A metabolic defect promotes obesity in mice lacking melanocortin-4 receptors.

Melanocortin-4 receptor (Mc4r)-null mice exhibit late-onset obesity. To determine whether aberrant metabolism contributes to the obesity, food consumption by Mc4r-null mice was restricted to (pair-fed to) that consumed by wild-type (WT) mice. Pair-fed Mc4r-null females maintained body weights intermediate to that of WT and nonpair-fed Mc4r-null females, whereas pairfeeding normalized the body weights of Mc4r-null male mice. Fat pad and circulating leptin levels were elevated in both male and female pair-fed Mc4r-null mice compared with WT mice. Oxygen consumption of Mc4r-null mice with similar body weights as WT controls was reduced by 20%. Locomotor activity of young nonobese Mc4r-null males was significantly lower than that of WT males; however, locomotion of young nonobese females was normal. Core body temperature of Mc4r-null mice was normal, and they responded normally to cold exposure. Young nonobese Mc4r-null females were unable to induce uncoupling protein 1 (UCP1) in brown adipose tissue in response to peripheral leptin administration, whereas UCP1 mRNA was increased by 60% in the WT females. These results indicate that Mc4r deficiency enhances caloric efficiency, similar to that seen in the agouti obesity syndrome and in melanocortin-3 receptor-null mice.

A novel selective melanocortin-4 receptor agonist reduces food intake in rats and mice without producing aversive consequences.

Studies using nonselective agonists and antagonists of melanocortin-3 receptor (MC3R) and MC4R point to the importance of the CNS melanocortin system in the control of food intake. We describe here a novel compound that is highly selective as an agonist at the MC4 receptor but has minimal activity at the MC3 receptor. When administered centrally to rats, this selective agonist increased Fos-like immunoreactivity in the paraventricular nucleus, central nucleus of the amygdala, nucleus of the solitary tract, and area postrema, a pattern of neuronal activation that is similar to that induced by a nonselective MC3/4R agonist. Additionally, it suppresses food intake when administered centrally to rats or peripherally to db/db mice that lack functional leptin receptors via a mechanism that is not accompanied by illness or other nonspecific effects. Conversely, a related compound that is a selective MC4R antagonist potently increased food intake when administered centrally in rats. These results support the hypothesis that the brain MC4R is intimately involved in the control of food intake and body weight and provide evidence that selective activation of MC4R causes anorexia that is not secondary to aversive effects.

Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues.

Central administration of neuropeptide Y (NPY) potently induces feeding and its abundance in the hypothalamus increases when energy stores fall. Consequently, NPY is considered to be a physiological effector of feeding behavior. Surprisingly, NPY-deficient (NPY-/-) mice feed and grow normally with ad libitum access to food and manifest a normal hyperphagic response after fasting, suggesting that other feeding effectors may compensate for the lack of NPY. Agouti-related protein (AgRP), a melanocortin receptor antagonist, can also stimulate feeding behavior when administered centrally and is coexpressed in a majority of hypothalmamic NPY-ergic neurons, making AgRP a candidate compensatory factor. To test this possibility, we evaluated AgRP mRNA and protein expression, as well as responsiveness to centrally administered AgRP in NPY-/- mice. These studies demonstrate that hypothalamic AgRP mRNA and immunoreactivity are upregulated with fasting and that these increases are not affected by NPY deficiency. Interestingly, NPY-/- mice are hypersensitive to central administration of AgRP(83-132), yet exhibit a normal response to centrally administered MTII, a melanocortin receptor agonist. These data suggest that if AgRP compensates for the lack of NPY in NPY-/- mice, it is not at the level of AgRP synthesis and may instead involve alterations in the postsynaptic signaling efficacy of AgRP. Moreover, the effects of AgRP are not limited to its actions at the melanocortin-4 receptor (MC4R), because MC4R-deficient (MC4R-/-) mice manifest a significant response to centrally administered AgRP. These data imply that AgRP has additional targets in the hypothalamus.

Melanocortin-4 receptor: a novel signalling pathway involved in body weight regulation.

For many years, genetically obese mouse strains have provided models for human obesity. The Avy/-agouti mouse, one of the oldest obese mouse models, is characterized by maturity-onset obesity and diabetes as a result of ectopic expression of the secreted protein hormone, agouti protein. Agouti protein is normally expressed in hair follicles to regulate pigmentation through antagonism of the melanocortin-1 receptor, but in-vitro studies have demonstrated that the hormone also has potent antagonist activity for the melanocortin-4 receptor (MC4-R). Subsequent development of the MC4-R knockout mouse model demonstrated that MC4-R plays a role in weight homeostasis as these mice recapitulated the metabolic defects of the agouti mouse. Further evidence for this hypothesis was obtained from pharmacological studies utilizing peptides with MC4-R agonist activity, that inhibited food intake (when administered intracerebrally). Additional studies with peptide antagonists have now implicated the MC4-R in the leptin signalling pathway. Finally, evidence that the MC4-R may play a role in human obesity has been obtained from the identification of a dis-functional variant of the receptor in genetically obese subjects.

Role of the CNS melanocortin system in the response to overfeeding.

The voluntary suppression of food intake that accompanies involuntary overfeeding is an effective regulatory response to positive energy balance. Because the pro-opiomelanocortin (POMC)-derived melanocortin system in the hypothalamus promotes anorexia and weight loss and is an important mediator of energy regulation, we hypothesized that it may contribute to the hypophagic response to overfeeding. Two groups of rats were overfed to 105 and 116% of control body weight via a gastric catheter. In the first group, in situ hybridization was used to measure POMC gene expression in the rostral arcuate (ARC). Overfeeding increased POMC mRNA in the ARC by 180% relative to levels in control rats. For rats in the second group, the overfeeding was stopped, and they were infused intracerebroventricularly with SHU9119 (SHU), a melanocortin (MC) antagonist at the MC3 and MC4 receptor, or vehicle. Although SHU (0.1 nmol) had no effect on food intake of control rats, intake of overfed rats increased by 265% relative to CSF-treated controls. This complete reversal of regulatory hypophagia not only maintained but actually increased the already elevated weight of overfed rats, whereas CSF-treated overfed rats lost weight. These results indicate that CNS MCs mediate hypophagic signaling in response to involuntary overfeeding and support the hypothesis that MCs are important in the central control of energy homeostasis.

Identification and functional analysis of novel human melanocortin-4 receptor variants.

Inactivation of the melanocortin-4 receptor (MC4-R) by gene-targeting results in mice that develop maturity-onset obesity, hyperinsulinemia, and hyperglycemia. These phenotypes resemble common forms of human obesity, which are late-onset and frequently accompanied by NIDDM. It is not clear whether sequence variation of the MC4-R gene contributes to obesity in humans. Therefore, we examined the human MC4-R gene polymorphism in 190 individuals ascertained on obesity status. Three allelic variants were identified, including two novel ones, Thr112Met and Ile137Thr. To analyze possible functional alterations, the variants were cloned and expressed in vitro and compared with the wild-type receptor. One of the novel variants, Ile137Thr, identified in an extremely obese proband (BMI 57), was found to be severely impaired in ligand binding and signaling, raising the possibility that it may contribute to development of obesity. Furthermore, our results also suggest that sequence polymorphism in the MC4-R coding region is unlikely to be a common cause of obesity in the population studied, given the low frequency of functionally significant mutations.

Melanocortin and leptin signaling systems: central regulation of catabolic energy balance.

The recent cloning of the ob gene (leptin) has revolutionized our understanding of obesity and the underlying factors that govern weight homeostasis. There is growing evidence that long term food intake regulation is controlled by the central nervous system by a number of peptide hormones in response to changes in leptin levels. Studies of these hormones, using both genetic and pharmacological approaches, have provided a foundation for decoding the molecular logic of the neuronal circuits which regulate food intake control and energy balance. A review of the current progress in the melanocortin-4 receptor pathway, with particular emphasis on its relation to leptin, neuropeptide Y and other obesity hormones known to modulate weight homeostasis, is presented.

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

The N-terminal domain of RGS4 confers receptor-selective inhibition of G protein signaling.

Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Gq and Gi alpha subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via Gq. Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10(4)-fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates Gq class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by Galphaq and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling.

A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo.

Agouti-related protein (Agrp) is present in rat and human hypothalamus and is structurally related to agouti protein. Overexpression of either of these proteins results in obesity. However the effect of exogenous Agrp and its in vivo interaction with alpha-melanocyte stimulating hormone (alphaMSH), the likely endogenous melanocortin 3 and 4 receptor (MC3-R and MC4-R) agonist, have not been demonstrated. We report that 1 nmol of Agrp(83-132), a C-terminal fragment of Agrp, when administered intracerebroventricularly (ICV) into rats, increased food intake over a 24-h period (23.0+/-1.4 g saline vs 32.9+/-2.3 g Agrp, p<0.05). The hyperphagia was similar to that seen when 1 nmol of the synthetic MC3-R and MC4-R antagonist SHU9119 was given i.c.v. (19.6+/-1.8 g saline vs 32.5+/-1.7 g SHU9119, p<0.001). Both Agrp(83-132) and SHU9119 blocked the reduction in 1-h food intake of i.c.v. alphaMSH at the beginning of the dark phase. This effect occurred independently of whether the antagonists were administered simultaneously, or nine hours prior, to the alphaMSH. We have also shown Agrp(83-132) is an antagonist at the MC3-R and MC4-R, with similar inhibition of cAMP activation to that previously reported for the full length peptide. In conclusion, Agrp(83-132) administered i.c.v. increases feeding with long lasting effects and is able to inhibit the action of alphaMSH. This interaction may be mediated by the MC3-R and/or MC4-R.

Central infusion of melanocortin agonist MTII in rats: assessment of c-Fos expression and taste aversion.

Like leptin (OB protein), central infusion of the nonspecific melanocortin agonist MTII reduces food intake for relatively long periods of time (i.e., 12 h; W. Fan, B. A. Boston, R. A. Kesterson, V. J. Hruby, and R. D. Cone, Nature; 385: 165-168, 1997). To test the hypothesis that MTII may influence ingestive behavior via mechanisms similar to those that mediate the effects of leptin, we infused a single dose of MTII into the third ventricle (i3vt) of Long-Evans rats and examined three dependent measures that have been studied following i3vt infusion of leptin: 1) effects on long-term food intake and body weight (48 h), 2) patterns of c-Fos expression in the brain, and 3) conditioned taste aversion learning. Similar to leptin, MTII reduced 48-h food intake (1.0 nmol dose), reduced body weight at 24 and 48 h (0.1 and 1.0 nmol doses, respectively), and induced c-Fos expression in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala. In contrast to leptin, MTII was found to produce conditioned taste aversions. These results are consistent with the hypothesis that MTII may influence regulatory behavior via mechanisms similar to those that mediate the effects of leptin.

Induction of c-fos in mouse bone marrow macrophages: a direct method for assessing bone marrow cell activation.

Intravenous LPS was used to induce bone marrow cell activation in c-fos/lacZ transgenic mice. Activated bone marrow cells were identified by staining with X-gal and cell lineage was determined using specific monoclonal antibodies. Four hours after intravenous injection of 100 micrograms LPS, 2.8 +/- 0.2% (28/1000) of total bone marrow cells was X-gal positive as compared to 0 +/- 0% (0/1000) of cells from untreated animals. CD8a, CD4 and CD45R antigens were not expressed on X-gal positive cells suggesting they were not from the T or B lymphocyte lineage. In contrast, Ly-6c, CD44 and Gr-1 antigens were expressed on 51 +/- 6%, 85 +/- 9% and 37 +/- 5% of X-gal positive cells, indicating that X-gal positive cells were from macrophage or granulocyte lineages. The presence of non-specific esterase and the lack of peroxidase activity in X-gal positive cells was used to conclude that X-gal positive cells were of macrophage lineage.

Essential fatty acids are antagonists of the leukotriene B4 receptor.

A series of essential fatty acids and fatty acid derivatives were evaluated for their ability to inhibit [3H] leukotriene B4 (LTB4) binding to pig neutrophil membranes. The fatty acids varied in chain length, extent of unsaturation, position of unsaturation, and isomerization. Generally, fatty acids with two or more unsaturated sites and chain lengths of 18-22 were potent inhibitors of [3H]LTB4 binding; both n-3 and n-6 fatty acids were inhibitory. The most potent compounds tested were homogammalinolenic acid and ricinelaidic acid which gave Ki values of 1 microM and 2 microM in the binding assay. Ricinelaidic acid was also tested for its ability to inhibit LTB4-mediated chemotaxis (IC50 = 10 microM) and LTB4-induced calcium fluxes (IC50 = 7 microM) in isolated human neutrophils. Ricinelaidic acid did not show agonist activity in these assays. In an in vivo model of LTB4-induced bronchoconstriction, ricinelaidic acid and homogammalinolenic acid gave 46% and 53% inhibition, respectively, at a 1 mg/kg i.v. dose. These results indicate that essential fatty acids are LTB4 receptor antagonists, which may account in part for their reported anti-inflammatory activities.

Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex.

The anatomic relationship between serotonergic (5-HT) axons and 5-HT2 receptors in the rat forebrain was determined by a combined analysis of transmitter immunocytochemistry and receptor autoradiography. High densities of 5-HT2 receptors, localized by the ligand N1-methyl-2-125I-LSD (125I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT2 receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT2 receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT2 receptors in layer Va of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT2 receptors and fine axons in other areas of the forebrain, suggesting that 5-HT2 receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT2 receptors may mediate the effects of dorsal but not median raphe projections.

Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes.

Insulin action in intact adipocytes leads to a rapid increase in the concentration of receptors for insulin-like growth factor (IGF) II on the adipocyte cell surface, and to a decrease in the [32P]phosphate content of these receptors on the plasma membrane [Corvera & Czech (1985) Proc. Natl. Acad. Sci. U.S.A. 82. 7314-7318]. It has been previously shown that the receptor for IGF-II can be phosphorylated on tyrosine residues by a kinase activity which is expressed in isolated adipocyte plasma membranes. It is now shown that IGF-II-receptor phosphorylation in vitro, in plasma membranes derived from insulin-treated cells, is markedly decreased compared with the phosphorylation of the receptor in membranes from control cells. This effect of insulin cannot be attributed to an increase in the activity of phosphotyrosyl phosphatase in the membranes. The tyrosine kinase that catalyses the phosphorylation of IGF-II receptors is associated with a fraction of the plasma membrane which is insoluble in Triton X-100. Removal of the Triton X-100-soluble components of the membrane markedly enhances receptor phosphorylation. Moreover, the expression of the inhibitory effect of insulin requires the presence of one or several Triton X-100-soluble components of the plasma membrane.

Serotonin 5-HT1C receptors are expressed at high density on choroid plexus tumors from transgenic mice.

Choroid plexus tumors develop spontaneously in adult transgenic mice carrying integrated copies of SV40 early region genes. In this communication, we report that these tumors exhibit the highest density of serotonin receptors (6600 fmol/mg protein) found in any tissue. 125I-LSD binding to choroid plexus tumors displays a pharmacological profile that matches the properties of 5-HT1C receptors in normal choroid plexus tissue. Autoradiographic localization of 125I-LSD binding in brain sections from transgenic mice shows high levels of labelling in the tumors, in correlation with immunohistochemical staining for SV40 large T antigen expression. Choroid plexus tumors from these transgenic mice provide an excellent model system for the study of serotonin 5-HT1C receptors.

Solubilization and characterization of the serotonin 5-HT1c site from pig choroid plexus.

A novel type of serotonergic binding site, termed the 5-HT1c site, was recently identified on choroid plexus epithelial cells. In the present study, we describe the solubilization of pig choroid plexus 5-HT1c sites by the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS). High affinity labeling of both membrane-bound and solubilized 5-HT1c sites was obtained by use of the serotonergic radioligand N1-methyl-2-[125I]lysergic acid diethylamide (125I-MIL). In solubilized preparations, 125I-MIL exhibited a dissociation constant (Kd) of 2.0 nM and a 60-75% ratio of specific to total binding. Approximately 45% of the membrane binding sites were solubilized by CHAPS as measured by a postlabeling, polyethylene glycol precipitation method. The CHAPS-solubilized 5-HT1c site fulfilled the accepted criteria for receptor solubilization. The affinities of a series of serotonergic antagonists for the 5-HT1c site showed little or no change upon solubilization of the site. Serotonin, however, showed a 20-fold increase in affinity for the 5-HT1c site after solubilization, which may indicate the loss of a modulatory component during detergent treatment. Both gel filtration and equilibrium sedimentation experiments indicate that the CHAPS-solubilized site is a large molecular weight complex. These studies demonstrate that the pig choroid plexus 5-HT1c site can be solubilized with retention of its binding activity in a form suitable for further purification and characterization.

125I-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells.

125I-Lysergic acid diethylamide (125I-LSD) binds with high affinity to serotonergic sites on rat choroid plexus. These sites were localized to choroid plexus epithelial cells by use of a novel high resolution stripping film technique for light microscopic autoradiography. In membrane preparations from rat choroid plexus, the serotonergic site density was 3100 fmol/mg of protein, which is 10-fold higher than the density of any other serotonergic site in brain homogenates. The choroid plexus site exhibits a novel pharmacology that does not match the properties of 5-hydroxytryptamine-1a (5-HT1a), 5-HT1b, or 5-HT2 serotonergic sites. 125I-LSD binding to the choroid plexus site is potently inhibited by mianserin, serotonin, and (+)-LSD. Other serotonergic, dopaminergic, and adrenergic agonists and antagonists exhibit moderate to weak affinities for this site. The rat choroid plexus 125I-LSD binding site appears to represent a new type of serotonergic site which is located on non-neuronal cells in this tissue.