Leptin mediates the increase in blood pressure associated with obesity.

Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin's effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity.

A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.

Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity.

The neuronal circuits involved in the regulation of feeding behavior and energy expenditure are soft-wired, reflecting the relative activity of the postsynaptic neuronal system, including the anorexigenic proopiomelanocortin (POMC)-expressing cells of the arcuate nucleus. We analyzed the synaptic input organization of the melanocortin system in lean rats that were vulnerable (DIO) or resistant (DR) to diet-induced obesity. We found a distinct difference in the quantitative and qualitative synaptology of POMC cells between DIO and DR animals, with a significantly greater number of inhibitory inputs in the POMC neurons in DIO rats compared with DR rats. When exposed to a high-fat diet (HFD), the POMC cells of DIO animals lost synapses, whereas those of DR rats recruited connections. In both DIO rats and mice, the HFD-triggered loss of synapses on POMC neurons was associated with increased glial ensheathment of the POMC perikarya. The altered synaptic organization of HFD-fed animals promoted increased POMC tone and a decrease in the stimulatory connections onto the neighboring neuropeptide Y (NPY) cells. Exposure to HFD was associated with reactive gliosis, and this affected the structure of the blood-brain barrier such that the POMC and NPY cell bodies and dendrites became less accessible to blood vessels. Taken together, these data suggest that consumption of an HFD has a major impact on the cytoarchitecture of the arcuate nucleus in vulnerable subjects, with changes that might be irreversible due to reactive gliosis.

Leptin action in the dorsomedial hypothalamus increases sympathetic tone to brown adipose tissue in spite of systemic leptin resistance.

Leptin regulates body weight in mice by decreasing appetite and increasing sympathetic nerve activity (SNA), which increases energy expenditure in interscapular brown adipose tissue (iBAT). Diet-induced obese mice (DIO) are resistant to the anorectic actions of leptin. We evaluated whether leptin still stimulated sympathetic outflow in DIO mice. We measured iBAT temperature as a marker of SNA. We found that obese hyperleptinemic mice have higher iBAT temperature than mice on regular diet. Conversely, obese leptin-deficient ob/ob mice have lower iBAT temperature. Additionally, leptin increased SNA in obese (DIO and ob/ob) and control mice, despite DIO mice being resistant to anorectic action of leptin. We demonstrated that neurons in the dorsomedial hypothalamus (DMH) of DIO mice mediate the thermogenic responses to hyperleptinemia in obese mammals because blockade of leptin receptors in the DMH prevented the thermogenic effects of leptin. Peripheral Melotan II (MTII) injection increased iBAT temperature, but it was blunted by blockade of DMH melanocortin receptors (MC4Rs) by injecting agouti-related peptide (AgRP) directly into the DMH, suggesting a physiological role of the DMH on temperature regulation in animals with normal body weight. Nevertheless, obese mice without a functional melanocortin system (MC4R KO mice) have an increased sympathetic outflow to iBAT compared with their littermates, suggesting that higher leptin levels drive sympathoexcitation to iBAT by a melanocortin-independent pathway. Because the sympathetic nervous system contributes in regulating blood pressure, heart rate, and hepatic glucose production, selective leptin resistance may be a crucial mechanism linking adiposity and metabolic syndrome.

Diet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons.

Despite high leptin levels, most obese humans and rodents lack responsiveness to its appetite-suppressing effects. We demonstrate that leptin modulates NPY/AgRP and alpha-MSH secretion from the ARH of lean mice. High-fat diet-induced obese (DIO) mice have normal ObRb levels and increased SOCS-3 levels, but leptin fails to modulate peptide secretion and any element of the leptin signaling cascade. Despite this leptin resistance, the melanocortin system downstream of the ARH in DIO mice is over-responsive to melanocortin agonists, probably due to upregulation of MC4R. Lastly, we show that by decreasing the fat content of the mouse's diet, leptin responsiveness of NPY/AgRP and POMC neurons recovered simultaneously, with mice regaining normal leptin sensitivity and glycemic control. These results highlight the physiological importance of leptin sensing in the melanocortin circuits and show that their loss of leptin sensing likely contributes to the pathology of leptin resistance.

Abdominal fat analyzed by DEXA scan reflects visceral body fat and improves the phenotype description and the assessment of metabolic risk in mice.

Clinical studies have demonstrated a strong relationship between visceral fat content and metabolic diseases, such as type 2 diabetes and liver steatosis. Obese mouse models are an excellent tool to study metabolic diseases; however, there are limited methods for the noninvasive measurement of fat distribution in mice. Although micromagnetic resonance imaging and microcomputed tomography are the "gold standards" in the measurement of fat distribution, more economical and accessible methods are required. Dual energy X-ray absorptiometry (DEXA) is an effective method in characterizing fat content; however, it cannot discriminate between visceral and subcutaneous fat depots. We demonstrate that an evaluation of abdominal fat content measured by DEXA through the selection of one localized abdominal area strongly correlates with visceral fat content in C57BL/6J mice. We found that DEXA is able to measure fat pad volume ex vivo with high accuracy; however, the measurement of visceral fat in vivo shows an overestimation caused by subcutaneous tissue interference. The overestimation is almost constant for a wide range of values, and thus it is possible to correct the data for a more accurate estimation of visceral fat content. We demonstrate the utility of this technique in characterizing phenotypes of several obese mouse models (ob/ob, db/db, MC4R-KO, and DIO) and evaluating the effect of treatments on visceral fat content in longitudinal studies. Additionally, we also establish abdominal obesity as a potential biomarker for metabolic abnormalities (liver fat accumulation, insulin resistance/diabetes) in mice, similar to that described in humans.

Changes of dipeptidyl peptidase IV (DPP-IV) in obese children with weight loss: relationships to peptide YY, pancreatic peptide, and insulin sensitivity.

AIM: Gastrointestinal (GI) hormones are involved in satiety regulation and in glucose metabolism. Most GI hormones are hydrolyzed and inactivated by the same enzyme, dipeptidyl peptidase IV (DPP-IV). We analyzed changes of DPP-IV after weight loss in obese children and its relationships to the GI hormones pancreatic peptide (PP), peptide YY (PYY), and insulin sensitivity. METHODS: We measured at baseline and one year later anthropometrics, percentage body fat based on skinfold thickness, DPP-IV, PP, PYY, insulin, and glucose concentrations in 18 obese children (mean age 10.9 years, 44% male, mean BMI 28.5 kg/m2) who participated in a one-year lifestyle intervention program based on physical activity, nutrition course, and behavioral therapy. Insulin sensitivity was calculated using QUICKI. RESULTS: Changes of DPP-IV correlated significantly to the changes of percentage body fat (r = 0.47) and BMI SDS (r = 0.60). In partial regression analysis adjusted for change in weight status, changes of DPP-IV correlated significantly to changes of PYY (r = -0.43), PP (r = -0.49), QUICKI (r = -0.53), and insulin (r = 0.57). The 10 children with substantial weight loss significantly reduced their DPP-IV and insulin concentrations, while QUICKI, PYY, and PP levels significantly increased. In children without substantial weight loss no significant changes were observed. CONCLUSIONS: These findings suggest that the increase of fasting PP and PYY in weight loss is influenced at least in part by a decrease of their cleavage enzyme DPP-IV. Further research is necessary to evaluate the mechanisms in weight loss leading to a decrease of DPP-IV activity and consequently to an improvement of insulin sensitivity.

Regulation of agouti-related protein messenger ribonucleic acid transcription and peptide secretion by acute and chronic inflammation.

Agouti-related protein (AgRP) is an orexigenic neuropeptide produced by neurons in the hypothalamic arcuate nucleus (ARC) that is a key component of central neural circuits that control food intake and energy expenditure. Disorders in energy homeostasis, characterized by hypophagia and increased metabolic rate, frequently develop in animals with either acute or chronic diseases. Recently, studies have demonstrated that proopiomelanocortin-expressing neurons in the ARC are activated by the proinflammatory cytokine IL-1beta. In the current study, we sought to determine whether inflammatory processes regulate the expression of AgRP mRNA and to characterize the response of AgRP neurons to IL-1beta. Here, we show by real-time RT-PCR and in situ hybridization analysis that AgRP mRNA expression in rodents is increased in models of acute and chronic inflammation. AgRP neurons were found to express the type I IL-1 receptor, and the percentage of expression was significantly increased after peripheral administration of lipopolysaccharide. Furthermore, we demonstrate that IL-1beta inhibits the release of AgRP from hypothalamic explants. Collectively, these data indicate that proinflammatory signals decrease the secretion of AgRP while increasing the transcription of the AgRP gene. These observations suggest that AgRP neurons may participate with ARC proopiomelanocortin neurons in mediating the anorexic and metabolic responses to acute and chronic disease processes.

Regulation of central melanocortin signaling by interleukin-1 beta.

Anorexia and involuntary weight loss are common and debilitating complications of a number of chronic diseases and inflammatory states. Proinflammatory cytokines, including IL-1 beta, are hypothesized to mediate these responses through direct actions on the central nervous system. However, the neural circuits through which proinflammatory cytokines regulate food intake and energy balance remain to be characterized. Here we report that IL-1 beta activates the central melanocortin system, a key neuronal circuit in the regulation of energy homeostasis. Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) were found to express the type I IL-1 receptor. Intracerebroventricular injection of IL-1 beta induced the expression of Fos protein in ARC POMC neurons but not in POMC neurons in the commissural nucleus of the tractus solitarius. We further show that IL-1 beta increases the frequency of action potentials of ARC POMC neurons and stimulates the release of alpha-MSH from hypothalamic explants in a dose-dependent fashion. Collectively, our data support a model in which IL-1 beta increases central melanocortin signaling by activating a subpopulation of hypothalamic POMC neurons and stimulating their release of alpha-MSH.

Peptide YY(3-36) inhibits morning, but not evening, food intake and decreases body weight in rhesus macaques.

Peptide YY(3-36) [PYY(3-36)] is a hormone that is released after meal ingestion that is currently being investigated for the treatment of obesity; however, there are conflicting reports of the effects of PYY(3-36) on energy balance in rodent models. To shed light on this controversy, we studied the effect of PYY(3-36) on food intake and body weight in a nonhuman primate. Intravenous PYY(3-36) infusions before a morning meal transiently suppressed the rate of food intake but did not suppress the evening meal or 24-h intake. Twice-daily or continuous intravenous PYY(3-36) infusions to supraphysiological levels (levels that exceeded normal physiological levels) again suppressed the rate of feeding for the morning but not the evening meal. Twice-daily intravenous PYY(3-36) infusions for 2 weeks significantly decreased body weight in all test animals (average weight loss 1.9%) without changing insulin response to glucose infusion. These results show that endogenous PYY(3-36) may alter morning but not evening meal intake, and supraphysiological doses are required for effective suppression of food intake.

α-MSH Stimulates Glucose Uptake in Mouse Muscle and Phosphorylates Rab-GTPase-Activating Protein TBC1D1 Independently of AMPK.

The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate -MSH stimulation in both wild type and AMPK deficient mice. We found that -MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that -MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation.

α-Melanocyte stimulating hormone promotes muscle glucose uptake via melanocortin 5 receptors.

OBJECTIVE: Central melanocortin pathways are well-established regulators of energy balance. However, scant data exist about the role of systemic melanocortin peptides. We set out to determine if peripheral α-melanocyte stimulating hormone (α-MSH) plays a role in glucose homeostasis and tested the hypothesis that the pituitary is able to sense a physiological increase in circulating glucose and responds by secreting α-MSH. METHODS: We established glucose-stimulated α-MSH secretion using humans, non-human primates, and mouse models. Continuous α-MSH infusions were performed during glucose tolerance tests and hyperinsulinemic-euglycemic clamps to evaluate the systemic effect of α-MSH in glucose regulation. Complementary ex vivo and in vitro techniques were employed to delineate the direct action of α-MSH via the melanocortin 5 receptor (MC5R)-PKA axis in skeletal muscles. Combined treatment of non-selective/selective phosphodiesterase inhibitor and α-MSH was adopted to restore glucose tolerance in obese mice. RESULTS: Here we demonstrate that pituitary secretion of α-MSH is increased by glucose. Peripheral α-MSH increases temperature in skeletal muscles, acts directly on soleus and gastrocnemius muscles to significantly increase glucose uptake, and enhances whole-body glucose clearance via the activation of muscle MC5R and protein kinase A. These actions are absent in obese mice, accompanied by a blunting of α-MSH-induced cAMP levels in skeletal muscles of obese mice. Both selective and non-selective phosphodiesterase inhibition restores α-MSH induced skeletal muscle glucose uptake and improves glucose disposal in obese mice. CONCLUSION: These data describe a novel endocrine circuit that modulates glucose homeostasis by pituitary α-MSH, which increases muscle glucose uptake and thermogenesis through the activation of a MC5R-PKA-pathway, which is disrupted in obesity.

Peptide YY is a regulator of energy homeostasis in obese children before and after weight loss.

CONTEXT: The gut hormone peptide YY(3-36) (PYY) reduces food intake via hypothalamic Y2 receptors in the brain. There is not much known about PYY in obese children. OBJECTIVE: The objective of this study was to investigate the role of PYY in the metabolic changes in obese children and its change during weight loss. SETTING: The study was performed at a university medical center. PARTICIPANTS: We studied 73 obese children and 45 age-matched normal-weight children. INTERVENTIONS: We determined fasting serum total PYY and leptin by RIA in obese and normal-weight children. Fasting PYY was also measured in 28 obese children before and after completion of a 1-yr outpatient weight reduction program. MAIN OUTCOME MEASURES: PYY, insulin, and body mass index were the main outcome measures. RESULTS: Obese children demonstrated significantly lower PYY levels than lean children (median, 67 vs. 124 pg/ml; P < 0.001). Fasting PYY correlated negatively to the degree of overweight. PYY levels did not differ significantly between boys and girls, nor between prepubertal and pubertal children. The group of patients participating in the outpatient weight reduction program was divided into four quartiles according to their changes in body mass index SD score over a 1-yr period. PYY increased significantly in patients with the most effective weight loss, but decreased in the subgroup of children with weight gain. CONCLUSIONS: PYY is negatively correlated to the degree of overweight, with reduced values in obese compared with normal-weight children. Decreased PYY levels could predispose subjects to develop obesity. Our results indicate that low pretreatment PYY levels that increase during weight loss may be a predictor of maintained weight loss.

The electrophysiology of feeding circuits.

Obesity is quickly becoming one of the most common and debilitating disorders of the developed world. More than 60% of American adults are now overweight or obese, predisposing them to a host of chronic diseases. To understand the etiology of obesity, and to discover new therapies for obesity, we must understand the components of energy balance. In simple terms, energy intake (feeding) must equal energy expenditure (physical activity, basal metabolism and adaptive thermogenesis) for body weight homeostasis. To maintain homeostasis, neurocircuitry must sense both immediate nutritional status and the amount of energy stored in adipose tissue, and must be able to provide appropriate output to balance energy intake and energy expenditure. The brain receives various signals that carry information about nutritional and metabolic status including neuropeptide PYY(3-36), ghrelin, cholecystokinin, leptin, glucose and insulin. Circulating satiety signals access the brain either by "leakage" across circumventricular organs or transport across the blood-brain barrier. Signals can also activate sensory vagal terminals that innervate the whole gastrointestinal tract.

A talk between fat tissue, gut, pancreas and brain to control body weight.

The incidence of obesity and its related disorders are increasing at a rate of pandemic proportions. Understanding the mechanisms behind the maintenance of energy balance is fundamental in developing treatments for clinical syndromes including obesity and diabetes. A neural network located in the nucleus of the solitary tract-area postrema complex in the hindbrain and the hypothalamus in the forebrain has long been implicated in the control of energy balance. In the hypothalamus this central neuronal network consists of small populations of nuclei with distinct functions such as the arcuate nucleus (ARH), the paraventricular nuclei of the hypothalamus (PVH), the dorsomedial (DMH), the ventromedial (VMH) and the lateral hypothalamus (LH). These hypothalamic areas form interconnected neuronal circuits that respond to fluctuations in energy status by altering the expression of neuropeptides, leading to changes in energy intake and expenditure. Regulation of these hypothalamic nuclei involves the actions of orexigenic peptides (ie ghrelin), which act to stimulate energy intake and decrease energy expenditure, and anorexigenic peptides (ie. leptin and insulin), which act to reduce energy intake and stimulate energy expenditure. Here we review the role of the ARH, DMH and PVH in the control of energy homeostasis and how recent advances in research technologies (Cre-loxP technology, optogenetics and pharmacogenetics) have shed light on the role of these hypothalamic nuclei in the control of energy balance. Such novel findings include the implication of ARH POMC and AgRP neurons in the browning of white adipose tissue to regulate energy expenditure as well as the likely existence of divergent hypothalamic pathways in the DMH and PVH in the control of food intake and energy expenditure.

Augmented serum levels of the IGF-I/IGF-binding protein-3 ratio in pre-menopausal patients with type I breast cysts.

OBJECTIVE: Gross cystic disease (GCD) is the most common benign breast pathology. Although breast cysts are not considered pre-malignant lesions, an increased risk of breast cancer has been reported for patients with type I cysts (Na(+)/K(+)<3). Furthermore, an augmented IGF-I/IGF-binding protein-3 (IGFBP-3) ratio has been described in breast cancer patients. The objective was to evaluate serum IGF-I and binding protein concentrations of type I and type II cyst patients as compared with healthy women. METHODS: Twenty-four patients with type I cysts, 17 with type II cysts and 25 healthy women were evaluated. Serum IGF-I, IGFBP-3 and IGFBP-1 concentrations were measured by IRMA. RESULTS: IGF-I concentrations were significantly higher in sera from patients with type I cysts than in patients with type II cysts. A highly significant decrease of IGFBP-3, the major IGFBP, was found in patients with type I cysts with respect to healthy women, whereas no significant difference was evident between the different cyst types. The IGF-I/IGFBP-3 ratio, an estimate of biologically active IGF-I, was very significantly higher in patients with type I cysts than in both type II patients and healthy women. IGFBP-1 levels were significantly lower in patients with type I than in controls and type II cysts. The IGF-I/IGFBP-1 ratio was significantly higher in patients with type I cysts than in type II bearers and healthy women. Estrogen levels correlated with IGF-I in patients and controls. CONCLUSIONS: The enhanced levels of IGF-I/IGFBP-3 found in patients with type I cysts could eventually be associated with the increased risk of breast cancer described for this group.

Estradiol prevents fat accumulation and overcomes leptin resistance in female high-fat diet mice.

In premenopausal and menopausal women in particular, suboptimal estrogens have been linked to the development of the metabolic syndrome as major contributors to fat accumulation. At the same time, estrogens have been described to have a role in regulating body metabolic status. We evaluated how endogenous or administered estrogens impact on the changes associated with high-fat diet (HFD) consumption in 2 different paradigms; ovarian-intact and in ovariectomized mice. When estradiol (E2) was cyclically administered to ovarian-intact HFD-fed mice for 12 weeks, animals gained significantly less weight than ovarian-intact vehicle controls (P < .01). This difference was mainly due to a reduced caloric intake but not to an increase in energy expenditure or locomotor activity. This E2 treatment regime to mice exposed to HFD was overall able to avoid the increase of visceral fat content to levels of those found in mice fed a regular chow diet. In the ovariectomized model, the main body weight and fat content reducing action of E2 was not only through decreasing food intake but also by increasing the whole-body energy expenditure, locomotor activity, and by inducing fat oxidation. Importantly, these animals became responsive to the anorexigenic effects of leptin in contrast to the vehicle-treated and the pair-fed control groups (P < .01). Further, in vitro hypothalamic secretion experiments revealed that treatment of obese mice with E2 is able to modulate the secretion of appetite-regulating neuropeptides; namely, E2 increased the secretion of the anorectic neuropeptide α-melanocyte-stimulating hormone and decreased the secretion of the orexigenic neuropetides neuropeptide Y and Agouti-related peptide. In conclusion, differences in response to E2 treatment of HFD-fed animals depend on their endogenous estrogenic status. Overall, E2 administration overcomes arcuate leptin resistance and partially prevents fat accumulation on these mice.

Excess of nerve growth factor in the ovary causes a polycystic ovary-like syndrome in mice, which closely resembles both reproductive and metabolic aspects of the human syndrome.

Polycystic ovarian syndrome (PCOS), the most common female endocrine disorder of unknown etiology, is characterized by reproductive abnormalities and associated metabolic conditions comprising insulin resistance, type 2 diabetes mellitus, and dyslipidemia. We previously reported that transgenic overexpression of nerve growth factor (NGF), a marker of sympathetic hyperactivity, directed to the ovary by the mouse 17α-hydroxylase/C17-20 lyase promoter (17NF mice), results in ovarian abnormalities similar to those seen in PCOS women. To investigate whether ovarian overproduction of NGF also induces common metabolic alterations of PCOS, we assessed glucose homeostasis by glucose tolerance test, plasma insulin levels, and body composition by dual-energy x-ray absorptiometry scan in young female 17NF mice and wild-type mice. 17NF mice exhibited increased body weight and alterations in body fat distribution with a greater accumulation of visceral fat compared with sc fat (P < .01). 17NF mice also displayed glucose intolerance (P < .01), decreased insulin-mediated glucose disposal (P < .01), and hyperinsulinemia (P < .05), which, similar to PCOS patients, occurred independently of body weight. Additionally, 17NF mice exhibited increased sympathetic outflow observed as increased interscapular brown adipose tissue temperature. This change was evident during the dark period (7 pm to 7 am) and occurred concomitant with increased interscapular brown adipose tissue uncoupling protein 1 expression. These findings suggest that overexpression of NGF in the ovary may suffice to cause both reproductive and metabolic alterations characteristic of PCOS and support the hypothesis that sympathetic hyperactivity may contribute to the development and/or progression of PCOS.