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,2
* Department of Animal and Dairy Science and Department of Foods and Nutrition, University of Georgia, Athens, GA 30602 and ** Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912
2 To whom correspondence should be addressed. E-mail: cbaile{at}uga.edu.
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ABSTRACT |
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KEY WORDS: • isoflavone • adipose tissue • 3T3-L1 adipocytes • apoptosis • weight loss
Genistein is an isoflavone from Glycine max (soybean), which is now widely studied due to its interesting effects on bone metabolism and tumor growth, as well as its use as a dietary supplement to attenuate the symptoms of menopause. Estrogen plays a vital role in adipose tissue regulation, particularly in postmenopausal women. Ovariectomy of rodents increases body fat, and estrogen replacement reverses that increase (1). Similarly, women often have increased body fat content after menopause, and estrogen treatment was shown to decrease body fat in postmenopausal women (2,3).
Genistein can act as a weak estrogen agonist or antagonist (4,5) due to the presence of a phenolic ring necessary to bind estrogen receptors (ER)3 (6–11), and genistein can increase uterine growth in both intact and ovariectomized rodents (12). Genistein has shown a high affinity for ERß (13,14), but genistein's action to reduce adipose tissue is attributed to the presence of ER
(15). ER knockout mice have increased body fat, adipocyte size and adipocyte number, and insulin resistance; glucose tolerance is impaired in both males and females (16) and they are resistant to genistein-induced fat reduction (15). In addition to its estrogen agonistic activity, genistein was also shown to affect gene expression of various proteins involved in cell cycle regulation, apoptosis, and proliferation and to inhibit protein tyrosine kinase, DNA topoisomerases, and ribosomal S6 kinases (17–19).
In a recent study, Naaz et al. (15) showed that in ovariectomized mice given dietary genistein, fat pad weights decreased dose dependently by 37–57% and lipoprotein lipase (LPL) mRNA was also decreased. In adipocytes genistein was shown to inhibit cell proliferation and increase lipolysis (20–23). Genistein treatment blocked differentiation of both preconfluent and postconfluent 3T3-L1 adipocytes (23). In isolated rat adipocytes, genistein was found to inhibit the conversion of acetate into lipid, inhibit basal lipogenesis, inhibit the conversion of glucose to lipids more than estradiol, and increase basal lipolysis (21). In bone marrow stromal cells, genistein decreased adipocyte number and lipid filling and decreased gene expression of LPL (24).
Until quite recently, it was thought that the total number of adipocytes remained fairly constant, whereas only the amount of lipid stored in adipocytes increased or decreased with changing energy demands. However, recent studies showed that like other cell types, adipocytes can be obtained and lost (25–27). Of patients who had undergone liposuction as a treatment for obesity, 80% maintained their postoperative weights up to 1 y post-treatment, suggesting that localized removal of adipose tissue can be an effective way to reduce total adipose mass (28). Furthermore, Kolonin et al. (29) showed that ablation of adipose tissue reversed obesity without causing adverse effects in dietary-induced obese mice. Our studies showed that weight loss can occur not only as a result of increasing lipolysis, but also by inducing adipocyte apoptosis (30,31). Similarly, we found that body fat loss in ob/ob mice treated with leptin is in part due to adipocyte ablation by apoptosis (32). These results point to adipocyte apoptosis as a novel mechanism for the treatment of obesity.
Because genistein was shown to decrease body weight (BW) (33,34) and fat pad weight (15), and because it induces apoptosis in certain cell types (35–37), we examined the effect of genistein on apoptosis of preadipocytes and mature adipocytes in vitro. Further, we investigated whether dietary genistein can cause loss of body fat in aged, ovariectomized mice and if so, whether adipose apoptosis contributes to the genistein-mediated reduction of adipose tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Materials and cell culture. Genistein (99% pure) was purchased from Indofine Chemical. 3T3-L1 mouse embryo fibroblasts were obtained from American Type Culture Collection and cultured as described elsewhere (38).
For mature adipocytes, cells were seeded (5000 cells/well), grown to confluence, induced to differentiate, and grown to maturation. Briefly, cells were cultured in DMEM (GIBCO, BRL Life Technologies) containing 10% bovine calf serum (BCS) until confluent. Two days after confluence (D0), the cells were stimulated to differentiate with DMEM containing 10% fetal bovine serum (FBS), 167 nmol/L insulin, 0.5 µmol/L isobutylmethylxanthine, and 1 µmol/L dexamethasone for 2 d (D2). Cells were then maintained in 10% FBS/DMEM medium with 167 nmol/L insulin for another 2 d (D4), followed by culturing with 10% FBS/DMEM medium for an additional 4 d (D8), at which time >90% of cells were mature adipocytes with accumulated fat droplets. For preadipocytes, seeding density of 2500 cells/well was used and cells were cultured in DMEM containing 10% BCS for 24 h before treatment and incubated with genistein at various concentrations (0, 1, 10, 100, and 400 µmol/L) for 12, 24, and 48 h (8 replicates). All media contained 100 kU/L penicillin, 100 mg/L streptomycin, and 292 mg/L glutamine (Invitrogen). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.
Cell viability assay. After incubation, mature adipocytes were assayed using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) following the manufacturer's instructions. The absorbance was measured at 490 nm in a plate reader (µ QuantTM Bio-Tek Instruments) to determine the formazan concentration, which is proportional to the number of living cells in culture.
Apoptosis detection. A single-stranded DNA ELISA kit was used for detection of apoptosis in 3T3-L1 cells (ApoStrandTM, BIOMOL). This assay is based on the selective denaturation of DNA in apoptotic cells by formamide, which reflects changes in chromatin associated with apoptosis, such as chromatin condensation and digestion of protein stabilizing DNA (39). The denatured DNA was detected with a mixture of primary antibody and peroxidase-labeled secondary antibody. After treatment with genistein, mature adipocytes and preadipocytes were fixed for 30 min and dried in an oven at 56°C for 20 min. Formamide was added to the cells and they were heated at 56°C for 30 min. Blocking solution was then added and cells were incubated with antibody mixture for 30 min and rinsed with 1X wash buffer. After washing, cells were incubated with 100 µL of peroxidase substrate and absorbance was read using an ELISA plate reader at 405 nm.
Expt. 2. Effects of dietary genistein on body composition, food intake, BW, and adipose tissue apoptosis of ovariectomized mice
Animals and diets.
All experimental procedures were conducted in accordance with the NIH guidelines and were approved by the Animal Care and Use Committee for The University of Georgia before initiating the studies. Ovariectomized C57/BL6 female mice (n = 30) were 
9 mo old at arrival from Harlan. Initially they were group-caged and fed a standard pelleted rodent diet (Prolab RMH 2500, PMI International); 1 wk later, mice were individually caged, given free access to water and switched to a semipurified phytoestrogen-free casein-based diet (AIN93M) described elsewhere (40) with corn oil replacing soybean oil.
After 2 wk of consuming the semipurified phytoestrogen-free casein-based diet, mice were divided into 3 experimental groups, using a weight-matched complete block design. Genistein treatments were randomly assigned and mice (n = 10) were fed diets containing 0 (control), 150, or 1500 mg/kg genistein (Indofine Chemical) for 21 d. Food intake was measured weekly and BW was measured on d 1 (first day of experimental diet feeding) and d 4, 8, 11, 15, 18, and 22. On d 22, mice were killed by CO2 asphyxiation and decapitated.
Trunk blood was collected for measurement of insulin, leptin, and glucose concentrations. Body composition was determined with a PIXImusTM densitometer (GE Medical Systems Lunar), which uses dual-energy X-ray absorptiometry (DEXA) to measure lean tissue and body fat (41,42) This method is based on the differential attenuation of low- and high-energy X-ray due to the difference in average atomic number of lean tissue and fat. The ratio of high- to low-energy X-ray attenuation coefficient (DEXA ratio) is used to calculate body composition by the software. Parametrial (PM), inguinal (ING), and retroperitoneal (RP) fat pads were removed bilaterally, frozen immediately in liquid nitrogen, transferred, and stored at –80°C for apoptosis assay. Gastrocnemius and soleus muscles were removed and weighed.
Serum leptin, insulin, and glucose concentrations. Glucose concentration was determined using the FreeStyle Blood Glucose monitoring system (TheraSense®). For other assays, blood was collected in a vial, allowed to clot on ice, and then centrifuged (2000 x g; 20 min) to obtain serum. Serum samples were stored at –80°C until assayed. Insulin and leptin concentrations were determined using the Luminex100TM instrumentation and a multiplex assay kit (Mouse Endocrine Immunoassay Panel) manufactured by LINCO Research.
DNA isolation and apoptosis assay.
Apoptosis was assayed in 2 ways. First, the DNA isolated from fat tissue was assayed on an agarose gel to identify a ladder pattern of internucleosomal DNA degradation, which is characteristic of apoptosis. Second, apoptosis was quantified as the ratio of fragmented:total DNA (43). Briefly, 100 mg of the PM, ING, and RP white adipose tissues was homogenized in lysis buffer (10 mmol/L Tris-HCl, 10 mmol/L EDTA; 0.5% Triton X-100, pH 8.0) and centrifuged at 14,000 x g for 15 min to separate fragmented DNA from genomic DNA. The supernatant, containing fragmented DNA, was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and the DNA was precipitated by adding polyacryl carrier (Molecular Research Center) and ethanol. Genomic (nonfragmented) DNA was extracted from the pellet with DNAzol and polyacryl carrier. DNA in each fraction was quantified by PicoGreen method (Molecular Probes) and fluorescence was measured using SpectroMax Gemini (Molecular Devices). Both fractions were loaded onto a 1.5% agarose gel and run at 110 V for 1.5 h. After electrophoresis, the gel was stained with SYBR Gold (Molecular Probes) and visualized under UV light using a FluorChem Imager 8000 (Alpha Innotech) with a SYBR Gold filter (520 nm). The percentage of DNA fragmentation was calculated as soluble DNA/(soluble + insoluble DNA) x 100.
Statistics. In Expt. 1, 2-way ANOVA (GLM procedure in Statistica, StatSoft, ver 6.1) was used to determine significance of treatment and time effects for viability and apoptosis assays. In Expt. 2, 2-way ANOVA was used to determine significance of treatment and time effects on BW, and 1-way ANOVA was used to determine significance of treatment effects for mean daily food intake, fat pad weight, and fat pad apoptosis. Least Significant Difference (LSD) was used to determine significance of differences between treatment means. Data presented are means ± SEM. Statistically significant differences are defined at the 95% confidence interval.
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RESULTS |
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Mature adipocyte viability.
There were significant effects of treatment (P < 0.01), time (P < 0.01), and treatment x time (P < 0.01) (2-way ANOVA). The viability of mature adipocytes was reduced by 400 µmol/L genistein at all time periods (Fig. 1). After treatment with 400 µmol/L genistein for 12, 24, and 48 h, cell viability was 50.3, 41.4, and 19.5% of control, respectively (P < 0.01 for each time period). Treatment with 1, 10, and 100 µmmol/L genistein did not reduce cell viability.
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Food intake, BW, and body composition. Daily food intake was not significantly affected by the 150 mg/kg dose of genistein (control: 3.63 ± 0.14 g/d; 150 mg/kg: 3.63 ± 0.12 g/d); however, the 1500 mg/kg dose of genistein decreased daily FI by 14% (3.16 ± 0.07 g/d; P < 0.01). For BW, there was a significant treatment effect (P < 0.01), but no significant time effect (2-way ANOVA). The treatment x time interaction was significant (P = 0.09). Body weight was decreased 9% compared with control by d 22 (P < 0.01; Fig. 3). By d 8, BW was lower in mice administered the 1500 mg/kg genistein dose than in controls, and the effect lasted until the end of the study (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It was shown that the effect of genistein on BW and fat pad weight was dose dependent (15). In our study, 1500 mg/kg dietary genistein reduced BW by 9% in contrast to no effect with 150 mg/kg genistein. This is consistent with the result of Naaz et al. (15), who reported that 1500 mg/kg genistein significantly decreased BW after 12 d of feeding and 300–1000 mg/kg doses did not differ from control. The authors found that dietary genistein for 12 d (500–1500 mg/kg) dose dependently reduced all fat pads by 37–57%. We found, however, that the decrease in adipose tissue weight of 19 and 22% occurred in the ING and PM fat pads, respectively, with no significant effect on the RP fat pads despite longer feeding periods (21 d). Discrepancies between our data and theirs could be due to the different developmental stages of the mice. Our study used 9-mo-old mice, compared with 25- 27-d-old juvenile mice in the experiment of Naaz et al. Considering that the effect of genistein treatment is more obvious in juvenile (25–27 d old) than adult (12–13 wk old) ovariectomized mice (15), the low responsiveness to genistein in our study could be attributed to the old age of the mice (9 mo), possibly due to reduced sensitivity of ER.
The relations among dietary isoflavones, weight loss, and fat content have not been explored adequately; however, in vivo studies support the role of isoflavones in the treatment of obesity. Isoflavone-rich diets result in improved lipid metabolism and antidiabetic effects in obese rats (44). When administered to CD1 male and female mice, oral genistein significantly decreased BW (33). It was also shown that soy isoflavone prevented body fat elevation and bone loss in ovariectomized mice (45). In recent years, substantial data from epidemiologic surveys and nutrition intervention studies have suggested the beneficial effect of soy isoflavone genistein on obesity in humans. It was shown that BMI was inversely related to 24-h urinary isoflavone excretion, the biomarker of soy intake, in a worldwide epidemiologic study (46).
Genistein intake has been associated with decreased BMI, weight, waist circumference, and total body fat mass in postmenopausal women (34). Supplementation of soy isoflavone increased bone mineral density and decreased body fat concomitantly with BMI reduction in middle-aged women (47). However, the mechanisms by which isoflavones exert their beneficial effect on body fat and obesity are unclear. Due to its structural similarity to endogenous estrogens, genistein can act as a weak estrogen and bind to the ER in various tissues, including adipose tissue. Naaz et al. (15) showed that genistein did not decrease adipose tissue in ovariectomized ER knockout mice, demonstrating that genistein's antilipogenic effect requires ER and regulation of estrogen-dependent processes. Experimental evidence suggests that genistein may also exert effects via non-ER–mediated mechanisms. Genistein modulates cell-signaling mechanisms and nuclear related cell proliferation and differentiation mechanisms by inhibiting protein tyrosine kinase (22), protein kinase (48), and topoisomerase II (49), and downregulating the nuclear factor-
B/Akt pathway (50). Additionally, the ability of genistein to induce apoptosis in several cancer cell types has been well documented (35–37,51), which led to its investigation for the treatment of cancers in animal models (52).
We showed that genistein induces adipocyte apoptosis in vivo and in vitro. This is the first paper to demonstrate that apoptosis may be a contributor to genistein's reducing effect on BW. In differentiated 3T3-L1 adipocytes, treatment with 10, 100, and 400 µmol/L genistein caused apoptosis after 24 h; 400 µmol/L genistein induced apoptosis at all time points and greatly reduced viability of the adipocytes, which reflects the vigorous damage to cells due to the great extent of apoptosis. In contrast, there was no significant effect on viability with lower doses of genistein despite apoptosis. This is caused by the methodological difference between apoptosis and viability assays. The MTS method used in the viability assay is based on the bioreduction of the MTS tetrazolium compound by NADPH/NADH produced by the mitochondrial electron chain reaction; thus, early stage DNA fragmentation detected by the ssDNA assay would not necessarily be accompanied by mitochondrial failure detected by the MTS assay.
In our in vivo experiment, 1500 mg/kg genistein increased DNA fragmentation in the ING fat pad (290%) of ovariectomized adult mice. However, DNA fragmentation was not detected in the RP or PM fat pads, suggesting that apoptosis by genistein may specific to the fat depot. There is evidence for adipose depot–related differences in responsiveness to lipolytic and apoptotic stimuli. For example, human preadipocytes from omental depots were more susceptible to tumor necrosis factor-–induced apoptosis than were preadipocytes from subcutaneous depots (27). Furthermore, in rats treated with leptin, apoptosis was increased in the ING fat pad, resulting in decreases in both cell number and volume, whereas apoptosis was not detected in the epididymal fat pad (53). However, because apoptotic cells are removed rapidly and efficiently by neighboring macrophages (54,55), it is difficult to correlate the presence or number of apoptotic cells at any one time point with the actual cumulative amount of tissue loss. In the present study, in addition to finding increased apoptosis as determined by the DNA fragmentation assay, we also found that the DNA from the ING fat pad showed a distinct apoptotic ladder pattern after gel electrophoresis. Therefore, both qualitative and quantitative data provide evidence that apoptosis occurred, at least in the ING fat pads of mice treated with the high dose of dietary genistein.
Dietary genistein was also shown to have direct effects on lipid metabolism in the liver and adipose tissue, decreasing triglycerides while increasing free fatty acids in serum (56). These in vivo data are further supported by in vitro studies showing that genistein induced lipolysis and inhibited de novo lipid synthesis in 3T3-L1 adipocytes (22,23) and rat adipocytes (21). Recently, it was suggested that genistein regulates lipid metabolism in adipose tissue via peroxisome proliferator-activated receptors (PPAR). Genistein blocked the expression of PPAR
and CCAAT/enhancer binding protein-, which are involved in adipogenesis and adipocyte differentiation (22), and it induced PPAR expression, which is involved in fatty acid catabolism (57).
A number of studies have been carried out to determine whether the amount of dietary genistein required to reduce adipose tissue mass can be achieved in humans. It was reported that serum genistein levels in mice fed 1500 mg/kg were 1–4 µmol/L depending on the analytical method used (1.12 ± 0.18 µmol/L by HPLC-UV and 3.81 ± 0.39 µmol/L by LC-electrospray-MS)(15). These values are higher than those reported in Japanese men (58) but within the range encountered in humans under various nutritional conditions. Other studies have found serum genistein levels between 1.1 and 4.5 µmol/L in both adults and infants fed soy-based meals (59, 60). The recent popularity of soy supplements makes it possible to consume amounts several fold greater than those obtained even with a high soy diet. Ingestion of recommended doses of isoflavone supplements by adults results in isoflavone levels similar to those in infants fed soy-based formula (61). These reports served as criteria for selecting doses in our study, and we assume that genistein levels in our study would have been in a range similar to that in the other studies using the same doses of genistein.
In summary, these studies showed that genistein is effective in decreasing BW and body fat in older ovariectomized female mice. Both in vitro and in vivo results also demonstrate that genistein is capable of inducing apoptosis of adipocytes, suggesting that at least part of the weight loss is due to ablation of fat cells, which could result in better maintenance of weight loss. The results of this study suggest that genistein may be useful in treating or preventing increased adiposity after menopause.
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FOOTNOTES |
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3 Abbreviations used: BCS, bovine calf serum; BW, body weight; DEXA, dual-energy X-ray absorptiometry; ER, estrogen receptor; FBS, fetal bovine serum; FI, food intake; ING, inguinal; LPL, lipoprotein lipase; PM, parametrial; PPAR, peroxisome proliferator-activated receptor; RP, retroperitoneal.
Manuscript received 6 May 2005. Initial review completed 2 June 2005. Revision accepted 4 November 2005.
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