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STUDY QUESTION: What is the underlying neuropathology in a cynomolgus macaque model of functional hypothalamic amenorrhoea (FHA) and can it be normalized to restore ovulation? SUMMARY ANSWER: Anovulatory monkeys exhibited increased hypothalamic norepinephrine (NE), kisspeptin and gonadotropin-releasing hormone (GnRH) in the early follicular phase, but administration of the NE reuptake inhibitor (NRI), reboxetine (REB), restored ovulation during stress and normalized NE, kisspeptin and GnRH. WHAT IS KNOWN ALREADY: Female cynomolgus macaques, like women, show individual reproductive sensitivity to modest psychosocial and metabolic stress. During stress, resilient females ovulate through two menstrual cycles whereas stress-sensitive (SS) macaques immediately cease ovulation. On Day 5 of a non-stressed menstrual cycle, resilient macaques have less NE synthesizing enzyme [dopamine ß-hydroxylase (DBH)], kisspeptin and GnRH innervation of the medial basal hypothalamus but more endogenous serotonin than SS macaques. Stress increased DBH/NE, kisspeptin and GnRH but did not alter serotonin. STUDY DESIGN, SIZE, DURATION: In a longitudinal design, 27 adult (7-13 years) female cynomolgus macaques (Macaca fascicularis) with three different levels of sensitivity to stress were monitored with daily vaginal swabs and frequent serum progesterone (P) measurements. Three 90-day experimental periods called 'Cycle Sets' were monitored. A Cycle Set consisted of one ovulatory menstrual cycle without stress, and two cycles, or 60 days, with modest stress. Each Cycle Set was followed by a rest period. During a Cycle Set, individuals were either untreated (placebo) or administered escitalopram (CIT) or REB. Ultimately, half of each sensitivity group was euthanized during stress with CIT or REB treatment and the hypothalamus was obtained. Neurobiological endpoints were compared between CIT and REB treatment groups in stress resilient and SS monkeys. PARTICIPANTS/MATERIALS, SETTING, METHODS: The monkeys were housed at the University of Pittsburgh primate facility for the duration of the experiments. Upon euthanasia, their brains and serum samples were shipped to the Oregon National Primate Research Center. The hypothalamus was examined with immunohistochemistry for the expression of DBH (a marker for NE axons), kisspeptin and GnRH. P was measured in the serum samples by radioimmunoassay. MAIN RESULTS AND THE ROLE OF CHANCE: Daily administration of REB restored ovulation in 9 of 10 SS animals during stress. Of note, REB significantly increased P secretion during stress in the most sensitive group (P = 0.032), which indicates ovulation. CIT lacked efficacy. REB significantly reduced DBH/NE, kisspeptin and GnRH axon density in the hypothalamus relative to CIT treatment (P = 0.003. 0.018 and 0.0001, respectively) on Day 5 of the menstrual cycle in resilient and sensitive groups. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The US FDA has not approved REB for human use, although it is used in Europe for the treatment of depression/anxiety as EdronaxTR. Whether REB could be useful for the treatment of FHA in women has not been determined. WIDER IMPLICATIONS FOR THE FINDINGS: The use of an NRI to treat FHA is a novel approach and the potential reinstatement of ovulation could be straightforward compared to current treatment protocols. The underlying neurobiology provides a compelling case for treating the origin of the pathology, i.e. elevated NE, rather than circumventing the hypothalamus altogether with gonadotropins, which have associated risks such as hyperstimulation syndrome or multiple births. STUDY FUNDING/COMPETING INTEREST(S): Portions of this study were supported by NIH grant HD062864 to C.L.B., NIH grant HD62618 to J.L.C. and C.L.B. and 1P51 OD011092 for the operation of the Oregon National Primate Research Center. There were no competing interests.

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The consumption of a diet high in fat and refined sugars has several health risks, including the development of cognitive decline and neurodegeneration. For women, menopause carries additional health risks that may interact with a high-fat diet in negative ways. Some symptoms of menopause, including cognitive impairments, can be modulated by hormone replacement therapy (HRT), but the hormonal formulation and the timing of the treatment relative to the onset of menopause are critical factors determining its efficacy. Little is known about how obesogenic, high-fat, high-sugar diets interact with HRT in menopause to affect cognition and neurodegeneration. Given the high prevalence of the consumption of an obesogenic Western-style diet, understanding how the effects of HRT are modulated by an obesogenic diet is critical for developing optimized therapeutic strategies for peri- and post-menopausal women. In this study, we investigated by magnetic resonance imaging (MRI) the effects of either immediate or delayed estradiol hormone therapy on cognition and neuroanatomy following ovo-hysterectomy (OvH) of aged, female rhesus macaques on an obesogenic diet. The macaques were followed for 2.5 years after ovo-hysterectomy, with four time points at which anatomical MRIs were acquired. Analysis of hippocampal volumes revealed an interaction between time point and treatment; hippocampal volumes in the delayed estrogen group, but not the immediate estrogen group, increased over time compared to those in untreated controls. Performance on a hippocampal-dependent spatial maze task showed improved performance in estrogen treated animals compared to OvH macaques given placebo. These results indicate that HRT may contribute to beneficial cognitive outcomes after menopause under an obesogenic diet.

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Whether hormone replacement therapy has beneficial metabolic effects in postmenopausal women remains controversial because of between-study differences in menopausal duration, estrogen formulations, and diet. Additionally, animal studies have not reflected the typical human obesogenic, Western-style diet (WSD). In this study, we determined the effects of immediate 17ß-estradiol (ImE) or delayed 17ß-estradiol treatment on weight and metabolism parameters in old ovo-hysterectomized rhesus macaques consuming a WSD over a 30-month period. The placebo and ImE groups exhibited progressive gains in weight and fat mass, which ImE initially attenuated but did not prevent. Progression of insulin resistance (IR) was lessened by ImE compared with placebo under both fasting and IV glucose-stimulated conditions, plateauing in all groups between 24 and 30 months. Consequently, relative euglycemia was maintained through lower stimulated insulin levels with ImE than with placebo. Bone mineral density decreased in the placebo group but was maintained in the ImE group, whereas bone mineral content was unaffected by placebo and increased with ImE. Daily activity was reduced while macaques consumed a WSD and was not affected by ImE. Over time, total cholesterol, triglyceride, very-low-density cholesterol, high-density lipoprotein cholesterol (HDL-C), non-HDL-C, and IL-8 levels increased or trended upward in all animals, with only the change in HDL-C affected by ImE. Delayed estrogen treatment (months 24 to 30) had no significant impact on body composition or glucometabolic parameters. In summary, detrimental WSD-induced changes in body composition and metabolism were only temporarily ameliorated by ImE, with the important exception of glucose homeostasis, which benefited from E replacement even as body composition worsened.

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Macaques have served as effective models of human disease, including pathological processes associated with obesity and the metabolic syndrome. This study approached several questions: (1) does a western-style diet (WSD) contribute to sedentary behavior or is sedentary behavior a consequence of obesity and (2) does estradiol (E) hormone therapy offset WSD or ameliorate sedentary behavior? We further questioned whether the timing of E administration (immediately following hysterectomy, ImE; or after a 2-year delay, DE) would impact behavior. Focal observations were taken on the animals in social housing over a period of 2.5 years before and after initiation of the WSD and hysterectomy. In addition, anxiety was assessed through the Human Intruder and Novel Object Tests. All animals gained weight, but ImE delayed the time to maximum weight achieved at 18 months. Over the course of the study, ImE-treated monkeys spent more time "alone" and less time in "close social" contact than placebo-controls. The DE-treated monkeys were not different from placebo-controls in these 2 outcomes. The placebo-control group exhibited more "self-groom" behavior, an indicator of anxiety, than did the ImE-treated group, and DE-treated animals approached levels observed in the ImE-treated animals. All animals exhibited an increase in "consume" behavior over time with no statistical difference between the groups. By the end of the protocol, the placebo-control group exhibited less activity compared to ImE + DE-treated animals combined. Animals also showed increased anxiety after starting on the WSD in the Human Intruder Test and the Novel Object Test. In summary, the data indicated that WSD per se promoted increased consummatory behavior, sedentary behavior, and anxiety-type behaviors, whereas ImE promoted activity. Thus, WSD may precipitate the behaviors observed in humans who then become obese, sedentary, anxious, and socially isolated. ImE replacement ameliorates some of these behaviors, but not all.

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The beneficial effects of bioidentical ovarian steroid hormone therapy (HT) during the perimenopause are gaining recognition. However, the positive effects of estrogen (E) plus or minus progesterone (P) administration to ovariectomized (Ovx) lab animals were recognized in multiple systems for years before clinical trials could adequately duplicate the results. Moreover, very large numbers of women are often needed to find statistically significant results in clinical trials of HT; and there are still opposing results being published, especially in neural and cardiovascular systems. One of the obvious differences between human and animal studies is diet. Laboratory animals are fed a diet that is low in fat and refined sugar, but high in micronutrients. In the US, a large portion of the population eats what is known as a "western style diet" or WSD that provides calories from 36% fat, 44% carbohydrates (includes 18.5% sugars) and 18% protein. Unfortunately, obesity and diabetes have reached epidemic proportions and the percentage of obese women in clinical trials may be overlooked. We questioned whether WSD and obesity could decrease the positive neural effects of estradiol (E) in the serotonin system of old macaques that were surgically menopausal. Old ovo-hysterectomized female monkeys were fed WSD for 2.5 years, and treated with placebo, Immediate E (ImE) or Delayed E (DE). Compared to old Ovx macaques on primate chow and treated with placebo or E, the WSD-fed monkeys exhibited greater individual variance and blunted responses to E-treatment in the expression of genes related to serotonin neurotransmission, CRH components in the midbrain, synapse assembly, DNA repair, protein folding, ubiquitylation, transport and neurodegeneration. For many of the genes examined, transcript abundance was lower in WSD-fed than chow-fed monkeys. In summary, an obesogenic diet for 2.5 years in old surgically menopausal macaques blunted or increased variability in E-induced gene expression in the dorsal raphe. These results suggest that with regard to function and viability in the dorsal raphe, HT may not be as beneficial for obese women as normal weight women.

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Like women, old female rhesus macaques undergo menopause and show many of the same age-associated changes, including perturbed activity/rest cycles and altered circulating levels of many hormones. Previous studies showed that administration of an estrogen agonist increased activity in female monkeys, that hormone therapy (HT) increased activity in postmenopausal women and that obesity decreased activity in women. The present study sought to determine if postmenopausal activity and circulating hormone levels also respond to HT when monkeys are fed a high-fat, high-sugar Western style diet (WSD). Old female rhesus macaques were ovo-hysterectomized (OvH) to induce surgical menopause and fed a WSD for 2 years. Half of the animals received estradiol-17ß (E), beginning immediately after OvH, while the other half received placebo. Animals in both groups showed an increase in body weight and a decrease in overall activity levels. These changes were associated with a rise in both daytime and nocturnal serum leptin concentrations, but there was no change in serum concentrations of either cortisol or dehydroepiandrosterone sulfate (DHEAS). These data suggest that 2 years of HT has little or no effect on locomotor activity or circadian hormone patterns in menopausal macaques fed an obesogenic diet.

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With the decline of ovarian steroids levels at menopause, many women experience an increase in anxiety and stress sensitivity. The locus coeruleus (LC), a central source of noradrenaline (NE), is activated by stress and is inhibited by ß-endorphin. Moreover, increased NE has been implicated in pathological anxiety syndromes. Hormone replacement therapy (HRT) in menopause appears to decrease anxiety and vulnerability to stress. Therefore, we questioned the effect of HRT on the inhibitory ß-endorphin innervation of the LC. In addition, we found that progesterone protects serotoninergic neurons in monkeys, leading us to question whether ovarian steroids are also neuroprotective in LC neurons in monkeys. Adult Rhesus monkeys (Macaca mulatta) were ovariectomized, and either treated with Silastic capsules that contained estradiol, estradiol+progesterone, progesterone alone or that were empty (ovariectomized; control). After 1month, the LC was obtained and processed for immunohistochemistry for ß-endorphin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). The density of ß-endorphin axons was determined with image analysis using ImageJ. The TUNEL-positive neurons were counted in the entire LC. Progesterone-alone significantly increased the density of the ß-endorphin axons in the LC (p<0.01). No significant differences between groups in the number of TUNEL-positive cells in the LC were found. In conclusion, we found that HRT increases the inhibitory influence of ß-endorphin in the LC, which could, in turn, contribute to reduce anxiety and increase stress resilience. In addition, we did not find compelling evidence of neurodegeneration or neuroprotection by HRT in the LC of Rhesus monkeys.

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Serotonin plays a key role in mood or affect, and dysfunction of the serotonin system has been linked to depression in humans and animal models. Depression appears prior to or coincident with overt symptoms of Alzheimer's disease (AD) in about 50% of patients, and some experts consider it a risk factor for the development of AD. In addition, AD is more prevalent in women, who also show increased incidence of depression. Indeed, it has been proposed that mechanisms underlying depression overlap the mechanisms thought to hasten AD. Women undergo ovarian failure and cessation of ovarian steroid production in middle age and the postmenopausal period correlates with an increase in the onset of depression and AD. This laboratory has examined the many actions of ovarian steroids in the serotonin system of non-human primates using a rhesus macaque model of surgical menopause with short or long-term estradiol (E) or estradiol plus progesterone (E+P) replacement therapy. In this mini-review, we present a brief synopsis of the relevant literature concerning AD, depression, and serotonin. We also present some of our data on serotonin neuron viability, the involvement of the caspase-independent pathway, and apoptosis-inducing factor in serotonin-neuron viability, as well as gene expression related to neurodegeneration and neuron viability in serotonin neurons from adult and aged surgical menopausal macaques. We show that ovarian steroids, particularly E, are crucial for serotonin neuron function and health. In the absence of E, serotonin neurons are endangered and deteriorating toward apoptosis. The possibility that this scenario may proceed or accompany AD in postmenopausal women seems likely.

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This study was initiated to determine whether the noradrenergic (NE) neurons of the locus coeruleus (LC) could mediate the stimulatory action of androgens on serotonin-related gene expression in male macaques. These experiments follow our observations that serotonin neurons lack androgen receptors (ARs), and yet respond to androgens. Male Japanese macaques (Macaca fuscata) were castrated for 5-7months and then treated for 3months with [1] placebo, [2] T (testosterone), [3] DHT (dihydrotestosterone; non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] FLUT (Flutamide; androgen antagonist) plus ATD (n=5/group). The noradrenergic (NE) innervation of the raphe was determined with immunolabeling of axons with an antibody to dopamine-ß-hydroxylase (DBH). Immunolabeling of tyrosine hydroxylase (TH) dendrites and corticotropin releasing hormone (CRH) axons innervating the LC was also determined. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. Androgen receptor (AR), estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) immunostaining was accomplished. Quantitative image analysis was applied and immunopositive neurons or axons with boutons were measured. Double-label of NE neurons for each receptor plus TH determined whether the receptors were localized in NE neurons. Androgens with or without aromatase activity significantly stimulated DBH axon density in the raphe (ANOVA, p=0.006), and LC dendritic TH (ANOVA, p<0.0001), similar to serotonin-related mRNA expression in the raphe. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups compared to placebo or FLUT+ATD-treated groups (ANOVA, p=0.0014). There was no difference in the number of positive-neurons stained for ERα or ERß. The CRH axon density in the LC was significantly reduced with aromatase inhibition, suggesting that CRH depends on estrogen, not androgens (ANOVA, p=0.0023). Double-immunohistochemistry revealed that NE neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, >80% of LC NE neurons contained ERα or ERß. In conclusion, the LC NE neurons may transduce the stimulatory effect of androgens on serotonin-related gene expression. Since LC NE neurons lack AR, the androgenic stimulation of dendritic TH and axonal DBH may be indirectly mediated by other neurons. Estrogen, either from metabolism of T or from de novo synthesis, appears necessary for robust CRH innervation of the LC, which differs from female macaques.

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With extended life spans in modern humans, menopause has become a significant risk factor for depression, anxiety, loss of cognitive functions, weight gain, metabolic disease, osteoporosis, cardiovascular disease, and neurodegenerative diseases. Clinical studies have found beneficial neural effects of ovarian steroid hormone therapy (HT) during the menopausal transition and data are emerging that it can be continued long term. To further understand molecular underpinnings of the clinical studies, we used quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to examine gene expression in the serotonergic dorsal raphe of old (>18 years) rhesus macaques, focusing on genes related to depression, cellular resilience, and neurodegenerative diseases. The animals were ovariectomized (Ovx, surgically menopausal) and subjected to either estradiol or estradiol plus progesterone HT, or to placebo, starting 2 months after Ovx and continuing for ∼ 4 years. Significant changes were observed in 36 of 48 genes examined that encode proteins supporting serotonin neurotransmission, synapse assembly, glutamate neurotransmission, DNA repair, chaperones, ubiquinases and transport motors, as well as genes encoding proteins that have potential to delay the onset of neuropathologies. The data reveal important gene targets for chronic HT that contribute to neural health. Alternatively, the loss of ovarian steroids may lead to loss of functions at the gene level that contribute to many of the observable neural deficits after menopause.

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We previously showed that tryptophan hydroxylase 2 (TPH2) and serotonin reuptake transporter (SERT) mRNAs are increased by the androgens, testosterone (T) and dihydrotestosterone (DHT) in serotonin neurons of male macaques. In addition, we observed that serotonin in axons of a terminal region were markedly decreased by aromatase inhibition and lack of estradiol (E) from metabolism of T. These observations implicated androgen receptors (AR) and estrogen receptors (ER) in the transduction of steroid hormone actions in serotonin neurons. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. We used single and double immunohistochemistry to quantitate and phenotypically localize AR, ERα and ERß in the dorsal raphe of male macaques. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with [1] placebo, [2] T, [3] DHT (non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] Flutamide (FLUT; androgen antagonist) plus ATD (n = 5/group). After single labeling of each receptor, quantitative image analysis was applied and receptor positive neurons were counted. Double-label of raphe neurons for each receptor plus TPH2 determined whether the receptors were localized in serotonin neurons. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups (p = 0.0014) compared to placebo or FLUT+ATD-treated groups. There was no difference in the number of positive-neurons stained for ERα or ERß⋅ Double-immunohistochemistry revealed that serotonin neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, approximately 40% of dorsal raphe serotonin neurons contained ERα or ERß⋅ In conclusion, the stimulatory effect of androgens on TPH2 and SERT mRNA expression is mediated indirectly by neighboring neurons contain AR. The stimulatory effect of E, derived from T metabolism, on serotonin transport is partially mediated directly via nuclear ERs.

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The administration of estradiol-17ß (E) to animal models after loss of ovarian steroid production has many beneficial effects on neural functions, particularly in the serotonin system in nonhuman primates (NHPs). E also has anorexic effects, although the mechanism of action is not well defined. In the US, obesity has reached epidemic proportions, and blame is partially directed at the Western style diet, which is high in fat and sugar. This study examined the interaction of E and diet in surgically menopausal nonhuman primates with a 2×2 block design. Marmosets (Callithrix jacchus; n=4/group) were placed on control-low fat diet (LFD; 14%kcal from fat) or high fat diet (HFD; 28%kcal from fat) 1month prior to ovariectomy (Ovx). Empty (placebo) or E-filled Silastic capsules were implanted immediately following Ovx surgery. Treatments extended 6months. The established groups were: placebo+LFD, E+LFD, placebo+HFD, or E+HFD. At necropsy, the brain was flushed with saline and harvested. The midbrain was dissected and a small block containing the dorsal raphe nucleus was processed for qRT-PCR using Evagreen (Biotinum). Genes previously found to impact serotonin neural functions were examined. Results were compared with 2-way ANOVA followed by Bonferroni post-hoc tests or Cohen's D analysis. There was a significant effect of treatment on tryptophan hydroxylase 2 (TPH2) across the groups (p=0.019). E stimulated TPH2 expression and HFD prevented E-stimulated TPH2 expression (p<0.01). Treatment differentially affected monoamine oxidase B (MAO-B) across the groups (p=0.05). E increased MAO-B with LFD, and this stimulatory effect was prevented by HFD (p<0.05). There was a significant difference between treatments in corticotrophin releasing factor-receptor 2 (CRF-R2) expression (p=0.012). E increased CRF-R2 and this stimulatory effect was blocked by HFD (p<0.01). Regardless of diet, E increased Fev mRNA (p=0.028) and decreased CRF-receptor 1 (CRF-R1) mRNA (p=0.04). HFD suppressed urocortin 1 (UCN1; stresscopin) expression (p=0.045) but E treatment had no effect. Monoamine oxidase A (MAO-A) was different due to treatment across the groups (p=0.028). MAO-A was increased in the E+HFD group (p<0.01) whereas previous studies showed E suppressed MAO-A in macaques. The serotonin reuptake transporter (SERT), the serotonin 1A receptor (5HT1A), estrogen receptor beta (ERß) and progestin receptor (PR) expressions were not different between groups. Estrogen receptor alpha (ERα) was undetectable. In summary, the data indicate that important actions of hormone therapy in the serotonin system may be lost in the context of a HFD.

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Clinical data suggest that atypical antipsychotics such as olanzapine (OLZ) induce significant metabolic changes that are serious side effects of their primary use. Since controlled human studies are problematic and rodent data may be poorly translatable, we have initiated development of a macaque model of OLZ-induced metabolic disease. In this preliminary feasibility study, we examined some metabolic effects of OLZ in a female macaque in the context of a standard low-calorie/fat monkey chow diet followed by a high-fat/sugar Western-style diet (WSD). A female Japanese macaque was administered OLZ (1.25 mg/day) for 6 months, with dietary changes at 2-month intervals as follows: OLZ+Restricted chow, OLZ+Unrestricted chow, OLZ+WSD, and placebo+WSD. Weight was assessed weekly. Glucose tolerance tests (GTT) and Dexascans were performed at baseline and every 2 months. Omental (OM) and subcutaneous (SQ) adipose tissue biopsies were obtained at baseline, after OLZ+Unrestricted chow and after OLZ+WSD to evaluate adipocyte size, lipolysis and insulin-stimulated free fatty acid uptake (FFA). A separate trial was conducted on 2 monkeys with 5 days of OLZ- or no-treatment followed by RT-PCR on rostral and medial basal hypothalamus. Weight increased on OLZ+Restricted chow and stabilized on OLZ+Unrestricted chow. OLZ+WSD did not significantly change the weight plateau. Weight declined upon withdrawal of OLZ with continued WSD. Body fat increased from 14% at baseline to 22%, 30%, 28% and 19% at 2, 4, 6 and 8 mo, respectively, indicating that body fat was elevated on OLZ regardless of diet and declined upon OLZ removal. Glucose tolerance and the insulin response during GTT were normal with OLZ+Restricted chow or OLZ+Unrestricted chow. Addition of WSD with OLZ impaired glucose clearance during GTT. Insulin remained in the normal range, but first phase insulin secretion was reduced. After removal of OLZ, but continued WSD, glucose clearance returned to normal, but this was associated with hyperinsulinemia. Adipocyte diameter was increased in OM and SQ fat by OLZ+chow and OLZ+WSD to a similar extent. (p<0.01, 2-way ANOVA). In OM, isoproterenol-stimulated lipolysis occurred at baseline. In both depots, isoproterenol-stimulated lipolysis occurred with OLZ+chow, but it was significantly blunted by addition of WSD (ANOVA p<0.0001; posthoc p<0.05). Insulin increased FFA uptake at baseline. OLZ +chow or OLZ+WSD increased basal FFA uptake and insulin-induced FFA uptake was blunted in both depots (posthoc p<0.05). There was a marked decrease in POMC gene expression, and increased AgRP and NPY expression in the hypothalamus. There was also a clear increase in serotonin (5HT) 2C, melanocortin (MCR4), and Leptin (LepR) receptor gene expression. These data support the hypotheses that OLZ acts on peripheral tissues as well as in the CNS; that changes in hypothalamic gene expression occur very rapidly and precede increased fat accumulation; that adipose tissue exhibits insulin resistance prior to alterations in GTT; that addition of WSD to OLZ precipitates hyperglycemia without an obvious insulin response; and that removal of OLZ and continued WSD resulted in normalized glucose clearance and elevated insulin. These data suggest complex and early responses to OLZ that may be exacerbated by WSD.

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Energy metabolism in humans is tuned to distinct sex-specific functions that potentially reflect the unique requirements in females for gestation and lactation, whereas male metabolism may represent a default state. These differences are the consequence of the action of sex chromosomes and sex-specific hormones, including estrogens and progesterone in females and androgens in males. In humans, sex-specific specialization is associated with distinct body-fat distribution and energy substrate-utilization patterns; i.e., females store more lipids and have higher whole-body insulin sensitivity than males, while males tend to oxidize more lipids than females. These patterns are influenced by the menstrual phase in females, and by nutritional status and exercise intensity in both sexes. This minireview focuses on sex-specific mechanisms in lipid and glucose metabolism and their regulation by sex hormones, with a primary emphasis on studies in humans and the most relevant pre-clinical model of human physiology, non-human primates.

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Estradiol (E) and progesterone (P) promote spinogenesis in several brain areas. Intracellular signaling cascades that promote spinogenesis involve RhoGTPases, glutamate signaling and synapse assembly. We found that in serotonin neurons, E ± P administration increases (a) gene and protein expression of RhoGTPases, (b) gene expression of glutamate receptors, and (c) gene expression of pivotal synapse assembly proteins. Therefore, in this study we determined whether structural changes in dendritic spines in the dorsal raphe follow the observed changes in gene and protein expression. Dendritic spines were examined with immunogold silver staining of a spine marker protein, postsynaptic density-95 (PSD-95) and with Golgi staining. In the PSD-95 study, adult Ovx monkeys received placebo, E, P, or E + P for 1 month (n = 3/group). Sections were immunostained for PSD-95 and the number of PSD-95-positive puncta was determined with stereology. E, P, and E + P treatment significantly increased the total number of PSD-95-positive puncta (ANOVA, P = 0.04). In the golgi study, adult Ovx monkeys received placebo, E or E + P for 1 month (n = 3-4) and the midbrain was golgi-stained. A total of 80 neurons were analyzed with Neurolucida software. There was a significant difference in spine density that depended on branch order (two-way ANOVA). E + P treatment significantly increased spine density in higher-order (3°-5°) dendritic branches relative to Ovx group (Bonferroni, P < 0.05). In summary, E + P leads to the elaboration of dendritic spines on dorsal raphe neurons. The ability of E to induce PSD-95, but not actual spines, suggests either a sampling or time lag issue. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and, in turn, increase serotonin neurotransmission throughout the brain.

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OBJECTIVE: To study the effect of moderate stress on corticotropin-releasing factor (CRF) components in the serotonergic midbrain region in a monkey model of functional hypothalamic amenorrhea. DESIGN: After characterization of stress sensitivity, monkeys were moved to a novel room and given 20% less chow for 5 days before euthanasia. SETTING: Primate research center. ANIMAL(S): Female cynomolgus macaques (Macaca fascicularis) characterized as highly stress resilient (HSR, n = 5), medium stress resilient (n = 4), or stress sensitive (SS, n = 4). INTERVENTION(S): Five days of diet in a novel room with unfamiliar conspecifics. MAIN OUTCOME MEASURE(S): Density of CRF axons in the serotonergic dorsal raphe nucleus; the number of urocortin 1 (UCN1) cells; the density of UCN1 axons; the expression of CRF receptor 1 (CRF-R1) and CRF-R2 in the dorsal raphe nucleus. RESULT(S): The CRF innervation was higher in HSR than in SS animals; UCN1 cell number was higher in HSR than in SS animals and UCN1 axon bouton density was not different; all opposite of nonstressed animals. The CRF-R1 was not different between the sensitivity groups, but CRF-R2 was higher in HSR than in SS animals. The relative expression of CRF-R1 and CRF-R2 was similar to nonstressed animals. CONCLUSION(S): The HSR animals respond to stress with an increase in CRF delivery to serotonin neurons. With stress, UCN1 transport decreases in HSR animals. The CRF receptor expression was similar with or without stress. These changes may contribute to resilience in HSR animals.

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Aggression in humans and animals has been linked to androgens and serotonin function. To further our understanding of the effect of androgens on serotonin and aggression in male macaques, we sought to manipulate circulating androgens and the activity of aromatase; and to then determine behavior and the endogenous availability of serotonin. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with (a) placebo; (b) testosterone (T); (c) T + Dutasteride (5a reductase inhibitor; AvodartTM); (d) T + Letrozole (nonsteroidal aromatase inhibitor; FemeraTM); (e) Flutamide + ATD (androgen antagonist plus steroidal aromatase inhibitor); or (f) dihydrotestosterone (DHT) + ATD (n = 5/group). Behavioral observations were made during treatments. At the end of the treatment period, each animal was sedated with propofol and administered a bolus of fenfluramine (5 mg/kg). Fenfluramine causes the release of serotonin proportional to endogenous availability and in turn, serotonin stimulates the secretion of prolactin. Therefore, serum prolactin concentrations reflect endogenous serotonin. Fenfluramine significantly increased serotonin/prolactin in all groups (p < .0001). Fenfluramine-induced serotonin/prolactin in the T-treated group was significantly higher than the other groups (p < .0001). Castration partially reduced the serotonin/prolactin response and Letrozole partially blocked the effect of T. Complete inhibition of aromatase with ATD, a noncompetitive inhibitor, significantly and similarly reduced the fenfluramine-induced serotonin/prolactin response in the presence or absence of DHT. Neither aggressive behavior nor yawning (indicators of androgen activity) correlated with serotonin/prolactin, but posited aromatase activity correlated significantly with prolactin (p < .0008; r² = 0.95). In summary, androgens induced aggressive behavior but they did not regulate serotonin. Altogether, the data suggest that aromatase activity supports serotonin production and that androgens increase aggression by another mechanism.

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Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. When stressed with a paradigm of relocation and diet for 60 days, or 2 menstrual cycles, highly stress resilient monkeys continue to ovulate during both stress cycles (HSR); medium stress resilient monkeys ovulate once (MSR) and stress sensitive monkeys do not ovulate for the entire 60 days (SS). This study examines serotonin-related gene expression in monkeys with different sensitivity to stress and exposed to 5 days of moderate stress. Monkeys were first characterized as HSR, MSR or SS. After resumption of menstrual cycles, each monkey was re-stressed for 5 days in the early follicular phase. The expression of 3 genes pivotal to serotonin neural function was assessed in the 3 groups of monkeys (n=4-5/group). Tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor mRNAs expression were determined at 4 morphological levels of the dorsal raphe nucleus with in situ hybridization (ISH) using digoxigenin-incorporated riboprobes. In addition, cFos was examined with immunohistochemistry. Positive pixel area and/or cell number were measured. All data were analyzed with ANOVA (3 groups) and with a t-test (2 groups). After 5 days of stress, TPH2, SERT, 5HT1A and cFos were significantly lower in the SS group than the HSR group (p<0.05, all). This pattern of expression was the same as the pattern observed in the absence of stress in previous studies. Therefore, the ratio of the HSR/SS expression of each serotonergic gene was calculated in the presence and absence of stress. There was little or no difference in the ratio of HSR/SS gene expression in the presence or absence of stress. Moreover, cFos expression indicates that overall, cell activation in the dorsal raphe nucleus and periaquaductal gray is lower in SS than HSR animals. These data suggest that the serotonin system may set the sensitivity or resilience of the individual, but serotonin-related gene expression may not rapidly respond to moderate stress in nonhuman primates.

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A body of knowledge implicates an increase in output from the locus ceruleus (LC) during stress. We questioned the innervation and function of the LC in our macaque model of Functional Hypothalamic Amenorrhea, also known as Stress-Induced Amenorrhea. Cohorts of macaques were initially characterized as highly stress resilient (HSR) or stress-sensitive (SS) based upon the presence or absence of ovulation during a protocol involving 2 menstrual cycles with psychosocial and metabolic stress. Afterwards, the animals were rested until normal menstrual cycles resumed and then euthanized on day 5 of a new menstrual cycle [a] in the absence of further stress; or [b] after 5 days of resumed psychosocial and metabolic stress. In this study, parameters of the LC were examined in HSR and SS animals in the presence and absence of stress (2×2 block design) using ICC and image analysis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of catecholamines; and the TH level was used to assess by inference, NE output. The pixel area of TH-positive dendrites extending outside the medial border of the LC was significantly increased by stress to a similar degree in both HSR and SS animals (p<0.0001). There is a significant CRF innervation of the LC. The positive pixel area of CRF boutons, lateral to the LC, was higher in SS than HSR animals in the absence of stress. Five days of moderate stress significantly increased the CRF-positive bouton pixel area in the HSR group (p<0.02), but not in the SS group. There is also a significant serotonin innervation of the LC. A marked increase in medial serotonin dendrite swelling and beading was observed in the SS+stress group, which may be a consequence of excitotoxicity. The dendrite beading interfered with analysis of axonal boutons. However, at one anatomical level, the serotonin-positive bouton area was obtained between the LC and the superior cerebellar peduncle. Serotonin-positive bouton pixel area was significantly higher in HSR than SS animals (p<0.04). There was no change in either group after 5 days of moderate stress. The ratio of serotonin/TH correlates with ovarian estrogen production with a sensitivity×stress interaction. Therefore, it appears that the serotonin system determines stress sensitivity and the NE system responds to stress. We hypothesize that elevated NE with low serotonin functionality ultimately leads to stress-induced infertility. In contrast, high serotonin functionality maintains ovulation in the presence of stress even with elevated NE.

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