A novel and selective melanin-concentrating hormone receptor 1 antagonist ameliorates obesity and hepatic steatosis in diet-induced obese rodent models.

Melanin-concentrating hormone (MCH), a cyclic neuropeptide expressed predominantly in the lateral hypothalamus, plays an important role in the control of feeding behavior and energy homeostasis. Mice lacking MCH or MCH1 receptor are resistant to diet-induced obesity (DIO) and MCH1 receptor antagonists show potent anti-obesity effects in preclinical studies, indicating that MCH1 receptor is a promising target for anti-obesity drugs. Moreover, recent studies have suggested the potential of MCH1 receptor antagonists for treatment of non-alcoholic fatty liver disease (NAFLD). In the present study, we show the anti-obesity and anti-hepatosteatosis effect of our novel MCH1 receptor antagonist, Compound A. Repeated oral administration of Compound A resulted in dose-dependent body weight reduction and had an anorectic effect in DIO mice. The body weight lowering effect of Compound A was more potent than that of pair-feeding. Compound A also reduced lipid content and the expression level of lipogenesis-, inflammation-, and fibrosis-related genes in the liver of DIO mice. Conversely, intracerebroventricular infusion of MCH caused induction of hepatic steatosis as well as increase in body weight in high-fat diet-fed wild type mice, but not MCH1 receptor knockout mice. The pair-feeding study revealed the MCH-MCH1 receptor system affects hepatic steatosis through a mechanism that is independent of body weight change. Metabolome analysis demonstrated that Compound A upregulated lipid metabolism-related molecules, such as acylcarnitines and cardiolipins, in the liver. These findings suggest that our novel MCH1 receptor antagonist, Compound A, exerts its beneficial therapeutic effect on NAFLD and obesity through a central MCH-MCH1 receptor pathway.

MCH receptor deletion does not impair glucose-conditioned flavor preferences in mice.

The post-oral actions of glucose stimulate intake and condition flavor preferences in rodents. Hypothalamic melanin-concentrating hormone (MCH) neurons are implicated in sugar reward, and this study investigated their involvement in glucose preference conditioning in mice. In Exp. 1 MCH receptor 1 knockout (KO) and C57BL/6 wildtype (WT) mice learned to prefer 8% glucose over an initially more-preferred non-nutritive 0.1% sucralose+saccharin (S+S) solution. In contrast, the KO and WT mice preferred S+S to 8% fructose, which is consistent with this sugar's weak post-oral reinforcing action. In Exp. 2 KO and WT mice were trained to drink a flavored solution (CS+) paired with intragastric (IG) infusion of 16% glucose and a different flavored solution (CS-) paired with IG water. Both groups drank more CS+ than CS- in training and preferred the CS+ to CS- in a 2-bottle test. These results indicate that MCH receptor signaling is not required for flavor preferences conditioned by the post-oral actions of glucose. This contrasts with other findings implicating MCH signaling in other types of sugar reward processing.

Importance of melanin-concentrating hormone receptor for the acute effects of ghrelin.

The hypothalamic peptide melanin-concentrating hormone (MCH) and the gastric hormone ghrelin take part in the regulation of energy homeostasis and stimulate food intake. In the present study, ghrelin was administered centrally to MCH-receptor knockout (MCHr KO) mice. MCHr KO mice and wild type (WT) controls both consumed more food when treated with ghrelin. After ghrelin administration, the serum levels of insulin increased only in WT mice whereas the serum levels of corticosterone increased both in WT and MCHr KO mice. The level of growth hormone (GH) mRNA in the pituitary gland was markedly increased in response to ghrelin injection in the WT mice but was unaffected in the MCHr KO mice. The different ghrelin responses could not be explained by a difference in growth hormone secretagogue receptor expression between MCHr KO and WT mice in the pituitary or hypothalamus. In summary, the MCHr is not required for ghrelin induced feeding. However, the MCHr does play a role for the effect of ghrelin on GH expression in the pituitary and serum insulin levels.

Osteoporosis in MCHR1-deficient mice.

It is well recognized that the hypothalamus is of central importance in the regulation of food intake and fat mass. Recent studies indicate that it also plays an important role in the regulation of bone mass. Melanin concentrating hormone (MCH) is highly expressed in the hypothalamus and has been implicated in regulation of energy homeostasis. We developed MCHR1 inactivated mice to evaluate the physiological role of this receptor. Interestingly, the MCHR1(-/-) mice have osteoporosis, caused by a reduction in the cortical bone mass, while the amount of trabecular bone is unaffected. The reduction in cortical bone mass is due to decreased cortical thickness. Serum levels of c-telopeptide, a marker of bone resorption, are increased in MCHR1(-/-) mice, indicating that the MCHR1(-/-) mice have a high bone turnover osteoporosis. In conclusion, the MCHR1(-/-) mice have osteoporosis, indicating that MCHR1-signalling is involved in a tonic stimulation of bone mass.

Reduced expression of brain-derived neurotrophic factor in mice deficient for pituitary adenylate cyclase activating polypeptide type-I-receptor.

In vitro pituitary adenylate cyclase activating polypeptide (PACAP) induces the expression of brain-derived neurotrophic factor (BDNF) via its specific receptor PAC1. Since BDNF has been implicated in learning paradigms and mice lacking functional PAC1 have deficits in hippocampus-dependent associative learning, we investigated whether PAC1 mutants show alterations in hippocampal expression of BDNF and its receptor TrkB. Semi-quantitative in situ-hybridization using exon-specific BDNF-probes revealed significantly reduced expression of the exon-III and exon-V-specific transcripts within the hippocampal CA3 region in PAC1-deficient mice. A similar trend was observed for the exon-I-specific transcript. The expression of the exon-III-specific transcript was also reduced within the dentate gyrus, while Trk B-expression did not differ between genotypes. Our data demonstrate that even in vivo PAC1-mediated signaling seems to play a pivotal role for the transcriptional regulation of BDNF.

Impaired long-term potentiation in vivo in the dentate gyrus of pituitary adenylate cyclase-activating polypeptide (PACAP) or PACAP type 1 receptor-mutant mice.

The present study was conducted to clarify a role of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type 1 receptor (PAC1R) in learning and memory function. We demonstrated long-term potentiation (LTP) in vivo in the dentate gyrus of PAC1R exon 2-deficient (PAC1R-/-) mice and heterozygous PACAP-deficient (PACAP+/-) mice using extracellular recording techniques. We used two paradigms of tetanic stimulation, suprathreshold and at threshold tetanus, which both induced LTP in vivo in PAC1R-/- and PACAP+/- mice. However, the population spike of 'at threshold' but not 'suprathreshold' LTP decreased significantly in PAC1R-/- and PACAP+/- mice. At threshold LTP of PACAP+/- mice was impaired greater than the one of PAC1R-/- mice. Thus, both PACAP and PAC1R could contribute to the establishment of LTP in a gene dosage-dependent manner, although PACAP rather than PAC1R might play a pivotal role in learning and memory function.

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

PAC1 receptor-mediated relaxation of longitudinal muscle of the mouse proximal colon.

Since pituitary adenylate cyclase-activating polypeptide (PACAP) was shown to partially mediate nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle of the proximal colon of ICR mice, we further studied the receptor subtype activated by PACAP by using a mutant mouse whose PAC1 receptors are markedly reduced. In wild-type mice, the PACAP-mediated component of NANC relaxation was 33%, but it was absent in the mutant mice. The potency of exogenous PACAP in inducing relaxation in the mutant mice was one hundredth of that in wild-type mice. VPAC1 and VPAC2 receptors were not suggested to have any role in the relaxation. These results suggest that PACAP mediates NANC relaxation of longitudinal muscle of mouse proximal colon via PAC1 receptors.

Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two mediators synthesized by immune cells, specially under inflammatory and antigen stimulation conditions. Reports have shown that neuropeptides attenuate the deleterious consequences of septic shock both by down-regulating the production of proinflammatory mediators and by stimulating the production of anti-inflammatory cytokines by activated macrophages. In this study, we used a knockout for the PACAP receptor (PAC1(-/-)) to demonstrate an important protective role for PAC1 receptor in endotoxic shock. Moreover, our results indicate that PAC1 receptor acts in vivo as an anti-inflammatory receptor, at least in part, by attenuating lipopolysaccharide (LPS)-induced production of proinflammatory IL-6, which appears to be the main cytokine regulating the expression of the majority of the acute phase protein genes, which are an important deleterious component of septic shock. Besides, our findings point to endogenously produced VIP and PACAP as participants of the natural anti-inflammatory machinery. Because VIP and PACAP are two attractive candidates for the development of therapies against acute and chronic inflammatory diseases, septic shock, and autoimmune diseases, this paper represents a contribution to the understanding of the mechanism of action of these anti-inflammatory agents.

Altered emotional behavior in PACAP-type-I-receptor-deficient mice.

PAC1 (pituitary adenylate cyclase activating polypeptide type I receptor) is a G-protein-coupled receptor that binds the strongly conserved neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) with a thousandfold higher affinity than the related peptide VIP (vasoactive intestinal peptide). PAC1 shows strong expression in brain areas which have been implicated in the emotional control of behavior, such as the amygdala, the hypothalamus, the locus coeruleus and the periaqueductal gray. To assess whether PAC1-mediated signaling has an impact on emotional behavior, we analysed two different mutant mouse lines with an ubiquitous or a forebrain-specific inactivation of PAC1 in several testing paradigms modelling general locomotor activity and anxiety-related behavior. We clearly demonstrate that mice with a ubiquitous but not with a forebrain-specific deletion of PAC1 exhibit elevated locomotor activity and strongly reduced anxiety-like behavior. We could not observe any gross alteration in circadian rhythmicity nor any enhanced sensitivity towards ethanol in the mutant mice. We previously demonstrated that PAC1 plays a crucial role in contextual fear conditioning. Therefore the finding that PAC1-deficient mice exhibit reduced anxiety is quite exciting, since the receptor and hence its ligand PACAP seem to be important for both, innate and learned fear.

Markedly reduced chronic nociceptive response in mice lacking the PAC1 receptor.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been proposed to have a role in nociception. Here we have used the formalin test, thermal laser stimulation and mechanical von Frey stimulation to investigate possible alteration of PAC1-/- mice nociceptive behaviour. Our finding, that PAC1-/- mice have a substantial, 75% decrease in nociceptive response during the late phase, provides clear evidence that the specific PACAP-receptor PAC1 is involved in the mediation of nociceptive responses during chronic conditions such as inflammation. PAC1-/- mice had small or no changes in the response to mechanical and thermal laser stimulation. This suggests a limited, if any, involvement of PAC1 in nociception after short-lasting stimuli. Injury-induced changes in DRG neuropeptide expression were more pronounced in PAC1-/- mice, implying neuroregulatory functions of PAC1.

Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice.

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning.

Dissociation between light-induced phase shift of the circadian rhythm and clock gene expression in mice lacking the pituitary adenylate cyclase activating polypeptide type 1 receptor.

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1(-)/-) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and PAC1(-)/- mice. However, in contrast to the phase advance seen in wild-type mice, PAC1(-)/- mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest that mPer1, mPer2, and c-fos are of less importance for light-induced phase shifts at night.

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

PAC1 receptor-deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic beta cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1(-/-)). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1(-/-) mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1(-/-) mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.