Effects of soy vs. casein protein on body weight and glycemic control in female monkeys and their offspringкод для вставкиСкачать
American Journal of Primatology 71:802–811 (2009) RESEARCH ARTICLE Effects of Soy vs. Casein Protein on Body Weight and Glycemic Control in Female Monkeys and their Offspring JANICE D. WAGNER1, MATTHEW J. JORGENSEN1, J. MARK CLINE1, CYNTHIA J. LEES1, ADRIAN A. FRANKE2, LI ZHANG1, MELISSA R. AYERS1, CARRIE SCHULTZ3, AND JAY R. KAPLAN1 1 Wake Forest University School of Medicine, Winston-Salem, North Carolina 2 Cancer Research Center of Hawaii, Honolulu, Hawaii 3 Purina LabDiet, Richmond, Indiana Nutritional interventions are important for reducing obesity and related conditions. Soy is a good source of protein and also contains isoflavones that may affect plasma lipids, body weight, and insulin action. Described here are data from a monkey breeding colony in which monkeys were initially fed a standard chow diet that is low fat with protein derived from soy. Monkeys were then randomized to a defined diet with a fat content similar to the typical American diet (TAD) containing either protein derived from soy (TAD soy) or casein–lactalbumin (TAD casein). The colony was followed for over two years to assess body weight, and carbohydrate and lipid measures in adult females (n 5 19) and their offspring (n 5 25). Serum isoflavone concentrations were higher with TAD soy than TAD casein, but not as high as when monkey chow was fed. Offspring consuming TAD soy had higher serum isoflavone concentrations than adults consuming TAD soy. Female monkeys consuming TAD soy had better glycemic control, as determined by fructosamine concentrations, but no differences in lipids or body weight compared with those consuming diets with TAD casein. Offspring born to dams consuming TAD soy had similar body weights at birth but over a two-year period weighed significantly less, had significantly lower triglyceride concentrations, and like adult females, had significantly lower fructosamine concentrations compared to TAD casein. Glucose tolerance tests in adult females were not significantly different with diet, but offspring eating TAD soy had increased glucose disappearance with overall lower glucose and insulin responses to the glucose challenge compared with TAD casein. Potential reasons for the additional benefits of TAD soy observed in offspring but not in adults may be related to higher serum isoflavone concentrations in offspring, presence of the diet differences throughout more of their lifespan (including gestation), or different tissue susceptibilities in younger animals. Am. J. Primatol. 71:802–811, 2009. r 2009 Wiley-Liss, Inc. Key words: cynomolgus monkeys; soy; isoflavones; glucose; insulin; lipids INTRODUCTION Obesity has become a worldwide epidemic and is a key risk factor for major chronic diseases such as type 2 diabetes (T2D) and cardiovascular disease (CVD). In women the incidence of T2D has been steadily increasing and is an even greater risk factor for CVD in women than in men [DeStefano et al., 1993; Kannel et al., 1990]. Also, of global importance, is the growing proportion of children who are obese, putting them at risk for numerous chronic and degenerative diseases as they age [Baker et al., 2007; Halm & Franke, 2004]. Pharmaceutical therapies to treat the morbidities and mortalities associated with diabetes are currently under investigation; however, nutritional interventions may also play an important role in primary prevention. Dietary soy has been proposed to be a ‘‘heart healthy’’ food supplement and the FDA approved a health claim for soy protein and soy-based food products, based largely on the r 2009 Wiley-Liss, Inc. evidence that soy consumption improves plasma lipid and lipoprotein concentrations and might reduce risk of CVD, yet does not appear to increase cancer risk [Setchell & Cassidy, 1999; Wagner et al., 2001]. Isoflavones, in particular genistein and daidzein, are abundant in soy protein and as they are structurally similar to estradiol, can bind to both Contract grant sponsor: National Center for Research Resources; Contract grant numbers: P40 RR021380; S10 RR020890. Correspondence to: Janice D. Wagner, DVM, Ph.D., Wake Forest University School of Medicine, Department of Pathology, Medical Center Boulevard, Winston-Salem, NC 27157– 1040. E-mail: email@example.com Received 18 November 2008; revised 4 May 2009; revision accepted 4 May 2009 DOI 10.1002/ajp.20716 Published online 29 May 2009 in Wiley InterScience (www. interscience.wiley.com). Soy Protein and Glycemic Measures / 803 estrogen receptor (ER) a and b, but with greater affinity for ER b [Kuiper et al., 1998; Morito et al., 2002]. Depending on their concentration, the concentration of endogenous estrogen, and ER number and type (e.g., ER a or b), they can act with estrogenlike activity or act as estrogen antagonists [Hwang et al., 2006; Setchell & Cassidy 1999]. Isoflavones, thus, have potential to be endocrine disrupters, especially if given during particular periods of development or reproductive stage [Cline & Wood, 2009; Hwang et al., 2006]. Genistein and daidzein have also been shown to bind to peroxisome proliferator-activated receptor (PPAR) gamma, similar to the pharmacologic agents, thiazolidinediones, which are used clinically to improve insulin resistance; suggesting the potential value of isoflavones as a nutritional approach to modulating insulin action [Dang et al., 2003; Mezei et al., 2003]. The earliest report of soybeans having beneficial effects in diabetes was in 1910 [Friedenwald & Ruhrah, 1910] where the consumption of soybeans decreased glycosuria in diabetics. More recently, soy protein with isoflavones has been associated with improved lipids and glycemic control in T2D postmenopausal women [Jayagopal et al., 2002]. In nondiabetic postmenopausal women, soy consumption was associated with lower fasting insulin concentrations and isoflavone intake was inversely associated with post-challenge insulin concentration [Goodman-Gruen & Kritz-Silverstein, 2001]. We have similarly observed improved lipid measures and decreased atherosclerosis extent [Adams et al., 2005; Clarkson et al., 2001] as well as increased insulin sensitivity in surgically postmenopausal female monkeys with soy [Wagner et al., 1997]. However, these effects on insulin have not been observed consistently in human [Hermansen et al., 2001] or nonhuman [Wagner et al., 2000, 2008] studies. Most rodent and nonhuman primate studies are carried out with animals fed commercial chows that derive a major portion of the protein from soy, resulting in quite high serum concentrations of isoflavones [Jensen & Ritskes-Hoitinga, 2007; Stroud et al., 2006]. Additionally, soy is used in infant formulas and the question of whether these formulas are safe in developing infants has been controversial for several years [Badger et al., 2002]. Recently, we started a breeding colony of cynomolgus monkeys that is fed two defined diets; one with the protein source from soy and the other from animal-based proteins, casein–lactalbumin. The long-term objective of this colony is to determine health effects of soy protein (compared with casein–lactalbumin) in the young, as well as how this may program disease outcomes as they age. In this initial report, we present differences in lipids, glycemic measures, and body weight in the adult females and their offspring fed these diets and show that early nutrition may play an important role in obesity and related conditions. METHODS Animal Studies The monkeys used in this study are part of a National Center for Research Resources (P40 RR021380)-supported cynomolgus monkey (Macaca fascicularis) breeding colony. The initial cohort of monkeys described here consisted of 19 adult female cynomolgus monkeys that were followed for over two years. During that time, 25 offspring (12 males and 13 females) were born with data collected on adults and offspring. Adults were initially fed (for a minimum of 1 year) a standard monkey chow (Table I), LabDiets Monkey Diet 5037 (Purina LabDiets, Richmond, IN) that is a low fat and low cholesterol, high carbohydrate diet with the protein derived from soy (baseline). Blood samples were collected after an overnight fast (for lipid and carbohydrate measures described below) and also 4 hr after eating (for serum isoflavones concentrations) before entering the breeding colony. They were randomly assigned to one of the two breeding groups. Both groups had their diets changed to approximate the typical American diet (TAD) with 35% of calories from fat, and a moderate amount of dietary cholesterol and less carbohydrate than standard monkey chow. One group continued to eat a diet with the protein derived from soy (TAD soy) while the other group ate a diet with the protein derived from casein–lactalbumin (TAD casein). Offspring remained in the natal groups with their moms during lactation and after weaning continuing to eat the TAD diet fed to their moms. Blood samples and body weights were obtained from the adults during the baseline and after consuming the TAD diets while all offspring were born to dams consuming TAD diets thus no measures during chow feeding are available. Both TAD diets satisfy National Research Council recommendations for nonhuman primates and were custom formulated by Purina LabDiets. Other than the protein source, diets were equal in macronutrients (Table I). TAD soy (LabDiets 5L0R) provides an isoflavone aglycone-equivalent dose of 180 mg/person/day on a caloric basis. The other diet (TAD casein, LabDiets 5L0P) is nearly devoid of soy and soy isoflavones. Both diets contain animal byproducts, with soy added as protein replacement in TAD soy to mimic a high soy supplementation diet. The isoflavone component present in the TAD soy diet consists of approximately 40–50% genistein, 40% daidzein, and 10–15% glycitein, in the native conjugated forms (Table I). This mixture is the naturally occurring ratio of isoflavones in soy products and is similar to that of the standard chow but with approximately 2.5 times the amount of total Am. J. Primatol. 804 / Wagner et al. TABLE I. Diet ingredients and macronutrient analysis Ingredients Casein Dried whey Dehulled soybean meal Ground soybean hulls Wheat flour Animal fat Ground corn Fructose Ground wheat Cellulose Wheat germ Dried whole egg Poultry fat Wheat middlings Corn gluten meal Dried beet pulp Dehydrated alfalfa meal Fish meal Sucrose Diet analysis Protein, % Fat, % Carbohydrate, % Fiber, % Cholesterol, mg/kcal Metabolizable energy, kcal/g Daidzein,a ppm (% of total isoflavones) Genistein,a ppm (% of total isoflavones) Glycitein,a ppm (% of total isoflavones) Total isoflavones,a ppm Chow LabDiets 5037 1 1 1 1 1 1 TAD soy LabDiets 5LOR TAD casein LabDiets 5LOP 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 36 46 8.7 0.17 3.56 142 (39.2) 181 (50.0) 39 (10.8) 362 18 36 46 8.7 0.16 3.56 17 (43.6) 16 (41.0) 6 (15.4) 39 1 1 1 1 1 1 18 13 69 5 0.023 3.00 55 (38.5) 65 (45.5) 23 (16.1) 143 Diets were formulated and analyzed by Purina LabDiet (Richmond, IN). a Aglycone units. isoflavones as all the protein is derived from soy in TAD soy. For scaling on caloric basis, monkeys are assumed to consume approximately 100 kcal per kg of body weight per day. Given an average body weight of 3.5 kg, the isoflavone dose is approximately 360 mg/monkey/day, which by allometric scaling to the typical US intake of 1,800 mg/person/day is equivalent to a human dose of 180 mg isoflavones per day, which is about 3–4 times the Japanese median daily isoflavones intake of about 50 mg isoflavones daily [Stroud et al., 2006]. Clinical Measures Animals were fasted overnight and sedated with ketamine hydrochloride (15 mg/kg intramuscularly) (Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) before blood collection and body weight measurement. Total cholesterol (TC), HDL cholesterol (HDLC), triglyceride (TG), and glucose concentrations were determined by enzymatic techniques (reagent kits SA1010-TC, SA2031-HDLC, SA1023-TG, SA1014GLU, respectively) on an Alfa Wassermann ACE Am. J. Primatol. Chemistry analyzer (West Caldwell, NJ). Plasma apoB-containing lipoprotein cholesterol (apoB-C) was determined by subtraction of HDLC from TC [Wagner et al., 2008]. Insulin (Mercodia, Uppsala, Sweden) and fructosamine (which assesses overall, fasting and postprandiol, glycemic control) (Roche Diagnostics, Indianapolis, IN) were determined as described [Kavanagh et al., 2008]. An intravenous glucose tolerance test (GTT) was done in adult females after consuming the TAD diet for at least 1 year (TAD casein, n 5 11; TAD soy, n 5 6) and in offspring that were on average 24 months of age (TAD casein, n 5 12; TAD soy, n 5 13). Plasma was collected for the GTT at baseline and 5, 10, 20, 30, and 60 min following a 750 mg/kg glucose infusion. The disappearance area under the curve (AUC) was calculated for glucose and insulin from all time points, and the disappearance rate (K value) for glucose was calculated from the linear portion of the curve [Wagner et al., 2008]. Blood samples for serum isoflavone concentrations were determined in 17 adult females 4 hr after a meal during baseline (when consuming chow) and Soy Protein and Glycemic Measures / 805 again after changing diets to TAD casein or TAD soy. Serum isoflavones were also determined in 18 of the 25 offspring born to mothers consuming the TAD diets (4 hr after feeding). Isoflavone analyses were done at the Cancer Research Center of Hawaii according to established procedures using liquid chromatography and mass spectrometry [Adams et al., 2005; Franke et al., 2002]. The major isoflavones (genistein, daidzein) and the metabolite, equol, were measured and the sum of these reported as total isoflavones. All procedures involving animals adhered to the American Society of Primatologists Principles for the Ethical Treatment of Nonhuman Primates and were conducted in compliance with state and federal laws of the US Department of Health and Human Services and guidelines established by the Wake Forest University Animal Care and Use Committee. Statistics All data are reported as mean7SEM. All analyses were either t-tests or analyses of variance, unless otherwise noted. Tests of heterogeneity of variance were performed for all analyses and all variables that failed these tests were log-transformed. In those cases, statistical tests are reported on the transformed variables, but the means and standard errors are reported for the nontransformed data. For data from offspring, initial analyses indicated no significant sex differences. Thus, all subsequent analyses included both males and females unless otherwise noted. Some of the offspring (N 5 7) had multiple measurements taken for glycemic and lipids. In those cases, the mean values for those animals were used for the analyses. Across all of the analyses there were no significant differences between the ages of the casein and soy subjects. This was true for both adult females and the offspring. RESULTS In both adults and their offspring, TAD soy diet resulted in significantly higher serum isoflavone concentrations than TAD casein diets (Fig. 1; adults: t(15) 5 6.31, Po0.001; offspring: t(16) 5 15.47, Po0.0001). Offspring consuming TAD casein had similar isoflavone concentrations to adults consuming TAD casein (t(16) 5 0.87, P 5 0.40), but offspring consuming TAD soy had higher concentrations than the adult females consuming TAD soy (t(15) 5 3.52, Po0.01). As shown previously [Stroud et al., 2006], standard chow consumption results in high serum isoflavone concentrations. In this study, serum isoflavone concentrations during chow feeding were higher than when adults were consuming TAD (F(1, 15) 5 41.73, Po0.0001). Statistical analyses were performed on total isoflavone concentrations, but as shown in Fig. 1, equol made up the majority of the isoflavones. Fig. 1. Total isoflavone concentrations (nM) in adult females (left) while eating standard monkey chow (baseline) and when eating the TAD with protein from casein–lactalbumin or soy; and offspring (right) when eating the TAD with protein from casein–lactalbumin or soy. Each bar represents the total isoflavone concentration, which was determined by the summing concentrations of the two primary dietary isoflavones (genistein and daidzein) and equol, the major metabolite of daidzein as noted in separate stacked bars. Statistical analyses were performed on total isoflavone concentrations. In both adults and offspring, TAD soy resulted in significantly higher serum isoflavone concentrations than TAD casein (adults: t(15) 5 6.31, Po0.001; offspring: t(16) 5 15.47, Po0.0001). Isoflavone concentrations during chow feeding were higher than when adults were consuming TAD (F(1, 15) 5 41.73, Po0.0001). Offspring consuming TAD casein had similar isoflavone concentrations to adults consuming TAD casein (t(16) 5 0.87, P 5 0.40). Offspring consuming TAD soy had higher concentrations than the adult females consuming TAD soy (t(15) 5 3.52, Po0.01). TAD, typical American diet. In adults, there were increases in body weight, TC, TG, apoB-C, and glucose with time (on average, adults were 5.3 years old during the baseline assessments and 7.75 years old during the TAD assessments with no significant differences between the diet groups) and change from baseline chow to TAD, but no difference between TAD casein and TAD soy (Table II). Fructosamine concentrations increased from baseline chow compared with TAD in all subjects (F(1, 17) 5 28.62, Po0.0001) but were significantly lower with TAD soy compared with TAD casein (F(1, 17) 5 9.10, Po0.01). The lower fructosamine concentrations with TAD soy while there were no changes in fasting glucose suggest lower postprandial glucose measures compared with TAD casein. There were no differences in HDLC or insulin concentrations with TAD diet groups. In the offspring, there were no differences in body weights at birth. However, by 1 year of age and continuing to 2 years of age, offspring consuming TAD casein weighed significantly more (significant main effect of age F(3, 36) 5 564.7, Po0.0001, and significant age diet interaction F(3, 36) 5 3.15, Po0.05) than those eating TAD soy (Fig. 2). Consistent with the increased body weight with Am. J. Primatol. 806 / Wagner et al. TABLE II. Clinical measurements in adult females during baseline chow and after consuming a TAD (Mean7SEM) Body weight (kg) Baseline chow TAD diet Plasma cholesterol (mg/dl) Baseline chow TAD diet Triglyceride (mg/dl) Baseline chow TAD diet HDL cholesterol (mg/dl) Baseline chow TAD diet ApoB-containing lipoproteins (mg/dl) Baseline chow TAD diet Glucose (mg/dl) Baseline chow TAD diet Insulin (mU/ml) Baseline chow TAD diet Fructosamine (mmol/l) Baseline chow TAD diet Casein (n 5 11) Soy (n 5 8) Significant effects 3.070.2 5.670.9 2.870.1 4.470.2 Baseline vs. TAD, Po0.001 14378 190724 14479 168717 Baseline vs. TAD, Po0.05 4774 5277 3474 57710 Baseline vs. TAD, Po0.05 6174 64710 7575 77714 NS 8377 126725 6977 9176 Baseline vs. TAD, Po0.05 6973 7473 6772 8376 Baseline vs. TAD, Po0.01 2174 2675 1973 41714 NS 16377 20676 14779 17477 Baseline vs. TAD, Po0.0001 Casein vs. soy, Po0.01 All data were analyzed using 2-way ANOVAs with time period (baseline chow vs. TAD diet) as a within-subjects factor and diet group (casein vs. soy) as a between-subjects factor. TAD, typical American diet. Body Weight by Age TABLE III. Clinical measurements consuming the TAD (Mean7SEM) 2.50 p<0.01 Body Weight (kg) 2.00 1.50 Casein (N=8) Soy (N=6) p=0.08 1.00 0.50 0.00 0-2_Weeks 6 Months 1 Year 2 Years Age Fig. 2. Change in body weight with age in offspring eating the TAD with protein from casein–lactalbumin or soy. TAD, typical American diet. TAD casein, these offspring also had impaired glycemic control as noted by higher fructosamine concentrations (F(1, 21) 5 6.04, Po0.05) but, as in adults, there was no difference in fasting glucose and insulin concentration (Table III). There was, however, a significant interaction between sex and diet for insulin (F(1, 21) 5 5.28, Po0.05) with females showing higher insulin in the TAD casein group (26.074.0 compared with 15.273.1 in the TAD soy group), while males showed higher insulin in the TAD soy group (18.471.8 compared with 14.274.9 Am. J. Primatol. Cholesterol (mg/dl) Triglyceride (mg/dl) HDL cholesterol (mg/dl) ApoB-C (mg/dl) Glucose (mg/dl) Insulin (mU/ml) Fructosamine (mmol/l) in offspring Casein (n 5 12) Soy (n 5 6) P 203715 5874 8775 116714 8573 2173 18676 202714 4574 9374 109713 8573 1772 17073 NS 0.03 NS NS NS NSa 0.02 All data were analyzed using independent groups t-tests. TAD, typical American diet. a See text for more details. in the TAD casein group). There were no differences in TC, HDLC, or apoB-C with diet, but TG concentrations were lower with TAD soy (F(1, 21) 5 5.43, Po0.05). Also, HDLC was significantly higher in females (98.374.3) compared with males (81.273.1, F(1, 21) 5 11.4, Po0.01). Analyses from the GTTs are presented in Table IV. GTTs were only performed during TAD consumption in adults and offspring (N 5 17 adults, N 5 25 offspring). Responses to the GTT were not different with diet in adult females (Table IV). In the offspring, the glucose AUC was significantly lower Soy Protein and Glycemic Measures / 807 TABLE IV. Measures from GTTs in adult females and offspring consuming a TAD (Mean7SEM) Adult females Glucose AUCa Insulin AUC K-value Offspring Glucose AUC Insulin AUC K-value Casein Soy (n 5 11) 7,08471,285 10,79771,006 4.770.5 (n 5 12; M 5 5, F 5 7) 8,3287641 2,4177183 3.370.3 (n 5 6) 6,29771,314 10,2677793 4.370.6 (n 5 13; M 5 7, F 5 6) 6,0307418 1,7337208 6.170.7 P NS NS NS o0.01 o0.05 o0.01 GTT, glucose tolerance test; TAD, typical American diet. a AUC 5 area under the curve. Adult female data were analyzed using independent groups t-tests. Two of the eight adult female subjects in the soy condition did not have GTT data. Offspring data were analyzed by ANOVA for diet effect (shown), sex and diet sex differences. There were no diet sex differences but there was a significant sex difference for glucose AUC (see text for details). higher in females (8,100.57529.4) compared with males (6,084.77585.7; t(23) 5 2.77, Po0.05), but there was no diet sex interaction. 350 TAD Casein (n=12) Glucose (mg/dL) 300 TAD Soy (n=13) 250 DISCUSSION 200 150 100 50 0 0 10 20 30 40 50 60 Time (min) TAD Casein (n=12) Insulin (µU/mL) 80 TAD Soy (n=13) 60 40 20 0 0 10 20 30 40 50 60 Time (min) Fig. 3. Changes in glucose (top) and insulin (bottom) responses to an intravenous GTT in offspring eating the TAD with protein from casein–lactalbumin or soy. TAD, typical American diet; GTT, glucose tolerance test. (t(23) 5 2.95, Po0.01) and the disappearance of glucose (K-value) was significantly faster with TAD soy (t(23) 5 3.84, Po0.0001, Table IV) as depicted in Fig. 3, top. The insulin responses to the glucose challenge were also significantly lower (AUC) in TAD soy (t(23) 5 2.45, Po0.05), as depicted in Fig. 3, bottom. The glucose AUC was also significantly A major finding of this study is that in both adult females and their offspring, monkeys consuming soy protein had better glycemic control, as determined by fructosamine concentrations, compared with those consuming diets with similar macronutrient content but with casein–lactalbumin as the protein source (Tables II and III). While there were no other treatment differences in the adults, offspring born to these dams had similar body weights at birth but over a two-year period weighed significantly less and had lower TG concentrations (Table III). Whereas GTTs in adult females were not significantly different, the offspring had faster glucose disappearance (K value) with overall lower glucose and insulin responses as determined by the AUC (Fig. 3, Table IV). The lower glucose AUCs in the face of lower insulin AUCs suggests less insulin resistance in these young animals with TAD soy. As expected, monkeys consuming TAD soy had higher serum isoflavones concentrations than those consuming TAD casein (Fig. 1). After ingestion, soy isoflavones are hydrolyzed by intestinal glucosidases, which release the aglycones (daidzein and genistein) that may be absorbed or further metabolized to other metabolites such as equol [Setchell et al., 1999]. The monkeys in this report, as in others [Stroud et al., 2006], have higher serum equol concentrations (due primarily from conversion of dietary daidzein to equol) than that found in people [Gu et al., 2006]. Despite higher total dietary isoflavones in TAD soy compared with chow (Table I), the serum concentrations were higher during chow consumption (baseline) than when consuming TAD soy (Fig. 1). This may be related to other dietary components affecting intestinal absorption. For example, high carbohydrate diets (such as chow) may increase intestinal fermentation and biotransformation as well as Am. J. Primatol. 808 / Wagner et al. increasing the formation of equol [Setchell & Cassidy, 1999]. An additional finding is that the offspring consuming the TAD soy had higher serum isoflavones than their mothers eating the same diet (Fig. 1). This is in agreement with the higher isoflavone bioavailability in children vs. adults as determined by challenges with soy nuts, considering body weight-adjusted isoflavone doses [Franke et al., 2006; Halm et al., 2007]. While the reason for this is unknown, it may be due to the maturing gut flora that can hydrolyze the isoflavone b glucosides more efficiently [Franke et al., 2006]. The offspring in this report ranged from 12 to 24 months of age and thus were no longer nursing. Future studies are planned to collect samples in younger offspring to further assess differences with age. It is important to note that sex-specific differences in metabolism of soy isoflavones have been published [Stroud et al., 2006]. In that report male monkeys had higher genistein, daidzein, and total isoflavone concentrations compared with premenopausal female monkeys fed the same diet. While we did not have sufficient numbers of adult males in the colony to assess differences in the adults, we did not find any effect of sex on serum isoflavones in the offspring. We have shown previously that soy protein containing isoflavones improved lipids and insulin sensitivity in ovariectomized females [Wagner et al., 1997]. In a subsequent study of premenopausal monkeys we found an increase in insulin secretion in response to a glucose challenge but no change in insulin sensitivity as determined by euglycemic clamp studies [Kavanagh et al., 2008]. In male monkeys we reported increased insulin secretion in response to a glucose challenge, but no change in glucose disappearance indicative of insulin resistance [Wagner et al., 2008]. We have postulated that the inconsistent findings with soy as it relates to insulin sensitivity may be due to the complex interactions between endogenous sex hormone concentrations and soy isoflavone concentrations. This is consistent with the in vitro response where isoflavone binding to the ER has been shown to be concentration dependent [Hwang et al., 2006]. In low estrogen concentrations, isoflavones have been shown to function as an ER agonist, but in higher estrogen concentrations they function as an ER antagonist. This may explain the mixed results for glycemic control with soy in men and women [Jayagopal et al., 2002; Hermansen et al., 2001] and the sex diet interaction with insulin in this report. This conclusion was also reached based on findings in mice where genistein resulted in worsening of heart failure in a transgenic mouse model of hypertrophic cardiomyopathy in male but not in female mice [Stauffer et al., 2006]. We are not aware of any reports of soy consumption and effects on carbohydrate metabo- Am. J. Primatol. lism in male or female human infants. There is some evidence of a transient endocrine disrupting effect of soy-based infant formula in marmosets [Tan et al., 2006]. However, body weights did not differ in this study, and glucose metabolism was not studied. Endogenous hormone concentrations have been shown to be affected by soy consumption. Sharpe et al.  reported that soy formulas compared with cow’s milk formulas resulted in lower testosterone in young male marmosets. Thus, endogenous hormone measurements are planned in this colony to determine if there is a relationship between sex hormones, isoflavone concentrations, and carbohydrate metabolism. A number of other mechanisms have been proposed for soy effects on glycemic control. Soy may affect insulin action via changes in body weight and body fat [for a review, see, Bhathena & Velasquez, 2002]. Soy protein has been shown to have reductions in body fat in monkeys; however, this was not as robust as estradiol [Wagner et al., 1997]. Arjmandi et al.  found similar effects in ovariectomized rats. Both estrogen and soy prevented the ovariectomy-induced body weight gain in these rats, with the greatest effect due to estradiol. Postmenopausal women consuming high levels of soy protein also had lower body mass indices [GoodmanGruen & Kritz-Silverstein, 2001]. The lower body weight of offspring consuming TAD soy may be an additional mechanism for the improved fructosamine and glucose disposal during the GTT. Further studies are planned to determine the effects of soy on body composition to determine if the changes in body weight reported here are due to changes in fat vs. lean tissue. Changes in body composition are significantly associated with determinants of in vivo insulin resistance [Stern & Haffner, 1986]. One potential mechanism involves the production of hormones or adipokines (e.g., leptin, adiponectin, TNFa, resistin, and plasminogen-activator inhibitor type 1 (PAI-1)) by adipose tissue, referred to as adipokines. Plasma concentrations of these peptides have been associated with insulin resistance; leptin, TNFa, and PAI-1 are associated positively, while adiponectin is associated negatively [Rasouli & Kern, 2008]. However, we have reported decreased adiponectin concentrations in male monkeys consuming soy [Wagner et al., 2008]. Other postulated mechanisms include changes in insulin receptor number, affinity, intracellular phosphorylation, alterations in the glucose transport apparatus, and lipolysis [Abler et al., 1992; Iritani et al., 1996; Kandulska et al.,1999; Mackowiak et al., 1999; Smith et al., 1993] and increases in b cell insulin secretion [Jonas et al., 1995; Sorenson et al., 1994] similar to estradiol [Costrini & Kalkhoff, 1971]. In vitro studies have shown that soy isoflavones affect expression of PPARs. Genistein and daidzein Soy Protein and Glycemic Measures / 809 have been shown to increase both PPARa and g-directed gene expression [Mezei et al., 2003]. Genistein (41 mm) was also shown to act as a ligand for PPARg in mesenchymal progenitor cells (precursor cells for osteoblasts and adipocytes), resulting in the upregulation of adipogenesis and downregulation of osteogenesis. Transfection experiments showed that activation of PPARg by genistein at micromolar concentrations downregulates its estrogenic transcriptional activity while activation of ERa and ERb by genistein downregulates PPARg transcriptional activity [Dang et al., 2003]. Contrary to these findings, during differentiation of 3T3-Li preadipocytes, high concentrations of genistein (100 mm) inhibited PPARg protein and TG accumulation [Harmon & Harp, 2001; Harmon et al., 2002]. These differences may be concentration dependent with the outcome dependent on the balance between activated ERs and PPARg [Dang et al., 2003] as well as the cell type and state of differentiation. As the activation and subsequent actions of both the ERs and PPARs are dependent on the isoflavone concentrations, the more robust findings in the offspring compared with the adults could be related to the higher isoflavone concentrations. Additional mechanisms for greater effects observed in offspring could relate to the life-time exposure to the soy isoflavones including throughout gestation. The soy isoflavone genistein alters DNA methylation in mice exposed in utero leading to altered adult phenotypes. Dolinoy et al.  demonstrated that in utero exposure to dietary genistein could modify coat color in mice by methylation of a regulatory sequence governing expression of the Agouti coat color gene, and of particular relevance here is that this also protected the offspring from adult obesity. This study demonstrated that methylation patterns in a retrotransposal element in the promoter of Agouti were hypermethylated at six CpG sites. These methylation patterns persisted into adulthood. This finding is consistent with the ‘‘Barker Hypothesis’’ [Barker, 1992; Barker & Bagby, 2005], which postulates that nutrition and/or environmental factors during prenatal and early development influence cellular plasticity, thereby altering future susceptibility to disease. This colony of monkeys will allow for continued studies to test this hypothesis. Interestingly, a recent report [Cederroth et al., 2008] found that in male mice that were fed a high soy isoflavone diet from conception of the dams had reduced whole body fat and improved insulin sensitivity compared with those fed soy-free diets; similar to the findings in this report. This was found, in part, to be mediated by activation of AMPactivated protein kinase (AMPK) in multiple tissues including adipose and skeletal muscle. The use of most commercial chows rich in soy isoflavones adds an uncontrolled variable to many studies; this phenomenon has been recognized and has for the last decade been controlled as a standard part of experimental design in rodent studies of endocrine effects [Thigpen et al., 1999]. As most primate resources feed standard monkey chow, this could result in a very different disease pattern than people in Western countries who eat very limited amounts of soy protein and thus have low isoflavone concentrations. Isoflavones may affect experimental outcomes such as endocrine function or disease pathways including reproduction [Delclos & Newbold, 2007], carcinogenicity [Lamartiniere et al., 1998], immune function [Sakai and Kogiso, 2008], cognition [Pan et al., 2000], osteoporosis [Arjmandi et al., 1996], and obesity, diabetes, and CVD [Adams et al., 2005; Wagner et al., 1997, 2001, 2008]. The high serum concentrations of isoflavones in the offspring suggest that these diets would likely influence early perinatal development and as suggested by the Barker hypothesis affect cellular plasticity and later risk of chronic disease [Barker, 1992; Barker & Bagby, 2005]. We are limited in this report due to the small number of dams, which may explain the fewer significant effects in lipids than previously noted [Clarkson et al., 2001; Wagner et al., 1997]. An additional cohort of animals is now under study. 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