The metabolism of isoflavones is rather complex. The two major isoflavones, genistein and daidzein, are present in soy as  -D-glycosides, namely genistin and daizin (Fig. 1). These glycoside forms are biologically inactive (Setchell, 1998). Once ingested, isoflavone glycosides are hydrolyzed by bacterial-glucosidases in the intestinal wall, resulting in the conversion to their corre-sponding bioactive aglycones (genistein and daidzein). Only the aglycone forms are absorbed by the intestinal tract and are there-fore biologically active. Daidzein can be further metabolized to equol andO-demethyangolensin, and genistein to p-ethyl phenol.
In fact, genistein, daidzein, equol andO-demethyangolensin are the major isoflavones detected in the blood and urine of humans and animals (Setchell, 1998). In rodents, equol is the major circulat-ing metabolite among isoflavones representcirculat-ing up to 70–90% of all circulating isoflavones. While all rodents are equol producers, only 30% of humans are able to metabolize daidzein into equol (Atkinson et al., 2005). It remains unclear whether the effectiveness of dietary phytoestrogens in reducing the risks of obesity, diabetes, and cardiovascular disease in humans correlates with the ability of individuals to metabolize daidzein into equol. However, it is a likely source of variability and therefore should be taken into account when performing clinical trials with soy or dietary phytoestrogens.
4. Soy consumption and phytoestrogen levels
In soybean, isoflavones are tightly associated with proteins.
As mentioned, the abundance of isoflavones varies according
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Fig. 1.The molecular structure of isoflavones resembles that of 17-estradiol. Isoflavones are found in vegetables and fruits in a biologically inactive glycoside form (genistin, daidzin and glycitin). After ingestion,-glucosidases from the intestine cleave the glucosyl residue and generate biologically active aglycones (genistein, daidzein and glycitein).
Daidzein can be further metabolized into equol.
to soy variety and culture conditions, but is also dependent on the way soybeans have been processed. Indeed, isoflavones can be dissociated from soy-proteins using alcohol extraction which significantly diminishes the amount of bound-isoflavones (Bhathena and Velasquez, 2002). This explains the substantial vari-ability of phytoestrogen content found in soy products [0.1–5 mg isoflavones/g of soy protein in mature and roasted soybeans, 0.3 mg/g soy protein in green soybeans and tempeh, 0.1–2 mg/g soy protein in tofu and some soy milk preparations (Bhathena and Velasquez, 2002)].
Numerous studies have included the investigation of the plasma concentration of phytoestrogens and their metabolites in humans and animals consuming a diet with or without soy (Adlercreutz et al., 1993a; Morton et al., 1994; Coward et al., 1996). In humans consuming soy-free diets, plasma concentration of isoflavones is usually in the nanomolar range≤40 nM (Morton et al., 1994; van Erp-Baart et al., 2003). In contrast, acute ingestion of dietary soy leads to a rapid increase in the plasma concentration of isoflavones up to the micromolar range (Adlercreutz et al., 1993b; Xu et al., 1994; King and Bursill, 1998; Watanabe et al., 1998). Pharmacoki-netic studies confirm that healthy adults absorb isoflavones rapidly and efficiently (Setchell et al., 2001). The fates of daidzein, genis-tein and their respective-glycosides are similar. The average time taken after ingesting the aglycones to reach peak plasma concentra-tions is 4–7 h, which is delayed to 8–11 hours for the corresponding
-glycosides. This suggests that the rate-limiting step for absorp-tion is the initial hydrolysis of the glycosidic moiety. The half-life for daidzein and genistein was reported to be 9.3 and 7.1 h respec-tively, indicating that isoflavones or their metabolites are rapidly excreted. Finally, factors that might influence isoflavone bioavail-ability include intestinal microflora, food matrix, the administered
dose, intestinal transit time and the chemical composition of the dietary isoflavones.
5. Phytoestrogens: complex hormetic compounds
In plants, the synthesis of phytoestrogens, such as soy isoflavones, generally coincides with environmental stresses such as pest infection, drought or lack of nutrients (Howitz and Sinclair, 2008). Recently it has been suggested that stress-induced plant compounds upregulate stress resistance pathways in animals. This phenomenon, called xenohormesis, proposes that chemical clues from autotrophs (e.g. plants) provide an advance warning about the deterioration of the environment, allowing heterotrophs (e.g.
mammals) to mount a preemptive defense response while condi-tions are still favorable (Howitz and Sinclair, 2008). This theory has been recently adopted to explain the health benefits provided by stress-induced phytochemicals such as polyphenols, a group which includes stilbenes (e.g. resveratrol found in red wine and peanuts), catechins (e.g. epigallocatechin-3-gallate or EGCG found in green tea), anthocyanidins, and most relevant to this review, isoflavones.
Similarly, we believe that the isoflavones genistein and daidzein mediate most of their biological effects through the modulation of key mammalian enzymes and receptors of stress-response path-ways or estrogen-dependent pathpath-ways, rather than through their well known antioxidant (Vedavanam et al., 1999) or tyrosine kinase inhibitory properties (Akiyama et al., 1987). The affinity of phy-toestrogens for estrogen receptors results in effects on a large number of estrogen-regulated systems, including the cardiovascu-lar, metabolic, reproductive, skeletal and central nervous systems.
A significant characteristic of isoflavones is their capacity to bind both to estrogen receptors (ERs)␣and(Kuiper et al., 1997, 1998),
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Table 1
Relative binding affinity (RBA) and relative transactivation activity (RTA) of various estrogenic compounds, isoflavones and endocrine disruptors in comparison with 17-estradiol.
Compound Relative binding affinity Relative transactivation
ER␣ ER ER␣ ER
17-estradiol 100 100 100 100
Diethylstilbestrol (DES) 236 221 117 69
Tamoxifen 4 3 6 2
Coumestrol 20 140 102 98
Isoflavones Genistein 4 87 198 182
Daidzein 0.1 0.5 97 80
Formononetin <0.01 <0.01 6 2
Biochanin A <0.01 <0.01 36 53
Ipriflavone <0.01 <0.01 11 3
Endocrine disruptors Bisphenol A 0.01 0.01 50 41
o,p-DDT 0.01 0.02 54 10
Nonylphenol 0.05 0.09 62 34
Methoxychlor <0.01 <0.01 9 2
The RBA of each competitor was calculated as the E2:competitor concentration ratio required to reduce the specific radioligand binding by 50% (ratio of IC50 values). The RBA value for E2 was arbitrarily set at 100. The RTA of each compound was calculated as the ratio of the luciferase reporter gene induction value, at a concentration of 1M of the relevant compound, relative to the luciferase reporter gene induction value using 17estradiol at 1M. The transactivation activity of 17estradiol was arbitrarily set at 100. Data adapted fromKuiper et al. (1998).
with preferential binding of genistein to ER(Table 1). Specific binding affinity to ERs enables isoflavones to elicit both estrogenic and antiestrogenic effects depending on the tissue, as well as on isoflavone and endogenous estradiol levels. For instance, in var-ious cell lines, in the presence of physiological levels (1 nM) of estradiol (E2), genistein acts as an anti-estrogen whereas at lev-els of E2 found in postmenopausal women (0.01 nM), genistein shows additive agonistic effects (Hwang et al., 2006). Genistein also has ER-mediated biphasic effects in intestinal cell prolifera-tion (Chen and Donovan, 2004). Thus, isoflavones, as stress-induced phytochemicals, may provide health benefits through their selec-tive estrogen receptor modulator (SERM) activities.
6. Role of estrogens in metabolism
Studies in humans and rodents have shown that ERs are impor-tant mediators of the action of estrogen on lipid and glucose metabolism. Estrogens have been reported to affect adiposity either directly, by modulating lipogenesis, lipolysis or adipogenesis, or indirectly, by modulating appetite or energy expenditure (Cooke and Naaz, 2004). As mentioned above, the concept of estrogens modulating metabolic features derived originally from the obser-vation that postmenopausal women develop visceral obesity and insulin resistance as a result of low levels of estrogens. Interest-ingly, hormonal replacement therapy normalizes these symptoms (Ahmed-Sorour and Bailey, 1980; Bailey and Ahmed-Sorour, 1980).
Estrogens also play an important role in glucose homeostasis, and are known to modulate insulin sensitivity (Godsland, 2005). Vari-ations in human glucose homeostasis are observed during the menstrual cycle, and diabetes becomes more resistant to treatment during the luteal phase (Case and Reid, 2001). Relevant genetic evidence that estrogen modifies metabolism can also be found in humans: individuals with mutations in thearomatasegene, an enzyme that converts androgens into estrogens, display truncal obesity, insulin resistance and hyperlipidemia (Carani et al., 1997).
Studies in rodents are far more advanced, and confirm the impor-tance of ER␣in modulating both lipid and glucose metabolism. Mice lacking either ER␣or thearomatasegene have increased adipos-ity and fatty livers (Heine et al., 2000; Jones et al., 2000; Takeda et al., 2003), confirming the role of estrogens in the regulation of adiposity. In aromatase knockout mice (ArKO), liver steatosis is associated with a decreased expression of genes responsible for fatty acid oxidation, but can be normalized with E2 treatment
(Nemoto et al., 2000). The function of ERis less clear—to date no overt metabolic phenotype has been described in mice lacking this receptor (ERKO), under normal dietary conditions (Ohlsson et al., 2000). Recently, it was found that, when challenged with a high fat diet,ERKO mice increase their weight and adiposiy to a greater extend than do wild-type mice (Foryst-Ludwig et al., 2008).
Estrogens have also been shown to modulate glucose metabolism in rodents. For instance in models of type 2 diabetes, ovariectomy induces hyperglycemia, whereas in males, estrogen perfusion reverses diabetes (Louet et al., 2004). Similarly, mice lack-ing either functional ER␣oraromatasedisplay glucose intolerance and insulin resistance (Heine et al., 2000; Jones et al., 2000; Takeda et al., 2003). Indeed, ER␣and ERare both expressed in muscle cells and are known to modulate the expression of the glucose transporter 4 (Glut4), a key molecule for the import of glucose in cells (Barros et al., 2006). The endocrine pancreatic function is also directly modulated by estrogen levels. In rats, circulating insulin levels have been shown to vary during the estrous cycle (Bailey and Matty, 1972). Estrogens and endocrine disruptors such as Bisphenol-A (BPA) have been reported to modulate insulin content in-cells via ER␣, but not via ER(Alonso-Magdalena et al., 2008).
These molecules also protect-cells from oxidative stress, apopto-sis, and streptozotocin-induced injuries in both genders, through ER␣(Le May et al., 2006). In addition, several studies reported that estrogens increase glucose-stimulated insulin secretion in isolated islets (Nadal et al., 1998; Adachi et al., 2005; Alonso-Magdalena et al., 2006, 2008; Le May et al., 2006; Martensson et al., 2008).
Finally, estrogens have been reported to modulate energy home-ostasis indirectly, through the central nervous system (CNS) and the hypothalamus. By sensing endocrine and metabolic signals, the hypothalamus engages distinct effector pathways, which result in behavioral, endocrine and metabolic changes, to maintain energy homeostasis. Several hormones are capable of influencing energy intake and expenditure. For instance, leptin and insulin modulate the activity of hypothalamic neurons in the arcuate nucleus, ulti-mately leading to changes in food intake (Schwartz et al., 2000). It has been shown recently that estrogens are among the hormones that modulate the energy balance. Deficiency in aromatase or ER␣, or ovariectomy, decreases physical activity and energy expendi-ture (Heine et al., 2000; Cooke et al., 2001; Meli et al., 2004), whereas treatment with E2 increases locomotor activity through an ER␣-dependent mechanism (Ogawa et al., 2003). Both estro-gen receptors are found in the hypothalamic nuclei and modulate
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food intake (Liang et al., 2002) and locomotor activity (Ogawa et al., 2003). Specific silencing of ER␣in the hypothalamus of female rodents leads to similar phenotypes to those observed in consti-tutive mutant mice, including obesity, hyperphagia and reduced energy expenditure (Musatov et al., 2007). Although it remains unknown whether or not these phenotypes are associated with changes in the expression of hypothalamic neuropeptides, these results show that hypothalamic ER␣is essential in the regulation of energy balance. Supporting a direct effect of E2 on the hypothalamic control of adiposity, it was shown that E2 modulates hypothalamic synapticity by bypassing the leptin receptor to act directly on the downstream Stat3 signaling in the hypothalamus, thus decreasing body weight (Gao et al., 2007).
Overall, estrogens appear to be crucial regulators of metabolic functions by directly and indirectly (via the CNS) modulating energy homeostasis. Whether or not dietary soy and phytoestrogens have effects on energy homeostasis through estrogen-mimics is still a matter of controversial debate, but is clearly a plausible hypothesis.
7. Effects of soy protein and phytoestrogens on human metabolism
The low frequency of obesity and related metabolic disorders in Asian populations has drawn attention towards soy, which is a characteristic component in asiatic diets. We searched the PubMed literature database for epidemiological and clinical studies evaluat-ing the effects of soy or isolated isoflavones on human metabolism, and the main results are summarized inTables 2–5.
Epidemiological studies have shown that type-2 diabetes is four times less prevalent in Japanese people in Tokyo than in Japanese-Americans in Seattle (Fujimoto et al., 1987, 1991). Consumption of more than 12.6 grams of soy protein per day is associated with a lower risk of glycosuria, a strong predictor of diabetes (Yang et al., 2004). Similarly, several studies have reported that isoflavone con-sumption by postmenopausal women correlated with lower body mass index (BMI), and higher HDL levels (Guthrie et al., 2000;
Goodman-Gruen and Kritz-Silverstein, 2001, 2003).
Clinical studies also suggest that soy protein or isoflavones may improve metabolic parameters. For instance, a metaanalysis of 38 trials (Anderson et al., 1995) as well as more recent reports (Crouse et al., 1999; Takatsuka et al., 2000; Teixeira et al., 2000; Gardner et al., 2001; Jayagopal et al., 2002; Greany et al., 2004) demonstrated a significant reduction in plasma concentrations of total and LDL cholesterol in humans exposed to soy proteins. In addition, post-menopausal Japanese women treated for 24 weeks with isoflavones exhibited a lower fat mass (Wu et al., 2006). Obese patients treated with soy protein isolates for 12 weeks had lower body weight and BMI, with decreased cholesterol and LDL levels in the blood (Allison et al., 2003). Additionally, a 6-month clinical trial was conducted
to compare the effects of isoflavones with that of conjugated estro-gens on blood glucose, insulin, and lipid profiles in postmenopausal Taiwanese women. The study revealed that during fasting both glu-cose and insulin levels were significantly reduced by soy isoflavones (100 mg/day) and conjugated estrogens (0.625 mg/day) (Cheng et al., 2004).
In contrast to the above mentioned trials, a significant number of studies reported an absence of beneficial effects of soy on classi-cal metabolic parameters such as body weight, serum lipid profiles, fat mass, blood glucose and insulin profiles (Yamashita et al., 1998;
Anderson and Hoie, 2005; Li et al., 2005; Hall et al., 2006; Ikeda et al., 2006; Anderson et al., 2007). These discrepancies make it difficult to draw firm conclusions regarding the beneficial effect of soy on glucose and lipid metabolism. When comparing these different clinical trials, the underlying causes of conflicting results are probably related to the variability of experimental designs and exposition protocols (route of administration, composition, dose, and duration), the capacity of individuals to produce equol and the genetic susceptibility. Clearly more standardized studies are needed to further evaluate these putative beneficial effects.
8. Actions of soy on metabolism in rodents
The current scientific evidence concerning the role of soy and isoflavones in rodents is based on studies where animals have been exposed either to purified isoflavones (injected or supplemented in the diet itself) or soy protein isolates (SPIs) (for a complete list of studies, see Tables 6–10). The difficulty in analyzing SPI studies arises from the fact that information on isoflavone levels and composition are quite often incomplete, making interpreta-tions problematic and comparisons hazardous. On the other hand, supplementation of the diet with soy proteins is more relevant than injection-based studies. This does, however, raise questions as to which compound is responsible for the observed effects and whether the relative benefits of soy are not in fact due to the poor performance of casein itself, which is usually used as control pro-tein.
In comparison to human studies which mainly focus on serum lipid analysis because of the clinical importance of atherosclero-sis and the risk in cardiovascular diseases, reports in rodents are rather oriented towards the assessment of soy-derived compounds on weight and fat loss, and in fewer cases, insulin sensitivity. Still concerning the effects of soy or isoflavones on serum lipid profiles, most rodent studies that have assessed these parameters under healthy or diabetic states point towards an improvement in total cholesterol (TC) and/or triglyceride (TG) levels after consumption of dietary soy, SPI, isoflavones or genistein (Kirk et al., 1998; Nagasawa et al., 2003; Ae Park et al., 2006; Penza et al., 2006; Cederroth et al., 2008; Nordentoft et al., 2008; Torre-Villalvazo et al., 2008). Only
Table 2
Epidemiological studies evaluating the effects of soy or isoflavones on human metabolism
Model Number of
individuals (total)
Dose Metabolic Effects References
Pre and postmenopausal women 323 Soy protein intake >12.61 g/day For >12.61 g/day: Lower risk in glycosuria in postmenopausal women with BMI
<25 kg/m2
Yang et al. (2004)
Pre and postmenopausal women 944 Fermented soy bean No effects on W or F/BMI Ikeda et al. (2006)
Postmenopausal women 208 Genistein intake >1 mg/day No effects on G, I, TC, LDL, TG Goodman-Gruen and
Kritz-Silverstein (2001) Lower F/BMI, increased HDL
Postmenopausal women 939 Isoflavone intake >0.236 mg/day No effects on F/BMI, TC, LDL, HDL, Lower TG de Kleijn et al. (2002) In these studies, serum isoflavone levels were not evaluated.Abbreviations: W, weight; F/BMI, fat or body mass index; G, serum glucose; I, serum insulin; TC, total cholesterol;
LDL, low density lipoprotein; HDL, high density lipoprotein; TG, triglycerides; FFA, free fatty acids; ND, not determined. Parameters listed here that do not appear in the table were not analyzed in the article in question.
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Table 3
Clinical studies performed using dietary supplementation with soy protein isolates (SPI)
Model Number of
individuals
Duration SPI and isoflavones intake Serum isoflavone Metabolic effects References
Young healthy normolipidemic men and women
Total = 22 13 days High isoflavone SPI (56 mg/day) vs low isoflavone SPI (2 mg/day)
Genistein: 0.75 No effects on TC, LDL, TG
Sanders et al. (2002)
Daidzein: 0.3 Increased HDL Equol: 0.1
Obese women 22 (21) 16 weeks SPI (15 g/day)
(150 mg/day isoflavones)
Postmenopausal women 100 (102) 12 months SPI (25.6 g/day) (99 mg/day isoflavones) vs milk
ND No effects on F/BMI Kok et al. (2005)
Moderate
hypercholesterolemic postmenopausal women
31 (33) 12 weeks SPI with isoflavone (80 mg) or without vs milk (42 g/day)
Total = 146 9 weeks SPI (25 g/day) (62 mg/day isoflavones) vs Casein (25 g/day)
ND No effects on HDL, TG Crouse et al. (1999)
Lower TC, LDL Normocholesterolinemic 71 (72) 6 weeks SPI with isoflavones
(44.3 mg/day) vs milk
ND No effects on W,
F/BMI, TC, LDL, HDL, TG
Greany et al. (2004)
Mildly hypercholesterolinemic 71 (72) 6 weeks SPI with isoflavones (44.3 mg/day) vs milk
ND No effects on W, F/BMI Greany et al. (2004)
Lower TC, LDL, TG Increased HDL
Obese men and women 50 (50) 12 weeks SPI vs control ND No effects on HDL Allison et al. (2003)
Lower W, BMI, TC, LDL
Obese male and women 17 (19) 16 weeks SPI vs meat ND No effects on W,
F/BMI, G, TC, LDL, HDL, TG
Yamashita et al. (1998)
Obese men and women 46 (36) 3 or 6 months SPI vs individual dietary intervention
ND No effects on I, TC,
LDL, HDL, TG
Li et al. (2005) Lower W, F/BMI, G
12 months SPI vs individual dietary
intervention
ND No effects on W,
F/BMI, G, I, TC, LDL, HDL, TG
Li et al. (2005)
Abbreviations: SPI, soy protein isolate; W, weight; F/BMI, fat or body mass index; G, serum glucose; I, serum insulin; TC, total cholesterol; LDL, low density lipoprotein; HDL, high density lipoprotein; TG, triglycerides; FFA, free fatty acids; ND, not determined. Parameters that do not appear in the table were not analyzed in the article in question.
Serum isoflavone values are expressed inM. For control groups, the number of individuals (n) is shown in parentheses.
one study shows an absence of effects on total cholesterol and TG
one study shows an absence of effects on total cholesterol and TG