Obesity and its related disorders, such as type 2 diabetes (T2D), cardiovascular diseases (CVD), high blood pressure, dyslipidemia [high levels of circulating triacylglycerols and low-density lipopro-tein (LDL) cholesterol, and low levels of high-density lipoprolipopro-tein (HDL) cholesterol], have recently become a major health problem reaching pandemic proportions (Engelgau et al., 2004). These diseases, commonly referred to as the Metabolic Syndrome (MS), are beginning to surpass malnu-trition and infectious diseases as the most significant contributor to ill health globally. In western societies, for the first time in modern history, life expectancy of newborns is declining as a result of these metabolic disorders (Olshansky et al., 2005). The rapid increase in obesity suggests that life-style factors such as high-calorie diets, physical inactivity and potentially environmental en-docrine disruption, rather than genetics, are the most plausible causes.
Although the source of metabolic disorders is often the diet itself, nutrition can also form part of the solution, in fact providing health benefits. Usually dietary intervention to control excess body weight, hyperglycemia and dyslipidemia has included low energy and low fat diets, but these are of limited efficacy due to the strict and long-term commitment required. However, long term health benefits can be gained from dietary proteins and bioactive non-nutrients, called phytochemicals, which could be either incorporated into the diet or be part of the food itself.
These phytochemicals are biologically active plant-derived compounds, which structurally and functionally mimic estrogens (Dixon, 2004). Phytoestrogens are found in numerous fruits and vegetables and are categorized into three classes, namely the isoflavones, lignans and coumestans.
While phytoestrogens are ubiquitous within the plant kingdom, isoflavones are mainly found the soybean the most important dietary source of phytoestrogens for humans, cattle and rodents.
Isoflavones have a non steroidal structure but possess a phenolic ring that enables them to bind the estrogen receptor (ER) and act either as estrogen agonists or antagonists (Makela et al., 1994;
Makela et al., 1995).
The fact that isoflavones have been shown to exert estrogenic effects raises the possibility that this class of phytochemicals may affect glucose and lipid metabolism. In fact, estradiol itself is a well known modulator of both obesity and glucose homeostasis. For instance, postmenopausal women develop visceral obesity and insulin resistance and are at an increased risk of diabetes but estrogen replacement therapy normalizes these abnormalities (Ahmed-Sorour and Bailey, 1980;
Bailey and Ahmed-Sorour, 1980; Gambacciani et al., 1997). From genetic studies in rodents, it has been shown that these effects are mediated by estrogen receptors (see below). This has caused researchers to focus on the identification of Selective Estrogen Receptor Modulators (SERMs) that could be of potential therapeutic interest for the treatment of metabolic disorders, without having negative effects. Studies in humans and rodents support the hypothesis that soy proteins or soy-derived phytoestrogens may be beneficial for the prevention of obesity and diabetes (Bhathena and Velasquez, 2002; Velasquez and Bhathena, 2007).
The complex interactions between soy proteins and isoflavones are fairly well understood. To understand these intricate relationships, one must assess the biological activity of soy components, both in isolation and in combination. So far, few studies have shown that pure soy-proteins or soy protein isolates (SPI) alone (in absence of isoflavones) can provide beneficial metabolic effects (Velasquez and Bhathena, 2007). The majority of the studies using SPI remain difficult
6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 21 to interpret because of the lack of clarity concerning the presence or absence of isoflavones in the diet. On the other hand, soy-derived phytoestrogens have received more attention mainly due to their benefits in decreasing age related diseases (e.g. osteoporosis, cardiovascular disease), or hormono-dependent cancers (e.g. prostate) (Setchell, 1998; Tham et al., 1998; Sacks et al., 2006).
Concerning metabolism, the American Food and Drug Administration (FDA) authorized in 1999 the labeling of health claims on food containing soy proteins, referring to the beneficial role of soy protein in reducing the risk of coronary heart disease (CHD). Attempts to show beneficial effects on metabolism in humans have been hotly debated, but studies in rodents may help in identifying the biologically relevant soy components and the intimate mechanisms involved. The purpose of this section is to examine the evidence regarding the use of soy and phytoestrogens in the prevention of obesity and diabetes mellitus in animals and humans. We also discuss the mechanisms by which soy and dietary phytoestrogens may affect glucose and lipid metabolism and improve the control of body weight and glucose homeostasis. To provide context and the requisite background information, we begin with a brief overview about soybeans, the nutritional composition of soy. We also present scientific evidence both in humans and rodents supporting or refuting the potential beneficial effects of soy and phytoestrogens on glucose and lipid metabolism.
6.2 Soybean composition
Soybean (Glycine max) is composed of macronutriments such as lipids, carbohydrates and pro-teins. Soybean lipids, which are deprived of cholesterol, contain about 15% of saturated fat, 61%
of polyunsaturated fat, and 24% of monounsaturated fat (USDA, 1979). Carbohydrates make up about 30% of the seed, with 15% being soluble carbohydrates (sucrose, raffinose, stachyose) and 15% insoluble carbohydrates (dietary fiber). The protein content of soybean varies from 36% to 46% depending on the variety (Garcia et al., 1997; Grieshop et al., 2001; Grieshop et al., 2003). Storage proteins are predominant, such as the 7S globulin (β-conglycinin) and 11S globulin (glycinin), which represent about 80% of total protein content, as well as less abundant storage proteins such as 2S, 9S, and 15S globulins (Garcia et al., 1997). Interestingly, β-conglycinin but not glycinin is capable of improving serum lipid profiles in mice and humans, in the absence of phytoestrogens (Moriyama et al., 2004; Kohno et al., 2006).
Soybean also contains micronutrients, which include isoflavones, phytates, saponins, phytos-terols, vitamins and minerals. Although beneficial effects of micronutrients such as saponins and phytosterols on cholesterol levels and absorption have been reported (Oakenfull, 2001; Lukaczer et al., 2006), there is an increasing body of literature suggesting that isoflavones may additionally have a beneficial role in lipid and glucose metabolism. Soybeans are the most abundant source of isoflavones in food. Studies have shown that there is a large variability in isoflavone content and composition in soybeans. This is function of the variety of soy grown, as well as environmental conditions (Wang and Murphy, 1994b; Caldwell et al., 2005). Abiotic and biotic stresses such as variation in temperature, drought or nutritional status, pest attack or light conditions may modify isoflavone content and composition. As a consequence, total isoflavone content may vary up to 3-fold with growth of the same soy cultivar in different geographical areas and years (Wang and Murphy, 1994a).
6.3 Absorption and metabolism of isoflavones
The metabolism of isoflavones is rather complex. The two major isoflavones, genistein and daidzein, are present in soy asβ-D-glycosides, namely genistin and daizin (seeFigure 3). These glycoside forms are biologically inactive (Setchell, 1998). Once ingested, isoflavone glycosides are hydrolyzed by bacterialβ-glucosidases in the intestinal wall, resulting in the conversion to their corresponding bioactive aglycones (genistein and daidzein). Only the aglycone forms are absorbed by the intestinal tract and are therefore biologically active. Daidzein can be further metabolized
6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 22
3.jpg
Figure 3: The molecular structure of isoflavones resembles that of 17β-estradiol. Isoflavones are found in vegetables and fruits in a biologically inactive glycoside form (genistin, daidzin and glycitin). After ingestion,β-glucosidases from the intestine cleave the glucosyl residue and generate biologically active aglycones (genistein, daidzein and glycitein). Daidzein can be further metabolized into equol. From Cederroth and Nef, 2009.
to equol and O-demethyangolensin, and genistein to p-ethyl phenol. In fact, genistein, daidzein, equol and O-demethyangolensin are the major isoflavones detected in the blood and urine of hu-mans and animals (Setchell, 1998). In rodents, equol is the major circulating metabolite among isoflavones perhaps representing 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.
6.4 Soy consumption and phytoestrogen levels
In soybean, isoflavones are tightly associated with proteins. As mentioned, the abundance of isoflavones varies according 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 variability 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 prepa-rations (Bhathena et al., 2002).
Numerous studies have included the investigation of the plasma concentration of phytoestro-gens and their metabolites in humans and animals consuming a diet with or without soy
(Adler-6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 23 creutz et al., 1993a; Morton et al., 1994; Coward et al., 1996). In humans consuming soy-free diets, plasma concentration of isoflavones are usually in the nanomolar range [≤40 nM see (Morton et al., 1994; Couse et al., 2000; 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 et al., 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, genistein and their respectiveβ-glycosides are similar. The average time taken after ingesting the aglycones to reach peak plasma concentrations is 4-7 hours, which is delayed to 8-11 hours for the correspondingβ-glycosides. This suggests that the rate-limiting step for absorption is the initial hydrolysis of the glycosidic moiety. The half life for daidzein and genistein was reported to be 9.3 and 7.1 hours respectively, indicating that isoflavones or their metabolites are rapidly excreted. Finally, factors that might influence isoflavone bioavailability include intestinal microflora, food matrix, the administered dose, intestinal transit time and the chemical composition of the dietary isoflavones.
6.5 Phytoestrogens: complex hormetic compounds
In plants, the synthesis of phytoestrogens, such as soy isoflavones, generally coincides with en-vironmental stresses such as pest infection, drought, lack of nutrients, and so on (Howitz et al., 2008). Recently it has been suggested that stress-induced plant compounds upregulate stress resistance pathways in animals. This phenomenon, called xenohormesis, proposes that chemi-cal cues 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 conditions are still favorable (Howitz et al., 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 section, isoflavones.
Similarly, we believe that the isoflavones genistein and daidzein mediate most of their biolog-ical effects through the modulation of key mammalian enzymes and receptors of stress-response pathways or estrogen-dependent pathways, rather than through their well known antioxidant (Ve-davanam et al., 1999) or tyrosine kinase inhibitory properties (Akiyama et al., 1987). The affinity of phytoestrogens for estrogen receptors results in effects on a large number of estrogen-regulated systems, including the cardiovascular, metabolic, reproductive, skeletal and central nervous sys-tems. A significant characteristic of isoflavones is their capacity to bind both to estrogen receptors (ERs)α andβ (Kuiper et al., 1997; Kuiper et al., 1998), with preferential binding of genistein to ERβ (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 various cell lines, in the presence of physiological levels (1nM) of estradiol (E2), genistein acts as an anti-estrogen whereas at levels 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 proliferation (Chen et al., 2004). Thus, isoflavones, as stress-induced phytochemicals, may provide health benefits through their selective estrogen receptor modulator (SERM) activities.
6.6 Role of estrogens in metabolism
Studies in humans and rodents have shown that ERs are important mediators of the action of estrogen on lipid and glucose metabolism. Estrogens have been reported to affect adiposity ei-ther directly, by modulating lipogenesis, lipolysis or adipogenesis, or indirectly, by modulating appetite or energy expenditure (for review see Cooke et al., 2004). As mentioned above, the
6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 24
Table 1: Relative binding affinity (RBA) and relative transactivation activity (RTA) of various estrogenic com-pounds, isoflavones and endocrine disruptors in comparison with 17β-estradiol. The RBA of each competitor was calculated as the E2:competitor concentration ratio required to reduce the specific radioligand binding by 50% (=ra-tio 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 1µM of the relevant compound, relative to the luciferase reporter gene induction value using 17β-estradiol at 1µM. The transactivation activity of 17β-estradiol was arbitrarily set at 100. From Cederroth and Nef, 2009.
concept of estrogens modulating metabolic features derived originally from the observation that postmenopausal women develop visceral obesity and insulin resistance as a result of low levels of estrogens. Interestingly, hormonal replacement therapy normalizes these symptoms (Ahmed-Sorour and Bailey, 1980; Bailey and Ahmed-(Ahmed-Sorour, 1980). Estrogens also play an important role in glucose homeostasis, and are known to modulate insulin sensitivity (Godsland, 2005).
Variations 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 thearomatase gene, 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 importance of ERαin modulating both lipid and glucose metabolism. Mice lacking either ERαor thearomatasegene have increased adiposity 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 knock-out 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 ERβ is less clear - to date no overt metabolic phenotype has been described in mice lacking this receptor (βERKO), under normal dietary conditions (Ohlsson et al., 2000). Recently, it was found that, when challenged with a high fat diet,βERKO mice increase their weight and adiposiy to a greater extend than do wild-type mice (Foryst-Ludwig et al., 2008).
6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 25 Estrogens have also been shown to modulate glucose metabolism in rodents. For instance in models of T2D, ovariectomy induces hyperglycemia, whereas in males, estrogen perfusion reverses diabetes (Louet et al., 2004). Similarly, mice lacking either functional ERα or textitaromatase display glucose intolerance and insulin resistance (Heine et al., 2000; Jones et al., 2000; Takeda et al., 2003). Indeed, ERα andβ are 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 et al., 1972). Estrogens and endocrine disruptors such as Bisphenol-A (BPA) have been reported to modulate insulin content inβ-cells via ERα, but not via ERβ (Alonso-Magdalena et al., 2008).
E2 also protect β-cells from oxidative stress, apoptosis, and streptozotocin-induced injuries in both genders, through ERα (Le May et al., 2006). Whether estrogens directly modulate insulin secretion is still a matter of debate.
Finally, estrogens have been reported to modulate energy homeostasis 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 hypotha-lamic neurons in the arcuate nucleus, ultimately 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 ERα, or ovariectomy, decreases physical activity and energy expenditure (Heine et al., 2000; Cooke et al., 2001; Meli et al., 2004), whereas treat-ment with E2 increases locomotor activity through an ERα-dependent mechanism (Ogawa et al., 2003). Both estrogen receptors are found in the hypothalamic nuclei and modulate food intake (Liang et al., 2002) and locomotor activity (Ogawa et al., 2003). Specific silencing of ERα in the hypothalamus of female rodents leads to similar phenotypes to those observed in constitutive mu-tant 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 ERαis 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 indi-rectly (via the CNS) modulating energy homeostasis. Whether or not dietary soy and phytoestro-gens have effects on energy homeostasis through estrogen-mimics is still a matter of controversial debate, but is clearly a plausible hypothesis.
6.7 Effects of soy protein and phytoestrogens on human metabolism
The low frequency of obesity and related metabolic disorders in Asian populations has drawn at-tention towards soy, which is a characteristic component of asiatic diets. 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; Fujimoto et al., 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 consumption by postmenopausal women correlated with lower body mass index (BMI), and higher HDL levels (Guthrie et al., 2000; Goodman-Gruen and Kritz-Silverstein, 2001; Goodman-Gruen and Kritz-Silverstein, 2003).
6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 26 Clinical studies also suggest that soy protein or isoflavones may improve metabolic parameters.
For instance, a meta-analysis 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, postmenopausal 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 estrogens on blood glucose, insulin, and lipid profiles in postmenopausal Taiwanese women. The study revealed that during fasting both glucose 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 classical 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
Li et al., 2005; Hall et al., 2006; Ikeda et al., 2006; Anderson et al., 2007). These discrepancies