Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Sulforaphane reduces obesity by reversing leptin resistance.

The ascending prevalence of obesity in recent decades is commonly associated with soaring morbidity and mortality rates, resulting in increased health-care costs and decreased quality of life. A systemic state of stress characterized by low-grade inflammation and pathological formation of reactive oxygen species (ROS) usually manifests in obesity. The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) is the master regulator of the redox homeostasis and plays a critical role in the resolution of inflammation. Here, we show that the natural isothiocyanate and potent NRF2 activator sulforaphane reverses diet-induced obesity through a predominantly, but not exclusively, NRF2-dependent mechanism that requires a functional leptin receptor signaling and hyperleptinemia. Sulforaphane does not reduce the body weight or food intake of lean mice but induces an anorectic response when coadministered with exogenous leptin. Leptin-deficient Lepob/ob mice and leptin receptor mutant Leprdb/db mice display resistance to the weight-reducing effect of sulforaphane, supporting the conclusion that the antiobesity effect of sulforaphane requires functional leptin receptor signaling. Furthermore, our results suggest the skeletal muscle as the most notable site of action of sulforaphane whose peripheral NRF2 action signals to alleviate leptin resistance. Transcriptional profiling of six major metabolically relevant tissues highlights that sulforaphane suppresses fatty acid synthesis while promoting ribosome biogenesis, reducing ROS accumulation, and resolving inflammation, therefore representing a unique transcriptional program that leads to protection from obesity. Our findings argue for clinical evaluation of sulforaphane for weight loss and obesity-associated metabolic disorders.

Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity.

The adipose tissue-derived hormone leptin can drive decreases in food intake while increasing energy expenditure. In diet-induced obesity, circulating leptin levels rise proportionally to adiposity. Despite this hyperleptinemia, rodents and humans with obesity maintain increased adiposity and are resistant to leptin's actions. Here we show that inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent leptin sensitizers and anti-obesity agents in diet-induced obese mice. Specifically, HDAC6 inhibitors, such as tubastatin A, reduce food intake, fat mass, hepatic steatosis and improve systemic glucose homeostasis in an HDAC6-dependent manner. Mechanistically, peripheral, but not central, inhibition of HDAC6 confers central leptin sensitivity. Additionally, the anti-obesity effect of tubastatin A is attenuated in animals with a defective central leptin-melanocortin circuitry, including db/db and MC4R knockout mice. Our results suggest the existence of an HDAC6-regulated adipokine that serves as a leptin-sensitizing agent and reveals HDAC6 as a potential target for the treatment of obesity.

Endogenous cannabinoids are required for MC4R-mediated control of energy homeostasis.

Hypothalamic regulation of feeding and energy expenditure is a fundamental and evolutionarily conserved neurophysiological process critical for survival. Dysregulation of these processes, due to environmental or genetic causes, can lead to a variety of pathological conditions ranging from obesity to anorexia. Melanocortins and endogenous cannabinoids (eCBs) have been implicated in the regulation of feeding and energy homeostasis; however, the interaction between these signaling systems is poorly understood. Here, we show that the eCB 2-arachidonoylglycerol (2-AG) regulates the activity of melanocortin 4 receptor (MC4R) cells in the paraventricular nucleus of the hypothalamus (PVNMC4R) via inhibition of afferent GABAergic drive. Furthermore, the tonicity of eCBs signaling is inversely proportional to energy state, and mice with impaired 2-AG synthesis within MC4R neurons weigh less, are hypophagic, exhibit increased energy expenditure, and are resistant to diet-induced obesity. These mice also exhibit MC4R agonist insensitivity, suggesting that the energy state-dependent, 2-AG-mediated suppression of GABA input modulates PVNMC4R neuron activity to effectively respond to the MC4R natural ligands to regulate energy homeostasis. Furthermore, post-developmental disruption of PVN 2-AG synthesis results in hypophagia and death. These findings illustrate a functional interaction at the cellular level between two fundamental regulators of energy homeostasis, the melanocortin and eCB signaling pathways in the hypothalamic feeding circuitry.

The melanocortin-3 receptor is a pharmacological target for the regulation of anorexia.

Ablation of hypothalamic AgRP (Agouti-related protein) neurons is known to lead to fatal anorexia, whereas their activation stimulates voracious feeding and suppresses other motivational states including fear and anxiety. Despite the critical role of AgRP neurons in bidirectionally controlling feeding, there are currently no therapeutics available specifically targeting this circuitry. The melanocortin-3 receptor (MC3R) is expressed in multiple brain regions and exhibits sexual dimorphism of expression in some of those regions in both mice and humans. MC3R deletion produced multiple forms of sexually dimorphic anorexia that resembled aspects of human anorexia nervosa. However, there was no sexual dimorphism in the expression of MC3R in AgRP neurons, 97% of which expressed MC3R. Chemogenetic manipulation of arcuate MC3R neurons and pharmacologic manipulation of MC3R each exerted potent bidirectional regulation over feeding behavior in male and female mice, whereas global ablation of MC3R-expressing cells produced fatal anorexia. Pharmacological effects of MC3R compounds on feeding were dependent on intact AgRP circuitry in the mice. Thus, the dominant effect of MC3R appears to be the regulation of the AgRP circuitry in both male and female mice, with sexually dimorphic sites playing specialized and subordinate roles in feeding behavior. Therefore, MC3R is a potential therapeutic target for disorders characterized by anorexia, as well as a potential target for weight loss therapeutics.

Leptin Receptor Signaling in Sim1-Expressing Neurons Regulates Body Temperature and Adaptive Thermogenesis.

Leptin signals to regulate food intake and energy expenditure under conditions of normative energy homeostasis. The central expression and function of leptin receptor B (LepRb) have been extensively studied during the past two decades; however, the mechanisms by which LepRb signaling dysregulation contributes to the pathophysiology of obesity remains unclear. The paraventricular nucleus of the hypothalamus (PVN) plays a crucial role in regulating energy balance as well as the neuroendocrine axes. The role of LepRb expression in the PVN in regard to the regulation of physiological function of leptin has been controversial. The single-minded homolog 1 gene (Sim1) is densely expressed in the PVN and in parts of the amygdala, making Sim1-Cre mice a useful model for examining molecular mechanisms regulating PVN function. In this study, we characterized the physiological role of LepRb in Sim1-expressing neurons using LepRb-floxed × Sim1-Cre mice. Sim1-specific LepRb-deficient mice were surprisingly hypophagic on regular chow but gained more weight upon exposure to a high-fat diet than did their control littermates. We show that Sim1-specific deletion of a single LepRb gene copy caused decreased surface and core body temperatures as well as decreased energy expenditure in ambient room temperatures in both female and male mice. Furthermore, cold-induced adaptive (nonshivering) thermogenesis is disrupted in homozygous knockout mice. A defective thermoregulatory response was associated with defective cold-induced upregulation of uncoupling protein 1 in brown adipose tissue and reduced serum T4. Our study provides novel functional evidence supporting LepRb signaling in Sim1 neurons in the regulation of body weight, core body temperature, and cold-induced adaptive thermogenesis.

Late onset obesity in mice with targeted deletion of potassium inward rectifier Kir7.1 from cells expressing the melanocortin-4 receptor.

Energy stores in fat tissue are determined in part by the activity of hypothalamic neurones expressing the melanocortin-4 receptor (MC4R). Even a partial reduction in MC4R expression levels in mice, rats or humans produces hyperphagia and morbid obesity. Thus, it is of great interest to understand the molecular basis of neuromodulation by the MC4R. The MC4R is a G protein-coupled receptor that signals efficiently through GαS , and this signalling pathway is essential for normal MC4R function in vivo. However, previous data from hypothalamic slice preparations indicated that activation of the MC4R depolarised neurones via G protein-independent regulation of the ion channel Kir7.1. In the present study, we show that deletion of Kcnj13 (ie, the gene encoding Kir7.1) specifically from MC4R neurones produced resistance to melanocortin peptide-induced depolarisation of MC4R paraventricular nucleus neurones in brain slices, resistance to the sustained anorexic effect of exogenously administered melanocortin peptides, late onset obesity, increased linear growth and glucose intolerance. Some MC4R-mediated phenotypes appeared intact, including Agouti-related peptide-induced stimulation of food intake and MC4R-mediated induction of peptide YY release from intestinal L cells. Thus, a subset of the consequences of MC4R signalling in vivo appears to be dependent on expression of the Kir7.1 channel in MC4R cells.

Regulation of energy rheostasis by the melanocortin-3 receptor.

Like most homeostatic systems, adiposity in mammals is defended between upper and lower boundary conditions. While leptin and melanocortin-4 receptor (MC4R) signaling are required for defending energy set point, mechanisms controlling upper and lower homeostatic boundaries are less well understood. In contrast to the MC4R, deletion of the MC3R does not produce measurable hyperphagia or hypometabolism under normal conditions. However, we demonstrate that MC3R is required bidirectionally for controlling responses to external homeostatic challenges, such as caloric restriction or calorie-rich diet. MC3R is also required for regulated excursion from set point, or rheostasis, during pregnancy. Further, we demonstrate a molecular mechanism: MC3R provides regulatory inputs to melanocortin signaling, acting presynaptically on agouti-related protein neurons to regulate γ-aminobutyric acid release onto anorexigenic MC4R neurons, exerting boundary control on the activity of MC4R neurons. Thus, the MC3R is a critical regulator of boundary controls on melanocortin signaling, providing rheostatic control on energy storage.

Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay.

The family of inward rectifying potassium channels (Kir channels) plays crucial roles in the regulation of heart rhythms, renal excretion, insulin release, and neuronal activity. Their dysfunction has been attributed to numerous diseases such as cardiac arrhythmia, kidney failure and electrolyte imbalance, diabetes mellitus, epilepsy, retinal degeneration, and other neuronal disorders. We have recently demonstrated that the melanocortin-4 receptor (MC4R), a Gαs-coupled GPCR, regulates Kir7.1 activity through a mechanism independent of Gαs and cAMP. In contrast to the many other members of the Kir channel family, less is known about the biophysical properties, regulation, and physiological functions of Kir7.1. In addition to using conventional patch clamp techniques, we have employed a high-throughput Tl+ flux assay to further investigate the kinetics of MC4R-Kir7.1 signaling in vitro. Here, we discuss the employment of the Tl+ flux assay to study MC4R -mediated regulation of Kir7.1 activity and to screen compounds for drug discovery.

Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake.

The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome.

60 YEARS OF POMC: Regulation of feeding and energy homeostasis by α-MSH.

The melanocortin peptides derived from pro-opiomelanocortin (POMC) were originally understood in terms of the biological actions of α-melanocyte-stimulating hormone (α-MSH) on pigmentation and adrenocorticotrophic hormone on adrenocortical glucocorticoid production. However, the discovery of POMC mRNA and melanocortin peptides in the CNS generated activities directed at understanding the direct biological actions of melanocortins in the brain. Ultimately, discovery of unique melanocortin receptors expressed in the CNS, the melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors, led to the development of pharmacological tools and genetic models leading to the demonstration that the central melanocortin system plays a critical role in the regulation of energy homeostasis. Indeed, mutations in MC4R are now known to be the most common cause of early onset syndromic obesity, accounting for 2-5% of all cases. This review discusses the history of these discoveries, as well as the latest work attempting to understand the molecular and cellular basis of regulation of feeding and energy homeostasis by the predominant melanocortin peptide in the CNS, α-MSH.

G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.

The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Electrophysiological analysis of circuits controlling energy homeostasis.

Since the discovery of leptin and the central melanocortin circuit, electrophysiological studies have played a major role in elucidating mechanisms underlying energy homeostasis. This review highlights the contribution of findings made by electrophysiological measurements to the current understanding of hypothalamic neuronal networks involved in energy homeostasis with a specific focus on the arcuate-paraventricular nucleus circuit.

Shining a light on energy homeostasis.

The hypothalamic arcuate nucleus is a complex structure containing both orexigenic and anorexigenic neurons, coordinately regulated by leptin and energy state. In their recent Nature Neuroscience study, Aponte et al. (2011) use optogenetic technology to provide a glimpse into the consequences of exclusive activation of either NPY/AgRP or POMC neurons.

Multinodal regulation of the arcuate/paraventricular nucleus circuit by leptin.

Melanocortin-4 receptor (MC4R) is critical for energy homeostasis, and the paraventricular nucleus of the hypothalamus (PVN) is a key site of MC4R action. Most studies suggest that leptin regulates PVN neurons indirectly, by binding to receptors in the arcuate nucleus or ventromedial hypothalamus and regulating release of products like α-melanocyte-stimulating hormone (α-MSH), neuropeptide Y (NPY), glutamate, and GABA from first-order neurons onto the MC4R PVN cells. Here, we investigate mechanisms underlying regulation of activity of these neurons under various metabolic states by using hypothalamic slices from a transgenic MC4R-GFP mouse to record directly from MC4R neurons. First, we show that in vivo leptin levels regulate the tonic firing rate of second-order MC4R PVN neurons, with fasting increasing firing frequency in a leptin-dependent manner. We also show that, although leptin inhibits these neurons directly at the postsynaptic membrane, α-MSH and NPY potently stimulate and inhibit the cells, respectively. Thus, in contrast with the conventional model of leptin action, the primary control of MC4R PVN neurons is unlikely to be mediated by leptin action on arcuate NPY/agouti-related protein and proopiomelanocortin neurons. We also show that the activity of MC4R PVN neurons is controlled by the constitutive activity of the MC4R and that expression of the receptor mRNA and α-MSH sensitivity are both stimulated by leptin. Thus, leptin acts multinodally on arcuate nucleus/PVN circuits to regulate energy homeostasis, with prominent mechanisms involving direct control of both membrane conductances and gene expression in the MC4R PVN neuron.

Regulation of thyrotropin-releasing hormone-expressing neurons in paraventricular nucleus of the hypothalamus by signals of adiposity.

Fasting-induced suppression of thyroid hormone levels is an adaptive response to reduce energy expenditure in both humans and mice. This suppression is mediated by the hypothalamic-pituitary-thyroid axis through a reduction in TRH levels expressed in neurons of the paraventricular nucleus of the hypothalamus (PVN). TRH gene expression is positively regulated by leptin. Whereas decreased leptin levels during fasting lead to a reduction in TRH gene expression, the mechanisms underlying this process are still unclear. Indeed, evidence exists that TRH neurons in the PVN are targeted by leptin indirectly via the arcuate nucleus, whereas correlative evidence for a direct action exists as well. Here we provide both in vivo and in vitro evidence that the activity of hypothalamic-pituitary-thyroid axis is regulated by both direct and indirect leptin regulation. We show that both leptin and α-MSH induce significant neuronal activity mediated through a postsynaptic mechanism in TRH-expressing neurons of PVN. Furthermore, we provide in vivo evidence indicating the contribution of each pathway in maintaining serum levels of thyroid hormone.

Neuropeptide W: an anorectic peptide regulated by leptin and metabolic state.

Neuropeptide W (NPW) is an anorectic peptide produced in the brain. Here, we showed that NPW was present in several hypothalamic nuclei, including the paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, lateral hypothalamus, and hypothalamic arcuate nucleus. NPW expression was significantly up-regulated in leptin-deficient ob/ob and leptin receptor-deficient db/db mice. The increase in NPW expression in ob/ob mice was abrogated to control levels after leptin replacement. Leptin induced suppressors of cytokine signaling-3 after phosphorylation of signal transducer and activator of transcription-3 in NPW-expressing neurons. In addition, we demonstrated that NPW reduces feeding via the melanocortin-4-receptor signaling pathway. We also showed that NPW activates proopiomelanocortin and inhibits neuropeptide Y neurons using loose-patch extracellular recording of these neurons identified by promoter-driven green fluorescent protein expression. This study indicates that NPW may play an important role in the regulation of feeding and energy metabolism under the conditions of leptin insufficiency.

PYY3-36 inhibits the action potential firing activity of POMC neurons of arcuate nucleus through postsynaptic Y2 receptors.

Intracerebroventricular administration of gut peptide PYY3-36 stimulates food intake. In contrast, peripheral administration inhibits food intake, suggesting that the peptide has the opposite effect by virtue of accessing a unique subset of brain sites. A previous study suggested that peripheral PYY3-36 activates anorexigenic POMC neurons in the arcuate nucleus, and this was proposed to be the mechanism underlying the peptide's anorexigenic activity. Here, we demonstrate in an electrophysiological slice preparation that, in contrast to the original model, PYY3-36 potently and reversibly inhibits POMC neurons via postsynaptic Y2 receptors. These data show a complex role for Y2 receptors in regulation of the NPY/POMC circuitry, as they are present as inhibitory receptors on both the orexigenic NPY neurons as well as the anorexigenic POMC neurons. Secondly, these data argue against a direct role of POMC neurons in mediating the anorexigenic response to administration of peripheral PYY3-36.

Muscarinic receptor modulation of slow afterhyperpolarization and phasic firing in rat supraoptic nucleus neurons.

A slow posttrain afterhyperpolarization (sAHP) was studied in rat magnocellular neurosecretory cells (MNCs) in vitro. The sAHP was isolated from other afterpotentials by blocking the depolarizing afterpotential (DAP) with Cs(+) and the medium afterhyperpolarization (mAHP) with apamin. The sAHP amplitude increased logarithmically with activity ( approximately 3 mV per e-fold increase in number of impulses) and, when firing stopped, decayed exponentially with a time constant of 2 sec. The sAHP was associated with increased membrane conductance, and its amplitude varied linearly with voltage, reversing at the K(+) equilibrium potential. The sAHP was blocked by Cd(2+) but not by charybdotoxin or iberiotoxin, blockers of intermediate- and big-conductance-type Ca(2+)-dependent K(+) (K(Ca)) channels. The sAHP was reversibly inhibited by muscarine, an effect antagonized by atropine, indicating involvement of muscarinic cholinergic receptors. Muscarine did not affect Ca(2+)-dependent features of action potentials, DAPs, or the mAHP in MNCs, indicating selective modulation of K(Ca) channels causing the sAHP. Muscarinic inhibition of the sAHP enhanced plateau potentials and increased the mean firing rate and duration of afterdischarges that followed spike trains evoked from voltages near threshold. Similarly, the frequency and duration of the spontaneous phasic bursts that characterize physiologically activated vasopressin-releasing MNCs were enhanced by muscarine. MNCs thus express apamin- and voltage-insensitive K(Ca) channels that mediate an sAHP. The activity dependence and kinetics of the sAHP cause it to mask DAPs in a manner that attenuates the amplitude of plateau potentials. Muscarinic inhibition of the sAHP provides an effective mechanism for promoting phasic firing in MNCs.

Flufenamic acid blocks depolarizing afterpotentials and phasic firing in rat supraoptic neurones.

Depolarizing afterpotentials (DAPs) that follow action potentials in magnocellular neurosecretory cells (MNCs) are thought to underlie the generation of phasic firing, a pattern that optimizes vasopressin release from the neurohypophysis. Previous work has suggested that the DAP may result from the Ca(2+)-dependent reduction of a resting K(+) conductance. Here we examined the effects of flufenamic acid (FFA), a blocker of Ca(2+)-dependent non-selective cation (CAN) channels, on DAPs and phasic firing using intracellular recordings from supraoptic MNCs in superfused explants of rat hypothalamus. Application of FFA, but not solvent (0.1 % DMSO), reversibly inhibited (IC(50) = 13.8 microM; R = 0.97) DAPs and phasic firing with a similar time course, but had no significant effects (P > 0.05) on membrane potential, spike threshold and input resistance, nor on the frequency and amplitude of spontaneous synaptic potentials. Moreover, FFA did not affect (P > 0.05) the amplitude, duration, undershoot, or frequency-dependent broadening of action potentials elicited during the spike trains used to evoke DAPs. These findings suggest that FFA inhibits the DAP by directly blocking the channels responsible for its production, rather than by interfering with Ca(2+) influx. They also support a role for DAPs in the generation of phasic firing in MNCs. Finally, the absence of a depolarization and increased membrane resistance upon application of FFA suggests that the DAP in MNCs may not be due to the inhibition of resting K(+) current, but to the activation of CAN channels.