Astrocytes Amplify Neuronal Dendritic Volume Transmission Stimulated by Norepinephrine.

In addition to their support role in neurotransmitter and ion buffering, astrocytes directly regulate neurotransmission at synapses via local bidirectional signaling with neurons. Here, we reveal a form of neuronal-astrocytic signaling that transmits retrograde dendritic signals to distal upstream neurons in order to activate recurrent synaptic circuits. Norepinephrine activates α1 adrenoreceptors in hypothalamic corticotropin-releasing hormone (CRH) neurons to stimulate dendritic release, which triggers an astrocytic calcium response and release of ATP; ATP stimulates action potentials in upstream glutamate and GABA neurons to activate recurrent excitatory and inhibitory synaptic circuits to the CRH neurons. Thus, norepinephrine activates a retrograde signaling mechanism in CRH neurons that engages astrocytes in order to extend dendritic volume transmission to reach distal presynaptic glutamate and GABA neurons, thereby amplifying volume transmission mediated by dendritic release.

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic-pituitary axis.

Cushing's disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelson's syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary-adrenal axis in clinically normal dogs. Dogs were administered 5mg/kg trilostane twice a day every day for 8 weeks (n=8) or 16 weeks (n=3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushing's disease.

Fetal hypoxemia on a molecular level: adaptive changes in the hypothalamic-pituitary-adrenal (HPA) axis and the lungs.

The development of diseases in later life, such as diabetes type II, hypertension and cardiovascular disease, is linked to abnormal intrauterine conditions that reduce birth weight. Obviously, fetal development can be disturbed so profoundly, that fetal programming is changed permanently. We have examined the effects of hypoxia, or more precisely hypoxemia, on the fetal hypothalamic-pituitary-adrenal (HPA) axis and lungs using molecular biology techniques in order to elucidate the underlying mechanisms. Chronically catheterized fetal sheep were subjected to a hypoxemia (48 h) without change in arterial pH or paCO2. Major changes occurred, although the degree of hypoxemia was just moderate. There was a transient increase in the fetal plasma ACTH-concentrations with an upregulation of the cortisol-concentrations, which was more pronounced in the older, hypoxemic fetuses (134-136 days of gestation) than in the younger, hypoxemic animals (126-130 days of gestation; term is 145 days). There was an unique, differential regulation for pro-opiomelanocortin messenger RNA (mRNA), the precursor molecule of e.g. ACTH, in the pars distalis and pars intermedia of the pituitary gland. This finding supported the increased bioactivity besides the increased concentrations for ACTH. Simultaneously, there was an increase in the mRNAs of the ACTH-receptor and of the steroid-synthesizing enzymes in the fetal adrenal gland of the older, hypoxemic fetuses. No changes in the fetal plasma androstenedione-concentrations were observed. Clearly, there was a selective increase of the cortisol-synthesis. Growth and maturation of the fetal lung might also have been affected, because of the increase in surfactant-protein A mRNA in the older, hypoxemic animals and the decrease in the insulin-like growth factor-I and its binding protein-5 mRNA in the younger, hypoxemic fetuses. In summary, even a moderate degree of hypoxemia was shown to affect the different levels of fetal organism profoundly, offering a pathophysiological basis for changes in fetal development.

Transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter.

The melanocortin-4 receptor (MC4-R) is an important regulator of energy homeostasis, and evidence suggests that MC4-R-expressing neurons are downstream targets of leptin action. MC4-Rs are broadly expressed in the CNS, and the distribution of MC4-R mRNA has been analyzed most extensively in the rat. However, relatively little is known concerning chemical profiles of MC4-R-expressing neurons. The extent to which central melanocortins act presynaptically or postsynaptically on MC4-Rs is also unknown. To address these issues, we have generated a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the MC4-R promoter, using a modified bacterial artificial chromosome. We have confirmed that the CNS distribution of GFP-producing cells is identical to that of MC4-R mRNA in wild-type mice and that nearly all GFP-producing cells coexpress MC4-R mRNA. For example, cells coexpressing GFP and MC4-R mRNA were distributed in the paraventricular hypothalamic nucleus (PVH) and the dorsal motor nucleus of the vagus (DMV). MC4-R promotor-driven GFP expression was found in PVH cells producing thyrotropin-releasing hormone and in cholinergic DMV cells. Finally, we have observed that a synthetic MC3/4-R agonist, MT-II, depolarizes some GFP-expressing cells, suggesting that MC4-Rs function postsynaptically in some instances and may function presynaptically in others. These studies extend our knowledge of the distribution and function of the MC4-R. The transgenic mouse line should be useful for future studies on the role of melanocortin signaling in regulating feeding behavior and autonomic homeostasis.

Alpha-melanocyte-stimulating hormone stimulates prolactin secretion through melanocortin-3 receptors expressed in mammotropes in the mouse pituitary.

The intermediate lobe of rodent pituitaries is involved in the regulation of prolactin (PRL) secretion from the anterior lobe. In a previous study, we demonstrated the stimulatory effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on PRL release and the expression of melanocortin-3 receptors (MC3-Rs) in cultured mouse pituitary cells. The aim of the present study was to clarify whether alpha-MSH directly stimulates PRL release through the MC3-Rs by determining the cell type of MC3-R-expressing cells in the mouse pituitary anterior lobe. Northern blot analysis revealed a 2.7-kb transcript for MC3-R mRNA in the anterior and neurointermediate lobes of pituitary glands of adult male and female mice. Dual cellular localization of MC3-R mRNA and PRL or growth hormone (GH) in the mouse pituitary glands was performed by in situ hybridization analysis of MC3-R mRNA followed by immunocytochemical detection of PRL or GH. MC3-R mRNA was detected in most mammotropes and some somatotropes. alpha-MSH increased PRL release and stimulated DNA replication in mammotropes, and these effects were blocked by SHU9119, an antagonist of MC3-R and MC4-R. These results indicate that alpha-MSH stimulates PRL release and proliferation of mammotropes through MC3-Rs, and suggest that alpha-MSH from intermediate lobes can regulate mammotrope functions in the mouse pituitary.

Functional role, structure, and evolution of the melanocortin-4 receptor.

The melanocortin (MC)-4 receptor participates in regulating body weight homeostasis. We demonstrated early that acute blockage of the MC-4 receptor increases food intake and relieves anorexic conditions in rats. Our recent studies show that 4-week chronic blockage of the MC-4 receptor leads to robust increases in food intake and development of obesity, whereas stimulation of the receptor leads to anorexia. Interestingly, the food conversion ratio was clearly increased by MC-4 receptor blockage, whereas it was decreased in agonist-treated rats in a transient manner. Chronic infusion of an agonist caused a transient increase in oxygen consumption. Our studies also show that the MC-4 receptor plays a role in luteinizing hormone and prolactin surges in female rats. The MC-4 receptor has a role in mediating the effects of leptin on these surges. The phylogenetic relation of the MC-4 receptor to other GPCRs in the human genome was determined. The three-dimensional structure of the protein was studied by construction of a high-affinity zinc binding site between the helices, using two histidine residues facing each other. We also cloned the MC-4 receptor from evolutionary important species and showed by chromosomal mapping a conserved synteny between humans and zebrafish. The MC-4 receptor has been remarkably conserved in structure and pharmacology for more than 400 million years, implying that the receptor participated in vital physiological functions early in vertebrate evolution.

The role of the melanocortin-3 receptor in cachexia.

Cachexia refers to a synergistic combination of a dramatic decrease in appetite and an increase in metabolism of fat and lean body mass. This combination is found in a number of chronic diseases and is an important determinant of mortality. In this paper, we provide evidence that in both acute and chronic disease models, blockade of the MC4-R results in a dramatic attenuation of cachexia. We have also demonstrated that blockade of the melanocortin-3 receptor (MC3-R) leads to enhanced disease-associated cachexia. Ultimately, this work may lead to investigation of drug therapy for this widespread medical problem.

Leptin and melanocortin signaling in the hypothalamus.

The regulation of body weight in humans is coordinated by the interplay between food intake and energy expenditure. The identification of the adipocyte-secreted hormone leptin as a key regulator on both of these processes has shed new light on the pathways involved in their regulation. Indeed, mutations in the gene's encoding leptin and its cognate receptor cause severe obesity in humans. Leptin's actions are mediated principally by target neurons in the hypothalamus where it acts to alter food intake, energy expenditure, and neuroendocrine-function. Recently, it has become clear that a number of critical neuropeptides are regulated by leptin in the hypothalamus. Among these is the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), which is produced in the arcuate nucleus and is a potent negative regulator of food intake. Like leptin, mutations in POMC or in central melanocortin receptors lead to obesity in humans. Thus, an understanding of the mechanisms by which the leptin and melanocortin pathways signal in the hypothalamus is critical in order to begin to clarify the pathways involved in regulating body weight in humans.

Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function.

The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.

The melanocortin receptors: lessons from knockout models.

Identifying the role of the melanocortin system in regulating energy homeostasis has relied on both genetic and pharmacological studies. The key findings included 1) that the coat color phenotype in the lethal yellow (A(Y)/a) mouse is due to antagonism of the melanocortin-1 receptor (MC1R) by the agouti gene product; 2) the MC3R and MC4R are expressed in CNS centers involved in energy homeostasis, and 3) the combined results of pharmacological studies showing that agouti is an antagonist of the MC4R and transgenic studies showing that inhibition or loss of the MC4R recapitulate the lethal yellow phenotype. Pro-opiomelanocortin (POMC), MC3R, and MC4R knockouts are obese and are now being used to further analyze melanocortin receptor function. The obesity phenotype observed in the MC3R and MC4R knockouts (KO) differ markedly. MC4RKO mice are hyperphagic, do not regulate pathways that increase energy expenditure (diet-induced thermogenesis) and physical activity in response to hyperphagia, and can develop type 2 diabetes. In contrast, MC3R deficient mice are not hyperphagic, have a normal metabolic response to increased energy consumption, and do not develop diabetes. The mechanism underlying the increased adiposity in the MC3R knockout remains unclear, but might be related to changes in nutrient partitioning or physical activity.

Salmon calcitonin - a potent inhibitor of food intake in states of impaired leptin signalling in laboratory rodents.

To compare the anorectic effectiveness of leptin and the amylin analogue salmon calcitonin (sCT), rodents were treated on 1 day with subcutaneous injections. In chow-fed C57Bl/6J mice, leptin and sCT reduced energy intake and acted additively. After C57Bl/6J mice had become leptin-resistant on being fed chocolate as a palatable high-caloric supplement to chow, their sCT-induced decrease in energy intake was more pronounced than in chow-fed mice with differential changes in the intake of chocolate (strong reduction) and chow (slight increase). Dose-response relationships for sCT-induced reductions in energy intake were analysed in chow-fed C57Bl/6J mice and two obese strains, ob/ob mice and melanocortin-4 receptor knockout (MC4-r-KO) mice, as well as in wild-type and fatty (fa/fa) rats. Compared to C57Bl/6J mice, reduction in food intake induced by sCT was attenuated in MC4-r-KO mice, and nearly absent in ob/ob mice, over the dose range investigated. Compared to C57Bl/6J mice, wild-type rats responded more sensitively to sCT and its efficiency was only slightly reduced in fatty (fa/fa) rats. Thus, while genetically induced failures of leptin signalling reduce the action of sCT, it effectively inhibits the intake of a palatable, high fat-high sugar diet even in states of diet-induced obesity with functional leptin resistance.

Pharmacological comparison of rat and human melanocortin 3 and 4 receptors in vitro.

The melanocortin 3 and 4 receptors are G-protein-coupled receptors found in the hypothalamus with important role in regulation of the energy balance. In this study, we performed pharmacological comparison of the rat and human melancortin (MC) 3 and MC4 receptors. We transiently expressed the genes for these receptors individually in a mammalian cell line and determined the binding affinities to several MSH peptides. The results showed no major difference between the rat and human MC3 receptors while the rat MC4 receptor had higher affinity to several peptides compared with the human MC4 receptor. NDP-, alpha-, beta-, gamma-MSH, ACTH(1-24), HS014 and MTII had from 5- to 34-fold higher affinity for the rat MC4 receptor, while SHU9119, HS024 and HS028 had similar affinity for both the MC4 receptors. Pharmacological species difference have earlier been reported for the MC1 and MC5 receptors but this is the first report showing important differences between the rat and human MC4 receptors.

Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus.

The pro-opiomelanocortin-derived peptides and the melanocortin receptors are implicated in various functions within the CNS including the regulation of food intake. In the present study, we used in situ hybridization, with specific 35S-labelled ovine riboprobes to map the expression of melanocortin receptor-3 (MC3-R) and -4 (MC4-R) mRNA in the diencephalon and brainstem of normal female sheep. Furthermore, we examined the effect of long-term alterations in energy balance on the distribution and expression of MC3-R and MC4-R mRNA in food-restricted and ad libitum-fed ovariectomized female sheep. The distribution of melanocortin receptors generally resembled that of the rat. A high number of MC3-R-labelled cells were seen in the ventral division of the lateral septum and the medial preoptic area. In the hypothalamus, a moderate number of MC3-R-labelled cells was observed in the lateral hypothalamic area while other nuclear groups had low to intermediate numbers of MC3-R-labelled cells. The distribution of MC4-R mRNA was generally similar to that of MC3-R mRNA in the septal/preoptic and hypothalamic regions, with a high number of labelled cells present in the intermediate division of the lateral septum. Within the hypothalamus, no MC4-R mRNA expression was observed in the arcuate nucleus. There was more widespread distribution of moderate to low numbers of MC4-R mRNA-expressing cells in the brainstem compared to that of MC3-R mRNA. Unlike findings in the rat, only a low number of cells expressed melanocortin receptor mRNA in the ovine hypothalamic nuclei associated with feeding behavior. The number of melanocortin receptor-labelled cells and the level of expression (silver grains/cell) in the hypothalamic feeding centers was similar in food-restricted and ad libitum-fed animals. These findings suggest that long-term alterations in metabolic status do not change the melanocortin receptor mRNA distribution and/or expression in the sheep hypothalamus.

Hypothalamic peptides as drug targets for obesity.

The importance of the melanocortin system in obesity has been confirmed by the recent discovery of mutations in the melanocortin MC4 receptor in morbidly obese patients and the finding that intranasal administration of a fragment of melanocortin decreases body fat in humans. Transgenic mice overexpressing melanin-concentrating hormone (MCH) are obese and a second MCH receptor has been identified. In addition, ghrelin, endocannabinoids and glucagon-like peptide 2 have been identified as potentially important central regulators of food intake.

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, photoperiodic regulation of puberty may be modulated by age, by photoperiodic history, and by changes in leptin signaling and the activity of the leptin-sensitive hypothalamic melanocortin system (POMC, MC3-R). A role for CART in photoperiodic regulation of growth is suggested, because the changes in CART gene expression preceded significant divergence of growth trajectories in the opposite photoperiods.

Development of a facile method for high throughput screening with reporter gene assays.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

The mouse adrenocorticotropin receptor gene: cloning and characterization of its promoter and evidence for a role for the orphan nuclear receptor steroidogenic factor 1.

To elucidate the mechanism underlying the tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R) in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The analysis of the cDNA 5'-end showed an untranslated region of 321 bp, and the ACTH-R gene was demonstrated to be composed of two exons of 113 and 112 bp lying upstream of the single coding exon. S1 nuclease protection assay showed two major transcription start sites separated by 4 bp; 1.8 kb of the 5'-flanking region inserted in a luciferase reporter vector had cell-specific promoter activity because it was functional only in mouse adrenocortical Y1 cells but not in mouse Leydig TM3 cells or fibroblast L cells. There was no dramatic change in the promoter activity in Y1 cells for all the deletions tested up to -113 bp upstream of the transcription start site. In contrast, expression in TM3 cells was switched on from deletion -908 bp, while remaining undetectable in L cells. Site-directed mutagenesis of a steroidogenic factor 1 (SF1)-like site at position -25 bp resulted in a significant reduction in luciferase expression by the promoter in Y1 cells. Gel shift analysis of this site indicated specific binding of a protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in L cells induced promoter activity of the construct p(908). In conclusion, cell-specific expression of the mouse ACTH-R appears to be controlled by at least two factors. One of them, most probably SF1, is responsible for steroidogenic cell-specific expression. The other as yet unknown factor binding between position -1236 bp and -908 bp acts as a strong inhibitory factor in nonadrenal steroidogenic cells, resulting in the adrenal-specific expression of ACTH-R.