Radiosynthesis and in Vivo Evaluation of [11C]A1070722, a High Affinity GSK-3 PET Tracer in Primate Brain.

Dysfunction of glycogen synthase kinase 3 (GSK-3) is implicated in the etiology of Alzheimer's disease, Parkinson's disease, diabetes, pain, and cancer. A radiotracer for functional positron emission tomography (PET) imaging could be used to study the kinase in brain disorders and to facilitate the development of small molecule inhibitors of GSK-3 for treatment. At present, there is no target-specific or validated PET tracer available for the in vivo monitoring of GSK-3. We radiolabeled the small molecule inhibitor [11C]1-(7-methoxy- quinolin-4-yl)-3-(6-(trifluoromethyl)pyridin-2-yl)urea ([11C]A1070722) with high affinity to GSK-3 (Ki = 0.6 nM) in excellent radiochemical yield. PET imaging experiments in anesthetized vervet/African green monkey exhibited that [11C]A1070722 penetrated the blood-brain barrier (BBB) and accumulated in brain regions, with highest radioactivity binding in frontal cortex followed by parietal cortex and anterior cingulate, and with the lowest bindings found in caudate, putamen, and thalamus, similarly to the known distribution of GSK-3 in human brain. Our studies suggest that [11C]A1070722 can be a potential PET radiotracer for the in vivo quantification of GSK-3 in brain.

Functional Hypothalamic Amenorrhea: An Endocrine Society Clinical Practice Guideline.

Cosponsoring Associations: The American Society for Reproductive Medicine, the European Society of Endocrinology, and the Pediatric Endocrine Society. This guideline was funded by the Endocrine Society. Objective: To formulate clinical practice guidelines for the diagnosis and treatment of functional hypothalamic amenorrhea (FHA). Participants: The participants include an Endocrine Society-appointed task force of eight experts, a methodologist, and a medical writer. Evidence: This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation approach to describe the strength of recommendations and the quality of evidence. The task force commissioned two systematic reviews and used the best available evidence from other published systematic reviews and individual studies. Consensus Process: One group meeting, several conference calls, and e-mail communications enabled consensus. Endocrine Society committees and members and cosponsoring organizations reviewed and commented on preliminary drafts of this guideline. Conclusions: FHA is a form of chronic anovulation, not due to identifiable organic causes, but often associated with stress, weight loss, excessive exercise, or a combination thereof. Investigations should include assessment of systemic and endocrinologic etiologies, as FHA is a diagnosis of exclusion. A multidisciplinary treatment approach is necessary, including medical, dietary, and mental health support. Medical complications include, among others, bone loss and infertility, and appropriate therapies are under debate and investigation.

Effect of soy isoflavones on thyroid hormones in intact and ovariectomized cynomolgus monkeys (Macaca fascicularis).

OBJECTIVE: Soy isoflavones are commonly used to alleviate menopause-related symptoms. Postmenopausal women are at increased risk for hypothyroidism, and there are concerns that isoflavones may be detrimental to thyroid health. The aim of this study was to examine the effects of soy protein and isoflavones on thyroid function and the relationship between thyroid function and ovarian function. METHODS: Adult female cynomolgus monkeys (Macaca fascicularis) were randomized to consume two diets differing only in protein source: casein-lactalbumin (n = 44) or soy protein with isoflavones (n = 41). After 34 months, all animals were ovariectomized via laparotomy. Half of the monkeys from each diet treatment group continued to consume their preovariectomy treatment phase diet (either isolated soy protein [n = 19] or casein-lactalbumin [n = 21]) for an additional 34 months. The remaining animals did not continue their diets and thus were not considered further. Circulating progesterone, triiodothyronine, thyroxine, and thyroid-stimulating hormone were measured at baseline. Thyroid hormones were remeasured during each treatment phase. RESULTS: Dietary soy increased triiodothyronine in preovariectomized monkeys and prevented a decline in thyroxine after surgical menopause (both P's < 0.05). Mean progesterone concentrations were positively correlated with triiodothyronine at baseline in preovariectomized monkeys (P < 0.01). CONCLUSIONS: Progesterone levels and triiodothyronine are positively correlated in macaques. Dietary soy increases triiodothyronine in preovariectomized monkeys and prevents a decline in thyroxine after surgical menopause. The outcomes observed in this study suggest that soy protein and isoflavone consumption does not adversely affect-and may even preserve-thyroid function in postmenopausal women.

Age-related degenerative functional, radiographic, and histological changes of the shoulder in nonhuman primates.

BACKGROUND: Nonhuman primates have similar shoulder anatomy and physiology compared to humans, and may represent a previously underutilized model for shoulder research. This study sought to identify naturally occurring bony and muscular degeneration in the shoulder of nonhuman primates and to assess relationships between structural and functional aspects of the shoulder and measures of physical function of the animals. We hypothesized that age-related degenerative changes in the shoulders of nonhuman primates would resemble those observed in aging humans. METHODS: Middle-aged (n = 5; ages 9.4-11.8 years) and elderly (n = 6; ages 19.8-26.4 years) female vervet monkeys were studied for changes in mobility and shoulder function, and radiographic and histologic signs of age-related degeneration. RESULTS: Four out of 6 (4/6) elderly animals had degenerative changes of the glenoid compared to 0/5 of the middle-aged animals (P = .005). Elderly animals had glenoid retroversion, decreased joint space, walked slower, and spent less time climbing and hanging than middle-aged vervets (P < .05). Physical mobility and shoulder function correlated with glenoid version angle (P < .05). Supraspinatus muscles of elderly animals were less dense (P = .001), had decreased fiber cross-sectional area (P < .001), but similar amounts of nuclear material (P = .085). Degenerative rotator cuff tears were not observed in any of the eleven animals. DISCUSSION AND CONCLUSION: The vervet monkey naturally undergoes age-related functional, radiographic and histological changes of the shoulder, and may qualify as an animal model for selected translational research of shoulder osteoarthritis.

Premenopausal antimüllerian hormone concentration is associated with subsequent atherosclerosis.

OBJECTIVE: The aim of this study was to determine if premenopausal ovarian reserve is associated with susceptibility for atherosclerosis. METHODS: Female cynomolgus macaques (n = 66, women's equivalent age = 45 y) consumed an atherogenic diet for ∼5 months before the measurement of a marker of ovarian reserve (antimüllerian hormone [AMH]), plasma lipids, follicular phase estradiol, and body weight (BW). Monkeys were then ovariectomized (OVX; n = 17), remained premenopausal (n = 20), or were induced to have reduced ovarian reserve (ROR, n = 29). After 26 additional months consuming the diet, atherosclerosis measurements and risk variables were reassessed. RESULTS: No differences in baseline AMH, plasma lipids, BW, and estradiol or postdiet lipids and BW were observed among the groups subsequently assigned to the OVX, premenopausal control, or reduced ovarian reserve conditions. Postdiet measurements of atherosclerosis extent did not differ among the groups. However, analysis of plaque size by tertile of baseline AMH revealed that plaques were largest in monkeys that began the experiment with the lowest baseline AMH, followed by those in the middle and high tertiles (plaque extent: low AMH, 0.76 ± 0.12 mm; mid AMH, 0.46 ± 0.1 mm; high AMH, 0.34 ± 0.08 mm; P = 0.02). Baseline AMH and plaque size were also correlated negatively (r = -0.31, P = 0.01). Plasma lipids were also correlated significantly with plaque extent (all P < 0.01) but not with AMH. CONCLUSIONS: We report for the first time an inverse relationship between a marker of ovarian reserve (AMH) and subsequent atherosclerosis risk.

Stimulatory adrenocortical effects of a selective estrogen receptor modulator in ovariectomized female macaques.

Here, we report the effects of estrogen and the selective estrogen receptor modulator (SERM) levormeloxifene on adrenocortical measures in ovariectomized female cynomolgus monkeys (Macaca fascicularis). Animals were randomized into one of five treatment groups, each containing 23 to 26 animals: (1) placebo, (2) 0.016 mg/kg 17ß-estradiol (E(2)), (3) 0.5 mg/kg levormeloxifene (L(1)), (4) 1.0 mg/kg levormeloxifene (L(2)), and (5) 5.0 mg/kg levormeloxifene (L(3)). Treatments were administered orally each day for 18 mo. All doses of levormeloxifene resulted in adrenal weights at least 50% greater than placebo (p < .0001 for all). The target dose of levormeloxifene (L(2)) resulted in higher serum concentrations of cortisol (+63%), dehydroepiandrosterone-sulfate (+73%), and androstenedione (+37%) compared with the placebo group (p < .05 for all). In contrast, E(2) resulted in no significant differences in adrenal weight or adrenocortical steroids. Oral E(2) and all SERM doses resulted in similar reductions in serum gonadotropins and at least threefold greater uterine weight versus placebo (p < .0001 for all). Results indicate that the SERM levormeloxifene, in contrast to E(2), may have robust stimulatory effects on adrenocortical hormones in a postmenopausal model. These findings warrant further investigation into long-term SERM effects on adrenocortical function.

Hippocampal responsiveness to 17ß-estradiol and equol after long-term ovariectomy: implication for a therapeutic window of opportunity.

A 'critical window of opportunity' has been proposed for the efficacy of ovarian hormone intervention in peri- and post-menopausal women. We sought to address this hypothesis using a long-term ovariectomized non-human primate (NHP) model, the cynomolgus macaque (Macaca fascicularis). In these studies, we assessed the ability of 17ß-estradiol and equol to regulate markers of hippocampal bioenergetic capacity. Results indicated that 17ß-estradiol treatment significantly increased expression of mitochondrial respiratory chain proteins complex-I and -III in the hippocampus when compared to non-hormone-treated animals. Expression of the TCA cycle protein succinate dehydrogenase α was decreased in animals treated with equol compared to those treated with 17ß-estradiol. There were no significant effects of either 17ß-estradiol or equol treatment on glycolytic protein expression in the hippocampus, nor were there significant effects of treatment on expression levels of antioxidant enzymes. Similarly, 17ß-estradiol and equol treatment had no effect on mitochondrial fission and fusion protein expression. In summary, findings indicate that while 17ß-estradiol induced a significant increase in several proteins, the overall profile of bioenergetic system proteins was neutral to slightly positively responsive. The profile of responses with the ERß-preferring molecule equol was consistent with overall nonresponsiveness. Collectively, the data indicate that long-term ovariectomy is associated with a decline in response to estrogens and estrogen-like compounds. By extension, the data are consistent with a primary tenet of the critical window hypothesis, i.e., that the brains of post-menopausal women ultimately lose their ability to respond positively to estrogenic stimulation.

The effect of dietary soy isoflavones before and after ovariectomy on hippocampal protein markers of mitochondrial bioenergetics and antioxidant activity in female monkeys.

Estrogen therapy can promote cognitive function if initiated within a 'critical window' during the menopausal transition. However, in the absence of a progestogen, estrogens increase endometrial cancer risk which has spurred research into developing estrogenic alternatives that have the beneficial effects of estrogen but which are clinically safer. Soy protein is rich in isoflavones, which are a class of potential estrogenic alternatives. We sought to determine the effects of two diets, one with casein-lactalbumin as the main protein source and the other with soy protein containing isoflavones, on protein markers of hippocampal bioenergetic capacity in adult female cynomolgus macaques (Macaca fascicularis). Further, we assessed the effects of dietary soy isoflavones before or after ovariectomy. Animals receiving soy diet premenopausally then casein/lactalbumin post-ovariectomy had higher relative hippocampal content of glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase subunit e1α. Post-ovariectomy consumption of soy was associated with higher succinate dehydrogenase α levels and lower levels of isocitrate dehydrogenase, both proteins involved in the tricarboxylic acid cycle, significantly decreased expression of the antioxidant enzyme peroxiredoxin-V, and a non-significant trend towards decreased manganese superoxide dismutase expression. None of the diet paradigms significantly affected expression levels of oxidative phosphorylation enzyme complexes, or of mitochondrial fission and fusion proteins. Together, these data suggest that long-term soy diet produces minimal effects on hippocampal expression of proteins involved in bioenergetics, but that switching between a diet containing primarily animal protein and one containing soy isoflavones before and after menopause may result in complex effects on brain chemistry.

The effect of diet and cardiovascular risk on ovarian aging in cynomolgus monkeys (Macaca fascicularis).

OBJECTIVE: The aim of this study was to determine the relationships among dietary protein source, cardiovascular risk, reproductive hormones, and ovarian aging. METHODS: Adult female cynomolgus monkeys (Macaca fascicularis) were assigned randomly to one of two diets containing saturated fat and cholesterol, differing only by protein source: (1) casein-lactalbumin (n = 29) or (2) soy protein with isoflavones (n = 32). Cardiovascular risk markers and reproductive hormones were measured at baseline and after 32 months of treatment, at which time the ovaries were removed and serially sectioned and ovarian follicles were counted in every 100th section. RESULTS: Casein-lactalbumin-fed monkeys had fewer primordial, primary, and secondary follicles (all P values < 0.05) than did their soy-fed counterparts. Antimüllerian hormone was significantly correlated with all follicle types (r values > or = 0.66, P < 0.001) for casein-fed monkeys and was significantly correlated with primary (rsoy = 0.47, P = 0.005) and secondary (rsoy = 0.45, P = 0.007) follicles in soy-fed monkeys. No significant associations were seen between any of the other reproductive hormones measured and follicle counts. Casein-lactalbumin-fed monkeys had a more atherogenic lipoprotein profile and increased atherosclerosis extent (P < 0.05), but despite these differences in cardiovascular risk between monkeys fed with casein-lactalbumin and monkeys fed with soy, none of the individual cardiovascular risk markers measured in this study explained the relationship between dietary protein source and follicle counts (linear regression, all P values > 0.05). CONCLUSIONS: Diet influences the rate of follicular depletion in cynomolgus macaques; however, the mechanism for this effect remains undetermined.

Endometrial profile of tamoxifen and low-dose estradiol combination therapy.

PURPOSE: Combination estrogen + progestin therapy has been associated with increased breast cancer risk in postmenopausal women. Selective estrogen receptor modulators (SERM) are potential alternatives to progestins, although the endometrial safety of estrogen + SERM co-therapies is not known. The goal of this study was to evaluate the endometrial profile of low-dose estradiol and the SERM tamoxifen alone and in combination. EXPERIMENTAL DESIGN: Twenty-four postmenopausal female cynomolgus macaques were randomized by social group to receive placebo, low-dose micronized estradiol (E(2); 0.25 mg/1,800 kcal), the SERM tamoxifen (Tam; 20 mg/1,800 kcal), or E(2) + Tam for 4 months in a parallel-arm design. RESULTS: Tamoxifen alone resulted in overlapping but distinct effects compared with E(2). Both E(2) and Tam increased uterine weight and endometrial thickness, whereas only E(2) increased endometrial proliferation. Morphologic effects were similar for Tam and E(2) + Tam, which both induced stromal fibrosis and cystic change. Tamoxifen inhibited E(2)-induced proliferation and expression of genes related to cell cycle progression while exhibiting mixed agonist and antagonist effects on gene markers of estrogen receptor activity. The gene expression profile for E(2) + Tam was distinct from either E(2) or Tam alone but dominated by the Tam effect for estrogen-regulated genes. Tam also attenuated E(2) effects on both vaginal maturation and cervical epithelial height. CONCLUSIONS: These findings characterize a novel phenotype resulting from estrogen + SERM co-therapy. The predominance of Tam effects on endometrial proliferation, morphology, and transcriptional profiles suggests that endometrial risks for E(2) + Tam may be similar to Tam alone.

Genomic diversity and interspecies host infection of alpha12 Macaca fascicularis papillomaviruses (MfPVs).

Alpha human papillomaviruses (HPVs) are among the most common sexually transmitted agents of which a subset causes cervical neoplasia and cancer in humans. Alpha-PVs have also been identified in non-human primates although few studies have systematically characterized such types. We cloned and characterized 10 distinct types of PVs from exfoliated cervicovaginal cells from different populations of female cynomolgus macaques (Macaca fascicularis) originating from China and Indonesia. These include 5 novel genotypes and 5 previously identified genotypes found in rhesus (Macaca mulatta) (RhPV-1, RhPV-a, RhPV-b and RhPV-d) and cynomolgus macaques (MfPV-a). Type-specific primers were designed to amplify the complete PV genomes using an overlapping PCR method. Four MfPVs were associated with cervical intraepithelial neoplasia (CIN). The most prevalent virus type was MfPV-3 (formerly RhPV-d), which was identified in 60% of animals with CIN. In addition, the complete genomes of variants of MfPV-3 and RhPV-1 were characterized. These variants are 97.1% and 97.7% similar across the L1 nucleotide sequences with the prototype genomes, respectively. Sequence comparisons and phylogenetic analyses indicate that these novel MfPVs cluster together within the alpha12 PV species closely related to the alpha9 (e.g., HPV16) and alpha11 species (e.g., HPV34), and all share a most recent common ancestor. Our data expand the molecular diversity of non-human primate PVs and suggest a recent expansion of alpha-PV species groups. Moreover, identification of an overlapping set of MfPVs in rhesus and cynomolgus macaques indicates that non-human primate alpha-PVs might not be strictly species-specific and may represent past interspecies infection.

Social subordination and polymorphisms in the gene encoding the serotonin transporter enhance estradiol inhibition of luteinizing hormone secretion in female rhesus monkeys.

Psychosocial factors, particularly social stress, may compromise reproduction. However, some individuals may be more susceptible to socially induced infertility. The present study used group-housed, adult, ovariectomized rhesus monkeys to test the hypothesis that exposure to psychosocial stress, imposed by social subordination, would enhance estradiol (E2)-negative feedback inhibition of LH. Because polymorphisms in the gene encoding the serotonin transporter (SLC6A4) may contribute to individual differences in response to adverse environments, we determined whether subordinate females with the short-promoter-length allele (s-variant) would show greater suppression of LH. Subordinate females, particularly those with the s-variant SLC6A4 genotype, received significantly higher rates of noncontact aggression from more dominant cage mates and had consistently lower body weights. Serum LH was not influenced by social status in the absence of E2. In contrast, subordinate females were hypersensitive to E2-negative feedback inhibition of LH. Furthermore, serum LH in subordinate females with s-variant SLC6A4 genotype was maximally suppressed by Day 4 of treatment, whereas nadir concentrations were not reached until later in treatment in other females. Finally, pharmacological elevation of serum cortisol potentiated E2-negative feedback inhibition in all females. The current data suggest that infertility induced by psychosocial stressors may be mediated by hypersensitivity to E2-negative feedback and that polymorphisms in the SLC6A4 gene may contribute to differences in reproductive compromise in response to chronic stress.

Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats: ovarian, clinical, and pathologic outcomes.

Young rats treated daily with intraperitoneal 4-vinylcyclohexene diepoxide (VCD) undergo selective destruction of primordial follicles, resulting in gradual ovarian failure resembling the menopausal transition in women. To determine whether VCD has similar effects on ovaries of older rats, adult and peripubertal Sprague-Dawley rats were injected intraperitoneally daily for 30 d with vehicle or VCD at 40 or 80 mg/kg. Body weight, food intake, complete blood counts, and markers of liver injury and renal function were measured during VCD treatment. Complete gross necropsy and microscopic observations were performed on day 31, and ovarian follicles were counted. At 80 mg/kg, VCD destroyed primordial and primary follicles to a similar extent in both adult and peripubertal animals, although adult rats likely started with fewer follicles and therefore approached follicle depletion. Treatment with VCD did not affect body weight, but food intake was reduced in both adult and peripubertal rats treated with 80 mg/kg VCD. Adult rats treated with 80 mg/kg VCD had neutrophilia and increased BUN and creatinine; in addition, 4 of these rats were euthanized on days 25 or 26 due to peritonitis. VCD treatment did not increase alanine aminotransferase levels, a marker of liver injury, although the 80-mg/kg dose increased liver weights. In conclusion, VCD effectively destroys small preantral follicles in adult Sprague-Dawley rats, making them a suitable model of the menopausal transition of women. However, because adult rats were more sensitive to the irritant properties of VCD, the use of a lower dose should be considered.

Ovarian dysfunction and the premenopausal origins of coronary heart disease.

OBJECTIVE: The menopausal loss of cyclic ovarian function is believed to contribute to coronary heart disease (CHD). However, ovarian function varies substantially throughout the premenopausal years, with disruptions in hormonal activity ranging from mild to profound. Here we propose our "precocious acceleration" hypothesis, which holds that to the extent cyclic ovarian function affords protection against CHD, even mild ovulatory abnormalities in young women will accelerate development of this disease. DESIGN: Data relating to the expression and incidence of premenopausal ovarian dysfunction in women and its relationship to CHD are reviewed. Also reviewed are the results from experiments conducted with socially housed cynomolgus monkeys (Macaca fascicularis), focused on the causes, occurrence, and pathobiological sequelae of premenopausal ovarian dysfunction. The implications of the foregoing material for understanding the health of peri- and postmenopausal women are then considered. RESULTS: Epidemiological and clinical studies indicate that common premenopausal reproductive abnormalities (manifested along a continuum from mild subclinical alterations to complete suppression) are associated with increased risk of CHD. This increased risk is probably mediated by different pathways, depending on whether the reproductive deficits relate to hypoestrogenemia (functional hypothalamic anovulatory syndrome) or hyperandrogenemia (polycystic ovary syndrome). Furthermore, although clinically obvious expressions of these syndromes affect perhaps 10% of premenopausal women, evidence suggests that a much larger number may experience subclinical dysfunction capable of increasing CHD risk. CONCLUSIONS: Epidemiological data and systematic studies in nonhuman primates provide initial support for the precocious acceleration hypothesis, suggesting that efforts to protect the health of postmenopausal women would best begin during the premenopausal years.

Origins and health consequences of stress-induced ovarian dysfunction.

Normal ovarian function is thought to protect women against coronary heart disease (CHD) and osteoporosis by delaying the pathobiological processes underlying these conditions. Supporting this proposition is the observation that, following menopause (i.e. the loss of cyclic ovarian function), these diseases accelerate and ultimately comprise a major portion of the health burden of older women. However, while all women eventually go through complete ovarian failure at menopause, many also experience episodes of cyclic ovarian disruption during their reproductive years (i.e. ages 18-40). These disruptions are relatively common and often are attributed to psychogenic factors (stress, anxiety, depression, or other emotional disturbance). This article hypothesizes that, to the extent that cyclic ovarian function affords protection against CHD and osteoporosis, ovulatory abnormalities associated with estrogen deficiency in young women - even if mild and subclinical - prematurely accelerate development of these two diseases of 'aging'. Consistent with this hypothesis are observations in group-housed, premenopausal monkeys confirming that reproductive deficits are commonly induced by psychosocial stress (social subordination), and, in the presence of a typical Western diet, accelerate the development of CHD and bone loss. Furthermore, in this model premenopausal disease extent predicts postmenopausal health outcomes irrespective of postmenopausal treatment, emphasizing the pathobiological importance of the premenopausal portion of the life cycle. Finally, data from both women and nonhuman primates suggest that reproductive deficits of the sort described here are adaptive when triggered appropriately, but detrimental when activated in an environment (e.g. sedentary lifestyle, high-fat diet) permissive to the development of chronic disease.

Effects of soy protein and isoflavones on insulin resistance and adiponectin in male monkeys.

Isoflavones may influence insulin action by means of their well-known receptor-mediated estrogenic activity. However, isoflavones also bind to peroxisome proliferator-activated receptors (PPARs) that are strongly associated with insulin action. Soy protein with its isoflavones has previously been shown to improve glycemic control in diabetic postmenopausal women and to improve insulin sensitivity in ovariectomized monkeys. The purpose of the current report was to extend our studies of dietary soy protein to male monkeys and determine effects of the soy isoflavones on insulin resistance. Two studies are reported here. Study one involved 91 male monkeys consuming 3 diets differing only by the source of protein (casein-lactalbumin, soy protein with a low isoflavone concentration, or soy protein with a high isoflavone concentration). Intravenous glucose tolerance tests were done, and plasma adiponectin and lipoprotein concentrations were determined after 25 months of study. Samples of visceral fat were obtained at 31 months for assessment of adiponectin and PPARgamma expression. The second study involved 8 monkeys in a Latin-square design that compared the effects of diets with casein/lactalbumin, soy protein with a high isoflavone concentration, or soy protein that was alcohol-washed to deplete the isoflavones. After 8 weeks of treatment, insulin sensitivity and plasma lipoproteins were assessed. At 10 weeks, a biopsy of the skeletal muscle was performed for determination of insulin receptor, PPARalpha, and PPARgamma content. The major findings were that consumption of isoflavone-containing soy protein dose-dependently increased insulin responses to the glucose challenge and decreased plasma adiponectin, whereas isoflavone-depleted soy protein decreased body weight and had no effect on plasma adiponectin concentrations. Muscle PPARalpha and gamma expression was also increased with the isoflavone-depleted soy relative to either casein or soy protein containing the isoflavones. Further studies are needed to determine the mechanisms involved in these effects of a high-soy isoflavone diet and to optimize dietary isoflavone content for maximal health benefits in male subjects.

Plasma lipid-dependent and -independent effects of dietary soy protein and social status on atherogenesis in premenopausal monkeys: implications for postmenopausal atherosclerosis burden.

OBJECTIVE: : Atherosclerosis developed during premenopausal years predicts postmenopausal atherosclerosis burden. The objective of this study was to determine the effects of dietary soy protein isolate (SPI) and social status on atherogenesis and arterial gene expression in a premenopausal monkey model. DESIGN: : Socially housed premenopausal cynomolgus macaques (n = 84) were fed an atherogenic diet deriving protein from casein/lactalbumin or SPI (containing 1.88 mg isoflavones/g). After 36 months of diet consumption, iliac artery biopsies were assessed for atherosclerosis and expression of mRNA transcripts related to inflammation, macrophage and T-cell content, and estrogen receptors (ERs). RESULTS: : SPI reduced plaque size (P < 0.05), total plasma cholesterol, non-high-density lipoprotein cholesterol (HDLc), and the total plasma cholesterol/HDLc ratio (all P < 0.003), while increasing triglycerides (P < 0.006) and HDLc (P < 0.0001). Arterial mRNA for CD68 (P < 0.001), CD3 (P < 0.02), and CD4 (P < 0.001) and inflammatory markers monocyte chemotactic protein-1, intercellular adhesion molecule-1, and interleukin-6 (all P < 0.0001) were also lower in the group receiving SPI. For most outcomes, this effect remained even after adjustments for plaque size and plasma lipid concentrations. Arterial ER-alpha was inversely associated with atherosclerosis (P < 0.02) and increased with SPI (P < 0.001). Subordinate monkeys had lower ER-beta (P < 0.02) and higher interleukin-6 (P < 0.05) transcripts but did not differ from dominant monkeys in extent of atherosclerosis (P > 0.9). CONCLUSIONS: : Premenopausal consumption of SPI had plasma lipid-independent beneficial effects on the pathobiological processes involved in atherosclerotic plaque development, thus potentially establishing the basis for reduced postmenopausal complications. Dominant social status provided similar, albeit less extensive, benefits in risk markers.

Bone mass and soy isoflavones in socially housed, premenopausal macaques.

BACKGROUND: Soy consumption is associated with a lower incidence of hip fracture in Asian than in Western women, an effect often attributed to estrogen-like compounds (isoflavones) in soy. It is not known whether premenopausal soy exposure initiated in adulthood can increase bone mass and thereby reduce fracture risk. OBJECTIVE: We aimed to determine whether a high-isoflavone soy diet influences bone mass in soy-naïve, premenopausal cynomolgus monkeys (Macaca fascicularis). DESIGN: Ninety-four skeletally mature females were randomly assigned to consume diets whose protein content came from either high-isoflavone soy or casein and lactalbumin. Animals were socially housed. Bone mass and circulating isoflavone concentrations were measured at baseline and 19 and 31 mo after the start of treatment; bone biomarkers were measured at baseline and 31 mo. RESULTS: There were no significant differences at any timepoint in whole-body bone mineral content between casein-fed (112.5 +/- 2.1, 119.2 +/- 1.9, and 120.7 +/- 2.1 g) and soy-fed (117.2 +/- 2.1, 122.4 +/- 2.0, and 125.4 +/- 2.3 g; P=0.12) monkeys. Similar results were seen for spinal bone mineral density (casein-fed: 0.46 +/- 0.01, 0.50 +/- 0.01, and 0.52 +/- 0.01 g/cm(2); soy-fed: 0.47 +/- 0.01, 0.51 +/- 0.01, and 0.52 +/- 0.01 g/cm(2); P=0.30) and bone biomarker measurements-bone-specific alkaline phosphatase (soy-fed: 82.3 +/- 4.1 and 63.2 +/- 3.4 ng/mL; casein-fed: 94.1 +/- 4.5 and 61.7 +/- 4.3 ng/mL; P=0.22) and C-terminal crosslink of type 1 collagen (soy-fed: 0.944 +/- 0.06 and 0.89 +/- 0.08 nmol/L; casein-fed: 0.97 +/- 0.07 and 0.78 +/- 0.06 nmol/L; P=0.20). CONCLUSION: A soy diet high in isoflavones does not significantly affect bone characteristics in initially soy-naïve premenopausal monkeys.

Concentrations of isoflavones in macaques consuming standard laboratory monkey diet.

The soy isoflavones genistein and daidzein, as well as the daidzein metabolite equol, have structural similarities to mammalian estrogens and bind with varying affinity to both known subtypes of the estrogen receptor. Consequently, prospective studies in both humans and animals have begun to evaluate the potential effects of isoflavones on estrogen receptor-mediated phenomena. However, many diets of laboratory-housed animals derive their protein from soy and thus likely contain substantial quantities of isoflavones. Exposing experimental subjects to these isoflavones via such diets could confound studies, particularly those evaluating the effects of estrogen or estrogen-like ligands. The aim of this study was to compare the levels of circulating concentrations of isoflavones and their metabolites in monkeys fed either a soy-free diet, a soy-based diet providing 130 mg of isoflavone (daidzein, genistein, and glycitein aglycon equivalents) daily, or a commercially available 'chow' diet containing an unspecified amount of soybean meal. Animals consuming the commercial diet had serum concentrations of daidzein, genistein, and glycitein that were significantly higher than those of animals fed a soy-free diet but similar to those of monkeys fed a soybased diet formulated to be high in isoflavones. Notably, animals fed the commercial diet also had serum equol concentrations that were similar to or, in some cases, in excess of serum concentrations in the animals fed the soy diet. These data argue for the use of soy-free diets in studies investigating estrogenic effects on physiologic or behavioral endpoints.