Melanin-concentrating Hormone Receptor Research Articles

P114 MCH LINKS IMMUNOMETABOLSIM TO INTESTINAL INFLAMMATION

Melanin concentrating hormone (MCH) was first identified as a hypothalamic neuropeptide regulating feeding behavior and energy balance. The MCH receptor, MCHR1, is present in brain as well as in several peripheral tissues including the gastrointestinal tract where its role is yet to be established. Increased MCH and MCHR1 expression in the mucosa of patients with inflammatory bowel disease (IBD) implies a role of MCH in disease pathogenesis. Our previous studies have shown that MCH-deficient mice develop attenuated colitis, a phenotype that was recapitulated when mice were treated with an anti-MCH antibody. The exact mechanisms by which MCH exerts proinflammatory effects remain under investigation, as well as the cell types involved and have been the focus of the present study. We found MCHR1 upregulation in CD4+ T cells isolated from the lamina propria of patients with Crohn’s disease, in contrast to those derived from peripheral blood. In vitro, MCHR1 was induced upon activation of CD4+ T-cells. Furthermore, treatment with MCH of mucosal explants from patients with IBD resulted in increased cytokine secretion in the culture supernatant (IFNg, IL-17, IL-2, IL-6, IL8, MCP1, TNFa). Most importantly, metabolomics analysis of Jurkat cells, a T-cell line expressing high basal levels of MCHR1 receptor, revealed that treatment with MCH increased glutamine, glutamate and a-ketoglutarate levels 4-, 2.5-, and 3.5-fold, respectively, as well as those of acetyl-CoA (3-fold) while glycolytic intermediates remained unaltered. These findings imply engagement of a “reductive carboxylation” cycle in the presence of MCH, the fuel of which is a-ketoglutarate instead of glucose. The role of a-ketoglutarate in driving T-effector activation and differentiation is well established. In further support of these observations, stimulation with MCH resulted in upregulation of the glutamine transporter SLC1A5 that determines intracellular glutamine levels, as well those of its metabolites glutamate and a-ketoglutarate. In relation to the immune phenotype, MCH-induced metabolic shifts correlated with increased proliferation and reduced apoptosis of Jurkat cells, as well as increased IL-2 secretion. In a more relevant context, treatment of isolated CD4+ T cells with MCH under polarized conditions resulted in 70% increase of IL-17+ cells, in contrast to 32% decrease of IL-4+ and 25% decrease of Foxp3+ cells. Collectively, these findings further support a deleterious effect of aberrant MCH signaling in IBD and potentially illustrate a new target in IBD therapeutics.

P114 MCH LINKS IMMUNOMETABOLSIM TO INTESTINAL INFLAMMATION

Melanin concentrating hormone (MCH) was first identified as a hypothalamic neuropeptide regulating feeding behavior and energy balance. The MCH receptor, MCHR1, is present in brain as well as in several peripheral tissues including the gastrointestinal tract where its role is yet to be established. Increased MCH and MCHR1 expression in the mucosa of patients with inflammatory bowel disease (IBD) implies a role of MCH in disease pathogenesis. Our previous studies have shown that MCH-deficient mice develop attenuated colitis, a phenotype that was recapitulated when mice were treated with an anti-MCH antibody. The exact mechanisms by which MCH exerts proinflammatory effects remain under investigation, as well as the cell types involved and have been the focus of the present study. We found MCHR1 upregulation in CD4+ T cells isolated from the lamina propria of patients with Crohn’s disease, in contrast to those derived from peripheral blood. In vitro, MCHR1 was induced upon activation of CD4+ T-cells. Furthermore, treatment with MCH of mucosal explants from patients with IBD resulted in increased cytokine secretion in the culture supernatant (IFNg, IL-17, IL-2, IL-6, IL8, MCP1, TNFa). Most importantly, metabolomics analysis of Jurkat cells, a T-cell line expressing high basal levels of MCHR1 receptor, revealed that treatment with MCH increased glutamine, glutamate and a-ketoglutarate levels 4-, 2.5-, and 3.5-fold, respectively, as well as those of acetyl-CoA (3-fold) while glycolytic intermediates remained unaltered. These findings imply engagement of a “reductive carboxylation” cycle in the presence of MCH, the fuel of which is a-ketoglutarate instead of glucose. The role of a-ketoglutarate in driving T-effector activation and differentiation is well established. In further support of these observations, stimulation with MCH resulted in upregulation of the glutamine transporter SLC1A5 that determines intracellular glutamine levels, as well those of its metabolites glutamate and a-ketoglutarate. In relation to the immune phenotype, MCH-induced metabolic shifts correlated with increased proliferation and reduced apoptosis of Jurkat cells, as well as increased IL-2 secretion. In a more relevant context, treatment of isolated CD4+ T cells with MCH under polarized conditions resulted in 70% increase of IL-17+ cells, in contrast to 32% decrease of IL-4+ and 25% decrease of Foxp3+ cells. Collectively, these findings further support a deleterious effect of aberrant MCH signaling in IBD and potentially illustrate a new target in IBD therapeutics.

Selective signaling pathway via feeding-related ciliary GPCR, melanin-concentrating hormone receptor 1

G-protein-coupled receptors (GPCRs), which constitute a highly diverse family of seven transmembrane receptors, respond to external signals and regulate a variety of cellular and physiological processes. GPCRs are encoded by about 800 different genes in human and they represent the largest family of drug targets in clinical trials, which accounts for about 30% of approved drugs acting on 108 unique GPCRs. Signaling through GPCRs can be optimized by enriching receptors, selective binding partners, and downstream effectors in discrete cellular environment. The primary cilium is a ubiquitous organelle that functions as a sensory antenna for surrounding physical and chemical stimuli. Primary cilium's compartment is as little as 1/10,000th of the total cell volume. Therefore, the ciliary membrane is highly enriched for specific signaling molecules, allowing the primary cilium to organize signaling in a highly ordered microenvironment. Recently, a set of non-olfactory GPCRs such as somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1) have been found to be selectively targeted to cilia on several mammalian cell types including neuronal cells both in vitro and in vivo approaches. Moreover, investigations into the pathophysiology have implicated GPCR ciliary signaling in a number of developmental and cellular pathways. Thus, cilia are now considered as an increasingly important connection for GPCR signaling. This review summarizes our current understanding of the signaling pathways though ciliary GPCR, especially feeding- and mood-related GPCR MCHR1, along with specific biological phenomenon as cilia length shortening.

Melanin-Concentrating Hormone (MCH) and MCH-R1 in the Locus Coeruleus May Be Involved in the Regulation of Depressive-Like Behavior

BackgroundPrevious anatomical and behavioral studies have shown that melanin-concentrating hormone is involved in the modulation of emotional states. However, little is known about brain regions other than the dorsal raphe nucleus that relate the melanin-concentrating hormone-ergic system to depressive states. Numerous studies have shown that the locus coeruleus is involved in the regulation of depression and sleep. Although direct physiological evidence is lacking, previous studies suggest that melanin-concentrating hormone release in the locus coeruleus decreases neuronal discharge. However, remaining unclear is whether the melanin-concentrating hormone-ergic system in the locus coeruleus is related to depressive-like behavior.MethodWe treated rats with an intra-locus coeruleus injection of melanin-concentrating hormone, intracerebroventricular injection of melanin-concentrating hormone, or chronic subcutaneous injections of corticosterone to induce different depressive-like phenotypes. We then assessed the effects of the melanin-concentrating hormone receptor 1 antagonist SNAP-94847 on depressive-like behavior in the forced swim test and the sucrose preference test.ResultsThe intra-locus coeruleus and intracerebroventricular injections of melanin-concentrating hormone and chronic injections of corticosterone increased immobility time in the forced swim test and decreased sucrose preference in the sucrose preference test. All these depressive-like behaviors were reversed by an intra-locus coeruleus microinjection of SNAP-94847.ConclusionsThese results suggest that the melanin-concentrating hormone-ergic system in the locus coeruleus might play an important role in the regulation of depressive-like behavior.

Effects of blue light spectra on retinal stress and damage in goldfish (Carassius auratus).

There have been a number of studies on the negative effects of blue light exposure in various species; however, little information is available on the impacts of blue light intensity and duration on fish. We investigated the effects of blue light spectra on stress in the retinas of goldfish, using a blue (460nm) light-emitting diode (LED) at three intensities (0.5, 1.0, and 1.5W/m2). The experiment was conducted for 4weeks, and sampling was performed at intervals of 1week. We measured changes in the expression of cortisol, and the concentrations of hydrogen peroxide (H2O2), melanin-concentrating hormone receptor (MCH-R), and caspase-3 in the retinas of goldfish. In addition, we measured histological changes in the retina. We used a transferase dUTP nick end labeling (TUNEL) assay to evaluate the apoptotic response to blue LED spectra. Levels of cortisol, H2O2, MCH-R, and caspase-3 increased with exposure time and light intensity. Histological analysis revealed that the thickness of melanin granules increased with exposure time and light intensity. The progressive TUNEL assay revealed many apoptotic cells after exposure to blue LED light, increasing with exposure time and light intensity. Irradiation with blue light for longer than 1week induced increased retinal stress and may induce apoptosis in the retinas of goldfish, even at a low intensity.

Melanin-concentrating hormone neurons contribute to dysregulation of rapid eye movement sleep in narcolepsy

The lateral hypothalamus contains neurons producing orexins that promote wakefulness and suppress REM sleep as well as neurons producing melanin-concentrating hormone (MCH) that likely promote REM sleep. Narcolepsy with cataplexy is caused by selective loss of the orexin neurons, and the MCH neurons appear unaffected. As the orexin and MCH systems exert opposing effects on REM sleep, we hypothesized that imbalance in this REM sleep-regulating system due to activity in the MCH neurons may contribute to the striking REM sleep dysfunction characteristic of narcolepsy. To test this hypothesis, we chemogenetically activated the MCH neurons and pharmacologically blocked MCH signaling in a murine model of narcolepsy and studied the effects on sleep-wake behavior and cataplexy. To chemoactivate MCH neurons, we injected an adeno-associated viral vector containing the hM3Dq stimulatory DREADD into the lateral hypothalamus of orexin null mice that also express Cre recombinase in the MCH neurons (MCH-Cre::OX-KO mice) and into control MCH-Cre mice with normal orexin expression. In both lines of mice, activation of MCH neurons by clozapine-N-oxide (CNO) increased rapid eye movement (REM) sleep without altering other states. In mice lacking orexins, activation of the MCH neurons also increased abnormal intrusions of REM sleep manifest as cataplexy and short latency transitions into REM sleep (SLREM). Conversely, a MCH receptor 1 antagonist, SNAP 94847, almost completely eliminated SLREM and cataplexy in OX-KO mice. These findings affirm that MCH neurons promote REM sleep under normal circumstances, and their activity in mice lacking orexins likely triggers abnormal intrusions of REM sleep into non-REM sleep and wake, resulting in the SLREM and cataplexy characteristic of narcolepsy.

A CreER mouse to study melanin concentrating hormone signaling in the developing brain.

SummaryThe neuropeptide, melanin concentrating hormone (MCH), and its G protein‐coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1‐Cre) exists, there is a need for an inducible Mchr1‐Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1‐Cre expression pattern are recapitulated by the Mchr1‐CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1‐CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1‐CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.

Acupuncture Alleviates Levodopa-Induced Dyskinesia via Melanin-Concentrating Hormone in Pitx3-Deficient aphakia and 6-Hydroxydopamine-Lesioned Mice.

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is currently the most effective medication for treating Parkinson's disease (PD) motor symptoms, its prolonged administration causes several adverse effects, including dyskinesia. To identify the mechanisms underlying the effects of acupuncture on L-DOPA-induced dyskinesia (LID), antidyskinetic effects of acupuncture were investigated in two mouse models of PD. Acupuncture stimulation at GB34 alleviated abnormal involuntary movements (AIMs) in Pitx3-deficient aphakia mice (ak/ak) following L-DOPA administration and these effects were reproduced in 6-hydroxydopamine (6-OHDA)-lesioned mice with LID. A transcriptome analysis of the hypothalamus revealed pro-melanin-concentrating hormone (Pmch) gene was highly expressed in acupuncture-treated mouse from ak/ak model of LID as well as 6-OHDA model of LID. Acupuncture combined with the administration of MCH receptor antagonist did not have any beneficial effects on dyskinesia in L-DOPA-injected ak/ak mice, but the intranasal administration of MCH attenuated LID to the same degree as acupuncture in both ak/ak and 6-OHDA mice with LID. A gene expression profile with a hierarchical clustering analysis of the dyskinesia-induced ak/ak mouse brain revealed an association between the mechanisms underlying acupuncture and MCH. Additionally, altered striatal responses to L-DOPA injection were observed after prolonged acupuncture and MCH treatments, which suggests that these treatment modalities influenced the compensatory mechanisms of LID. In summary, present study demonstrated that acupuncture decreased LID via hypothalamic MCH using L-DOPA-administered ak/ak and 6-OHDA mouse models and that MCH administration resulted in novel antidyskinetic effects in these models. Thus, acupuncture and MCH might be valuable therapeutic candidates for PD patients suffering from LID.

Melanin-concentrating hormone and orexin systems in rat nucleus incertus: Dual innervation, bidirectional effects on neuron activity, and differential influences on arousal and feeding

The rat nucleus incertus (NI) contains GABA/peptide-projection neurons responsive to orexin (hypocretin)/orexin receptor-2 (OX2) signalling. Melanin-concentrating hormone (MCH) and orexin neurons often innervate and influence common target areas. Therefore, we assessed the relationship between these hypothalamic peptidergic systems and rat NI, by investigating the presence of an MCH innervation and MCH receptor-1 (MCH1) expression, and neurophysiological and behavioural effects of MCH c.f. orexin-A (OXA), within the NI. We identified lateral hypothalamus (LH), perifornical and sub-zona incerta MCH neurons that innervate NI, and characterised the rostrocaudal distribution of MCH-containing fibres in NI. Single-cell RT-PCR detected MCH1 and OX2 mRNA in NI, and multiplex, fluorescent in situ hybridisation revealed distinct co-expression patterns of MCH1 and OX2 mRNA in NI neurons expressing vesicular GABA transporter (vGAT) mRNA. Patch-clamp recordings revealed 34% of NI neurons tested were hyperpolarised by MCH (1 μM), representing a distinct population from OXA-sensitive NI neurons (35%). Intra-NI OXA infusion (600 pmol) in satiated rats during the light/inactive phase produced increased locomotor activity and food (standard chow) intake, whereas intra-NI MCH infusion (600 pmol) produced only a trend for decreased locomotor activity and no effect on food intake. Furthermore, in satiated or pre-fasted rats tested during the dark/active phase, intra-NI infusion of MCH did not alter the elevated locomotor activity or higher food intake observed. However, quantification of neuropeptide-immunostaining revealed differential diurnal fluctuations in orexin and MCH trafficking to NI. Our findings identify MCH and orexin inputs onto divergent NI populations which may differentially influence arousal and motivated behaviours.

SNAPshots of the MCHR1: a Comparison Between the PET-Tracers [18F]FE@SNAP and [11C]SNAP-7941

PurposeThe melanin-concentrating hormone receptor 1 (MCHR1) has become an important pharmacological target, since it may be involved in various diseases, such as diabetes, insulin resistance, and obesity. Hence, a suitable positron emission tomography radiotracer for the in vivo assessment of the MCHR1 pharmacology is imperative. The current paper contrasts the extensive in vitro, in vivo, and ex vivo assessments of the radiotracers [18F]FE@SNAP and [11C]SNAP-7941 and provides comprehensive information about their biological and physicochemical properties. Furthermore, it examines their suitability for first-in-man imaging studies.ProceduresKinetic real-time cell-binding studies with [18F]FE@SNAP and [11C]SNAP-7941 were conducted on adherent Chines hamster ovary (CHO-K1) cells stably expressing the human MCHR1 and MCHR2. Small animal imaging studies on mice and rats were performed under displacement and baseline conditions, as well as after pretreatment with the P-glycoprotein/breast cancer resistant protein inhibitor tariquidar. After the imaging studies, detailed analyses of the ex vivo biodistribution were performed. Ex vivo metabolism was determined in rat blood and brain and analyzed at various time points using a quantitative radio-HPLC assay.Results[11C]SNAP-7941 demonstrates high uptake on CHO-K1-hMCHR1 cells, whereas no uptake was detected for the CHO-K1-hMCHR2 cells. In contrast, [18F]FE@SNAP evinced binding to CHO-K1-hMCHR1 and CHO-K1-hMCHR2 cells. Imaging studies with [18F]FE@SNAP and [11C]SNAP-7941 showed an increased brain uptake after tariquidar pretreatment in mice, as well as in rats, and exhibited a significant difference between the time-activity curves of the baseline and blocking groups. Biodistribution of both tracers demonstrated a decreased uptake after displacement. [11C]SNAP-7941 revealed a high metabolic stability in rats, whereas [18F]FE@SNAP was rapidly metabolized.ConclusionsBoth radiotracers demonstrate appropriate imaging properties for the MCHR1. However, the pronounced metabolic stability as well as superior selectivity and affinity of [11C]SNAP-7941 underlines the decisive superiority over [18F]FE@SNAP.

Depression-resistant Phenotype in Mice Overexpressing Regulator of G Protein Signaling 8 (RGS8)

Regulator of G protein signaling (RGS) proteins are negative regulators of heterotrimeric G proteins that act by accelerating Gα-mediated GTPase activity to terminate G protein-coupled receptor-associated signaling. RGS8 is expressed in several brain regions involved with movement and mood. To investigate the role of RGS8 in vivo, we generated transgenic mice overexpressing brain RGS8 (RGS8tg). RGS8 gene and protein expressions were examined by real-time PCR and immunohistochemistry, respectively, and a significant increase in RGS8 protein was detected in the hippocampal CA1 region compared with wild-type mice (WT). We characterized the phenotypic traits, and found that RGS8tg showed decreased depressive-like behavior in the forced swimming test (FST). Previously, RGS8 was identified as a potent negative regulator of melanin-concentrating hormone receptor 1 (MCHR1), whose activation is mainly involved in energy homeostasis and emotional processing. Interestingly, acute oral administration of MCHR1 antagonist SNAP94847 did not have antidepressant-like effects on RGS8tg in the FST, but did show antidepressant effects on WT. In contrast, selective noradrenaline reuptake inhibitor desipramine had a significant effect on RGS8tg in the FST. MCHR1 is enriched in a subset of primary cilia, as sensory organelles that mediate extracellular signaling. Immunohistochemical analyses revealed significant elongation of MCHR1-positive cilia in the CA1 region of RGS8tg compared with WT. Taken together, these findings suggest that RGS8 participates in modulation of depression-like behavior through ciliary MCHR1 expressed in the CA1 region. The present study may support the possible modulation of RGS8 function in mood disorders.

Left-right pigmentation pattern of Japanese flounder corresponds to expression levels of melanocortin receptors (MC1R and MC5R), but not to agouti signaling protein 1 (ASIP1) expression

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.

Iodine‐Catalyzed Synthesis of Chalcogenophenes by the Reaction of 1,3‐Dienyl Bromides and Potassium Selenocyanate/Potassium Sulfide (KSeCN/K2S)

AbstractThe methods available for the synthesis of chalcogenophenes, in general, are associated with drawbacks of harsh conditions, use of costly metals, broad applicability, tedious purification process and low yield. To avoid these drawbacks a transition metal‐free iodine‐catalyzed reaction of aryl‐susbstituted 1,3‐dienyl bromides with potassium selenocyanate/potassium sulfide (KSeCN/K2S) leading to the corresponding selenophenes and thiophenes has been developed. Iodine is relatively benign, less expensive and readily available. Several diversely substituted selenophenes and thiophenes have been obtained by this procedure in high yields. Using this procedure 2‐(4‐chlorophenyl)thiophene, a key intermediate for the synthesis of a melanin concentrating hormone receptor ligand involved in the treatment of eating disorders, weight gain, obesity, depression and anxiety has been synthesized. Although the reaction is one‐pot essentially it proceeds in two steps involving a selenocyanate/thiolate intermediate leading to the selenophene/thiophene. The simple operation, use of inexpensive reagents and a metal‐free process make this procedure more attractive for an easy access to substituted selenophenes and thiophenes.magnified image

DNA sequencing and copy number variation analysis of MCHR2 in a cohort of Prader Willi like (PWL) patients

Prader Willi Syndrome (PWS) is a syndromic form of obesity caused by a chromosomal aberration on chromosome 15q11.2-q13. Patients with a comparable phenotype to PWS not carrying the 15q11.2-q13 defect are classified as Prader Willi like (PWL). In literature, PWL patients do frequently harbor deletions at 6q16, which led to the identification of the single-minded 1 (SIM1) gene as a possible cause for the presence of obesity in these patients. However, our previous work in a PWL cohort showed a rather limited involvement of SIM1 in the obesity phenotype. In this paper, we investigated the causal role of the melanin-concentrating hormone receptor 2 (MCHR2) gene in PWL patients, as most of the reported 6q16 deletions also encompass this gene and it is suggested to be active in the control of feeding behavior and energy metabolism. Copy number variation analysis of the MCHR2 genomic region followed by mutation analysis of MCHR2 was performed in a PWL cohort. Genome-wide microarray analysis of 109 patients with PWL did not show any gene harboring deletions on chromosome 6q16. Mutation analysis in 92 patients with PWL demonstrated three MCHR2 variants: p.T47A (c.139A>G), p.A76A (c.228T>C) and c.*16A>G. We identified a significantly higher prevalence of the c.228T>C C allele in our PWL cohort compared to previously published results and controls of the ExAC Database. Overall, our results are in line with some previously performed studies suggesting that MCHR2 is not a major contributor to human obesity and the PWL phenotype.

Environmentally Friendly Synthesis of Indoline Derivatives using Flow‐Chemistry Techniques

Flow chemistry proved to be a valuable technique to improve the synthesis route to melanin‐concentrating hormone receptor 1 (MCHr1) antagonists with the 1H,2H,3H,4H,5H‐[1,4]diazepino[1,7‐a]indole scaffold. A one‐step route for the heterogeneous catalytic hydrogenation of ethyl 4‐(2‐nitrophenyl)‐3‐oxobutanoate for the synthesis of ethyl 2‐(2,3‐dihydro‐1H‐indol‐2‐yl)acetate was developed, and the use of common reducing chemicals was avoided. N‐Alkylation of the indoline nitrogen atom was also optimized by using a purpose‐built flow reactor and by design of experiment (DoE). Applying an optimal set of parameters allowed us to decrease the amount of carcinogenic 1,2‐dibromoethane used by a factor of 10. Additionally, nearly complete conversion was achieved in a fraction of the original reaction time (30 min vs. 4 d); therefore, the productivity (space‐time yield) of the flow‐reactor system was proven to be ca. 200 times higher than that of the batch process.

Melanin concentrating hormone modulates oxytocin-mediated marble burying

Repetitive and perseverative behaviors are common features of a number of neuropsychiatric diseases such as Angelman's syndrome, Tourette's syndrome, obsessive-compulsive disorder, and autism spectrum disorders. The oxytocin system has been linked to the regulation of repetitive behavior in both animal models and humans, but many of its downstream targets have still to be found. We report that the melanin-concentrating hormone (MCH) system is a target of the oxytocin system in regulating one repetitive behavior, marble burying. First we report that nearly 60% of MCH neurons express oxytocin receptors, and demonstrate using rabies mediated tract tracing that MCH neurons receive direct presynaptic input from oxytocin neurons. Then we show that MCH receptor knockout (MCHR1KO) mice and MCH ablated animals display increased marble burying response while central MCH infusion decreases it. Finally, we demonstrate the downstream role of the MCH system on oxytocin mediated marble burying by showing that central infusions of MCH and oxytocin alone or together reduce it while antagonizing the MCH system blocks oxytocin-mediated reduction of this behavior. Our findings reveal a novel role for the MCH system as a mediator of the role of oxytocin in regulating marble-burying behavior in mice.

Cytoskeleton-related regulation of primary cilia shortening mediated by melanin-concentrating hormone receptor 1

Primary cilia are specialized microtubule-based organelles. Their importance is highlighted by the gamut of ciliary diseases associated with various syndromes including diabetes and obesity. Primary cilia serve as signaling hubs through selective interactions with ion channels and conventional G-protein-coupled receptors (GPCRs). Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a key regulator of feeding, is selectively expressed in neuronal primary cilia in distinct regions of the mouse brain. We previously found that MCH acts on ciliary MCHR1 and induces cilia shortening through a Gi/o-dependent Akt pathway with no cell cycle progression. Many factors can participate in cilia length control. However, the mechanisms for how these molecules are relocated and coordinated to activate cilia shortening are poorly understood. In the present study, we investigated the role of cytoskeletal dynamics in regulating MCH-induced cilia shortening using clonal MCHR1-expressing hTERT-RPE1 cells. Pharmacological and biochemical approaches showed that cilia shortening mediated by MCH was associated with increased soluble cytosolic tubulin without changing the total tubulin amount. Enhanced F-actin fiber intensity was also observed in MCH-treated cells. The actions of various pharmacological agents revealed that coordinated actin machinery, especially actin polymerization, was required for MCHR1-mediated cilia shortening. A recent report indicated the existence of actin-regulated machinery for cilia shortening through GPCR agonist-dependent ectosome release. However, our live-cell imaging experiments showed that MCH progressively elicited cilia shortening without exclusion of fluorescence-positive material from the tip. Short cilia phenotypes have been associated with various metabolic disorders. Thus, the present findings may contribute toward better understanding of how the cytoskeleton is involved in the GPCR ligand-triggered cilia shortening with cell mechanical properties that underlies clinical manifestations such as obesity.

In vivo evaluation of radiotracers targeting the melanin-concentrating hormone receptor 1: [11C]SNAP-7941 and [18F]FE@SNAP reveal specific uptake in the ventricular system

The MCHR1 is involved in the regulation of energy homeostasis and changes of the expression are linked to a variety of associated diseases, such as diabetes and adiposity. The study aimed at the in vitro and in vivo evaluation of [11C]SNAP-7941 and [18F]FE@SNAP as potential PET-tracers for the MCHR1. Competitive binding studies with non-radioactive derivatives and small-animal PET/CT and MRI brain studies were performed under baseline conditions and tracer displacement with the unlabelled MCHR1 antagonist (±)-SNAP-7941. Binding studies evinced high binding affinity of the non-radioactive derivatives. Small-animal imaging of [11C]SNAP-7941 and [18F]FE@SNAP evinced high tracer uptake in MCHR1-rich regions of the ventricular system. Quantitative analysis depicted a significant tracer reduction after displacement with (±)-SNAP-7941. Due to the high binding affinity of the non-labelled derivatives and the high specific tracer uptake of [11C]SNAP-7941 and [18F]FE@SNAP, there is strong evidence that both radiotracers may serve as highly suitable agents for specific MCHR1 imaging.

Translational Modeling to Guide Study Design and Dose Choice in Obesity Exemplified by AZD1979, a Melanin-concentrating Hormone Receptor 1 Antagonist.

In this study, we present the translational modeling used in the discovery of AZD1979, a melanin‐concentrating hormone receptor 1 (MCHr1) antagonist aimed for treatment of obesity. The model quantitatively connects the relevant biomarkers and thereby closes the scaling path from rodent to man, as well as from dose to effect level. The complexity of individual modeling steps depends on the quality and quantity of data as well as the prior information; from semimechanistic body‐composition models to standard linear regression. Key predictions are obtained by standard forward simulation (e.g., predicting effect from exposure), as well as non‐parametric input estimation (e.g., predicting energy intake from longitudinal body‐weight data), across species. The work illustrates how modeling integrates data from several species, fills critical gaps between biomarkers, and supports experimental design and human dose‐prediction. We believe this approach can be of general interest for translation in the obesity field, and might inspire translational reasoning more broadly.

Systematic Data Mining Reveals Synergistic H3R/MCHR1 Ligands.

In this study, we report a ligand-centric data mining approach that guided the identification of suitable target profiles for treating obesity. The newly developed method is based on identifying target pairs for synergistic positive effects and also encompasses the exclusion of compounds showing a detrimental effect on obesity treatment (off-targets). Ligands with known activity against obesity-relevant targets were compared using fingerprint representations. Similar compounds with activities to different targets were evaluated for the mechanism of action since activation or deactivation of drug targets determines the pharmacological effect. In vitro validation of the modeling results revealed that three known modulators of melanin-concentrating hormone receptor 1 (MCHR1) show a previously unknown submicromolar affinity to the histamine H3 receptor (H3R). This synergistic activity may present a novel therapeutic option against obesity.