Effects of chronic exposure to triclosan on reproductive and thyroid endpoints in the adult Wistar female rat.

Triclosan (TCS), an antibacterial, has been shown to be an endocrine disruptor in the rat. Previously, subchronic TCS treatment to female rats was found to advance puberty and potentiate the effect of ethinyl estradiol (EE) on uterine growth when EE and TCS were co-administered prior to weaning. In the pubertal study, a decrease in serum thyroxine (T4) concentrations with no significant change in serum thyroid-stimulating hormone (TSH) was also observed. The purpose of the present study was to further characterize the influence of TCS on the reproductive and thyroid axes of the female rat using a chronic exposure regimen. Female Wistar rats were exposed by oral gavage to vehicle control, EE (1 µg/kg), or TCS (2.35, 4.69, 9.375 or 37.5 mg/kg) for 8 months and estrous cyclicity monitored. Although a divergent pattern of reproductive senescence appeared to emerge from 5 to 11 months of age between controls and EE-treated females, no significant difference in cyclicity was noted between TCS-treated and control females. A higher % control females displayed persistent diestrus (PD) by the end of the study, whereas animals administered with positive control (EE) were predominately persistent estrus (PE). Thyroxine concentration was significantly decreased in TCS-administered 9.375 and 37.5 mg/kg groups, with no marked effects on TSH levels, thyroid tissue weight, or histology. Results demonstrate that a long-term exposure to TCS did not significantly alter estrous cyclicity or timing of reproductive senescence in females but suppressed T4 levels at a lower dose than previously observed.

Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits.

Brown adipose tissue (BAT) thermogenesis is critical to maintain homoeothermia and is centrally controlled via sympathetic outputs. Body temperature and BAT activity also impact energy expenditure, and obesity is commonly associated with decreased BAT capacity and sympathetic tone. Severely obese mice that lack leptin or its receptor (LepRb) show decreased BAT capacity, sympathetic tone, and body temperature and thus are unable to adapt to acute cold exposure (Trayhurn et al., 1976). LepRb-expressing neurons are found in several hypothalamic sites, including the dorsomedial hypothalamus (DMH) and median preoptic area (mPOA), both critical sites to regulate sympathetic, thermoregulatory BAT circuits. Specifically, a subpopulation in the DMH/dorsal hypothalamic area (DHA) is stimulated by fever-inducing endotoxins or cold exposure (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Using the retrograde, transsynaptic tracer pseudorabies virus (PRV) injected into the BAT of mice, we identified PRV-labeled LepRb neurons in the DMH/DHA and mPOA (and other sites), thus indicating their involvement in the regulation of sympathetic BAT circuits. Indeed, acute cold exposure induced c-Fos (as a surrogate for neuronal activity) in DMH/DHA LepRb neurons, and a large number of mPOA LepRb neurons project to the DMH/DHA. Furthermore, DMH/DHA LepRb neurons (and a subpopulation of LepRb mPOA neurons) project and synaptically couple to rostral raphe pallidus neurons, consistent with the current understanding of BAT thermoregulatory circuits from the DMH/DHA and mPOA (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Thus, these data present strong evidence that LepRb neurons in the DMH/DHA and mPOA mediate thermoregulatory leptin action.

Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons.

Leptin, the adipose-derived hormonal signal of body energy stores, acts via the leptin receptor (LepRb) on neurons in multiple brain regions. We previously identified LepRb neurons in the lateral hypothalamic area (LHA), which are distinct from neighboring leptin-regulated melanin-concentrating hormone (MCH)- or orexin (OX)-expressing cells. Neither the direct synaptic targets of LHA LepRb neurons nor their potential role in the regulation of other LHA neurons has been determined, however. We thus generated several adenoviral and transgenic systems in which cre recombinase promotes the expression of the tracer, WGA (wheat germ agglutinin), and used these in combination with LepRb(cre) mice to determine the neuronal targets of LHA LepRb neurons. This analysis revealed that, although some LHA LepRb neurons project to dopamine neurons in the ventral tegmental area, LHA LepRb neurons also densely innervate the LHA where they directly synapse with OX, but not MCH, neurons. Indeed, few other LepRb neurons in the brain project to the OX-containing region of the mouse LHA, and direct leptin action via LHA LepRb neurons regulates gene expression in OX neurons. These findings thus reveal a major role for LHA leptin action in the modulation of OX neurons, suggesting the importance of LHA LepRb neurons in the regulation of OX signaling that is crucial to leptin action and metabolic control.

Ventral tegmental area leptin receptor neurons specifically project to and regulate cocaine- and amphetamine-regulated transcript neurons of the extended central amygdala.

Leptin acts via its receptor (LepRb) to regulate neural circuits in concert with body energy stores. In addition to acting on a number of hypothalamic structures, leptin modulates the mesolimbic dopamine (DA) system. To determine the sites at which LepRb neurons might directly influence the mesolimbic DA system, we examined the distribution of LepRb neurons and their projections within mesolimbic brain regions. Although the ventral tegmental area (VTA) contains DA LepRb neurons, LepRb neurons are absent from the amygdala and striatum. Also, LepRb-EGFPf mice (which label projections from LepRb neurons throughout the brain) reveal that few LepRb neurons project to the nucleus accumbens (NAc). In contrast, the central amygdala (CeA) and its rostral extension receive copious projections from LepRb neurons. Indeed, LepRb-specific anterograde tracing demonstrates (and retrograde tracing confirms) that VTA LepRb neurons project to the extended CeA (extCeA) but not the NAc. Consistently, leptin promotes cAMP response element-binding protein phosphorylation in the extCeA, but not NAc, of leptin-deficient animals. Furthermore, transgenic mice expressing the trans-synaptic tracer wheat germ agglutinin in LepRb neurons reveal the innervation of CeA cocaine- and amphetamine-regulated transcript (CART) neurons by LepRb neurons, and leptin suppresses the increased CeA CART expression of leptin-deficient animals. Thus, LepRb VTA neurons represent a subclass of VTA DA neurons that specifically innervates and controls the extCeA; we hypothesize that these neurons primarily modulate CeA-directed behaviors.

Direct innervation of GnRH neurons by metabolic- and sexual odorant-sensing leptin receptor neurons in the hypothalamic ventral premammillary nucleus.

Leptin acts via its receptor (LepRb) on specific CNS neurons to signal the adequacy of long-term energy stores, thereby permitting the expenditure of resources on energy-intensive processes such as reproduction. The ventral premammillary nucleus of the hypothalamus (PMv), which has been implicated in the stimulation of gonadotropin release by olfactory cues, contains numerous LepRb neurons, suggesting a potential role for LepRb PMv neurons in transmitting both metabolic and odorant signals to the neuroendocrine reproductive system. Indeed, Fos immunoreactivity and electrophysiologic recordings revealed the direct activation of LepRb PMv neurons by leptin, and exposure to odors from mice of the opposite sex promoted Fos immunoreactivity (Fos-IR) in many LepRb PMv neurons. To determine the regions innervated by the LepRb PMv neurons, we used two novel cre-activated tract-tracing systems in Lepr(cre) animals; data from these systems and from standard tracing techniques revealed that LepRb PMv neurons project to a subset of the regions, including the preoptic area, that are innervated by the PMv as a whole. Furthermore, the retrograde accumulation in LepRb PMv neurons of a trans-synaptic tracer from GnRH neurons revealed the direct innervation of GnRH neurons by many LepRb PMv neurons. Thus, LepRb PMv neurons sense metabolic and sexual odorant cues and project to the rostral hypothalamus to directly innervate GnRH neurons. These results are consistent with a role for LepRb PMv neurons in regulating the reproductive axis in response to metabolic and odorant stimuli.

Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function.

The adipose-derived hormone, leptin, acts via its receptor (LRb) to convey the status of body energy stores to the brain, decreasing feeding and potentiating neuroendocrine energy expenditure. The failure of high levels of leptin in most obese individuals to promote weight loss defines a state of diminished responsiveness to increased leptin, termed leptin resistance. Leptin stimulates the phosphorylation of several tyrosine residues on LRb to mediate leptin action. We homologously replaced LRb in mice with a receptor with a mutation in one of these sites (Tyr985) in order to examine its role in leptin action and signal attenuation in vivo. Mice homozygous for this mutation are neuroendocrinologically normal, but females demonstrate decreased feeding, decreased expression of orexigenic neuropeptides, protection from high-fat diet-induced obesity, and increased leptin sensitivity in a sex-biased manner. Thus, leptin activates autoinhibitory signals via LRb Tyr985 to attenuate the anti-adiposity effects of leptin, especially in females, potentially contributing to leptin insensitivity in obesity.

A potential role for hypothalamomedullary POMC projections in leptin-induced suppression of food intake.

Melanocortin-3/4 receptor ligands administered to the caudal brain stem potently modulate food intake by changing meal size. The origin of the endogenous ligands is unclear, because the arcuate nucleus of the hypothalamus and the nucleus of the solitary tract (NTS) harbor populations of proopiomelanocortin (POMC)-expressing neurons. Here we demonstrate that activation of hypothalamic POMC neurons leads to suppression of food intake and that this suppression is prevented by administration of a melanocortin-3/4 receptor antagonist to the NTS and its vicinity. Bilateral leptin injections into the rat arcuate nucleus produced long-lasting suppression of meal size and total chow intake. These effects were significantly blunted by injection of SHU-9119 into the fourth ventricle, although SHU-9119 increased meal size and food intake during the first, but not the second, 14-h observation period. Leptin effects on meal size and food intake were abolished throughout the 40-h observation period by injection of SHU-9119 into the NTS at a dose that by itself had no effect. Neuron-specific tracing from the arcuate nucleus with a Cre-inducible tract-tracing adenovirus in POMC-Cre mice showed the presence of labeled axons in the NTS. Furthermore, density of alpha-melanocyte-stimulating hormone-immunoreactive axon profiles throughout the NTS was decreased by approximately 70% after complete surgical transection of connections with the forebrain in the chronic decerebrate rat model. The results further support the existence of POMC projections from the hypothalamus to the NTS and suggest that these projections have a functional role in the control of food intake.

Urinary metalloproteinases: noninvasive biomarkers for breast cancer risk assessment.

Matrix metalloproteinases (MMP) and a disintegrin and metalloprotease 12 (ADAM 12) can be detected in the urine of breast cancer patients and provide independent prediction of disease status. To evaluate the potential of urinary metalloproteinases as biomarkers to predict breast cancer risk status, urine samples from women with known risk marker lesions, atypical hyperplasia and lobular carcinoma in situ (LCIS), were analyzed. Urine samples were obtained from 148 women: 44 women with atypical hyperplasia, 24 women with LCIS, and 80 healthy controls. MMP analysis was done using gelatin zymography and ADAM 12 analysis was done via immunoblotting with monospecific antibodies and subsequent densitometric measurement. Positive urinary MMP-9 levels indicated a 5-fold risk of atypical hyperplasia and >13-fold risk of LCIS compared with normal controls. Urinary ADAM 12 levels were significantly elevated in women with atypical hyperplasia and LCIS from normal controls, with receiver operating characteristic curve analysis showing an area under the curve of 0.914 and 0.950, respectively. To assess clinical applicability, a predictive index was developed using ADAM 12 in conjunction with Gail risk scores for women with atypia. Scores above 2.8 on this ADAM 12-Gail risk prediction index score are predictive of atypical hyperplasia (sensitivity, 0.976; specificity, 0.977). Our data suggest that the noninvasive detection and analysis of urinary ADAM 12 and MMP-9 provide important clinical information for use as biomarkers in the identification of women at increased risk of developing breast cancer.

The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat.

Triclosan (TCS), an antibacterial, has been shown to be an endocrine disruptor in the rat. We reported previously that TCS potentiated the estrogenic effect of ethinyl estradiol (EE) on uterine growth in rats exposed to EE and TCS in the uterotrophic assay, whereas TCS alone had no effect. To further characterize this potentiation, we evaluated the effect of co-exposure with lower doses of EE that are comparable to the concentrations in hormone replacement regimens and began to assess the mechanisms by which this potentiation occurs. Changes in uterine weight, epithelial cell growth, and estrogen-sensitive gene expression were assessed. TCS expectedly enhanced the uterotrophic response to EE, however at significantly lower doses of EE. Similarly, TCS increased the EE-induced stimulation of epithelial cell height following cotreatment. Cotreatment also enhanced the estrogen-induced change in gene expression, which was reversed with an ER antagonist. Furthermore, the TCS-induced potentiation was independent of ER activation, as no effects were observed in the ER TA assay.

Molecular mapping of the neural pathways linking leptin to the neuroendocrine reproductive axis.

Negative energy balance and insufficient adipose energy stores decrease the production of leptin, thereby diminishing the leptin-supported secretion of GnRH from the hypothalamus and promoting decreased reproductive function. Leptin acts via its receptor (LepRb) to support the neuroendocrine reproductive axis, but the nature and location of the relevant LepRb neurons remain poorly understood. Possibilities include the direct or indirect action of leptin on hypothalamic GnRH neurons, or on kisspeptin (Kiss1) neurons that are major regulators of GnRH neurons. To evaluate these potential mechanisms, we employed immunohistochemical analysis of the female brain from various molecular mouse models and sheep. Our analysis revealed no LepRb in GnRH neurons or in anteroventral periventricular Kiss1 neurons, and very limited (0-6%) colocalization with arcuate nucleus Kiss1 cells, suggesting that leptin does not modulate reproduction by direct action on any of these neural populations. LepRb neurons, primarily in the hypothalamic ventral premammillary nucleus and a subregion of the preoptic area, lie in close contact with GnRH neurons, however. Furthermore, an unidentified population or populations of LepRb neurons lie in close contact with arcuate nucleus and anteroventral periventricular Kiss1 neurons. Taken together, these findings suggest that leptin communicates with the neuroendocrine reproductive axis via multiple populations of LepRb neurons that lie afferent to both Kiss1 and GnRH neurons.

Leptin does not directly affect CNS serotonin neurons to influence appetite.

Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sert(cre)) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sert(cre) promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sert(cre)-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.

Hyperphagia and obesity in female mice lacking tissue inhibitor of metalloproteinase-1.

Certain matrix metalloproteinases and their regulators, the tissue inhibitors of metalloproteinases (TIMPs), are involved in development and remodeling of adipose tissue. In studying Timp1() mice, which have a null mutation in Timp1 (Timp1(-/-)), we observed that females exhibit increased body weight by 3 months of age due to increased total body lipid and adipose tissue. Whereas Timp1(-/-) mice have increased size and number of adipocytes, they also display increased food intake despite hyperleptinemia, suggesting that alterations in hypothalamic leptin action or responsiveness may underlie their weight gain. Indeed, leptin promotes the expression of Timp1 mRNA in the hypothalamus, and leptin signaling via signal transducer and activator of transcription-3 mediates the expression of hypothalamic Timp1. Furthermore, Timp1(-/-) mice demonstrate increased food intake and altered expression of certain hypothalamic neuropeptide genes prior to elevated weight gain. Thus, whereas previous data suggested roles for matrix metalloproteinases and TIMPs in the regulation of adipose tissue, these data reveal that Timp1 mRNA is induced by leptin in the hypothalamus and that expression and action of Timp1 contributes to the regulation of feeding and energy balance.

Leptin acts via leptin receptor-expressing lateral hypothalamic neurons to modulate the mesolimbic dopamine system and suppress feeding.

The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area (VTA) and other components of the mesolimbic dopamine (DA) system to control motivation, including the incentive to feed. The anorexigenic hormone leptin modulates the mesolimbic DA system, although the mechanisms underlying this control have remained incompletely understood. We show that leptin directly regulates a population of leptin receptor (LepRb)-expressing inhibitory neurons in the LHA and that leptin action via these LHA LepRb neurons decreases feeding and body weight. Furthermore, these LHA LepRb neurons innervate the VTA, and leptin action on these neurons restores VTA expression of the rate-limiting enzyme in DA production along with mesolimbic DA content in leptin-deficient animals. Thus, these findings reveal that LHA LepRb neurons link anorexic leptin action to the mesolimbic DA system.

Loss of cytokine-STAT5 signaling in the CNS and pituitary gland alters energy balance and leads to obesity.

Signal transducers and activators of transcription (STATs) are critical components of cytokine signaling pathways. STAT5A and STAT5B (STAT5), the most promiscuous members of this family, are highly expressed in specific populations of hypothalamic neurons in regions known to mediate the actions of cytokines in the regulation of energy balance. To test the hypothesis that STAT5 signaling is essential to energy homeostasis, we used Cre-mediated recombination to delete the Stat5 locus in the CNS. Mutant males and females developed severe obesity with hyperphagia, impaired thermal regulation in response to cold, hyperleptinemia and insulin resistance. Furthermore, central administration of GM-CSF mediated the nuclear accumulation of STAT5 in hypothalamic neurons and reduced food intake in control but not in mutant mice. These results demonstrate that STAT5 mediates energy homeostasis in response to endogenous cytokines such as GM-CSF.

The role of leptin in the regulation of neuroendocrine function and CNS development.

Leptin, a hormone produced by adipocytes in proportion to fat stores, signals the sufficiency of energy reserves to the brain to control feeding and metabolism. Leptin represents a vital link between metabolic and neuroendocrine pathways, and adequate circulating leptin levels are required to permit the expenditure of energy on reproduction, growth, and other energy-intensive endocrine outputs. Leptin mediates its effects by acting upon a distributed network of CNS neurons that express the signaling form of the leptin receptor (LRb). Nutritional status early in development influences a lifelong metabolic program that modulates risk for diabetes, obesity and other elements of the metabolic syndrome. Recent evidence has demonstrated a number of important roles for leptin in the regulation of neural development and metabolic programming. In this review, we discuss leptin action, the neural circuits on which leptin acts, and our nascent understanding of how early leptin exposure may influence neural development and the predisposition to metabolic diseases.