vascular smooth muscle cell proliferation.
J Biol Chem
272:
24684-24690,
1997
20.
McIntyre, BS,
and
Sylvester PW.
Genistein and erbstatin inhibition of normal mammary epithelial cell proliferation is associated with EGF-receptor down-regulation.
Cell Prolif
31:
35-46,
1998[ISI][Medline].
21.
Miksicek, RJ.
Commonly occurring plant flavonoids have estrogenic activity.
Mol Pharmacol
44:
37-43,
1993[Abstract].
22.
Molteni, A,
Brizio-Molteni L,
and
Persky V.
In vitro hormonal effects of soybean isoflavones.
J Nutr
125:
751S-756S,
1995[Medline].
23.
Nogowski, L,
Mackowiak P,
Kandulska K,
Szkudelski T,
and
Nowak KW.
Genistein-induced changes in lipid metabolism of ovariectomized rats.
Ann Nutr Metab
42:
360-366,
1998[ISI][Medline].
24.
Robak, J,
and
Gryglewski RJ.
Flavonoids are scavengers of superoxide anions.
Biochem Pharmacol
37:
837-841,
1988[ISI][Medline].
25.
Rubin, CS,
Hirsch A,
Fung C,
and
Rosen OM.
Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells.
J Biol Chem
253:
7570-7578,
1978[ISI][Medline].
26.
Ruh, MF,
Zacharewski T,
Connor K,
Howell J,
Chen I,
and
Safe S.
Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity.
Biochem Pharmacol
50:
1485-1493,
1995[ISI][Medline].
27.
Schleicher, RL,
Lamartiniere CA,
Zheng M,
and
Zhang M.
The inhibitory effect of genistein on the growth and metastasis of a transplantable rat accessory sex gland carcinoma.
Cancer Lett
136:
195-201,
1999[ISI][Medline].
28.
Scott, RE,
Florine DL,
Wille JJJ,
and
Yun K.
Coupling of growth arrest and differentiation at a distinct state in the G1 phase of the cell cycle: GD.
Proc Natl Acad Sci USA
79:
845-849,
1982[Abstract].
29.
Shimabukuro, M,
Koyama K,
Chen G,
Wang M,
Trieu F,
Lee Y,
Newgard CB,
and
Unger RH.
Direct antidiabetic effect of leptin through triglyceride depletion of tissues.
Proc Natl Acad Sci USA
94:
4637-4641,
1997
30.
Shisheva, A,
and
Shechter Y.
Quercetin selectively inhibits insulin receptor function in vitro and the bioresponses of insulin and insulinomimetic agents in rat adipocytes.
Biochemistry
31:
8059-8063,
1992[ISI][Medline].
31.
Smith, PJ,
Wise LS,
Berkowitz R,
Wan C,
and
Rubin CS.
Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes.
J Biol Chem
263:
9402-9408,
1988
32.
Tontonoz, P,
Hu E,
Graves RA,
Budavari AI,
and
Spiegelman BM.
mPPAR -2: tissue-specific regulator of an adipocyte enhancer.
Genes Dev
8:
1224-1234,
1994[Abstract].
33.
Wang, HY,
and
Malbon CC.
Gs repression of adipogenesis via Syk.
J Biol Chem
274:
32159-32166,
1999
34.
Yamashita, Y,
Kawada S,
and
Nakamo H.
Induction of mammalian topoisomerase II dependent DNA cleavage by nonintercalative flavonoids, genistein and orobol.
Biochem Pharmacol
39:
737-744,
1990[ISI][Medline].