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Nutrition Research 30 (2010) 1–13 www.nrjournal.com

Antidiabetic effects of fermented products on type 2 diabetes ⁎ Dae Young Kwona, James W. Daily IIIb, Hyun Jin Kima, Sunmin Parkc, aEmerging Innovative Technology Research Division, Korean Food Research Institutes, Sungnam bDaily Manufacturing Inc., Rockwell, NC, USA cDepartment of Food and Nutrition, Obesity/Diabetes Research Institutes, Hoseo University, Asan Received 30 September 2009; revised 12 November 2009; accepted 15 November 2009

Abstract

Historically, the incidence of type 2 diabetes has been lower in Asian populations compared with those in Western countries. One possible reason for the lower incidence among Asians is that they consume fermented soybean products, which are unique to the traditional Asian diet. Some have hypothesized that dietary and soy peptides in fermented soybean foods consumed in traditional Asian diets may help prevent and slow the progression of type 2 diabetes. This review evaluates the existing evidence from animal studies and clinical and epidemiologic investigations on fermented in the prevention and treatment of type 2 diabetes. Nutritional studies performed in animals and intervention studies with humans suggest that the ingestion of with isoflavones improves glucose control and reduces insulin resistance. Korean fermented soybean products such as , kochujang, and chungkookjang contain alterations in the structures and content of isoflavonoids and small bioactive peptides, which are produced during fermentation. Several studies revealed improvements in insulin resistance and insulin secretion with the consumption of these fermented products. Therefore, fermented soybean products may help prevent or attenuate the progression of type 2 diabetes. Although the lack of human intervention trials does not permit definitive conclusions, the evidence does suggest that fermented soy products may be better for preventing or delaying the progression of type 2 diabetes compared with nonfermented soybeans. © 2010 Elsevier Inc. All rights reserved. Keywords: β-Cell function; β-Cell mass; Fermented soybean products; Insulin resistance; Isoflavonoids; Peptides; Type 2 diabetes Abbreviations: IGF-1, insulin-like growth factor 1; IRS-2, insulin receptor substrate 2; OVX, ovariectomy; PPAR, peroxisome- proliferator–activated receptors; SERMs, selective estrogen receptor modulators.

1. Introduction disorder characterized by both insulin deficiency and peripheral insulin resistance [1]. The pancreatic β cell adapts Because type 2 diabetes in Western populations is to increased nutrient availability and insulin resistance by typically accompanied by obesity and hyperinsulinemia, increasing its function and mass [2]. These processes are insulin secretion has received less emphasis than do insulin orchestrated by signals derived from nutrient metabolism, resistance. However, recent studies have revealed the hormones, and cytokines. The failure of β-cell adaptation importance of insulin secretion capacity to prevent type 2 results in type 2 diabetes [3]. Thus, β-cell dysfunction and diabetes. Type 2 diabetes is a heterogeneous metabolic deficient mass pancreatic mass play crucial roles in the development of insulin resistance and subsequent β-cell ⁎ compensation failure and diabetes [4]. Corresponding author. Department of Food and Nutrition, Hoseo β University, Chungnam-Do 336-795, . Tel.: +82 41 540 5633; fax: +82 The primary defect in type 2 diabetes is increased -cell 41 548 0670. apoptosis, which decreases β-cell mass [5]. Therefore, the E-mail address: [email protected] (S. Park). progressive nature of type 2 diabetes is due to β-cell failure.

0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2009.11.004 2 D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 Attenuating insulin resistance, improving β-cell function, and that fermentation changes those bioactive compounds in and increasing β-cell mass are important for preventing and ways that potentiate the antidiabetic properties. delaying the progression of type 2 diabetes. Herein we discuss how fermented soybean products, Soybeans (Glycine max MERILL) have long been isoflavones, and soy peptides improve glucose metabolism, important protein sources, complementing grain proteins, insulin resistance, insulin secretion, and β-cell mass. This in Asian countries. Asians typically consume 9 to 30 g report reviews epidemiologic and experimental evidence soybeans per day, with individual and regional variations describing the effectiveness and possible mechanisms that (Table 1) [6]. In addition to protein, soybeans also contain support and challenge the hypothesis. Some human studies various nutrients and functional components including have been conducted to investigate the antidiabetic effects isoflavonoids [7]. Because isoflavonoids have structural of soybeans and soy isoflavonoids, but a few studies have similarities to endogenous estrogen, they exert estrogenic or investigated fermented soybeans. Thus, this review includes antiestrogenic action with weak binding affinity to the a discussion of soybeans and their compositional changes estrogen receptors, where they act as agonists or competitive that occur during fermentation to support the antidiabetic antagonists [8]. Estrogen is reportedly beneficial for effects and mechanism of action of these fermented preventing and treating type 2 diabetes by attenuating soybean products. insulin resistance, improving insulin secretion, and increas- ing β-cell mass [9]. Therefore, soy isoflavonoids may improve glucose homeostasis through estrogenic action. In 2. Fermented soybean products addition, soy proteins may also improve glucose metabo- Various types of fermented soybean foods are consumed lism. Hence, soybeans may help prevent type 2 diabetes and in Asian countries such as Korea, , , , delay its progression. and . The most common Korean fermented soybean Fermentation is one of the major processes used in the foods are chungkookjang, doenjang, kochujang, and soy production of food from soybeans. is . Natto and are Japanese versions of chungkook- indigenous to the cuisines of East and . jang and doenjang, respectively. China also has various Korean soy foods are increasingly present on the worldwide fermented soybean products such as , , market, and because kochujang and fermented soybean sweet noodle sauce, tauchu, yellow soybean paste, and pastes (deonjang and chungkookjang) were registered in dajiang. Doenjang, kochujang, and are fermented CODEX on July 4, 2009, they are now internationally with varying microorganisms when traditionally made, accepted foods. Most fermented soybean pastes are salty and because fermentation conditions and ambient microbiota savory and some are spicy. They are often used as are different among regional environments (Fig. 1). To make to flavor foods such as stir-fries, stews, and these products by the traditional method, cooked soybeans soups. Differences in their colors and flavors are due to are formed into blocks and fermented outdoors for 20 to 60 different production methods such as the conditions of days by microorganisms naturally present in the environ- fermentation; the addition of flour, pulverized , ment. The fermented blocks are known as meju and are used , or ; and the presence of different microflora such to prepare the soy pastes and sauce. Meju can be fermented as bacteria or molds used in their production, as well as for less than 5 days by inoculating with microorganisms such whether the soybeans are roasted (as in chunjang) or aged (as as Aspergillus sp; however, when fermented by traditional in tauchu) before being ground. The fermentation of these methods meju is typically fermented primarily by Bacilli soybean products changes the bioactive components, such as species during the early stages of fermentation, followed by isoflavonoids and peptides, in ways which may alter their Aspergillus species, which predominate during the remaining efficacy in the treatment of type 2 diabetes. The intake of fermentation period. is the major soybeans and fermented soybeans may be associated with microorganism in the final product of meju when it is made the antecedently lower incidence of type 2 diabetes in Asians in the traditional way. Chungkookjang is a short-term [10]. This review is based on the hypothesis that bioactive fermented soybean product similar to Japanese natto, compounds found in soybean have antidiabetic properties whereas doenjang, kochujang, and soy sauce undergo long- term fermentation as do Chinese tauchu and Japanese miso. Table 1 Consumption of soybeans and prevalence of in various countries 3. Changes in soybean components by fermentation Countries Soybean Breast cancer Prostates cancer consumption (g/d) prevalence prevalence 3.1. Nutritional and functional compounds in soybeans Japan 29.5 60 35 Korea 19.9 26 5 Soybean products have been designated as one of world's Hong Kong 10.3 84 29 healthiest foods due to being an excellent source of high- China 9.3 47 28 quality protein as well as providing various health benefits. United States minor 224 157 The protein content of soybean is 32% to 42% (depending on Prevalence is defined as the number of diagnosed per 100 000. the variety and growth conditions) of which approximately D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 3

Fig. 1. The preparation of fermented Korean foods made from soybeans.

80% is composed of 2 storage globulins, 7S globulin (β- method for improving nutritional and functional properties conglycinin) and 11S globulin (glycinin), having various of soybeans due to the increased content of small bioactive functional and physicochemical properties [11-13]. Soybean compounds. The large protein, lipid, and carbohydrate products are considered a good substitute for animal protein, molecules in raw soybean are broken down by enzymatic and their nutritional value is almost equivalent to that of hydrolysis during fermentation to small molecules such as animal protein because soy proteins contain most of the peptides, amino acids, fatty acids, and , which are essential amino acids for human nutrition. Soybean products responsible for the unique sensory and functional proper- have less saturated fat and various phytochemicals, which ties of the final products. Short-term fermented soy foods possess numerous health benefits [14]. In particular, the such as chungkookjang and , which are fermented association of high-quality protein and phytochemicals, with Bacillus subtilis and Rhizopus oligosporus, respec- especially isoflavones, is unique among plant-based proteins tively, for less than 72 hours have a much greater because isoflavones are not widely distributed in plants other concentration of large molecules than do long-term than legumes [15]. fermented foods including doenjang and miso, which are Soybeans contain 0.1 to 5 mg total isoflavones per gram, fermented for more than 6 months with Aspergilus and primarily , , and [15]. These Bacillus species from rice straw and koji, respectively nonsteroidal compounds, commonly known as soy phytoes- (Table 3) [11,17-23]. trogens, are naturally present as the β-glucosides , The degradation of lipids and carbohydrates proceeds , and , representing 50% to 55%, 40% to 45%, especially rapidly during the initial stage of fermentation. and 5% to 10% of the total isoflavone content, respectively Dry matter declines continuously during fermentation time, [16]. Total isoflavonoid contents are varied among different 70% to 80% of which is due to decreases in crude lipid and soy products (Table 2) [14-16]. In addition to high-quality carbohydrate, which are used as the major energy sources for protein and isoflavones, soybeans contain high levels of the microorganisms [24]; the remaining loss is largely due to unsaturated fatty acids, dietary fiber, and minerals. fermentative hydrolysis of approximately 25% of the soy protein, thereby producing ammonia. Of this hydrolyzed protein, 65% remains in the fermented products as amino 3.2. Changes in functional components during fermentation acids and peptides, 25% is assimilated into the mold The qualitative and quantitative composition of soybean biomass, and 10% is oxidized [24]. After the initial stage components is dramatically changed by physical and of fermentation, however, soy proteins are rapidly degraded enzymatic processes during the preparation of soy-based and only 9% to 17% of the crude protein remains at the end foods [11,17-23]. Fermentation is an excellent processing stage of long-term fermentation. 4 D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 Table 2 and fermentation [26,27]. During preparation of fermented Isoflavone contents in various soy products soy foods, 6-ο-malonylglucosides, the most prevalent Foods Contents Foods Contents soybean isoflavones, are converted to 6-ο-acetylglucosides (mg%)/100 g (mg%)/100 g or β-glucosides by heating, and β-glucosides are unconju- Miso 42.6 ± 9.2 Japanese soybeans 118.5 ± 22.2 gated by the action of β-glucosidases secreted by fermenta- Natto 58.9 ± 7.4 Korean soybeans 145.0 ± 10.7 tion microorganisms [16]. Most isoflavones are not Nonfat soy protein 177. 9 ± 12.6 American soybeans 128.4 ± 11.7 enzymatically hydrolyzed during short-term fermentation, Soymilk in USA 9.6 ± 1.8 Bean sprout 40.7 ± 8.3 ο Soymilk in Asia 25.2 ± 1.2 Tempe 43.5 ± 8.3 in contrast to long-term fermentation in which 6- - Doenjang 31.5 ± 9.3 23.6 ± 6.3 malonylglucoside content declines with increasing fermen- Isolated soy protein 97.4 ± 11.1 Ganjang 1.0 tation time with concomitant increases in unconjugated aglycones (genistein and daizein). In chungkookjang, the aglycones are 21 times higher than in soybean [17]. Among the various small metabolites derived from Moreover, the chemical profiles of various minor compo- macromolecules, the changes in amino acid and peptide nents related to health benefits and nutritional quality of concentrations were especially prominent, although qualita- products are also affected by fermentation [28,29]. Unlike tive and quantitative changes in individual peptides were not the production of small molecules by degradation of large studied. Amino acids increased or remained almost constant ones during fermentation, the content of some compounds is with increased fermentation time, but glutamate, the richest changed by artificial addition during the process. amino acid in soybean, was obviously decreased by Adding in particular increases the sodium content in fermentation, suggesting that microorganisms might use it soy paste of doenjang and miso, 9-9.7 g/100 g and 3.7 g/ as a preferred nitrogen source [25]. 100 g, respectively, whereas potassium content in doenjang, Isoflavones, which are mostly present as 6-ο-malonylglu- tempeh, and miso is decreased by fermentation. However, coside and β-glucoside conjugates and associated with chungkookjang and tempeh do not have higher sodium proteins in soybean, are also broken down by heat treatment contents because salt is not added [30-32]. Although

Table 3 Components of soybean and various soy fermented foods Soybean Doenjang Miso Chungkookjang Tempeh Reference [11,15,19,30] [15,16,19,21,32] [18,19,33] [15,20,33] [17,19,30] Protein (%) 20-42 12 10-17 41 23-55 Lipid (%) 18-22 4-11 3-11 26 14-23 Carbohydrate (%) 35 11 15-38 24 10-30 Isoflavone (mg/100 g) Genistin 36-86 1-2 3-27 87-91 6-19 Daidzin 15-57 0.3-0.6 3-18 79-93 2-3 Glycitin 2-6 0.1 1-2 10-12 0.1-0.4 Malonyl genistin 123-186 0.5-0.8 0.1-3 20-22 39-42 Genstein 0.2-5 0.1 11-47 3-4 7-10 Daidzein 0.3-5 0.1-3.7 9-36 4-7 7-8 Glycitein 0.1-0.6 0.4-0.7 1 11-13 0.5-0.7 Amino acid (g/100 g) Aspartate 1.4-1.7 1.2 1.8-4.6 3.4-3.8 1.5 Glutamate 3.8-4.8 3 4.9-9.9 4.4-5.5 2.3 Serine 1.1-1.4 0.8 2.0-5.7 1.5-1.8 0.8 Glycine 0.7-0.9 0.6 1.4-4.7 1.3-1.5 0.6 Arginine 1.5-1.9 1.2 0.5-3.7 1.9-2.1 0.9 Alanine 0.8-1.0 0.8 – 1.5-1.6 0.6 Proline 1.1-1.3 1.2 2.4-6.4 1.8-2.0 0.6 Histidine 0.7-0.9 0.4 3.0-9.0 0.9 0.4 Valine 0.8-1.0 0.7 2.0-5.0 2.0-3.0 0.6 Methionine 0.2-0.3 0.2 1.8-9.0 0.3-0.4 0.2 Cystein 0.5-0.6 1 0.01-0.03 0.3-0.4 0.2 Isoleucine 1.0-1.3 0.9 3.6-7.3 1.5-1.6 0.7 Leucine 1.6-2.0 0.4 3.2-5.9 2.5-2.7 1.1 Phenylalanine 1.1-1.4 0.7 1.7-3.3 1.7-1.8 0.5 Tyrosine 0.8-1.0 0.9 1.5-2.7 0.9-1.0 0.5 Lysine 1.4-1.7 0.8 2.4-4.5 2.1-2.7 0.8 Threonine 0.8-1.0 0.7 1.4-2.6 1.2-1.4 0.5 Mineral (g/100 g) Na 0.02 9.1-9.7 3.7 0.03 0.006 K 2.3 0.8-1 0.2 2.4 0.4 D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 5 doenjang and miso contain high contents of salt, they have hydrolyzed to the aglycone forms by lactase enzyme in the not increased sodium intakes in Asians because they are used small intestine and by β-glucosidases associated with gut as seasoning in place of salt. Thus, doenjang and miso may bacteria, especially Bifobacterium strains [44-46], and that not increase salt-related hypertension. intestinal absorption occurs throughout the small intestine but most isoflavone absorption occurs in the large intestine. Delayed activity of the enzymatic release of aglycone is 4. Absorption and bioavailability of soy isoflavones probably responsible for the late peak appearance of the and proteins isoflavones in the blood (4-8 hours after ingestion). Therefore, it is possible that isoflavonoids in fermented Soybeans contain numerous biologically active compo- soybeans may have enhanced absorption compared with nents. However, those most studied for antidiabetic proper- those in nonfermented soybeans. It is interesting that most of ties are the 2 major isoflavones, genistein and daidzein, and the studies never recovered at least half of the isoflavones in soy proteins. The structural changes that occur in soy either urine or feces, and it is likely that much of the ingested proteins and isoflavones may be responsible for the isoflavones are degraded into other metabolites in the gut. confusing and contradictory results of the research into the The possible bioactivities of isoflavone metabolites have not biologic activities of soy foods, which include many been well studied, with the exception of the daidzein different commonly used fermented and unfermented metabolite, [47-49]. foods, extracts, and supplements. The complexity of isoflavone metabolism makes it difficult to characterize the absorption and metabolism of 4.1. Isoflavones these compounds. Several factors contribute to the under- A study in Japanese men and women by Yang et al [33] standing the complex biologic processes influencing how the found that soy isoflavone aglycones are better absorbed than chemical structures are handled. Animal models and humans their corresponding glucosides. This study found that both may be very different in how they metabolize isoflavones. genistein and daidzein were much better absorbed, with peak For example, isoflavone aglycones are absorbed in the plasma concentrations almost 4 times higher, than were their stomach of rats [34], something that has not been described glucoside counterparts. Peak plasma concentrations also in humans. There may also be many variations among occurred 2 hours earlier for aglycones than for glucosides. It humans because it is known that children absorb isoflavones seems reasonable that aglycones in foods would be better more efficiently than do adults [50,51]. In addition to the absorbed because isoflavones must be in the aglycone form effects of age on isoflavone absorption, there may also be to be absorbed from the gut [34]. However, many other effects due to different food matrices and gut bacterial investigators have not reported results consistent with Yang populations. These might explain why some studies have et al [33]. Most of the isoflavones in foods are glycosylated shown better absorption of agyclones [33], but others have [35]; therefore, isoflavone absorption is delayed until not [52]. Populations with very different diets may absorb glycosylated isoflavones can be hydrolyzed, but they are and metabolize isoflavones differently. Currently, it seems ultimately absorbed at the same levels in rats [36]. Likewise, unlikely that fermenting soy foods increases the absorption Zubik and Meydani [37] found little difference in either total of isoflavones directly; however, it is possible that absorption or rate of absorption in humans. fermentation produces isoflavone metabolites with potential The lack of consensus on the comparative absorption of biologic activities and that the probiotic effects of fermented isoflavone glucosides and aglycones continues, but a recent foods may result in a gut bacterial population that assists in study suggested better absorption of daidzein when con- isoflavone hydrolysis and absorption. sumed as a glucoside [38]. In that study, the fractional absorption of the daidzein aglycone was 11.6 versus 38.9 for 4.2. Soy peptides produced by fermentation the glucoside. However, simply comparing glucoside with aglycone forms of isoflavones may not be adequate because Bioactive oligpeptides from fermented soy is an many other factors such the food matrix, intestinal bacteria, emerging area of research with great promise. It is well and others may be important determinants of absorption and known that the savory flavor of fermented and nonfer- bioavailability. Studies of the effects of food matrices have mented soy foods, such as soy sauce and hydrolyzed also been contradictory [39-43]; however, because there are vegetable protein, is a result of the release of small many different food matrices in typical diets, it is an even peptides and amino acids from the fermentative digestion more complex evaluation than glucoside versus aglycone or acidic hydrolysis of soy proteins [53,54]. Soy protein is comparisons. Despite the lack of consensus in these reported to have numerous beneficial effects in humans, important areas, some conclusions can be drawn about the including improvements in body composition and insulin absorption and bioavailability of isoflavones. First, it is well secretion [54], but it is not known if peptides released from established that isoflavones are absorbed as aglycones in digestion of the proteins contribute to the observed effects. various areas of the intestine. Second, it has been repeatedly , a 43-amino-acid peptide from soy, has been demonstrated that the glycosylated isoflavones can be shown to have numerous biologic properties including 6 D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 anticancer and anti-inflammatory activities, some of which changes in insulin resistance are related to body fat. Several may depend on epigenetic mechanisms by inhibiting the studies showed that ovariectomy (OVX) did not increase acetylation of histones [55-57]. serum leptin levels even with a high-fat diet due to decreased Fermented soy peptides from miso [58] and doenjang [59] leptin synthesis and also showed an impaired leptin signaling have been shown to possess angiotensin-converting enzyme pathway [72,73]. This increase in body weight and body fat inhibitory activity and tripeptides (Val-Pro-Pro and Ile-Pro- is possibly related to impairment of leptin synthesis and Pro) from casein-added miso paste are reported to act as signaling pathway. However, leptin cannot explain the antihypertensive agents in spontaneously hypertensive rats weight gains completely because Burguera et al [73] [58]. Peptides from soy are currently the subject of revealed that leptin treatment was only partially effective investigation for new drugs and functional food ingredients in modulating appetite and limiting weight gain in OVX rats. for gut health and modulating the intestinal absorption of Increased body fat is a crucial trigger in elevating nutrients [60]. Research into bioactive soy peptides is still in insulin resistance in estrogen insufficient states [74]. its infancy, but holds great promise. Weight loss itself reverses insulin resistance and insulin hypersecretion by enhancing first-phase insulin secretion [75,76]. However, it is not clear whether weight loss itself 5. Type 2 diabetes and estrogen or restricted diets modulate insulin secretion capacity and 5.1. The phathophysiology of type 2 diabetes pancreatic β-cell mass. Choi et al [9] showed that weight loss as a result of restricted diets increased first-phase Insulin resistance is a characteristic feature of type 2 insulin secretion in OVX rats, but decreased second-phase diabetes mellitus. Insulin resistance is typically accompanied insulin secretion at 120 minutes during a hyperglycemia by hyperinsulinemia and islet hyperplasia and hypertrophy clamp was not overcome. Estrogen replacement reversed to maintain normoglycemia [61-63]. When insulin secretion the decrease in second-phase as well as first-phase insulin cannot compensate for insulin resistance, type 2 diabetes secretion [65,75]. Estrogen is a key mediator for glucose- develops. Insulin resistance affects β-cell function with stimulated insulin secretion and the expansion and biphasic states, initially enhancing insulin output by maintenance of β-cell mass in rats and mice [71,76]. The stimulating islet hyperplasia, but subsequently reversing mechanism is not fully understood, but synergistically with these compensatory changes when extreme insulin resistance glucose, estrogen closes KATP channels through a cyclic has been sustained for a long period [62,63]. The mechanism guanosine monophosphate–dependent phosphorylation by which β-cells become incapable of satisfying insulin process. As a consequence, a calcium signal stimulates demand has never been revealed in humans. One possibility insulin secretion [77]. Glucose-stimulated insulin secretion is pancreatic β-cell exhaustion with decreased β-cell mass. A can be delayed and suppressed in estrogen-insufficient postmortem study indicated that the decompensation for states. Estrogen also increased β-cell mass by potentiating insulin resistance is associated with decreased β-cell mass in insulin/insulin-like growth factor 1 (IGF-1) signaling [9]. patients with diabetes [64]. Because obese type 2 diabetic Insulin receptor substrate 2 (IRS2) is a crucial mediator for patients have greater insulin secretion capacity with β-cell β-cell growth and survival through its regulation of insulin/ hypertrophy, it takes time to develop diabetes from glucose IGF-1 signaling. intolerance. However, nonobese type 2 diabetes is a rapidly Insulin receptor substrate 2 knockout mice revealed that developing insulin deficiency and β-cell insufficiency in insulin secretion cannot compensate for insulin resistance which insulin resistance is induced during condition such as when there is decreased β-cellcellmass,andasa aging, , and obesity. Therefore, diabetes needs consequence, they develop severe diabetes [62,78].Itis to be treated by relieving insulin resistance and potentiating important to increase islet mass during a high-insulin- β-cell function and mass. resistant state to prevent progression to diabetes. Induction of IRS2 expression via the activation of cAMP–response 5.2. Estrogen effects on type 2 diabetes element-binding protein in Min6 cells and islets [78,79] Although aging plays an important role in insulin accounted for the fact that the upstream region of IRS2 gene resistance and β-cell dysfunction in both men and women coding contains a cAMP response element. When elevated [65,66], the prevalence and progression are lower in levels of IRS2 potentiated an insulin/IGF-1 signaling premenopausal women in comparison to men despite their cascade, it increased duodenal homeobox factor 1 expression higher body fat, but their incidence markedly increases with in β cells. The increased duodenal homeobox factor 1 higher fat accumulation after menopause. This phenomenon expression in islets was consistent with the proliferation of β suggests that estrogen prevents insulin resistance and β-cell cells, resulting in increased mass [9,64]. A possible dysfunction. There is substantial evidence that postmeno- mechanism for enhancement of β-cell function and mass pausal estrogen replacement can improve insulin sensitivity by estrogen and phytoestrogens in islets is shown in Fig. 2. [67,68], although the data are not consistent [69,70]. Based on these findings, estrogen is a key mediator for However, rather recent studies have shown that estrogen maintaining glucose homeostasis in females through im- replacement modulates β-cell function and mass [9,71]. The proving insulin sensitivity and β-cell function and mass. D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 7

Fig. 2. A possible mechanism for the enhancement of β-cell function and the survival of these cells. The dashed lines represent the possible mechanism for enhanced glucose-stimulated insulin secretion and β-cell mass by enhancing proliferation and decreasing apoptosis.

Therefore, phytoestrogens such as isoflavonoids may is an effective agent for preventing diet- and OVX-induced improve glucose homeostasis through improving estrogen obesity [87]. signaling pathways. Animal and human studies have been conducted to investigate antidiabetic effects of soybeans and their action (Table 4) [48,88-94]. The effects of soybeans, including isoflavonoids and soy proteins, on glucose metabolism are 6. Effects of soybeans in glucose metabolism inconsistent, and the mechanisms have not been exten- Soybean isoflavonoid and protein consumption alleviate sively studied. The consumption of isolated isoflavonoids some of the symptoms associated with type 2 diabetes, such (114 mg/d) for 3 months did not affect insulin sensitivity as insulin resistance and glycemic control by some yet- as assessed by an oral 2-hour glucose tolerance test (75 g) in unrecognized mechanism [7,80,81]. However, not all studies a crossover study of postmenopausal women, even though have reported the same results [82,83]. Isoflavonoids may serum ghrelin levels were decreased by the isoflavonoid improve antidiabetic actions through estrogen receptors. The treatment, indicating some changes in appetite [95].In estrogen receptor α is emerging as a key molecule involved addition, insulin secretion, visceral fat, total body fat, and in glucose and lipid metabolism. Isoflavonoids are phytoes- lean body mass were not different among postmenopausal trogens that naturally occur with soy proteins and are women who consumed soy protein for 3 months compared structurally and functionally similar to estradiol [84]. with those that consumed casein protein [82]. However, Estrogen receptors α and β are both present in β cells. The some studies showed positive effects. For example, 46 role of estrogen receptor β is still unknown, but estrogen postmenopausal women taking isolated isoflavone extracts receptor α plays an important role in the regulation of insulin had significantly increased high-density lipoprotein choles- biosynthesis, insulin secretion, and β-cell survival. Isofla- terol and a decrease in apolipoprotein B, the primary vones such as genistein and daidzein bind weakly to estrogen apolipoprotein in low-density lipoprotein particles [96,97]. receptor α and more strongly to estrogen receptor β, and they Unlike human studies, recent experiments have shown that may possess organ-specific estrogenic and antiestrogenic isoflavonoids in soybeans enhance insulin secretion and effects [85,86]. Because some agents are antiestrogenic in insulin sensitivity in experimental animals such as KKAy breast and endometrial cells but have estrogenic actions in diabetic rats, streptozotocin-induced diabetic rats, and CD1 other tissues such as adipose tissues and muscles, these mice [89,93,94,98], and that soy protein attenuates insulin agents are classified as selective estrogen receptor modula- resistance in male Sprague-Dawley rats [99]. However, the tors (SERMs). These SERMs have recently been studied for effect of isoflavonoids and soy protein remains unclear, their metabolic and therapeutic properties and are known to although several studies have revealed mechanisms by improve energy and lipid metabolism in an estrogen-like which soy isoflavones may impact glucose metabolism. fashion in rodent models. The SERM, Acolbifene (EM-652), The consumption of soy protein isolates activates a pure antiestrogen in human breast and uterine cancer cells, peroxisome-proliferator–activated receptors (PPARs) and 8 D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13

Table 4 Comparison of antidiabetic outcomes after consumption of nonfermented soybeans Model Bioactive components and dose used Duration End point Reference Adult males and 40 g soy protein isolates containing 57 d Lower serum LDL, LDL/HDL, [88] postmenopausal females 88 mg isoflavonoid aglycones and apolipoprotein B/apolipoprotein A-I 100 mg/kg STZ diabetic rats 20% high-isoflavone soy protein (HIS), 8 wk Lower serum glucose levels and increased [93] (type 1 diabetes) 20% low-isoflavone soy protein (LIS) insulin secretion by HIS KKAy diabetic mice 50% high-content isoflavone soy protein 9 wk Reduce fasting plasma glucose and insulin; [89] increase insulin sensitivity CD1 male and female mice High soy-containing diet 4 mo Reduced serum insulin levels and pancreatic [94] and offsprings insulin content; improved insulin sensitivity Type 2 diabetic patients 0.28 g soy protein/kg body weight 4 y Reduced serum total , LDL, triglyceride, [90] with nephropathy and C-reactive protein Postmenopausal women Soy protein plus 132 mg isoflavones 4 wk Reduced total and LDL cholesterol and triglycerides; [83] no effect of isoflavones alone Obese Zucker rat 20% soy protein 120 d Improved renal function and proteinuria; reduced [91] tubular dilation, glomerulosclerosis, intersticial fibrosis, and extracapilar proliferation Male patients with type 2 Isolated soy protein 8 wk Decreased urinary albumin excretion and increased HDL [92] diabetes and nephropathy LDL indicates low-density lipoprotein; HDL, low-density lipoprotein; STZ, streptozotocin. liver X receptor signaling and inhibits sterol regulatory fed cholesterol. Thus, fermented soybean products may be element-binding protein-1c signaling, contributing to insulin more effective for controlling glucose metabolism due to sensitization and improved lipid homeostasis in rats after increased isoflavonoid aglycones. consumption of diets high in fat and cholesterol in Kwon et al [104,105] showed that chungkookjang experimental animals [7,79,99]. In addition, soy-fed CD-1 improved glucose homeostasis by enhancing hepatic mice exhibited enhanced insulin sensitivity, especially in insulin sensitivity and insulinotropic actions in 90% white adipose tissue, due to potentiation of phosphorylation pancreatectomized rats, a type 2 diabetic animal model. of AMP-activated protein kinase and acetyl-CoA carboxyl- In addition, 0.3 g water extract of touchi, fermented ase and upregulation of the expression of genes implicated in soybean, decreased fasting and postprandial blood glucose peroxisomal fatty acid oxidation and mitochondrial biogen- levels and HbA1C in KKAy diabetic animals and mild esis and in skeletal muscles by increasing glucose uptake type 2 diabetic patients [106,107]. In comparison to [94]. However, these rodents had reduced serum insulin cooked soybeans, chungkookjang decreased hepatic glu- levels and pancreatic insulin content. In contrast, Veloso et al cose output in a hyperinsulinemic state by potentiating [100] demonstrated that a soybean diet enhanced the insulin signaling via induction of IRS2 in diabetic rats, secretory pattern of β cells, at least in part, by activating indicating the attenuation of hepatic insulin resistance. In the cAMP/protein kinase A signaling cascade. Thus, the addition, chungkookjang enhanced glucose-stimulated effect and mechanism of soybean-based diets on insulin insulin secretion in a hyperglycemic clamp study in secretion remains controversial. diabetic rats and increased pancreatic β-cell mass via increased proliferation and decreased apoptosis. This may be associated with activating estrogen receptors in β cells 7. Effects of fermented soybean products on (Fig. 2). Kim et al [108] also showed similar results in glucose metabolism C57BL/KsJ-db/db mice. Chungkookjang supplementation induced a significant decrease in blood glucose and 7.1. Effects of short-term fermented soybean paste on glycosylated hemoglobin levels and improved insulin glucose metabolism tolerance compared with the diabetic control group via Several animal studies and a few human studies have increasing serum insulin and pancreatic insulin contents. evaluated the effects of fermented soybeans on glucose Therefore, chungkookjang delayed diabetic symptoms in metabolism (Table 5) [101-108]. After fermentation, iso- type 2 diabetic rats, and this was related to increased flavonoid glycones are changed into isoflavonoid aglycones, isoflavonoid aglycones such as daidzein and genistein and which seem to have greater activity than do isoflavonoid small peptides. glycones. Kawakami et al [103] demonstrated that the level Although no studies have evaluated the effects of specific of serum total isoflavones in the isoflavone aglycone–rich smaller peptides made from soybean fermentation on diet was significantly higher than that of the isoflavone glucose regulation, these peptides may have significant glycoside–rich diet in male Sprague-Dawley rats. Moreover, effects on glucose metabolism because the changes in an isoflavone aglycone–rich diet reduced liver and serum isoflavonoids cannot explain the improvement of glucose total cholesterol levels, and liver triglyceride levels in rats homeostasis. Only daidzein-rich fractions of chungkookjang D.Y. Kwon et al. / Nutrition Research 30 (2010) 1–13 9

Table 5 Comparison of antidiabetic outcomes after consumption of fermented soybean products Model Dose used Duration End point Reference Healthy subjects Combination of 50 g natto, 1 d Decreased peak glucose and insulin concentrations [101] 60 g Japanese yams, 40 g okras and the incremental areas under the curve for glucose and insulin more than 0-120 min during OGTT 90% pancreatectomized rats 5% kochujang powder 8 wk Improved glucose tolerance by enhancing hepatic [102] insulin sensitivity C57BL/KsJ-db/db mice 5% chungkookjang 8 wk Reduced blood glucose and glycosylated hemoglobin; [106] improved insulin tolerance; increased serum and pancreatic insulin levels 90% pancreatectomized rats 40% chungkookjang 8 wk Improved insulin sensitivity and insulin signaling in [104,105] the liver; enhanced glucose-stimulated insulin secretion insulin/IGF-1 signaling in islets Male KKAy mice 0.4% water-soluble touchi-extract 60 d Reduced fasting and postprandial blood glucose levels; [107] decreased index of liver function such as GOT and GPT Mild type 2 diabetic humans 0.3 g water-soluble touchi-extract 3 mo Decreased fasting blood glucose and HbA1C [108] in every meal OGTT indicates oral glucose tolerance test; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase. elevated PPAR-γ activation by approximately 50% of the reduced by approximately 50% during the fermentation rosiglitazone activity, a member of the nuclear hormone process, kochujang has been shown to reduce body weight, receptor superfamily and a central regulator of insulin and visceral fat, and serum leptin levels without modulating glucose metabolism [109]. In addition, the smaller peptides energy intake in diabetic rats [102]. It also improves glucose (less than 3 kD) in chungkookjang increased PPAR-γ tolerance by enhancing insulin sensitivity. The improvement activity by approximately 59% of rosiglitazone, which in hepatic insulin sensitivity lowered hepatic glucose output indicates that it works as a mild PPAR-γ agonist from our and triglyceride accumulation and increased glycogen preliminary study. However, there was no activation of storage. The possible mechanism is the potentiated phos- PPAR-γ by larger peptides (more than 3 kD). In addition, phorylation of signal transducer and activator of transcrip- certain types of peptides isolated from the breakdown of tion-3 → AMP-activated protein kinase → acetyl CoA soybeans or black beans by microbes have shown antihy- carboxylase and the reduced phosphoenol pyruvate carboxy pertensive and anti-inflammatory properties, but no specific kinase expression [113]. However, the putative effects of peptide has revealed antidiabetic actions [57,58]. Thus, capsaicin on insulin secretion capacity remain controversial further studies are needed to determine the antidiabetic [111,113]. Few studies have evaluated the effects of effects of specific peptides. kochujang on glucose-stimulated insulin secretion, although Kwon et al [102] showed that kochujang supplementation does not modify glucose-stimulated insulin secretion in 7.2. Effects of long-term fermented soybean paste on diabetic rats. Thus, kochujang may have greater efficacy for glucose metabolism preventing and delaying diabetic progression than capsaicin, Unlike chungkookjang, long-term fermented soybeans but further study is needed. with added salt, such as doenjang, have not been demonstrated to affect glucose homeostasis. However, doenjang has been shown to have greater antimutagenic 8. Summary and conclusion and anticancer activities than other fermented soybean foods such as chungkookjang (Korean-style natto) and miso We presented limited but supporting evidence that soy (Japanese-style doenjang) and nonfermented soybeans. foods (soy protein and phytoestrogens) are beneficial for Anticancer and antimetastatic properties of doenjang were decreasing the risk of onset and progression of insulin enhanced as aging time progressed [110]. However, resistance and type 2 diabetes and that the effectiveness is kochujang containing meju and red pepper may affect enhanced by fermentation. However, available data from energy, lipid, and glucose metabolism. Red pepper, a major human studies on soybeans do not offer complete evidence, component of kochujang, and its active principle capsaicin and further research is required before a firm conclusion can are known to enhance energy and lipid metabolism, possibly be made about the benefits if soy and phytoestrogens in by increasing catecholamine secretion from the adrenal glucose metabolism. Furthermore, no human intervention medulla through the activation of the sympathetic nervous studies have been performed to directly investigate the effect system [111,112]. The decreased numbers of adipocytes may of fermented soybeans on diabetes. Phytoestrogens and improve glucose tolerance by the enhancement of insulin proteins in soybeans seem to have beneficial actions both on sensitivity. Although capsaicin content in red pepper was glucose metabolisms, and additional micronutrients such as 10 D.Y. 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