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Effects of soy vs. casein protein on body weight and glycemic control in female monkeys and their offspring

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American Journal of Primatology 71:802–811 (2009)
Effects of Soy vs. Casein Protein on Body Weight and Glycemic Control
in Female Monkeys and their Offspring
Wake Forest University School of Medicine, Winston-Salem, North Carolina
Cancer Research Center of Hawaii, Honolulu, Hawaii
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
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
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.
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.
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]
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
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
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
Dried whey
Dehulled soybean meal
Ground soybean hulls
Wheat flour
Animal fat
Ground corn
Ground wheat
Wheat germ
Dried whole egg
Poultry fat
Wheat middlings
Corn gluten meal
Dried beet pulp
Dehydrated alfalfa meal
Fish meal
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
LabDiets 5037
TAD soy
LabDiets 5LOR
TAD casein
LabDiets 5LOP
142 (39.2)
181 (50.0)
39 (10.8)
17 (43.6)
16 (41.0)
6 (15.4)
55 (38.5)
65 (45.5)
23 (16.1)
Diets were formulated and analyzed by Purina LabDiet (Richmond, IN).
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.
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.
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
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
Baseline vs. TAD, Po0.001
Baseline vs. TAD, Po0.05
Baseline vs. TAD, Po0.05
Baseline vs. TAD, Po0.05
Baseline vs. TAD, Po0.01
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)
Body Weight (kg)
Casein (N=8)
Soy (N=6)
6 Months
1 Year
2 Years
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)
(n 5 12)
(n 5 6)
All data were analyzed using independent groups t-tests. TAD, typical
American diet.
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
Glucose AUC
Insulin AUC
(n 5 11)
(n 5 12; M 5 5, F 5 7)
(n 5 6)
(n 5 13; M 5 7, F 5 6)
GTT, glucose tolerance test; TAD, typical American diet.
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.
TAD Casein (n=12)
Glucose (mg/dL)
TAD Soy (n=13)
Time (min)
TAD Casein (n=12)
Insulin (µU/mL)
TAD Soy (n=13)
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. [2002] 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. [1996] 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
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. [2006] 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. This expanding resource colony will
provide monkeys that have had defined diets during
gestation, lactation, and the remainder of life to help
address the importance of diet and chronic disease.
Funding for this study was provided by National
Center for Research Resources (P40 RR021380
[J. D. W.] and S10 RR020890 [A. A. F.]). 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.
The authors thank Joel Collins, Mickey Flynn, Kathy
Kaplan, Kerry Kakazu, Maryanne Post, Aida Sajuthi,
and Trish Warren for the technical assistance.
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