Melanin-concentrating hormone receptor 1 is discarded by exosomes after internalization.

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a G protein-coupled receptor, is poised for interaction with its ligands on the plasma membrane. Analyses of MCHR1 knockout mice suggest that this receptor could be a therapeutic target for the treatment of appetite disorders, glucose metabolism, psychiatric disorders, and inflammation. Binding of MCH to MCHR1 initiates calcium signaling, which is subsequently attenuated through receptor internalization. However, the ultimate destiny of the receptor post-internalization remains unexplored. In this study, we report the extracellular secretion of MCHR1 via exosomes. The recruitment of MCHR1 to exosomes occurs subsequent to its internalization, which is induced by stimulation with the ligand MCH. Although a highly glycosylated form of MCHR1, potentially representing a mature form, is selectively recruited to exosomes, the MCHR1 transferred into other cells does not exhibit functionality. The truncation of MCHR1 at the C-terminus not only impairs its response to MCH but also hinders its recruitment to exosomes. These findings imply that functional MCHR1 could be secreted extracellularly via exosomes, a process that may represent a mechanism for the termination of intracellular MCHR1 signaling.

PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide's stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide's stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors' conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets.Communicated by Ramaswamy H. Sarma.

Early Alterations of PACAP and VIP Expression in the Female Rat Brain Following Spinal Cord Injury.

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.

Discovery of melanin-concentrating hormone receptor 1 in brown adipose tissue.

Although extensive research on brown adipose tissue (BAT) has stimulated optimism in the battle against obesity and diabetes, BAT physiology and organ crosstalk are not fully understood. Besides BAT, melanin-concentrating hormone (MCH) and its receptor (MCHR1) play an important role in energy homeostasis. Because of the link between hypothalamic MCH neurons and sympathetic BAT activation via ß-adrenoceptors, we investigated the expression and physiological role of the MCHR1 in BAT. MCHR1 was detected in rodent and human BAT with RT-qPCR and western blot analyses. In vivo imaging in rats used the glucose analog [18 F]FDG and the MCHR1-tracer [11 C]SNAP-7941. We found that the ß3-adrenoceptor (ADRB3) agonist CL316,243 increased [11 C]SNAP-7941 uptake in BAT. Additionally, a pharmacological concentration of SNAP-7941-a low-affinity ADRB3 ligand-stimulated [18 F]FDG uptake, reflecting BAT activation. In cultured human adipocytes, CL316,243 induced MCHR1 expression, further supporting a direct interaction between MCHR1 and ADRB3. These findings characterized MCHR1 expression in rodent and human BAT for the first time, including in vitro and in vivo data demonstrating a link between MCHR1 and the ß3-adrenergic system. The presence of MCHR1 in BAT emphasizes the role of BAT in energy homeostasis and may help uncover treatment approaches for obesity.

The Rat Mammary Gland as a Novel Site of Expression of Melanin-Concentrating Hormone Receptor 1 mRNA and Its Protein Immunoreactivity.

Lactation is a complex physiological process, depending on orchestrated central and peripheral events, including substantial brain plasticity. Among these events is a novel expression of pro-melanin-concentrating hormone (Pmch) mRNA in the rodent hypothalamus, such as the ventral part of the medial preoptic area (vmMPOA). This expression reaches its highest levels around postpartum day 19 (PPD19), when dams transition from lactation to the weaning period. The appearance of this lactation-related Pmch expression occurs simultaneously with the presence of one of the Pmch products, melanin-concentrating hormone (MCH), in the serum. Given the relevance of the MPOA to maternal physiology and the contemporaneity between Pmch expression in this structure and the weaning period, we hypothesized that MCH has a role in the termination of lactation, acting as a mediator between central and peripheral changes. To test this, we investigated the presence of the MCH receptor 1 (MCHR1) and its gene expression in the mammary gland of female rats in different stages of the reproductive cycle. To that end, in situ hybridization, RT-PCR, RT-qPCR, nucleotide sequencing, immunohistochemistry, and Western blotting were employed. Although Mchr1 expression was detected in the epidermis and dermis of both diestrus and lactating rats, parenchymal expression was exclusively found in the functional mammary gland of lactating rats. The expression of Mchr1 mRNA oscillated through the lactation period and reached its maximum in PPD19 dams. Presence of MCHR1 was confirmed with immunohistochemistry with preferential location of MCHR1 immunoreactive cells in the alveolar secretory cells. As was the case for gene expression, the MCHR1 protein levels were significantly higher in PPD19 than in other groups. Our data demonstrate the presence of an anatomical basis for the participation of MCH peptidergic system on the control of lactation through the mammary gland, suggesting that MCH could modulate a prolactation action in early postpartum days and the opposite role at the end of the lactation.

New perspectives on GPCRs: GPCR heterodimer formation (melanocortin receptor) and GPCR on primary cilia (melanin concentrating hormone receptor).

GPCRs are the largest family of receptors accounting for about 30% of the current drug targets. However, it is difficult to fully elucidate the mechanisms regulating intracellular GPCR signal regulation. It is thus important to consider and investigate GPCRs with respect to endogenous situations. Our group has been investigating GPCRs involved in body color (teleost and amphibian) and eating (vertebrate). Here, I review two independent GPCR systems (heterodimer formation and primary ciliated GPCR) that can be breakthroughs in GPCR research. In teleosts, MCRs form heterodimers, which significantly reduce their affinity for acetylated ligands. In mammals, MCHR1 is localized in the ciliary membrane and shortens the length of the primary cilia through a unique signal from the ciliary membrane. Considering these two new GPCR concepts is expected to advance the overall view of the GPCR system.

Melanin-concentrating hormone (MCH) in the median raphe nucleus: Fibers, receptors and cellular effects.

Serotonergic neurons of the median raphe nucleus (MnR) and hypothalamic melanin-concentrating hormone (MCH)-containing neurons, have been involved in the control of REM sleep and mood. In the present study, we examined in rats and cats the anatomical relationship between MCH-containing fibers and MnR neurons, as well as the presence of MCHergic receptors in these neurons. In addition, by means of in vivo unit recording in urethane anesthetized rats, we determined the effects of MCH in MnR neuronal firing. Our results showed that MCH-containing fibers were present in the central and paracentral regions of the MnR. MCHergic fibers were in close apposition to serotonergic and non-serotonergic neurons. By means of an indirect approach, we also analyzed the presence of MCHergic receptors within the MnR. Accordingly, we microinjected MCH conjugated with the fluorophore rhodamine (R-MCH) into the lateral ventricle. R-MCH was internalized into serotonergic and non-serotonergic MnR neurons; some of these neurons were GABAergic. Furthermore, we determined that intracerebroventricular administration of MCH induced a significant decrease in the firing rate of 53 % of MnR neurons, while the juxtacellular administration of MCH reduced the frequency of discharge in 67 % of these neurons. Finally, the juxtacellular administration of the MCH-receptor antagonist ATC-0175 produced an increase in the firing rate in 78 % of MnR neurons. Hence, MCH produces a strong regulation of MnR neuronal activity. We hypothesize that MCHergic modulation of the MnR neuronal activity may be involved in the promotion of REM sleep and in the pathophysiology of depressive disorders.

Natural hypothalamic circuit dynamics underlying object memorization.

Brain signals that govern memory formation remain incompletely identified. The hypothalamus is implicated in memory disorders, but how its rapidly changing activity shapes memorization is unknown. During encounters with objects, hypothalamic melanin-concentrating hormone (MCH) neurons emit brief signals that reflect object novelty. Here we show that targeted optogenetic silencing of these signals, performed selectively during the initial object encounters (i.e. memory acquisition), prevents future recognition of the objects. We identify an upstream inhibitory microcircuit from hypothalamic GAD65 neurons to MCH neurons, which constrains the memory-promoting MCH cell bursts. Finally, we demonstrate that silencing the GAD65 cells during object memory acquisition improves future object recognition through MCH-receptor-dependent pathways. These results provide causal evidence that object-associated signals in genetically distinct but interconnected hypothalamic neurons differentially control whether the brain forms object memories. This gating of memory formation by hypothalamic activity establishes appropriate behavioral responses to novel and familiar objects.

The role of Olfaction in MCH-regulated spontaneous maternal responses.

Melanin concentrating hormone (MCH) is involved in the initiation of maternal behavior during the postpartum period. Virgin females also display some aspects of maternal care independent of the hormonal and neurochemical changes associated with pregnancy and parturition. Maternal behavior in virgin females is triggered by pups-generated chemosensory signals. We therefore examined the role of MCH in maternal-related behaviors in virgin mice and whether it involves chemosensory mechanisms. We used mice with germline knock-out of MCH receptor (MCHR1 KO) and mice with conditional ablation of MCH neurons (MCH cKO) using Cre-inducible diphtheria toxin (iDTR) system. We report that germline deletion of MCHR1 and ablation of MCH neurons impair spontaneous maternal behavior that is induced upon pups' exposure. The latency and duration to retrieve pups by MCHR1 KO and MCH cKO mice are longer than their control littermate mice. In support of this finding, we found that in the three-chamber social test, both MCHR1 KO and MCH cKO mice display a lack of interest in interacting with pups. Strikingly, however, we found that while MCHR1 KO mice were unable to detect pups' chemosensory signals and displayed impairment in general olfactory discrimination, MCH cKO mice exhibited normal olfactory function. Our findings indicate that the lack of MCHR1 or of normal MCH levels causes defects in maternal behavior in non-sensitized virgin mice, and that disruption of the olfactory signaling might not count for these defects.

Arginine vasotocin (AVT)/mesotocin (MT) receptors in chickens: Evidence for the possible involvement of AVT-AVPR1 signaling in the regulation of oviposition and pituitary prolactin expression.

Two structurally related peptides, arginine vasotocin (AVT) and mesotocin (MT), are reported to regulate many physiological processes, such as anti-diuresis and oviposition in birds, and their actions are likely mediated by four AVT/MT receptors (AVPR1A, AVPR1B, MTR and AVPR2b), which are orthologous/paralogous to human AVPR1A, AVPR1B, OXTR and AVPR2 respectively. However, our knowledge regarding the functions of these avian AVT/MT receptors has been limited. Here, we examined the functionality and expression of these receptors in chickens and investigated the roles of AVT in the anterior pituitary. Our results showed that 1) AVPR1A, AVPR1B and AVPR2b could be preferentially activated by AVT, monitored by cell-based luciferase reporter assays and/or Western blot, indicating that they are AVT-specific receptors (AVPR1A; AVPR1B) or AVT-preferring receptor (AVPR2b) functionally coupled to intracellular calcium, MAPK/ERK and cAMP/PKA signaling pathways. In contrast, MTR could be activated by AVT and MT with similar potencies, indicating that MTR is a receptor common for both peptides; 2) Using qPCR, differential expression of the four receptors was found in chicken tissues including the oviduct and anterior pituitary. In particular, only AVPR1A is abundantly expressed in the uterus, suggesting its involvement in mediating AVT-induced oviposition. 3) In cultured chick pituitary cells, AVT could stimulate ACTH and PRL expression and secretion, an action likely mediated by AVPR1B and/or AVPR1A abundantly expressed in anterior pituitary. Collectively, our data helps to elucidate the roles of AVT/MT in birds, such as the 'oxytocic action' of AVT, which induces uterine muscle contraction during oviposition.

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.

Expression of genes for melanotropic peptides and their receptors for morphological color change in goldfish Carassius auratus.

To evaluate the association of the melanotropic peptides and their receptors for morphological color change, we investigated the effects of changes in background color, between white and black, on xanthophore density in the scales and expression levels of genes for hormonal peptides and corresponding receptors (MCH-R2, MC1R, and MC5R) in goldfish (Carassius auratus). The xanthophore density in both dorsal and ventral scales increased after transfer from a white to black background. However, xanthophore density in dorsal scales increased after transfer from a black to white background, and that of ventral scales decreased after transfer from a black to black background, which served as the control. In the white-reared fish, melanin-concentrating hormone (mch) mRNA content in the brain was higher than that in black-reared fish, whereas proopiomelanocortin a (pomc-a) mRNA content in the pituitary was lower than that in the black-reared fish. Agouti-signaling protein (asp) mRNA was detected in the ventral skin but not in the dorsal skin. No difference was observed in the asp mRNA content between fish reared in white or black background, suggesting that ASP might not be associated with background color adaptation. In situ hybridization revealed that both mc1r and mc5r were expressed in the xanthophores in scales. The mRNA content of mc1r in scales did not always follow the background color change, whereas those of mc5r decreased in the white background and increased in the black background, suggesting that mc5r might be a major factor reinforcing the function of MSH in morphological color changes. White backgrounds increased mch mRNA content in the brain, but decreased mch-r2 mRNA content in the scales. These altered expression levels of melanotropin receptors might affect reactivity to melanotropins through long-term adaptation to background color.

Anti-Müllerian Hormone (AMH) May Stall Ovarian Cortex Function Through Modulation of Hormone Receptors Other Than the AMH Receptor.

OBJECTIVE: To test whether recombinant anti-Müllerian hormone (AMH) can inhibit ovarian cortex function by modulating the expression of other hormone receptors. MATERIALS AND METHODS: Pilot experimental study with ovarian cortex obtained from 5 patients. Immediately after explant, the ovarian cortex specimens were divided into 5 equal fragments. One fragment was flash-frozen (uncultured) and 4 were incubated for 48 hours at 37°C in a pH-adjusted gamete buffer medium with increasing AMH concentrations of 0, 5, 25, and 50 ng/mL. After incubation, all specimens were rinsed and flash-frozen for polymerase chain reaction (PCR) executed in triplicates. We utilized real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine messenger RNA (mRNA) levels of AMH and its receptor Anti-Müllerian Hormone-Receptor 2 (AMH-R2), follicle stimulating hormone receptor (FSH-R), luteinizing hormone receptor (LH-R), inhibin B, and insulin-like growth factor 1 receptor 1 (IGF1-R1) in ovarian cortex tissue. In addition, we performed Ki-67 immunostaining to evaluate cell proliferation in the treatment groups. RESULTS: Absence of recombinant human AMH (rAMH) caused upregulation of all markers. Exposure to increasing rAMH concentrations caused tissue AMH expression downregulation ( P = .024), while AMH-R2 ( P = .005), FSH-R ( P = .009), LH-R ( P = .003), and inhibin B ( P = .001) mRNA expression followed a bell-shaped response with an increased expression at low dose, followed by a decreased expression at higher doses. Expression of IGF1-R1 was independent ( P = .039) of rAMH exposure. The Ki-67 immunostaining showed an increased cell proliferation in the media control compared to the uncultured and the tissue cultured with rAMH. CONCLUSIONS: Culture with increasing rAMH concentrations caused downregulation of its own, as well as other hormone receptors, and a decreased ovarian cortex cell proliferation. These results help understanding the inhibitory effects of AMH on follicular development.

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.

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.

Characterization of melanin-concentrating hormone (MCH) and its receptor in chickens: Tissue expression, functional analysis, and fasting-induced up-regulation of hypothalamic MCH expression.

Melanin-concentrating hormone (MCH) is a neuropeptide expressed in the brain and exerts its actions through interaction with the two known G protein-coupled receptors, namely melanin-concentrating hormone receptor 1 and 2 (MCHR1 and MCHR2) in mammals. However, the information regarding the expression and functionality of MCH and MCHR(s) remains largely unknown in birds. In this study, using RT-PCR and RACE PCR, we amplified and cloned a MCHR1-like receptor, which is named cMCHR4 according to its evolutionary origin, and a MCHR2 from chicken brain. The cloned cMCHR4 was predicted to encode a receptor of 367 amino acids, which shares high amino acid identities with MCHR4 of ducks (90%), western painted turtles (85%), and coelacanths (77%), and a comparatively low identity to human MCHR1 (58%) and MCHR2 (38%), whereas chicken MCHR2 encodes a putative C-terminally truncated receptor and is likely a pseudogene. Using cell-based luciferase reporter assays or Western blot, we further demonstrated that chicken (and duck) MCHR4 could be potently activated by chicken MCH1-19, and its activation can elevate calcium concentration and activate MAPK/ERK and cAMP/PKA signaling pathways, indicating an important role of MCHR4 in mediating MCH actions in birds. Quantitative real-time PCR revealed that both cMCH and cMCHR4 mRNA are expressed in various brain regions including the hypothalamus, and cMCH expression in the hypothalamus of 3-week-old chicks could be induced by 36-h fasting, indicating that cMCH expression is correlated with energy balance. Taken together, characterization of chicken MCH and MCHR4 will aid to uncover the conserved roles of MCH across vertebrates.

Effects of alpha-melanocyte-stimulating hormone on mitochondrial energy metabolism in rats of different age-groups.

Hypothalamic alpha-melanocyte-stimulating hormone (α-MSH) is a key catabolic mediator of energy homeostasis. Its anorexigenic and hypermetabolic effects show characteristic age-related alterations that may be part of the mechanism of middle-aged obesity and geriatric anorexia/cachexia seen in humans and other mammals. We aimed to investigate the role of α-MSH in mitochondrial energy metabolism during the course of aging in a rodent model. To determine the role of α-MSH in mitochondrial energy metabolism in muscle, we administered intracerebroventricular (ICV) infusions of α-MSH for 7-days to different age-groups of male Wistar rats. The activities of oxidative phosphorylation complexes I to V and citrate synthase were determined and compared to those of age-matched controls. We also quantified mitochondrial DNA (mtDNA) copy number and measured the expression of the master regulators of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPARγ). The peptide reduced weight gain in juvenile rats to one fifth of that of controls and increased the weight loss in older animals by about five fold. Mitochondrial DNA copy number inversely correlated with changes in body weight in controls, but not in α-MSH-treated animals. The strong increase in body weight in young rats was associated with a low mtDNA copy number and high PPARγ mRNA levels in controls. Expression of PGC-1α and PPARγ declined with age, whereas OXPHOS and citrate synthase enzyme activities were unchanged. In contrast, α-MSH treatment suppressed OXPHOS enzyme and citrate synthase activity. In conclusion, our results showed age-related differences in the metabolic effects of α-MSH. In addition, administration of α-MSH suppressed citrate synthase and OXPHOS activities independent of age. These findings suggest that α-MSH exposure may inhibit mitochondrial biogenesis.

A novel role for pigment genes in the stress response in rainbow trout (Oncorhynchus mykiss).

In many vertebrate species visible melanin-based pigmentation patterns correlate with high stress- and disease-resistance, but proximate mechanisms for this trait association remain enigmatic. Here we show that a missense mutation in a classical pigmentation gene, melanocyte stimulating hormone receptor (MC1R), is strongly associated with distinct differences in steroidogenic melanocortin 2 receptor (MC2R) mRNA expression between high- (HR) and low-responsive (LR) rainbow trout (Oncorhynchus mykiss). We also show experimentally that cortisol implants increase the expression of agouti signaling protein (ASIP) mRNA in skin, likely explaining the association between HR-traits and reduced skin melanin patterning. Molecular dynamics simulations predict that melanocortin 2 receptor accessory protein (MRAP), needed for MC2R function, binds differently to the two MC1R variants. Considering that mRNA for MC2R and the MC1R variants are present in head kidney cells, we hypothesized that MC2R activity is modulated in part by different binding affinities of the MC1R variants for MRAP. Experiments in mammalian cells confirmed that trout MRAP interacts with the two trout MC1R variants and MC2R, but failed to detect regulation of MC2R signaling, possibly due to high constitutive MC1R activity.

Investigation of the effect of α-melanocyte-stimulating hormone on proliferation and early stages of differentiation of human induced pluripotent stem cells.

We have studied the influence of α-melanocyte-stimulating hormone (α-MSH) on proliferation and early stages of differentiation of human induced pluripotent stem cells (iPSc). We have demonstrated that α-MSH receptor genes are expressed in undifferentiated iPSc. The expression levels of MCR1, MCR2, and MCR3 increased at the embryoid body (EB) formation stage. The formation of neural progenitors was accompanied by elevation of MCR2, MCR3, and MCR4 expression. α-MSH had no effect on EB generation and iPSc proliferation at concentrations ranging from 1 nM to 10 µM. At the same time, α-MSH increased the generation of neural rosettes in human iPSc cultures more than twice.

Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells.

We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPKp42/44 and AKTser473. Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.