Endospanin 1 silencing in the hypothalamic arcuate nucleus contributes to sustained weight loss of high fat diet obese mice.

Leptin targets specific receptors (OB-R) expressed in the hypothalamus to regulate energy balance. Leptin decreases food intake in normal weight individuals, but this effect is blunted in obese subjects who are characterized by a state of leptin resistance. The prevention of leptin resistance is one of the major goals of obesity research. Recently, we identified endospanin 1 as a negative regulator of OB-R, which by interacting with OB-R retains the receptor inside the cell. We show here that in obese mice endospanin 1 is upregulated in the hypothalamic arcuate nucleus (ARC), the major brain structure involved in body weight regulation, suggesting that endospanin 1 is implicated in obesity development and/or the installation of leptin resistance. In contrast, silencing of endospanin 1 with lentiviral vectors in the ARC of obese mice fully restores leptin responsiveness when combined with a switch to ad libitum fed chow diet. The recovery of central leptin sensitivity is accompanied by sustained body weight loss and amelioration of blood lipid parameters and steatosis. Collectively, our results define endospanin 1 as a novel therapeutic target against obesity.

Melatonin: Facts, Extrapolations and Clinical Trials.

Melatonin is a fascinating molecule that has captured the imagination of many scientists since its discovery in 1958. In recent times, the focus has changed from investigating its natural role as a transducer of biological time for physiological systems to hypothesized roles in virtually all clinical conditions. This goes along with the appearance of extensive literature claiming the (generally) positive benefits of high doses of melatonin in animal models and various clinical situations that would not be receptor-mediated. Based on the assumption that melatonin is safe, high doses have been administered to patients, including the elderly and children, in clinical trials. In this review, we critically review the corresponding literature, including the hypotheses that melatonin acts as a scavenger molecule, in particular in mitochondria, by trying not only to contextualize these interests but also by attempting to separate the wheat from the chaff (or the wishful thinking from the facts). We conclude that most claims remain hypotheses and that the experimental evidence used to promote them is limited and sometimes flawed. Our review will hopefully encourage clinical researchers to reflect on what melatonin can and cannot do and help move the field forward on a solid basis.

Melatonin receptors, heterodimerization, signal transduction and binding sites: what's new?

Melatonin is a neurohormone that has been claimed to be involved in a wide range of physiological functions. Nevertheless, for most of its effects, the mechanism of action is not really known. In mammals, two melatonin receptors, MT1 and MT2, have been cloned. They belong to the G-protein-coupled receptor (GPCR) superfamily. They share some specific short amino-acid sequences, which suggest that they represent a specific subfamily. Another receptor from the same subfamily, the melatonin-related receptor has been cloned in different species including humans. This orphan receptor also named GPR50 does not bind melatonin and its endogenous ligand is still unknown. Nevertheless, this receptor has been shown to behave as an antagonist of the MT1 receptor, which opens new pharmacological perspectives for GPR50 despite the lack of endogenous or synthetic ligands. Moreover, MT1 and MT2 interact together through the formation of heterodimers at least in cells transfected with the cDNA of these two receptors. Lastly, signalling complexes associated with MT1 and MT2 receptors are starting to be deciphered. A third melatonin-binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). Inhibition of QR2 by melatonin may explain melatonin's protective effect that has been reported in different animal models and that is generally associated with its well-documented antioxidant properties.

Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells.

The influence of cell confluence on the ß-adrenoceptor (ß-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. ß-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the ß-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100µM) induced a similar saturating response in both LD and HD cells. A ß1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a ß2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a ß-AR ligand, identified two binding sites in HD cells, corresponding to ß1- and ß2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to ß2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10µM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1µM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the ß-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both ß1- and ß2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only ß2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the ß1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular ß-AR signalling.

Pineal and cortical melatonin receptors MT1 and MT2 are decreased in Alzheimer's disease.

The pineal hormone melatonin is involved in physiological transduction of temporal information from the light dark cycle to circadian and seasonal behavioural rhythms, as well as possessing neuroprotective properties. Melatonin and its receptors MT1 and MT2, which belong to the family of G protein-coupled receptors, are impaired in Alzheimer's disease (AD) with severe consequences to neuropathology and clinical symptoms. The present data provides the first immunohistochemical evidence for the cellular localization of the both melatonin receptors in the human pineal gland and occipital cortex, and demonstrates their alterations in AD. We localized MT1 and MT2 in the pineal gland and occipital cortex of 7 elderly controls and 11 AD patients using immunohistochemistry with peroxidase-staining. In the pineal gland both MT1 and MT2 were localized to pinealocytes, whereas in the cortex both receptors were expressed in some pyramidal and non-pyramidal cells. In patients with AD, parallel to degenerative tissue changes, there was an overall decrease in the intensity of receptors in both brain regions. In line with our previous findings, melatonin receptor expression in AD is impaired in two additional brain areas, and may contribute to disease pathology.

Cloning and functional analysis of a polymorphic variant of the ovine Mel 1a melatonin receptor.

We have isolated a novel variant of the Mel 1a melatonin receptor from an ovine PT cDNA library. Relative to the reported sequence for the Mel 1a melatonin receptor there are 8 changes in the DNA sequence. Only 3 of these result in amino acid substitutions, one in extracellular loop 3 and two in the carboxy-terminal tail. We have designated the novel variant of the sheep Mel 1a receptor Mel 1a(beta), and correspondingly the previously reported variant Mel 1a(alpha). As minor changes in the primary amino acid sequence of G-protein-coupled receptors can influence their functional characteristics we have accordingly characterized this novel variant of the Mel 1a melatonin receptor. This melatonin receptor displays high affinity binding and inhibits the cAMP second messenger pathway in transfected L-cells demonstrating that this receptor is fully functional. PCR analysis shows Mel 1a(beta) is present in several breeds of sheep and suggests that the Mel 1a(beta) receptor was established early in the evolution of the sheep species.

Novel isoforms of Mel1c melatonin receptors modulating intracellular cyclic guanosine 3',5'-monophosphate levels.

Two cDNAs encoding novel isoforms of Xenopus laevis melatonin receptors were cloned using PCR primers specific for the X. laevis-melanophore Mel1c melatonin receptor described in a recent publication. The novel isoforms were highly homologous to the described frog Mel1c cDNA, although the C-terminal tail of both was shorter by 65 amino acid residues. Nucleotide sequences of these novel isoforms, called Mel1c(alpha) and Mel1c(beta), differed from each other by only 35 nucleotides and six amino acid residues. Studies on several animals of various Xenopus species indicate that Mel1c(alpha) and Mel1c(beta) receptors may correspond to allelic variants of the same locus. Studies on cells transfected with both receptor cDNAs showed the expression of high-affinity 2-[125I]iodomelatonin binding sites. Agonist stimulation of Mel1c(alpha) receptor was associated with the inhibition of cAMP accumulation stimulated by forskolin (IC50 approximately 10(-10) M) in HeLa, Ltk-, and human embryonic kidney 293 (HEK 293) cells. Mel1c(beta) receptor modulated cAMP in HeLa and HEK 293 cells but not in Ltk- cells. Both receptors inhibited, in a dose-dependent manner, cGMP accumulation in all three cell lines incubated with a phosphodiesterase inhibitor. This effect was localized upstream of soluble guanylyl cyclase and was blocked by pertussis toxin treatment. However, IC50 values (approximately 10(-10) M for Mel1c(beta) and 10(-9) to 10(-7) M for Mel1c(alpha)) and maximal inhibition levels showed that Mel1c(alpha) receptors are much less efficiently coupled to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between Mel1c(alpha) and Mel1c(beta) receptors are located within cytoplasmic regions potentially involved in signal transduction. The existence of coupling differences is in agreement with the observation that expression of both receptors is evolutionally conserved in native tissue. In conclusion, two novel, potentially allelic, isoforms of Xenopus Mel1c melatonin receptors display identical ligand-binding characteristics, but different potencies in modulating cAMP and cGMP levels through G(i)/G(o)-dependent pathways. Furthermore, to our knowledge, this study provides the first data on the modulation of intracellular cGMP levels by cloned melatonin receptors.

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins.

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

Functional expression of MT2 (Mel1b) melatonin receptors in human PAZ6 adipocytes.

Several reports have demonstrated that the pineal hormone, melatonin, plays an important role in body mass regulation in mammals. To date, however, the target tissues and relevant biochemical mechanisms involved remain uncharacterized. As adipose tissue is the principal site of energy storage in the body, we investigated whether melatonin could also act on this tissue. Semiquantitative RT-PCR analysis revealed the expression of MT1 and MT2 melatonin receptor mRNAs in the human brown adipose cell line, PAZ6, as well as in human brown and white adipose tissue. Binding analysis with 2-[(125)I]iodomelatonin ((125)I-Mel) revealed the presence of a single, high affinity binding site in PAZ6 adipocytes with a binding capacity of 7.46 +/- 1.58 fmol/mg protein and a K(d) of 457 +/- 5 pM. Both melatonin and the MT2 receptor-selective antagonist, 4-phenyl-2-propionamidotetraline, competed with 2-[(125)I]iodomelatonin binding, with respective K(i) values of 3 x 10(-11) and 1.5 x 10(-11) M. Functional expression of melatonin receptors in PAZ6 adipocytes was indicated by the melatonin-induced, dose-dependent inhibition of forskolin-stimulated cAMP levels and basal cGMP levels with IC(50) values of 2 x 10(-9) and 3 x 10(-10) M, respectively. Modulation of the cGMP pathway by melatonin further supports functional expression of MT2 receptors, as this pathway was shown to be specific for that subtype in humans. In addition, long-term melatonin treatment of PAZ6 adipocytes was found to decrease the expression of the glucose transporter Glut4 and glucose uptake, an important parameter of adipocyte metabolism. These results suggest that melatonin may act directly at MT2 receptors on human brown adipocytes to regulate adipocyte physiology.

Desensitization of the beta-adrenergic response in human brown adipocytes.

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.

Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein-coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels.

Binding assays using 2-[125I]iodomelatonin revealed high-affinity, guanosine 5'-O-(3-thiotriphosphate) sensitive, melatonin binding sites (B(max) 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT-PCR techniques identified these sites as human Mel1a melatonin receptors. Challenge of HEK293 cells with 1 microM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over-express the human Mel1a melatonin receptor (B(max) > 400 fmol/mg protein) melatonin dose-dependently inhibited stimulated cyclic AMP levels (IC50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein-coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.

Enhancement of immunoassay sensitivity by molecular modification of competitors.

The potential of weak competitors to enhance the sensitivity of competitive immunoassays is described. Several triazine derivatives have been analyzed for their use as competitors. Their binding properties were determined using an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific to triazines. Selected derivatives were immobilized onto the surface of a fibre optic sensor and atrazine was determined in a competitive manner using a fluorescein-labelled antibody. Using the weak binding competitor 11-(4-ethylamino-6-methylthio-s-triazine-2-yl)undecanoic acid (TE11S) the detection limit for atrazine could be lowered 100-fold in comparison to 2-aminohexylamino-4-ethylamino-6-isopropylamino-s-triazine (AHA), the previously used competitor.

Differential signaling of human Mel1a and Mel1b melatonin receptors through the cyclic guanosine 3'-5'-monophosphate pathway.

Cyclic guanosine 3'-5'-monophosphate (cGMP) has recently been shown to constitute a second messenger for Xenopus laevis melatonin Mel1c receptors. To verify whether cGMP levels are also modulated by mammalian melatonin receptors, we cloned the genes encoding the human Mel1a and Mel1b receptor subtypes and expressed them in human embryonic kidney cells. Pharmacological profiles and inhibition of forskolin-stimulated adenosine 3'-5'-cyclic monophosphate levels by melatonin confirmed functional expression of high-affinity melatonin receptors. Mel1b receptor-transfected cells modulated cGMP levels in a dose-dependent manner via the soluble guanylyl cyclase pathway. In contrast, Mel1a receptors had no effect on cGMP levels. These results demonstrate that mammalian melatonin receptors modulate cGMP levels and reveal for the first time differences in signaling between melatonin receptor subtypes, which may explain the necessity to express different receptor subtypes.

Cerebrovascular melatonin MT1-receptor alterations in patients with Alzheimer's disease.

The pineal hormone melatonin has two major functions: as a transducer of the circadian day-night signal across the seasons, and as a vasoactive substance regulating cerebral circulation. The vasoconstrictive effects of melatonin have been postulated to be mediated by the melatonin 1a-receptor (MT1). The objective of this study was to provide the first immunohistochemical evidence for the localization of vascular MT1 in human control hippocampus compared to Alzheimer's disease (AD) patients, since regional blood flow impairments contribute to the neurodegenerative course of the disease. Both superficial and intrahippocampal arteries revealed MT1 immunoreactivity in adventitia in controls, which was distinctly increased in AD patients. The increased MT1 in AD may indicate a regulatory response to impaired melatonin levels in those patients, contributing to the regulation of cerebral circulation.

Melatonin MT-1-receptor immunoreactivity in the human eye.

AIM: To examine the distribution of melatonin 1a (MT1) receptors in the human eye. METHODS: Seven normal human eyes were examined by immunohistochemical staining of paraffin sections, using an anti-MT1 primary antibody and an ABC detection system. RESULTS: MT1 receptor immunoreactivity (MT1-IR) was detected primarily in the inner segments of rods and cones and in retinal ganglion cells. In addition, MT1-IR was present in the adventitia of retinal arteries and veins, including the papillary region, but absent in ciliary and choroidal vessels. Mild staining of corneal endothelial cells and keratocytes was observed in all but two eyes. CONCLUSION: MT1-IR is present in various ocular tissues with the highest density in photoreceptor cells and ganglion cells. The physiological function of these receptors deserves further investigation.

Melatonin and energy homeostasis: peripheral versus central regulation.

Melatonin, the hormone of darkness, has been known for a long time to be a major regulator of energy homeostasis in hibernating animals. Much less is known about the role of melatonin in energy homeostasis in non-hibernating animals, including humans. In mammals, two specific melatonin receptor subtypes, MT1 and MT2, have been cloned and are known to be expressed at central and peripheral sites. Although a central regulation of energy homeostasis has been widely accepted for hibernating animals, the exact site of melatonin action remains still poorly defined. Central effects appear to be predominantly mediated by the MT1 subtype. Recently, several groups showed that melatonin may also have a direct effect on peripheral tissues involved in energy homeostasis such as pancreatic beta cell, hepatocytes and adipocytes. Both, the MT1 and MT2 subtypes appear to be involved. The respective contribution of central and peripheral effects of melatonin on energy homeostasis in vivo must be established in future studies.

[Signaling by melatonin receptors]. / Signalisation des récepteurs de la mélatonine.

Melatonin is a hormone involved in various physiological processes such as the circadian cycle, hormone release and immune response. High-affinity melatonin receptors are classified in two pharmacologically distinct groups: Mel1 and Mel2. These receptors have first been localized in different organs and brain structures and some subtypes have since been cloned. Inhibition of adenylyl cyclase by Mel1 receptors is the best investigated signalling pathway but cannot be entirely responsible for the diversity of melatonin-induced phenomena. Phospholipase C, potassium ion channels and guanylyl cyclases have also been reported to be modulated by melatonin. This review updates present knowledge of the characterization and signalization of melatonin receptors.

Association of the intronic rs2072621 polymorphism of the X-linked GPR50 gene with affective disorder with seasonal pattern.

This case-control study found an association between Seasonal Affective Disorder (SAD) and a single nucleotide polymorphism (intronic rs2072621) of the gene encoding GPR50 (an orphan member of the G protein-coupled melatonin receptor subfamily) in females. This may represent a gender-specific risk factor and a molecular link between melatonin and SAD.

Neuroanatomical distribution of the orphan GPR50 receptor in adult sheep and rodent brains.

GPR50, formerly known as melatonin-related receptor, is one of three subtypes of the melatonin receptor subfamily, together with the MT(1) and MT(2) receptors. By contrast to these two high-affinity receptor subtypes and despite its high identity with the melatonin receptor family, GPR50 does not bind melatonin or any other known ligand. Specific and reliable immunological tools are therefore needed to be able to elucidate the physiological functions of this orphan receptor that are still largely unknown. We have generated and validated a new specific GPR50 antibody against the ovine GPR50 and used it to analyse the neuroanatomical distribution of the GPR50 in sheep, rat and mouse whole brain. We demonstrated that GPR50-positive cells are widely distributed in various regions, including the hypothalamus and the pars tuberalis of the pituitary, in all the three species studied. GPR50 expressing cells are abundant in the dorsomedial nucleus of the hypothalamus, the periventricular nucleus and the median eminence. In rodents, immunohistochemical studies revealed a broader distribution pattern for the GPR50 protein. GPR50 immunoreactivity is found in the medial preoptic area (MPA), the lateral septum, the lateral hypothalamic area, the bed nucleus of the stria terminalis, the vascular organ of the laminae terminalis and several regions of the amygdala, including the medial nuclei of amygdala. Additionally, in the rat brain, GPR50 protein was localised in the CA1 pyramidal cell layer of the dorsal hippocampus. In mice, moderate to high numbers of GPR50-positive cells were also found in the subfornical organ. Taken together, these results provide an enlarged distribution of GPR50 protein, give further insight into the organisation of the melatoninergic system, and may lay the framework for future studies on the role of the GPR50 in the brain.