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Section on Obesity (P.P., D.J.T., E.M.-F.), Research Division, Joslin Diabetes Center, and the Division of Endocrinology (I.T.), Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Eleftheria Maratos-Flier, Research Division, Section on Obesity, Joslin Diabetes Center, 1 Joslin Place, Boston, Massachusetts 02215. E-mail: terry.maratos-flier{at}joslin.harvard.edu.
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Abstract |
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Introduction |
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Recently, two high-affinity receptors for MCH have been identified. The first receptor, MCHR1, was identified by several groups (11, 12, 13) and is homologous to the previously cloned orphan somatostatin-like receptor SLC-1 (14). A second receptor, designated MCHR2, exhibits 38% identity to the MCHR1 and is expressed in humans and dogs but thus far has not been identified in rodents (15, 16, 17, 18). Distribution of the receptors throughout the central nervous system (CNS) corresponds well to the projections of MCH neurons. Both receptors are expressed broadly in the cortex and the basal ganglia. Strongest expression is seen in the piriform cortex and the ventral striatum including the shell of the nucleus accumbens and the olfactory tubercle. Hippocampus and amygdala also express significant amounts of the MCHRs (16, 19, 20). Ablating the expression of the MCHR1 in mice leads to a lean phenotype, and, although these mice are not hypophagic, this finding confirms the importance of MCHR1 in energy homeostasis (21).
Both receptors belong to the superfamily of the G protein-coupled receptors (GPCRs), which are the most common receptors mediating the action of peptide neurotransmitters in the CNS. Signaling pathways activated by GPCRs, such as the cAMP-protein kinase A (PKA), phospholipase C (PLC), and MAPK pathways, play an important role in regulating neuronal functions ranging from fast modulation of channel activity (22, 23) to regulation of neuronal growth and changes in gene expression (24, 25).
Information about intracellular pathways activated by the two MCHRs is limited. MCHR1 couples to multiple G proteins including Gi, Go, and Gq (26). Consistent with this coupling, MCHR1 inhibits cAMP production stimulated by forskolin and increases intracellular Ca2+ levels (11, 12, 26, 27). In addition, MCHR1 is expressed in 3T3-L1 preadipocytes and adipocytes and affects leptin expression possibly through activation of ERK1/2 and p70S6 kinase (28). MCHR2 increases intracellular Ca2+ levels apparently through exclusive coupling to Gq and is therefore unable to inhibit cAMP synthesis in mammalian cells (16).
In this study, we used heterologous cells stably expressing the human MCHR1 to characterize in more detail the enzymatic activities necessary for signaling by the receptor. In addition, we examined the interactions between the MCHR1 and other hormonal systems, especially those that increase the cAMP levels. Our results indicate that the interaction between traditionally opposing pathways is more complex than previously thought and depends on specific intracellular signaling mediators. More specifically, whereas MCH blocks forskolin- or isoproterenol-dependent stimulation of cAMP synthesis, it unexpectedly increases ERK phosphorylation to levels above those observed with forskolin or isoproterenol alone, a phenomenon also associated with prolonged duration of action. This synergy appears to be mediated through pertussis toxin-independent pathways and requires several enzymatic activities including protein kinase C (PKC), PKA, and Src kinase kinases as well as PLC activity. Finally, using metabolically active brain slices, we have observed similar enhancement in ERK phosphorylation in the olfactory tubercle/piriform cortex, indicating that similar synergy occurs in MCH-responsive cells within the central nervous system.
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Materials and Methods |
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Cell culture and signaling experiments
HEK293 cells stably expressing the human (h)MCHR1 were maintained in DMEM/10% fetal bovine serum supplemented with G418 (Invitrogen). For all signaling experiments, the cells were plated in six-well plates at a density of 0.5 x 106/well. The next day, the cells were switched to serum-free media (DMEM/0.1% BSA). After overnight (O/N) serum starvation, the cells were stimulated with different combinations of ligands for the indicated time. Cells were washed once with ice-cold PBS and harvested in cell lysis buffer [20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM NaPPi, 1 mM ß-glycerophosphate, 1 mM Na3VO4] supplemented with various protease inhibitors. The kinase inhibitors were added 30 min before stimulation except for pertussis toxin pretreatment, which was O/N. Protein concentration of the extracts was determined with the Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay. Twenty-five micrograms of total protein were analyzed by SDS-PAGE on a 10% mini-protean gel (Bio-Rad Laboratories, Inc.) and transferred onto nitrocellulose (Protran, Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). Immunoblotting was performed according to the manufacturer’s recommendations (Cell Signaling Technology). The blots were then developed with chemiluminescent reagent (Renaissance, NEN Life Science Products, Boston, MA).
cAMP ELISA
cAMP levels were determined 30 min after ligand stimulation of the HEK293/hMCHR1 cells using a cAMP ELISA kit according to the manufacturer’s recommendations (Stratagene, La Jolla, CA).
Transfection assays
Transfections were performed in 24-well plates seeded with 0.2 x 106 cells/well the previous day. Typically, 200 ng of the cAMP-responsive or the control reporter plasmids, 200 ng of a thymidine kinase-luciferase vector for normalization, and 400 ng of pBluescriptSK as a carrier plasmid were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen). Four hours later, the medium was changed to DMEM/0.1% BSA and the cells were serum starved O/N. Fresh serum-free medium supplemented with various ligands was added to the wells the next day. Six hours later, the medium was collected and the amount of secreted alkaline phosphatase (SEAP) activity was determined with the chemiluminescent detection system according to the manufacturer’s recommendations (CLONTECH Laboratories, Inc., Palo Alto, CA). Transfection efficiency was evaluated by measuring luciferase activity using the MLX luminometer (Dynatech Corp. Technologies, Chantilly, VA).
Immunoprecipitation and kinase assays
For the immunoprecipitation experiments, HEK293 cells were stimulated with ligands as described above and harvested according to manufacturer’s protocol (Upstate Biotechnology, Inc., Lake Placid, NY). Active Raf-1 or B-Raf was immunoprecipitated O/N from 0.5 mg of total cell extracts, and the precipitate was incubated with inactive MAPK kinase (MEK)1 and ERK1, both expressed as glutathione-S-transferase (GST) fusions, in the presence of [32P]ATP, at 30 C for 20 min. The supernatant was then analyzed by SDS-PAGE and visualized by autoradiography. The extent of MEK and ERK1 phosphorylation was quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Due to very low endogenous Raf-1 activity recovered in the immunoprecipitates, expression vector for Raf-1 (pUSEAMP-Raf-1, Upstate Biotechnology, Inc.) was transfected in HEK293 cells before the extracts were used in the kinase assay.
Preparation of rat brain slices
Rat brain slices were prepared from 2- to 3-wk-old rats. Briefly, the rats were killed by CO2 inhalation and their brains were rapidly isolated and cooled in ice-cold oxygenated Krebs-Ringer solution [10 mM D-glucose, 26 mM NaHCO3, 114 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 10 mM HEPES, 1.3 mM MgSO4, 2.4 mM CaCl2 (pH 7.4)]. Tissue containing the area of interest was then sliced at 0.3-mm thickness with the OTS-4000 oscillating tissue slicer (Electron Microscopy Sciences, Fort Washington, PA). The slices were subsequently dissected manually to isolate the frontal cortex, the olfactory tubercle/piriform cortex, and the hippocampus from the surrounding tissue. The slices were then incubated for 1 h at 37 C with two changes of the oxygenated buffer. Various combinations of ligands were added to the medium, and the slices were rapidly frozen on dry ice after stimulation for 5 min. Total protein extracts was prepared using the cell lysis buffer, and the extracts were analyzed for the presence of phosphorylated ERK1/2 as described above.
Quantitation of results
Each experiment with inhibitors was performed at least three times. Immunoblots were scanned, and the density of the bands was analyzed with ImageQuant 5.1 software. Phosphorylation of ERK in response to isoproterenol and MCH was set at 100%.
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Results |
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). Similar results are also obtained by transiently expressing a cAMP-responsive reporter plasmid (CRE-SEAP) into the HEK293/hMCHR1 cells. After a 6-h incubation, forskolin stimulates the activity of the reporter gene approximately 25-fold above vehicle alone. This reporter activity is dramatically decreased by 75% upon addition of both forskolin and MCH, compared with forskolin alone (Fig. 1B). Finally, MCH alone has no effect on the reporter activity.
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). ERK phosphorylation is clearly seen at 10 nM of MCH and reaches a plateau at about 100 nM of MCH. The response to MCH addition is both rapid and transient. Two minutes after MCH addition, ERK phosphorylation is maximal and, 10 min later, it returns to the nonstimulated levels (Fig. 2B). No increase in c-Jun N-terminal kinase and p38 kinase phosphorylation is observed with MCH treatment (data not shown).
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). The strong increase in ERK phosphorylation suggests a synergistic interaction between the two pathways. In addition, the duration of ERK phosphorylation was also increased. With addition of either MCH or forskolin, the phosphorylated signal declined to basal levels within 10 min. In contrast, this signal persisted for at least 20 min when both ligands were added to the medium (Fig. 3A). Similar synergy was seen with MCH and the ß-adrenergic receptor agonist isoproterenol (Fig. 3B). Quantitation of three experiments performed under identical conditions shows that the level of synergy was more than three times higher, when a combination of forskolin and MCH or isoproterenol and MCH were used, than the sum of ERK phosphorylation induced by these ligands alone (Fig. 3B).
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). Surprisingly, the synergistic activation of ERK by MCH and forskolin or MCH and isoproterenol was also not significantly affected (Fig. 4A).
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). H89 also decreased the synergistic activation of ERK by MCH and forskolin (50% inhibition) but did not affect the synergism between MCH and isoproterenol (Fig. 4B).
Pretreatment with the PLC inhibitor U73122 caused a significant decrease in ERK phosphorylation by forskolin and MCH alone (50–60% inhibition) as well as MCH combination with forskolin or isoproterenol (Fig. 4C). Finally, the PKC inhibitor bisindolylmaleimide completely blocked the ability of forskolin, isoproterenol, or MCH to increase ERK1 phosphorylation (
90% inhibition) and completely inhibited the synergistic effect of the ligands as well (Fig. 4D). As a nonspecific negative control, we show that inhibition of PKC does not affect the activation of the cAMP/PKA pathway by forskolin and isoproterenol as determined by phosphorylation of cAMP response element binding protein at Ser133 (Fig. 4E).
SRC tyrosine kinase is necessary for signaling by MCH and forskolin
Tyrosine kinases such as the EGF receptor kinase and SRC tyrosine kinase have been implicated in signaling via several GPCRs (31, 32). We therefore determined whether any of the above activities is necessary for the phosphorylation of ERK by MCH alone and in combination with forskolin or isoproterenol. Pretreatment of the cells with the nonspecific tyrosine kinase inhibitor genistein resulted in a significant decrease of ERK phosphorylation in response to forskolin (66% inhibition), isoproterenol (71% inhibition), and to stimulation by forskolin and MCH (64%) (Fig. 5A). Next, we tested the effect of specific inhibitors of either EGF receptor or SRC kinase on our system. Inhibition of the EGF receptor kinase activity by the specific inhibitor AG1478 had no effect on ERK phosphorylation when the ligands are used either alone or in combination (Fig. 5B), whereas AG1478 effectively blocked the phosphorylation of ERK in these cells by EGF (Fig. 5D). In contrast, pretreatment of cells with the specific SRC tyrosine kinase inhibitor PP2 inhibited ERK phosphorylation by all ligands (Fig. 5C). Decrease was observed in all treatments; however, some did not reach statistical significance. In this context, forskolin and isoproterenol were more sensitive to inhibition of SRC activity (78%) than MCH alone (50%). Pretreatment of cells with PP2 had no effect on EGF-mediated ERK phosphorylation (Fig. 5D). Finally, none of these inhibitors affected phosphorylation of ERK by PMA (Fig. 5D).
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(top panel) shows the extent of ERK phosphorylation in response to different ligands. The middle and bottom panels show a clear separation of the signaling pathways that lead to ERK1/2 phosphorylation. EGF stimulates Raf-1 activity 3.0-fold (middle panel) and B-Raf activity 2.0-fold (bottom panel), as measured by MEK1 and ERK2 phosphorylation. In contrast, forskolin and MCH mainly activate B-Raf (1.7- and 1.9-fold, respectively, bottom panel) but not Raf-1 activity (1.1- and 1.3-fold, respectively, middle panel). Simultaneous addition of both ligands results in further increase of B-Raf (2.9-fold, bottom panel) but not Raf-1 activity (1.1-fold, middle panel), compared with control extracts. We therefore conclude that the synergistic activation of ERK by forskolin and MCH proceeds mainly through B-Raf-dependent pathways. A summary of the potential pathways that leads to activation of ERK in response to forskolin, isoproterenol, and MCH stimulation is illustrated in Fig. 6B.
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). The slices were treated with MCH, forskolin, or both ligands for 5 min and analyzed by immunoblotting with phospho-ERK-specific antibody. Baseline phosphorylation of ERK was low in both the cortex and in the olfactory tubercle/piriform cortex; however, there was appreciable baseline activity in the hippocampus (Fig. 7B). Although forskolin activates ERK1/2 in all three areas, MCH-stimulated ERK phosphorylation was observed only in the olfactory tubercle/piriform cortex area. Scanning densitometry of the gel revealed that, when added together, MCH and forskolin were additive rather than synergistic in this area of the brain. Phosphorylation of ERK2 was increased 2.3-fold by forskolin, 1.75-fold by MCH, and 3.2-fold by both ligands compared with control-treated slices. In the hippocampus and neocortex, MCH alone was without an independent effect, and no further interaction between MCH and forskolin was seen. Similar results were seen in three separate experiments using brain slice preparations from different animals.
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Discussion |
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Signaling in the CNS is particularly complex as cells continuously receive multiple stimuli from various sources, thus making analysis of the integration and output of these pathways difficult. For example, cAMP and Ca2+ affect the activity of several ion channels and thereby modulate the excitability of neurons (22, 23). Hence, the initial identification of potential signaling pathways in cell lines is important. Identification of these pathways is also important for defining actions on neurons beyond direct signaling events such as the regulation of growth, migration, and apoptosis.
In this study, we have assessed the potential interaction of MCH with factors that activate adenylate cyclase, either directly or by coupling to Gs in both a cultured cell system and in brain. As previously reported, we found opposing effects of MCH and adenylate cyclase activators, such as forskolin and isoproterenol, on cAMP generation. However, we found that MCH, acting together with either forskolin or isoproterenol, had an unexpected synergistic effect on ERK. This synergy was seen both in the magnitude of phosphorylation and in the half-life of phosphorylated ERK.
Furthermore, using a panel of inhibitors, we found that MCH acts via different pathways when activating ERK1/2 alone vs. when activating synergistically with activators of adenylate cyclase. The direct effect of MCH on ERK phosphorylation is pertussis toxin dependent. In contrast, the synergistic activation, which leads to a 4-fold higher activation than that seen with any of the ligands alone, is not affected by pertussis toxin. This indicates that the synergistic effect is not mediated by coupling to Gi, but rather by coupling to Gq. Thus, whereas coupling of MCHR1 to Gq is not sufficient to activate ERK, interactions between the Gq and Gs act to amplify the action of MCH in this pathway.
This finding was unexpected because cAMP typically activates ERK in HEK293 cells in a PKA-dependent manner, and the synergistic interactions observed here have not previously been reported (33). In contrast, the inhibitory actions of cAMP and PKA on ERK phosphorylation have been reported in several cell lines, such as NIH3T3 fibroblasts (29, 30). Phosphorylation of ERK by forskolin or isoproterenol alone requires PKA and is blocked by the PKA inhibitor H89. Interestingly, PKA blockade appears to significantly diminish the synergy between forskolin and MCH, whereas PKA blockade has no effect on the synergism of isoproterenol and MCH. This finding suggests that alternative pathways activated by the Gs-coupled ß-adrenergic receptor may contribute to the synergy, because it is known that the ß
subunits of the G proteins can activate PLC directly. Additional pathways from the ß-adrenergic receptor include activation of SRC by PKA-dependent and -independent pathways (see below) (34). Binding of arrestin upon activation and subsequent receptor internalization may also be involved (35).
PKC appears to play an important role in mediating both the direct and synergistic activation of ERK. Treatment of cells with the PKC inhibitor bisindolylmaleimide abolished ERK activation. We also examined the role of PLCß in the synergistic interaction. MCH is known to increase intracellular calcium, an effect mediated through Gq. In turn, PLCß can activate a host of other intracellular signaling mediators such as PKC and Ras and Rap. This led us to examine the effect of the PLC inhibitor U73122 on the synergy. We found that U73122 partially inhibited the ability of MCH and forskolin to individually phosphorylate ERK and also markedly reduced the synergistic activation of ERK, consistent with a role for PLC.
Activation of GPCRs may also promote activation of tyrosine kinases (36). For example, trans-activation of EGF receptor (EGFR) by GPCR agonists have been reported (37, 38). In addition, some reports show that SRC kinase is required for activation of ERK by GPCRs (31, 32, 34). Our results indicate that in HEK293 cells, SRC but not EGFR kinase activity is required for activation of ERK by MCHR1 as well as forskolin and ß-adrenergic receptor agonists. A recent report demonstrated that SRC kinase can be activated by PKA and that it also associates with the adaptor CRK proteins. SRC kinase also associates with the nucleotide exchanger for Rap1, C3G. This activation is necessary for the inhibitory actions of cAMP on ERK activation and proliferation of fibroblasts (39). It is possible that the same pathways may be necessary for mediating activation of ERK in HEK293 cells. Consistent with this hypothesis, both PKA and SRC activities are required for ERK phosphorylation by forskolin in HEK293 cells (34) and for the observed synergy between forskolin and MCH in our system.
The mechanism of this synergy is not clear, and a number of potential pathways may be involved. It is known that cAMP alone can activate ERK in cells expressing high levels of B-Raf kinase, such as cells of neuronal origin, as well as HEK293 cells. It is also known that in these cells, forskolin and ß-adrenergic agonists activate ERK through the small GTPase Rap1, which interacts with the B-Raf kinase (33). This pathway may predominate in some cell types. For example, PC12 cells treated with nerve growth factor show activation of both Ras and Rap1. However, only activation of Rap1 is required for cell survival (40). Several upstream activators of Rap1, designated Rap-GEFs, have been identified. These include C3G, mentioned earlier, Epac, which is directly activated by cAMP, and CalDAG-GEF activated by calcium and diacylglycerol, which interacts with the adaptor proteins CRK (41, 42). Support that these pathways may be involved in the synergy we have observed comes from the differential activation of Raf-1 vs. B-Raf in immunoprecipitates of stimulated cell extracts. Thus, combination of MCH and forskolin leads only to activation of B-Raf and not Raf-1.
Finally, we show that activation of ERK by MCHR1 in HEK293 cells may be relevant to signaling in the CNS as we observe MCH-induced ERK activation in ex vivo brain slices.
Activation of ERK by MCH alone was observed in the olfactory regions (olfactory tubercle and piriform cortex), and combination of MCH with forskolin had an additive effect on ERK phosphorylation in the same regions. In the neocortex and hippocampus, MCH alone had no effect on ERK phosphorylation, and no additional increase was seen when MCH was combined with forskolin. The absence of an additive rather than a synergistic effect in the brain slice system suggests that data from complex tissues may be more difficult to interpret in comparison to data from single-cell cultures. The results also suggest site-specific actions of MCH. Cell-cell interactions are likely to play a significant role in brain signaling processes, and the pathways engaged by MCH may be differentially modulated by local interneurons. Because MCH is important in regulating feeding, it is of interest that we have observed activation of ERK in brain areas known to integrate olfactory stimuli, e.g. the piriform cortex and the olfactory tubercle.
Communication between GPCRs is multiple and complex. GPCRs may form homodimers or heterodimers with either receptors in the same family or receptors in other families. Dimerization may lead to synergism, antagonism, changes in ligand binding, or redistribution of the effector G proteins. The level of complexity is increased by the interactions of the various molecules involved in the signaling cascades. Understanding factors that regulate these pathways is important, because ERK is a key mediator in many processes. In the CNS, it is involved in neuronal survival and cognitive processes such as long-term memory formation. Indeed, ablation of ERK1 in the CNS leads to changes in memory and in synaptic plasticity (43).
We have demonstrated a complex interaction between MCHR1 and Gs-coupled signaling pathways. MCHR1, when acting alone, activates ERK through its coupling to Gi. In contrast, the synergistic activation of ERK by MCH and cAMP-dependent pathways is mediated through coupling of MCHR1 to Gq. In addition, we have shown that ERK activation by MCH occurs in an anatomically specific manner in the CNS and especially in a region of the brain that we believe is important in mediating MCH action. Finding evidence of MCH signaling in the olfactory tubercle/piriform cortex is of particular interest because it provides a potential connection between feeding behavior and olfaction. The processes mediating appetite and satiety are extremely complex, and it is now clear that animals eat for reasons beyond simple metabolic need. For example, laboratory rodents can be made obese by offering a high-fat diet and highly palatable chow. Further examination of the role of MCH signaling in olfactory areas should provide molecular insights into the link between eating and smell.
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Footnotes |
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Abbreviations: CNS, Central nervous system; EGF, epidermal growth factor; EGFR, EGF receptor; GPCR, G protein-coupled receptor; GST, glutathione-S-transferase; MCH, melanin-concentrating hormone; MCHR, MCH receptor; MEK, MAPK kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12 myristate 13 acetate; O/N, overnight; SEAP, secreted alkaline phosphatase.
Received November 1, 2002.
Accepted for publication April 14, 2003.
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and Gß require distinct Src-dependent pathways to activate Rap1 and Ras. J Biol Chem 277:43024–43032This article has been cited by other articles:
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  E Kokkotou, D O Espinoza, D Torres, I Karagiannides, S Kosteletos, T Savidge, M O'Brien, and C Pothoulakis Melanin-concentrating hormone (MCH) modulates C difficile toxin A-mediated enteritis in mice Gut, January 1, 2009; 58(1): 34 - 40. [Abstract] [Full Text] [PDF]
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 Y. Rao, M. Lu, F. Ge, D. J. Marsh, S. Qian, A. H. Wang, M. R. Picciotto, and X.-B. Gao Regulation of Synaptic Efficacy in Hypocretin/Orexin-Containing Neurons by Melanin Concentrating Hormone in the Lateral Hypothalamus J. Neurosci., September 10, 2008; 28(37): 9101 - 9110. [Abstract] [Full Text] [PDF]
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 E. Kokkotou, A. C. Moss, D. Torres, I. Karagiannides, A. Cheifetz, S. Liu, M. O'Brien, E. Maratos-Flier, and C. Pothoulakis Melanin-concentrating hormone as a mediator of intestinal inflammation PNAS, July 29, 2008; 105(30): 10613 - 10618. [Abstract] [Full Text] [PDF]
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 P. Pissios, U. Ozcan, E. Kokkotou, T. Okada, C. W. Liew, S. Liu, J. N. Peters, G. Dahlgren, J. Karamchandani, Y. C. Kudva, et al. Melanin Concentrating Hormone Is a Novel Regulator of Islet Function and Growth Diabetes, February 1, 2007; 56(2): 311 - 319. [Abstract] [Full Text] [PDF]
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 F. Francke, R. J. Ward, L. Jenkins, E. Kellett, D. Richter, G. Milligan, and D. Bachner Interaction of Neurochondrin with the Melanin-concentrating Hormone Receptor 1 Interferes with G Protein-coupled Signal Transduction but Not Agonist-mediated Internalization J. Biol. Chem., October 27, 2006; 281(43): 32496 - 32507. [Abstract] [Full Text] [PDF]
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 P. Pissios, R. L. Bradley, and E. Maratos-Flier Expanding the Scales: The Multiple Roles of MCH in Regulating Energy Balance and Other Biological Functions Endocr. Rev., October 1, 2006; 27(6): 606 - 620. [Abstract] [Full Text] [PDF]
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 G. Segal-Lieberman, H. Rubinfeld, M. Glick, N. Kronfeld-Schor, and I. Shimon Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E982 - E988. [Abstract] [Full Text] [PDF]
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J. Y. Jeon, R. L. Bradley, E. G. Kokkotou, F. E. Marino, X. Wang, P. Pissios, and E. Maratos-Flier MCH-/- Mice Are Resistant to Aging-Associated Increases in Body Weight and Insulin Resistance Diabetes, February 1, 2006; 55(2): 428 - 434. [Abstract] [Full Text] [PDF]
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 Y. Saito, M. Tetsuka, S. Saito, K. Imai, A. Yoshikawa, H. Doi, and K. Maruyama Arginine Residue 155 in the Second Intracellular Loop Plays a Critical Role in Rat Melanin-Concentrating Hormone Receptor 1 Activation Endocrinology, August 1, 2005; 146(8): 3452 - 3462. [Abstract] [Full Text] [PDF]
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 E. Kokkotou, J. Y. Jeon, X. Wang, F. E. Marino, M. Carlson, D. J. Trombly, and E. Maratos-Flier Mice with MCH ablation resist diet-induced obesity through strain-specific mechanisms Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R117 - R124. [Abstract] [Full Text] [PDF]
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 A.-K. Wermter, K. Reichwald, T. Buch, F. Geller, C. Platzer, K. Huse, C. Hess, H. Remschmidt, T. Gudermann, G. Preibisch, et al. Mutation analysis of the MCHR1 gene in human obesity Eur. J. Endocrinol., June 1, 2005; 152(6): 851 - 862. [Abstract] [Full Text] [PDF]
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 U. A. Unmehopa, J. J. van Heerikhuize, W. Spijkstra, J. W. Woods, A. D. Howard, E. Zycband, S. D. Feighner, D. L. Hreniuk, O. C. Palyha, X.-M. Guan, et al. Increased Melanin Concentrating Hormone Receptor Type I in the Human Hypothalamic Infundibular Nucleus in Cachexia J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2412 - 2419. [Abstract] [Full Text] [PDF]
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D. Georgescu, R. M. Sears, J. D. Hommel, M. Barrot, C. A. Bolanos, D. J. Marsh, M. A. Bednarek, J. A. Bibb, E. Maratos-Flier, E. J. Nestler, et al. The Hypothalamic Neuropeptide Melanin-Concentrating Hormone Acts in the Nucleus Accumbens to Modulate Feeding Behavior and Forced-Swim Performance J. Neurosci., March 16, 2005; 25(11): 2933 - 2940. [Abstract] [Full Text] [PDF]
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 W. F. Schwindinger, K. E. Giger, K. S. Betz, A. M. Stauffer, E. M. Sunderlin, L. J. Sim-Selley, D. E. Selley, S. K. Bronson, and J. D. Robishaw Mice with Deficiency of G Protein {gamma}3 Are Lean and Have Seizures Mol. Cell. Biol., September 1, 2004; 24(17): 7758 - 7768. [Abstract] [Full Text] [PDF]
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