Effects of a novel potent melanin-concentrating hormone receptor 1 antagonist, AZD1979, on body weight homeostasis in mice and dogs.

BACKGROUND AND PURPOSE: Melanin-concentrating hormone (MCH) is an orexigen, and while rodents express one MCH receptor (MCH1 receptor), humans, non-human primates and dogs express two MCH receptors (MCH1 and MCH2 ). MCH1 receptor antagonists have been developed for the treatment of obesity and lower body weight in rodents. However, the mechanisms for the body weight loss and whether MCH1 receptor antagonism can lower body weight in species expressing both MCH receptors are not fully understood. EXPERIMENTAL APPROACH: A novel recently identified potent MCH1 receptor antagonist, AZD1979, was studied in wild type and Mchr1 knockout (KO) mice and by using pair-feeding and indirect calorimetry in diet-induced obese (DIO) mice. The effect of AZD1979 on body weight was also studied in beagle dogs. KEY RESULTS: AZD1979 bound to MCH1 receptors in the CNS and dose-dependently reduced body weight in DIO mice leading to improved homeostasis model assessment-index of insulin sensitivity. AZD1979 did not affect food intake or body weight in Mchr1 KO mice demonstrating specificity for the MCH1 receptor mechanism. In DIO mice, initial AZD1979-mediated body weight loss was driven by decreased food intake, but an additional component of preserved energy expenditure was apparent in pair-feeding and indirect calorimetry studies. AZD1979 also dose-dependently reduced body weight in dogs. CONCLUSION AND IMPLICATIONS: AZD1979 is a novel potent MCH1 receptor antagonist that affects both food intake and energy expenditure. That AZD1979 also lowers body weight in a species expressing both MCH receptors holds promise for the use of MCH1 receptor antagonists for the treatment of human obesity.

Discovery of (3-(4-(2-Oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone (AZD1979), a Melanin Concentrating Hormone Receptor 1 (MCHr1) Antagonist with Favorable Physicochemical Properties.

A novel series of melanin concentrating hormone receptor 1 (MCHr1) antagonists were the starting point for a drug discovery program that culminated in the discovery of 103 (AZD1979). The lead optimization program was conducted with a focus on reducing lipophilicity and understanding the physicochemical properties governing CNS exposure and undesired off-target pharmacology such as hERG interactions. An integrated approach was taken where the key assay was ex vivo receptor occupancy in mice. The candidate compound 103 displayed appropriate lipophilicity for a CNS indication and showed excellent permeability with no efflux. Preclinical GLP toxicology and safety pharmacology studies were without major findings and 103 was taken into clinical trials.

Partial agonist activity of R3(BΔ23-27)R/I5 at RXFP3--investigation of in vivo and in vitro pharmacology.

Relaxin family peptide receptor 3 (RXFP3) is a G-protein coupled receptor mainly expressed in the brain and involved in appetite regulation. Previous studies in lean Wistar rats during the light phase have shown that the chimeric peptide R3(BΔ23-27)R/I5 suppresses food intake stimulated by an RXFP3 agonist, but has no effect on food intake when administered alone. We wanted to further investigate if R3(BΔ23-27)R/I5 on its own is able to antagonize the basal tone of the relaxin-3/RXFP3 system and therefore characterized the pharmacology of R3(BΔ23-27)R/I5 in vivo and in vitro. R3(BΔ23-27)R/I5 was intracerebroventricularly (ICV) injected in diet induced obese (DIO) Wistar rats and food intake was automatically measured during the dark phase when feeding drive is high. In our hands, R3(BΔ23-27)R/I5 alone did not have a significant effect on food intake during 24h following administration. Consistent with previous results, relaxin-3 stimulated food intake in satiated lean rats. R3(BΔ23-27)R/I5 was characterized in vitro using [(35)S]-GTPγS binding and cAMP assays, both assessing Gαi-protein mediated signalling, and dynamic mass redistribution (DMR) assays capturing the integrated cell response. R3(BΔ23-27)R/I5 showed partial agonist activity in all three functional assays. Thus, since R3(BΔ23-27)R/I5 displays partial RXFP3 agonist properties in vitro, further in vivo studies including additional tool compounds are needed to address if antagonizing relaxin-3/RXFP3 basal tone is a therapeutically relevant mechanism to regulate food intake and body weight.

A modeling approach for compounds affecting body composition.

Body composition and body mass are pivotal clinical endpoints in studies of welfare diseases. We present a combined effort of established and new mathematical models based on rigorous monitoring of energy intake (EI) and body mass in mice. Specifically, we parameterize a mechanistic turnover model based on the law of energy conservation coupled to a drug mechanism model. Key model variables are fat-free mass (FFM) and fat mass (FM), governed by EI and energy expenditure (EE). An empirical Forbes curve relating FFM to FM was derived experimentally for female C57BL/6 mice. The Forbes curve differs from a previously reported curve for male C57BL/6 mice, and we thoroughly analyse how the choice of Forbes curve impacts model predictions. The drug mechanism function acts on EI or EE, or both. Drug mechanism parameters (two to three parameters) and system parameters (up to six free parameters) could be estimated with good precision (coefficients of variation typically <20 % and not greater than 40 % in our analyses). Model simulations were done to predict the EE and FM change at different drug provocations in mice. In addition, we simulated body mass and FM changes at different drug provocations using a similar model for man. Surprisingly, model simulations indicate that an increase in EI (e.g. 10 %) was more efficient than an equal lowering of EI. Also, the relative change in body mass and FM is greater in man than in mouse at the same relative change in either EI or EE. We acknowledge that this assumes the same drug mechanism impact across the two species. A set of recommendations regarding the Forbes curve, vehicle control groups, dual action on EI and loss, and translational aspects are discussed. This quantitative approach significantly improves data interpretation, disease system understanding, safety assessment and translation across species.

In vivo imaging of lipid storage and regression in diet-induced obesity during nutrition manipulation.

Changes in adipose tissue distribution and ectopic fat storage in, liver and skeletal muscle tissue impact whole body insulin sensitivity in both humans and experimental animals. Numerous mouse models of obesity, insulin resistance, and diabetes exist; however, current methods to assess mouse phenotypes commonly involve direct harvesting of the tissues of interest, precluding the possibility of repeated measurements in the same animal. In this study, we demonstrate that whole body 3-D imaging of body fat composition can be used to analyze distribution as well as redistribution of fat after intervention by repeated assessment of intrahepatocellular lipids (IHCL), intra-abdominal, subcutaneous, and total adipose tissue (IAT, SAT, and TAT) and brown adipose tissue (BAT). C57BL/6J mice fed a cafeteria diet for 16 wk were compared with mice fed standard chow for 16 wk and mice switched from café diet to standard chow after 12 wk. MRI determinations were made at 9 and 15 wk, and autopsy was performed at 16 wk. There was a strong correlation between MRI-calculated weights in vivo at 15 wk and measured weights at 16 wk ex vivo for IAT (r = 0.99), BAT (r = 0.93), and IHCL (r = 0.97). IHCL and plasma insulin increased steeply relative to body weight at body weights above 45 g. This study demonstrates that the use of 3-D imaging to assess body fat composition may allow substantial reductions in animal usage. The dietary interventions indicated that a marked metabolic deterioration occurred when the mice had gained a certain fat mass.

The metabotropic glutamate mGluR5 receptor agonist CHPG stimulates food intake.

The metabotropic glutamate receptor 5 (mGluR5) has been suggested to modulate energy balance. For example, mGluR5 antagonists inhibit food intake in rodents and mGluR5 knockout mice resist diet-induced obesity. However, nonspecific effects can reduce food intake. Thus, to further support the role of mGluR5 in feeding behaviour, we evaluated if the mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) would induce the opposite effect, i.e. increased food intake in rats. Intracerebroventricularly injected CHPG (0.5-1.5 micromol) induced a dose-dependent stimulation of food intake (349% increase at 2 h with 1.5 micromol). The mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (10 mg/kg intraperitoneally) reduced 24 h food intake, without altering CHPG-induced feeding. These findings further support a physiologically relevant role of mGluR5 in appetite regulation.

Central NMU signaling in body weight and energy balance regulation: evidence from NMUR2 deletion and chronic central NMU treatment in mice.

To investigate the role of the central neuromedin U (NMU) signaling system in body weight and energy balance regulation, we examined the effects of long-term intracerebroventricular (icv) infusion of NMU in C57Bl/6 mice and in mice lacking the gene encoding NMU receptor 2. In diet-induced obese male and female C57BL/6 mice, icv infusion of NMU (8 microg x day(-1) x mouse(-1)) for 7 days decreased body weight and total energy intake compared with vehicle treatment. However, these parameters were unaffected by NMU treatment in lean male and female C57BL/6 mice fed a standard diet. In addition, female (but not male) NMUR2-null mice had increased body weight and body fat mass when fed a high-fat diet but lacked a clear body weight phenotype when fed a standard diet compared with wild-type littermates. Furthermore, female (but not male) NMUR2-null mice fed a high-fat diet were protected from central NMU-induced body weight loss compared with littermate wild-type mice. Thus, we provide the first evidence that long-term central NMU treatment reduces body weight, food intake, and adiposity and that central NMUR2 signaling is required for these effects in female but not male mice.

Intestinal, adipose, and liver inflammation in diet-induced obese mice.

Chronic inflammation and increased visceral adipose tissue (VAT) are key elements of the metabolic syndrome. Both are considered to play a pathogenic role in the development of liver steatosis and insulin resistance. The aim of the present study was to investigate the hypothesis that an inflamed intestine, induced both by diet and chemical irritation, could induce persistent inflammation in VAT. Female C57BL/6JOlaHsd mice were used. In study I, groups of mice (n = 6 per group) were given an obesity-inducing cafeteria diet (diet-induced obesity) or regular chow only (control) for 14 weeks. In study II, colitis in mice (n = 8) was induced by 3% dextran sulfate sodium in tap water for 5 days followed by 21 days of tap water alone. Healthy control mice (n = 8) had tap water only. At the end of the studies, all mice were killed; and blood and tissues were sampled and processed for analysis. Body weight of diet-induced obese mice was greatly increased, with evidence of systemic inflammation, insulin resistance, and liver steatosis. Tissue inflammation indexed by proinflammatory cytokine expression was recorded in liver, mesenteric fat, and proximal colon/distal ileum, but not in subcutaneous or perigonadal fat. In dextran sulfate sodium-induced colitis mice, mesenteric fat was even more inflamed than the colon, whereas a much milder inflammation was seen in liver and subcutaneous fat. The studies showed both diet- and colitis-initiated inflammation in mesenteric fat. Fat depots contiguous with intestine and their capacity for exaggerated inflammatory responses to conditions of impaired gut barrier function may account for the particularly pathogenic role of VAT in obesity-induced metabolic disorders.

Opposing effects of adiponectin receptors 1 and 2 on energy metabolism.

The adipocyte-derived hormone adiponectin regulates glucose and lipid metabolism and influences the risk for developing obesity, type 2 diabetes, and cardiovascular disease. Adiponectin binds to two different seven-transmembrane domain receptors termed AdipoR1 and AdipoR2. To study the physiological importance of these receptors, AdipoR1 gene knockout mice (AdipoR1(-/-)) and AdipoR2 gene knockout mice (AdipoR2(-/-)) were generated. AdipoR1(-/-) mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure. However, AdipoR2(-/-) mice were lean and resistant to high-fat diet-induced obesity associated with improved glucose tolerance and higher spontaneous locomotor activity and energy expenditure and reduced plasma cholesterol levels. Thus, AdipoR1 and AdipoR2 are clearly involved in energy metabolism but have opposing effects.

Variations in opioid peptide levels during the estrous cycle in Sprague-Dawley rats.

The estrous cycle, with its various hormonal conditions, may provide us with the means of understanding how endocrine states relate to opioid mechanisms. There has been increasing experimental support for interaction between sex steroids and opioid peptides in the central nervous system. Here, we describe fluctuations in endogenous brain immunoreactive (ir) peptide levels during various phases of the estrous cycle in the female Sprague-Dawley rat. Ir levels of dynorphin A, dynorphin B, Leu-enkephalin-Arg(6), Met-enkephalin-Arg(6)Phe(7) and nociceptin/orphanin FQ were measured in the pituitary gland and in 10 areas of the brain during the diestrus, proestrus and estrus phase. In several areas of the brain, basal levels of endogenous opioid peptides showed variation during the course of the estrous cycle. Significant differences were found between the diestrus state and the proestrus and/or estrus conditions, particularly in the nucleus accumbens, caudate putamen and the substantia nigra. The ir levels of the endogenous peptide nociceptin/orphanin FQ became altered in only one of the areas measured, indicating less variance during the estrous cycle. Correlation analyses revealed that significant associations between dynorphin A or dynorphin B and Leu-enkephalin-Arg(6) were found more often during estrus than during the diestrus and proestrus conditions. The ratio between the ir levels of Leu-enkephalin-Arg(6), a cleavage product of the enzymatic conversion of dynorphin peptides into shorter peptides in vivo, and dynorphin peptides was calculated. The significantly lower ratio between Leu-enkephalin-Arg(6) and dynorphin B in diestrus than in proestrus and estrus also indicates cyclic fluctuations in the enzymatic cleavage of dynorphin. These findings are discussed in relation to the possible role of interactions between sex steroids and opioid peptide mechanisms during the normal estrous cycle.

Melanin-concentrating hormone receptor 1 deficiency increases insulin sensitivity in obese leptin-deficient mice without affecting body weight.

The hypothalamic peptide melanin-concentrating hormone (MCH) plays important roles in energy homeostasis. Animals overexpressing MCH develop hyperphagia, obesity, and insulin resistance. In this study, mice lacking both the MCH receptor-1 (MCHr1 knockout) and leptin (ob/ob) double-null mice (MCHr1 knockout ob/ob) were generated to investigate whether the obesity and/or the insulin resistance linked to the obese phenotype of ob/ob mice was attenuated by ablation of the MCHr1 gene. In MCHr1 knockout ob/ob mice an oral glucose load resulted in a lower blood glucose response and markedly lower insulin levels compared with the ob/ob mice despite no differences in body weight, food intake, or energy expenditure. In addition, MCHr1 knockout ob/ob mice had higher locomotor activity and lean body mass, lower body fat mass, and altered body temperature regulation compared with ob/ob mice. In conclusion, MCHr1 is important for insulin sensitivity and/or secretion via a mechanism not dependent on decreased body weight.

Rats with steroid-induced polycystic ovaries develop hypertension and increased sympathetic nervous system activity.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, abdominal obesity, hyperandrogenism, hypertension, and insulin resistance. METHODS: Our objectives in this study were (1) to estimate sympathetic-adrenal medullary (SAM) activity by measuring mean systolic blood pressure (MSAP) in rats with estradiol valerate (EV)-induced PCO; (2) to estimate alpha1a and alpha2a adrenoceptor expression in a brain area thought to mediate central effects on MSAP regulation and in the adrenal medulla; (3) to assess hypothalamic-pituitary-adrenal (HPA) axis regulation by measuring adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels in response to novel-environment stress; and (4) to measure abdominal obesity, sex steroids, and insulin sensitivity. RESULTS: The PCO rats had significantly higher MSAP than controls, higher levels of alpha1a adrenoceptor mRNA in the hypothalamic paraventricular nucleus (PVN), and lower levels of alpha2a adrenoceptor mRNA in the PVN and adrenal medulla. After exposure to stress, PCO rats had higher ACTH and CORT levels. Plasma testosterone concentrations were lower in PCO rats, and no differences in insulin sensitivity or in the weight of intraabdominal fat depots were found. CONCLUSION: Thus, rats with EV-induced PCO develop hypertension and increased sympathetic and HPA-axis activity without reduced insulin sensitivity, obesity, or hyperandrogenism. These findings may have implications for mechanisms underlying hypertension in PCOS.

Effects of maternal separation on voluntary ethanol intake and brain peptide systems in female Wistar rats.

In previous studies, changes in adult ethanol intake after early environmental experiences, such as short and prolonged maternal separation, have been described in male rats. The aim of this study was to further investigate long-term effects of maternal separation on voluntary ethanol intake as well as brain opioid and nociceptin/orphanin FQ (N/OFQ) peptides in female Wistar rats. During postnatal days (PNDs) 1-21, rat pups were subjected to 15 min (MS15) or 360 min (MS360) of daily maternal separation, or were kept under normal animal facility rearing (AFR) conditions. At 10 weeks of age, ethanol intake was measured using a two-bottle free choice paradigm and dynorphin B (DYNB), Met-enkephalin-Arg6-Phe7 (MEAP) and N/OFQ levels were analyzed. MS15 resulted in changes in hypothalamus (DYNB), medial prefrontal cortex and nucleus accumbens (MEAP), and amygdala (N/OFQ). MS360 induced alterations in medial prefrontal cortex (MEAP) and amygdala (N/OFQ). MS15 and MS360 had no effect on ethanol intake. However, 4 weeks of ethanol intake affected peptide levels differently in MS15, MS360 and AFR rats and resulted in attenuation of the separation-induced differences. These results show that even though maternal separation has no effect on voluntary ethanol intake in female rats, the ethanol-induced effects on peptide levels depend on the early environmental setting.

Maternal separation has no effect on voluntary ethanol intake in female Wistar rats.

Adverse experiences, early in life or during adulthood, can increase the vulnerability for development of drug dependence. Investigators have shown that short and prolonged periods of maternal separation during the postnatal period can affect voluntary ethanol intake in male rats. Recent study findings have indicated sex-dependent effects of maternal separation, and, in the current study, the effects of maternal separation on acquisition of voluntary ethanol intake in female Wistar rats were investigated. Rat pups were subjected to daily maternal separation for 15 min (MS15) or 360 min (MS360) during the first 3 weeks of life or reared under normal animal facility rearing (AFR) conditions. During 25 days, starting at 10 weeks of age, the effects of maternal separation on acquisition of voluntary ethanol intake were investigated. In contrast with previous study results for male rats, neither MS15 nor MS360 affected acquisition of voluntary ethanol intake in female rats. A stressful situation in adulthood, restraint stress, resulted in a significant increase in ethanol intake during the restraint period compared with baseline levels in the animals reared under normal AFR conditions, an effect that persisted throughout the postrestraint period. In rats subjected to MS15 or MS360, a significant increase in ethanol intake was shown during the postrestraint period compared with baseline levels. The current study findings therefore give further evidence for sex differences in the consequences of maternal separation. Compared with previous findings in male rats, acquisition of ethanol intake was not affected, and restraint-induced effects were less pronounced but more prolonged, in female rats.

Long-term effects of short and long periods of maternal separation on brain opioid peptide levels in male Wistar rats.

Environmental manipulations early in life may induce persistent alterations in adult behaviour and physiology. The underlying neural mechanisms of these responses are not yet clear. We have previously reported long-term changes in brain opioid peptide levels in male and female Sprague-Dawley rats after short periods (15 min, known as neonatal handling) of maternal separation (MS) until weaning. To study this further, we investigated behavioural and neurochemical effects of repeated MS in male Wistar rats. The rat pups were separated from their dams in litters for either 360 min (MS360) or 15 min (MS15) daily from postnatal day 1 to 21 or exposed to normal animal facility rearing. Behavioural analysis showed that MS360 rats had increased ultrasonic calls on postnatal day 5 compared to MS15 rats, but not on postnatal day 6. Moreover, the MS360 rats had more animals with higher frequency of calls at day 5 than 6 than the MS15 rats. Analysis of the opioid peptides dynorphin B and Met-enkephalin-Arg(6)Phe(7) with radioimmunoassay 7 weeks after the MS procedure, revealed long-term neurochemical changes in several brain areas and in the pituitary gland. Immunoreactive dynorphin B and Met-enkephalin-Arg(6)Phe(7) levels were affected in the hypothalamus and dynorphin B levels in the neurointermediate pituitary lobe, amygdala, substantia nigra and the periaqueductal gray. Together, these findings show that repeated periods of MS early in life in male Wistar rats affect the development of the ultrasonic call response and induce long-lasting and possibly permanent alterations in the opioid peptide systems.

Long-term effects on brain opioid and opioid receptor like-1 receptors after short periods of maternal separation in rats.

Short periods of maternal separation of neonatal rats are known to induce attenuated behavioural and neuroendocrine responses to stress in adult life. The present study was carried out to evaluate whether 15 min separation from the dam during postnatal days 1-21 (MS15) can induce long-term changes in brain opioid (kappa- and delta-receptors) and opioid receptor-like 1 (ORL1) densities in male Sprague-Dawley rats. Receptor autoradiography indicated that MS15 rats had increased delta-receptor density in the basomedial amygdala compared to animal facility reared rats 2 months after MS15. No differences in brain kappa- or ORL1-receptor density were found. The results indicate that a manipulation early in life can induce persistent neurochemical changes in the delta-opioid receptor system, which suggests involvement of delta-opioid receptors in the altered emotional processing in these rats.

Effects of melanocortin receptor ligands on ethanol intake and opioid peptide levels in alcohol-preferring AA rats.

Melanocortin (MC) peptides are suggested to play a role in opiate dependence, where they antagonise the addictive properties of opiates. To further study the involvement of the MCs in drug dependence, we analysed the effects of the MC(4)-receptor antagonist HS014 (1 nmol/rat), and the non-selective MC-receptor agonist MTII (1 nmol/rat), using i.c.v. administration, on ethanol intake in alcohol-preferring AA rats. The rats had access to ethanol during 40 days, resulting in a mean ethanol intake of 6.6 g/kg/day, before treatment. One group received only artificial cerebrospinal fluid solution. MTII caused a reduction in ethanol intake and ethanol preference, whereas HS014 was without effect. No effect on water intake was observed. A decrease in food intake was detected after MTII, whereas HS014 induced an increase in food intake. Analysis of dynorphin B and Met-enkephalin-Arg(6)Phe(7) immunoreactive levels revealed that MTII and HS014 altered opioid peptide levels in several brain areas and the pituitary gland of the rats with an established ethanol intake. This is the first report showing that manipulation of the MC-receptor system changes ethanol intake in chronically ethanol-drinking AA rats. In addition, manipulation of the MC system modulates ethanol-induced changes in opioid peptide levels.

Effects of maternal separation on brain nociceptin/orphanin FQ peptide levels in male Wistar rats.

Environmental manipulation early in life may induce persistent alterations in adult behaviour and physiology. In this study, we investigated the long-term effects of daily maternal separation, Days 1-21, on brain immunoreactive nociceptin/orphanin FQ (ir-N/OFQ) levels in male Wistar rats. The rat pups were separated in litters for 360 min (MS360) or 15 min (H15). Control rats were left undisturbed until weaning. Peptide levels were measured at 10 weeks of age. In the hypothalamus and periaqueductal gray, MS360 induced an increase in ir-N/OFQ levels in comparison with control rats. H15 rats had increased ir-N/OFQ levels in the hypothalamus and the medial prefrontal cortex compared with control animals. The rats were also tested at two occasions in an elevated plus-maze. An increased anxiety-like behaviour was shown in MS360 rats at weaning, whereas a decreased anxiety response was found at 9 weeks of age compared with control rats. The study shows that early life experiences induce long-term effects on behaviour, as well as brain N/OFQ levels.

Nociceptin/orphanin FQ tissue concentration in the rat brain. Effects of repeated ethanol administration at various post-treatment intervals.

The aim of this study was to study short- and long-term effects of repeated ethanol administration on nociceptin/orphanin FQ (N/OFQ) tissue concentrations in rat brain with radioimmunoassay. Animals were given either ethanol (intraperitoneal) or saline for 13 consecutive days. N/OFQ levels were examined at 30 min, 5 days and 21 days after the last dose on day 13. Ethanol-treated rats had significantly decreased N/OFQ tissue concentration in the hippocampus at 30 min after the last dose. N/OFQ levels were decreased in the cingulate cortex at 5 days after cessation of ethanol administration whereas no significant changes were found at 21 days. There were no significant changes in N/OFQ tissue concentrations at any time point studied in the mesolimbic dopamine (DA) system, a brain area associated with ethanol-induced activation. However, the results indicate that repeated ethanol administration may induce short- and long-term changes in N/OFQ tissue concentrations in other brain regions innervated with dopaminergic neurons.