GPR39 Knockout Mice Research Articles

GPR55 is expressed in glutamate neurons and functionally modulates drug taking and seeking in rats and mice

G protein-coupled receptor 55 (GPR55) has been thought to be a putative cannabinoid receptor. However, little is known about its functional role in cannabinoid action and substance use disorders. Here we report that GPR55 is predominantly found in glutamate neurons in the brain, and its activation reduces self-administration of cocaine and nicotine in rats and mice. Using RNAscope in situ hybridization, GPR55 mRNA was identified in cortical vesicular glutamate transporter 1 (VgluT1)-positive and subcortical VgluT2-positive glutamate neurons, with no detection in midbrain dopamine (DA) neurons. Immunohistochemistry detected a GPR55-like signal in both wildtype and GPR55-knockout mice, suggesting non-specific staining. However, analysis using a fluorescent CB1/GPR55 ligand (T1117) in CB1-knockout mice confirmed GPR55 binding in glutamate neurons, not in midbrain DA neurons. Systemic administration of the GPR55 agonist O-1602 didnt impact ∆9-THC-induced analgesia, hypothermia and catalepsy, but significantly mitigated cocaine-enhanced brain-stimulation reward caused by optogenetic activation of midbrain DA neurons. O-1602 alone failed to alter extracellar DA, but elevated extracellular glutamate, in the nucleus accumbens. In addition, O-1602 also demonstrated inhibitory effects on cocaine or nicotine self-administration under low fixed-ratio and/or progressive-ratio reinforcement schedules in rats and wildtype mice, with no such effects observed in GPR55-knockout mice. Together, these findings suggest that GPR55 activation may functionally modulate drug-taking and drug-seeking behavior possibly via a glutamate-dependent mechanism, and therefore, GPR55 deserves further study as a new therapeutic target for treating substance use disorders.

Discoveries of GPR39 as an evolutionarily conserved receptor for bile acids and of its involvement in biliary acute pancreatitis.

Acute pancreatitis (AP) is one of the most common gastrointestinal diseases. Bile acids (BAs) were proposed to be a cause of AP nearly 170 years ago, though the underlying mechanisms remain unclear. Here, we report that two G protein-coupled receptors, GPR39 and GHSR, mediated cellular responses to BAs. Our results revealed GPR39 as an evolutionarily conserved receptor for BAs, particularly 3-O-sulfated lithocholic acids. In cultured cell lines, GPR39 is sufficient for BA-induced Ca2+ elevation. In pancreatic acinar cells, GPR39 mediated BA-induced Ca2+ elevation and necrosis. Furthermore, AP induced by BAs was significantly reduced in GPR39 knockout mice. Our findings provide in vitro and in vivo evidence demonstrating that GPR39 is necessary and sufficient to mediate BA signaling, highlighting its involvement in biliary AP pathogenesis, and suggesting it as a promising therapeutic target for biliary AP.

Abstract 18781: Targeting GPR39 in Pericytes for Preventing No Reflow After Acute Myocardial Infarction

Background: No reflow (NR) continues to contribute towards morbidity and mortality in acute myocardial infarction (AMI). Currently, there is no treatment specific to NR. We demonstrated that NR is caused by pericyte-inducted capillary constriction and identified the G-protein coupled receptor, GPR39, through which the vasoconstrictor 15-Hydroxyeicosatetraenoic acid (15-HETE) results in pericyte contraction during myocardial ischemia. GPR39 knockout mice demonstrate a marked reduction in NR after AMI associated with less pericyte-induced capillary constriction. Consequently, we developed a drug candidate, VC108, that inhibits GPR39 with high specificity (antagonist potency, fpki=7.9) and no effect on 87 other receptors involved in cardiovascular regulation. Methods: Forty-nine Wild-type Sprague Dawley rats (3-4 weeks old) of both sexes underwent 1 hour of coronary occlusion followed by 1 hr of reperfusion. Either vehicle (n=13) or VC108 (in-vivo half-life of 2 hr) at 3 different doses: 0.3 (n=11), 0.5 (n=13), and 1.0 mg/kg (n-12) was injected 5 min before reperfusion. Risk volume (as %of LV volume) was derived from 4-6 short axis slices using Evans Blue staining and NR volume was measured using Thioflavin S. Results: There was no significant difference in the risk volume in animals treated with vehicle and the 3 doses of VC108 (Figure 1). In contradistinction, compared to vehicle, VC108 showed marked reduction in NR at all doses (Figure 2) with a trend towards lower NR volume at the highest dose. Conclusion: Pharmacological inhibition of GPR39 results in marked reduced in NR after AMI. These findings open up a novel approach to treating AMI.

Chronic memantine disrupts spatial memory and up-regulates Htr1a gene expression in the hippocampus of GPR39 (zinc-sensing receptor) KO male mice

GPR39 is a receptor involved in zincergic neurotransmission, and its role in regulating psychological functions is an active area of research. The purported roles of GPR39 at the cellular level include regulation of inflammatory and oxidative stress response, and modulation of GABAergic and endocannabinoid neurotransmission. GPR39 knock-out (KO) mice exhibit episodic-like and spatial memory (ELM and SM, respectively) deficits throughout their lifetime, and are similar in that respect to senescent wild-type (WT) conspecifics. Since a role for zinc has been postulated in neurodegenerative disorders, in this study we investigated the possibility of a pharmacological rescue of both types of declarative memory with memantine - a noncompetitive NMDAR antagonist used for slowing down dementia; or, a putative GPR39 agonist – TC-G 1008.First, we tested adult WT and GPR39KO male mice under acute 5 mg/kg memantine or vehicle treatment in an object recognition task designed to simultaneously probe the “what?”, “where?” and “when?” components of ELM. Next, we investigated the impact of chronic memantine or TC-G 1008 on ELM and SM (Morris water maze, MWM) in both WT and GPR39KO mice. Following chronic experiments, we assessed with qRT-PCR hippocampal gene expression of targets previously associated with GPR39.We report: no effects of acute memantine on ELM; a tendency to improve the “where?” component of ELM in both WT and GPR39 KO mice following 12 days of memantine; and, a disruption of SM in GPR39KO mice after 24 days of memantine treatment. The latter result was associated with upregulation of Htr1a hippocampal expression.

TC-G 1008 facilitates epileptogenesis by acting selectively at the GPR39 receptor but non-selectively activates CREB in the hippocampus of pentylenetetrazole-kindled mice

The pharmacological activation of the GPR39 receptor has been proposed as a novel strategy for treating seizures; however, this hypothesis has not been verified experimentally. TC-G 1008 is a small molecule agonist increasingly used to study GPR39 receptor function but has not been validated using gene knockout. Our aim was to assess whether TC-G 1008 produces anti-seizure/anti-epileptogenic effects in vivo and whether the effects are mediated by GPR39. To obtain this goal we utilized various animal models of seizures/epileptogenesis and GPR39 knockout mice model. Generally, TC-G 1008 exacerbated behavioral seizures. Furthermore, it increased the mean duration of local field potential recordings in response to pentylenetetrazole (PTZ) in zebrafish larvae. It facilitated the development of epileptogenesis in the PTZ-induced kindling model of epilepsy in mice. We demonstrated that TC-G 1008 aggravated PTZ-epileptogenesis by selectively acting at GPR39. However, a concomitant analysis of the downstream effects on the cyclic-AMP-response element binding protein in the hippocampus of GPR39 knockout mice suggested that the molecule also acts via other targets. Our data argue against GPR39 activation being a viable therapeutic strategy for treating epilepsy and suggest investigating whether TC-G 1008 is a selective agonist of the GPR39 receptor.

Gpr88 Deletion Impacts Motivational Control Without Overt Disruptions to Striatal Dopamine

BackgroundDisrupted motivational control is a common—but poorly treated—feature of psychiatric disorders, arising via aberrant mesolimbic dopaminergic signaling. GPR88 is an orphan G protein–coupled receptor that is highly expressed in the striatum and therefore well placed to modulate disrupted signaling. While the phenotype of Gpr88 knockout mice suggests a role in motivational pathways, it is unclear whether GPR88 is involved in reward valuation and/or effort-based decision making in a sex-dependent manner and whether this involves altered dopamine function. MethodsIn male and female Gpr88 knockout mice, we used touchscreen-based progressive ratio, with and without reward devaluation, and effort-related choice tasks to assess motivation and cost/benefit decision making, respectively. To explore whether these motivational behaviors were related to alterations in the striatal dopamine system, we quantified expression of dopamine-related genes and/or proteins and used [18F]DOPA positron emission tomography and GTPγ[35S] binding to assess presynaptic and postsynaptic dopamine function, respectively. ResultsWe showed that male and female Gpr88 knockout mice displayed greater motivational drive than wild-type mice, which was maintained following reward devaluation. Furthermore, we showed that cost/benefit decision making was impaired in male, but not female, Gpr88 knockout mice. Surprisingly, we found that Gpr88 deletion had no effect on striatal dopamine by any of the measures assessed. ConclusionsOur results highlight that GPR88 regulates motivational control but that disruption of such behaviors following Gpr88 deletion occurs independently of gross perturbations to striatal dopamine at a gene, protein, or functional level. This work provides further insights into GPR88 as a drug target for motivational disorders.

The orphan receptor GPR88 controls impulsivity and is a risk factor for Attention-Deficit/Hyperactivity Disorder.

The neural orphan G protein coupled receptor GPR88 is predominant in the striatum and cortex of both rodents and humans, and considered a potential target for brain disorders. Previous studies have shown multiple behavioral phenotypes in Gpr88 knockout mice, and human genetic studies have reported association with psychosis. Here we tested the possibility that GPR88 contributes to Attention Deficit Hyperactivity Disorder (ADHD). In the mouse, we tested Gpr88 knockout mice in three behavioral paradigms, best translatable between rodents and humans, and found higher motor impulsivity and reduced attention together with the reported hyperactivity. Atomoxetine, a typical ADHD drug, reduced impulsivity in mutant mice. Conditional Gpr88 knockout mice in either D1R-type or D2R-type medium spiny neurons revealed distinct implications of the two receptor populations in waiting and stopping impulsivity. Thus, animal data demonstrate that deficient GPR88 activity causally promotes ADHD-like behaviors, and identify circuit mechanisms underlying GPR88-regulated impulsivity. In humans, we performed a family-based genetic study including 567 nuclear families with DSM-IV diagnosis of ADHD. There was a minor association for SNP rs2036212 with diagnosis, treatment response and cognition. A stronger association was found for SNP rs2809817 upon patient stratification, suggesting that the T allele is a risk factor when prenatal stress is involved. Human data therefore identify GPR88 variants associated with the disease, and highlight a potential role of life trajectories to modulate GPR88 function. Overall, animal and human data concur to suggest that GPR88 signaling should be considered a key factor for diagnostic and treatment of ADHD.

The GPR88 agonist RTI-13951-33 reduces alcohol drinking and seeking in mice.

GPR88 is an orphan G‐protein‐coupled receptor that is considered a potential target to treat neuropsychiatric disorders, including addiction. Most knowledge about GPR88 function stems from knockout mouse studies, and in vivo pharmacology is still scarce. Here we examine the effects of the novel brain‐penetrant agonist RTI‐13951‐33 on several alcohol‐related behaviours in the mouse. In the intermittent‐access‐two‐bottle‐choice paradigm, the compound reduced excessive voluntary alcohol drinking, while water drinking was intact. This was observed for C57BL/6 mice, as well as for control but not Gpr88 knockout mice, demonstrating efficacy and specificity of the drug in vivo. In the drinking‐in‐the‐dark paradigm, RTI‐13951‐33 also reduced binge‐like drinking behaviour for control but not Gpr88 knockout mice, confirming the alcohol consumption‐reducing effect and in vivo specificity of the drug. When C57BL/6 mice were trained for alcohol self‐administration, RTI‐13951‐33 decreased the number of nose‐pokes over a 4‐h session and reduced the number of licks and bursts of licks, suggesting reduced motivation to obtain alcohol. Finally, RTI‐13951‐33 did not induce any place preference or aversion but reduced the expression of conditioned place preference to alcohol, indicative of a reduction of alcohol‐reward seeking. Altogether, data show that RTI‐13951‐33 limits alcohol intake under distinct conditions that require consummatory behaviour, operant response or association with contextual cues. RTI‐13951‐33 therefore is a promising lead compound to evaluate GPR88 as a therapeutic target for alcohol use disorders. More broadly, RTI‐13951‐33 represents a unique tool to better understand GPR88 function, disentangle receptor roles in development from those in the adult and perhaps address other neuropsychiatric disorders.

High fat-fed GPR55 null mice display impaired glucose tolerance without concomitant changes in energy balance or insulin sensitivity but are less responsive to the effects of the cannabinoids rimonabant or Δ(9)-tetrahydrocannabivarin on weight gain.

BackgroundThe insulin-sensitizing phytocannabinoid, Δ(9)-tetrahydrocannabivarin (THCV) can signal partly via G-protein coupled receptor-55 (GPR55 behaving as either an agonist or an antagonist depending on the assay). The cannabinoid receptor type 1 (CB1R) inverse agonist rimonabant is also a GPR55 agonist under some conditions. Previous studies have shown varied effects of deletion of GPR55 on energy balance and glucose homeostasis in mice. The contribution of signalling via GPR55 to the metabolic effects of THCV and rimonabant has been little studied.MethodsIn a preliminary experiment, energy balance and glucose homeostasis were studied in GPR55 knockout and wild-type mice fed on both standard chow (to 20 weeks of age) and high fat diets (from 6 to 15 weeks of age). In the main experiment, all mice were fed on the high fat diet (from 6 to 14 weeks of age). In addition to replicating the preliminary experiment, the effects of once daily administration of THCV (15 mg kg−1 po) and rimonabant (10 mg kg−1 po) were compared in the two genotypes.ResultsThere was no effect of genotype on absolute body weight or weight gain, body composition measured by either dual-energy X-ray absorptiometry or Nuclear Magnetic Resonance (NMR), fat pad weights, food intake, energy expenditure, locomotor activity, glucose tolerance or insulin tolerance in mice fed on chow. When the mice were fed a high fat diet, there was again no effect of genotype on these various aspects of energy balance. However, in both experiments, glucose tolerance was worse in the knockout than the wild-type mice. Genotype did not affect insulin tolerance in either experiment. Weight loss in rimonabant- and THCV-treated mice was lower in knockout than in wild-type mice, but surprisingly there was no detectable effect of genotype on the effects of the drugs on any aspect of glucose homeostasis after taking into account the effect of genotype in vehicle-treated mice.ConclusionsOur two experiments differ from those reported by others in finding impaired glucose tolerance in GPR55 knockout mice in the absence of any effect on body weight, body composition, locomotor activity or energy expenditure. Nor could we detect any effect of genotype on insulin tolerance, so the possibility that GPR55 regulates glucose-stimulated insulin secretion merits further investigation. By contrast with the genotype effect in untreated mice, we found that THCV and rimonabant reduced weight gain, and this effect was in part mediated by GPR55.

Beta‐caryophyllene, a Volatile Phytocannabinoid, Attenuates Cocaine Self‐administration and Relapse in Rats

Cocaine abuse continues to be a serious health problem worldwide. Despite intense research, there is still no FDA‐approved medication to treat cocaine use disorder. The recent search for effective therapeutics has been focused on the endocannabinoid system because of its critical role in the neurobiology of drug addiction. In the last decade, there has been a growing interest in beta‐caryophyllene (BCP), a volatile phytocannabinoid present in high proportions in cannabis and large number of spice and food plants including black pepper, thyme and cloves. This dietary additive that has a CB2 receptor (CB2R) agonist profile, has shown to produce promising therapeutic effects for multiple neuropsychiatric disorders. Surprisingly, the therapeutic potential of BCP in the treatment of drug abuse and addiction has not been explored. Here, using gold standard animal models of drug abuse, we systematically evaluated the potential utility of BCP against cocaine‐related behaviors in rodents and its mechanisms of action. In a series of experiments we found that BCP attenuated cocaine‐enhanced electrical brain‐stimulation reward and optogenetic intracranial self‐stimulation driven by activation of dopamine (DA) neurons in DAT‐cre mice, both outcomes indicating reduced reward. Intriguingly, when administered systemically or orally, BCP attenuated cocaine self‐administration in rats, suggesting an ability to reduce drug abuse. BCP also reduced drug‐primed reinstatement of cocaine seeking and cocaine conditioned place preference, indicative of its preventative effects against relapse. When BCP was substituted for cocaine, rats ceased responding, suggesting BCP has low liability abuse. In regards to the receptor mechanisms, our pharmacological approach (along with our transgenic approach utilizing with CB1, CB2, GPR55 knockout mice) indicates that BCP produces attenuating effects on cocaine self‐administration that are mediated by non‐cannabinoid receptors. We are now exploring the involvement of peroxisome proliferator‐activated receptor (PPAR) and transient receptor potential vanilloid 1 cation channels (TRPV1) in BCP action against cocaine. Our findings suggest that BCP shows exceptional promise as a therapeutic candidate in the treatment of cocaine use disorder. Importantly, given its good oral bioavailability and the advantage of being an already FDA‐approved non‐toxic dietary additive, BCP is a valuable candidate for drug repurposing programs in translational medicine.

Abstract WP327: Modulation of Microvascular Blood Flow and Stroke Outcome via GPR39

Background: Ischemic stroke is a leading cause of morbidity and mortality. Efficacy of current thrombolytic and endovascular therapies are not uniform in all patients, and depends on age, stroke severity and diabetic status. This is in part attributed to impaired microvascular reperfusion, which limits the benefit from large-vessel recanalization. Microvascular blood flow is regulated by a number of molecules including the P450 eicosanoids which are arachidonic acid-derived lipid signaling molecules whose levels are known to be altered during stroke. We have recently identified GPR39 as a dual sensor for two vasoactive P450 eicosanoids: the dilator 14,15-epoxyeicosatrienoate (EET) and constrictor 15-hydroxyeicosatetranoate (HETE). GPR39 is expressed in arteriolar vascular smooth muscle cells and peri-capillary pericytes. Thus, GPR39 is uniquely positioned to sense the balance of vasoactive eicosanoids and modulate microvascular blood flow during and following stroke. To investigate the role of GPR39 in stroke, we have generated a global GPR39 knock-out (KO) mouse via CRISPR/Cas9 deletion of the receptor’s first exon, eliminating expression of GPR39. We tested the hypothesis that GPR39 KO mice sustain larger infarcts after transient focal cerebral ischemia compared to wild-type (WT) littermates with intact GPR39. Methods: A 60-min middle cerebral artery occlusion (MCAO) was induced in 3-month old male and female mice using a silicone coated filament introduced through an external carotid stump under isoflurane anesthesia. A total of 15 KO (8 males, 7 females) and 8 WT (4 males and 4 females) mice were used for the study. Brains were harvested at 24 hours of reperfusion, sliced coronally in 2-mm segments, sections stained with triphenyltetrazolium chloride ( TTC ), and infarcted areas measured after accounting for edema. Results: Hemispheric infarct was 44.7% ± 3.31% in male GPR39 KO mice compared to 34.7% ± 5.23% in WT male littermates (mean ± sem, p=0.07). In females, infarct size was 30.3% ± 5.58% in GPR39 KO mice compared to 33.9% ± 1.45% in WT littermates (p = 0.34). Conclusions: Our preliminary results suggest that GPR39 plays a sexually-dimorphic protective role in ischemic stroke, and that GPR39 may serve as a therapeutic target in stroke.

The orphan receptor GPR88 blunts the signaling of opioid receptors and multiple striatal GPCRs.

GPR88 is an orphan G protein-coupled receptor (GPCR) considered as a promising therapeutic target for neuropsychiatric disorders; its pharmacology, however, remains scarcely understood. Based on our previous report of increased delta opioid receptor activity in Gpr88 null mice, we investigated the impact of GPR88 co-expression on the signaling of opioid receptors in vitro and revealed that GPR88 inhibits the activation of both their G protein- and β-arrestin-dependent signaling pathways. In Gpr88 knockout mice, morphine-induced locomotor sensitization, withdrawal and supra-spinal analgesia were facilitated, consistent with a tonic inhibitory action of GPR88 on µOR signaling. We then explored GPR88 interactions with more striatal versus non-neuronal GPCRs, and revealed that GPR88 can decrease the G protein-dependent signaling of most receptors in close proximity, but impedes β-arrestin recruitment by all receptors tested. Our study unravels an unsuspected buffering role of GPR88 expression on GPCR signaling, with intriguing consequences for opioid and striatal functions.

1161-P: SCO-267, a GPR40 Full Agonist, Improves Glycemic and Body Weight Control More Than That by Fasiglifam in Rat Models of Diabetes and Obesity

GPR40/FFAR1 is a G-protein-coupled receptor expressed in pancreatic islets and enteroendocrine cells. SCO-267 is a novel full agonist of GPR40 being developed as a treatment for type 2 diabetes and obesity. The efficacy of SCO-267 was investigated in cells and animal models of diabetes and obesity. In animal studies, the efficacy of SCO-267 was compared with that of fasiglifam, a clinically effective GPR40 agonist. SCO-267 treatment activated Gq signaling in both high and low GPR40-expressing CHO cells, stimulated insulin secretion in MIN6 cells, and induced GLP-1 release in GLUTag cells. In rats, SCO-267 increased insulin, glucagon, GLP-1, GIP, and PYY secretion, without hypoglycemia. In the neonatal streptozotocin (N-STZ)-1.5 rat, a diabetic model with impaired beta-cell function, compared to fasiglifam, SCO-267 was more effective in stimulating insulin and GLP-1 secretion, and improving glucose tolerance. In a single dose study using N-STZ-1.5 rats, 0.3 mg/kg SCO-267 (Cmax 0.023 μg/mL) and 3 mg/kg fasiglifam (Cmax 6.2 μg/mL) exhibited similar glucose-lowering efficacies. In a two-week dosing study of N-STZ-1.5 rats, glucose tolerance was more effectively improved by 1 mg/kg SCO-267 (Cmax, 0.139 μg/mL; AUC0-24hr, 0.626 μg h/mL) than by 10 mg/kg fasiglifam (Cmax, 39.8 μg/mL; AUC0-24hr, 254.6 μg h/mL). Diet-induced obese rats treated with SCO-267 for 2 weeks showed elevated plasma GLP-1 and PYY, reduced food intake, and decreased body weight, whereas fasiglifam-treated rats were weight-neutral. The reduction in body weight by SCO-267 was abolished in GPR40 knockout mice. Plasma protein binding of SCO-267 was similar across species (99.6-99.7%), indicating that similar compound exposure may induce therapeutic effects in humans. SCO-267 showed good pharmacokinetic and safety profiles in preclinical studies. These results suggest that SCO-267 treatment may result in effective glycemic and body weight control in patients. Disclosure Y. Moritoh: Employee; Self; SCOHIA PHARMA, Inc. H. Ueno: Employee; Self; Takeda Pharmaceutical Company Limited. R. Ito: Employee; Self; Takeda Pharmaceutical Company Limited. S. Abe: Employee; Self; SCOHIA PHARMA, Inc. H. Miyashita: Employee; Self; Takeda Pharmaceutical Company Limited. H. Ogino: Employee; Self; Takeda Pharmaceutical Company Limited. M. Ookawara: Employee; Self; SCOHIA PHARMA, Inc. Y. Ishimura: Employee; Self; Takeda Pharmaceutical Company Limited. Y. Miyamoto: None. T. Yoshihara: Employee; Self; Takeda Pharmaceutical Company Limited. Employee; Spouse/Partner; Takeda Pharmaceutical Company Limited. Y. Tsujihata: Employee; Self; Takeda Pharmaceutical Company Limited. K. Takeuchi: Employee; Self; Takeda Pharmaceutical Company Limited. N. Nishigaki: Employee; Self; Takeda Pharmaceutical Company Limited. Y. Yamada: Board Member; Self; SCOHIA PHARMA, Inc. M. Watanabe: Employee; Self; SCOHIA PHARMA, Inc. Funding Takeda Pharmaceutical Company Limited; SCOHIA PHARMA, Inc.

Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake.

The orphan G-protein-coupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88 knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To date, the biological effects of GPR88 activation are still unknown due to the lack of a potent and selective agonist appropriate for in vivo investigation. In this study, we report the discovery of the first potent, selective, and brain-penetrant GPR88 agonist RTI-13951-33 (6). RTI-13951-33 exhibited an EC50 of 25 nM in an in vitro cAMP functional assay and had no significant off-target activity at 38 GPCRs, ion channels, and neurotransmitter transporters that were tested. RTI-13951-33 displayed enhanced aqueous solubility compared to (1 R,2 R)-2-PCCA (2) and had favorable pharmacokinetic properties for behavioral assessment. Finally, RTI-13951-33 significantly reduced alcohol self-administration and alcohol intake in a dose-dependent manner without effects on locomotion and sucrose self-administration in rats when administered intraperitoneally.

FFAR1 Agonism Restores Insulin Secretion in Rodents, Human Islets, and Diabetic Monkeys

The free fatty acid receptor 1 (FFAR1) mediates fatty acid-induced insulin secretion from pancreatic β-cells. Numerous ligands have been investigated for their antidiabetic actions. The most advanced, fasiglifam (TAK-875), is a site 1 binding agonist that stimulates IPone and improves glycemic control in diabetic patients via glucose-dependent insulin secretion (GSIS). Recently, FFAR1 agonists were discovered that bind to site 3, stimulate IPone and cAMP and result in both insulin and glucagon-like peptide-1 (GLP-1) secretion. We hypothesized that this novel pharmacology would lead to superior glucose lowering relative to fasiglifam in diabetic monkeys. A selective FFAR1 agonist (CpdA) was discovered that activates rodent, cynomolgus and human FFAR1 receptors in vitro, triggering similar IPone and cAMP production. Incubation with CpdA but not fasiglifam restored insulin secretion in human islets from diabetic donors. Cpd A stimulates GLP-1 secretion in wild type but not GPR40 knockout mice, additionaly, as well as stimulates insulin secretion in ZDF rats. The ability of CpdA to control blood glucose was evaluated in diabetic cynomolgus monkeys during an oral glucose tolerance test after a single dose and repeated dosing for 7 days. Notably, the restoration of insulin secretion elicited by CpdA in diabetic human islets was recapitulated in vivo in diabetic monkeys and resulted in superior acute and chronic improvement in glucose tolerance relative to fasiglifam. In conclusion, administration of CpdA restores insulin secretion and produces robust improvement in glucose metabolism in diabetic rodents and monkeys. Thus, FFAR1 agonists represent a potential treatment for type 2 diabetes. Disclosure J. Liu: None. S.P. Lee: Employee; Self; Janssen Research & Development. T. Martin: Employee; Self; Janssen Research & Development. Stock/Shareholder; Self; Janssen Research & Development. B. Rady: Employee; Self; Janssen Research & Development. S. Meegalla: None. H. Huang: None. M.P. Winters: None. L.D. Norquay: Employee; Self; Janssen Research & Development. J. Qi: None. F. Du: None. A.R. Nawrocki: Employee; Self; Janssen Pharmaceuticals, Inc.. M. Player: None. A. Pocai: None.

Increased Alcohol Seeking in Mice Lacking Gpr88 Involves Dysfunctional Mesocorticolimbic Networks

BackgoundAlcohol use disorder (AUD) is devastating and poorly treated, and innovative targets are actively sought for prevention and treatment. The orphan G protein–coupled receptor GPR88 is enriched in mesocorticolimbic pathways, and Gpr88 knockout mice show hyperactivity and risk-taking behavior, but a potential role for this receptor in drug abuse has not been examined. MethodsWe tested Gpr88 knockout mice for alcohol-drinking and -seeking behaviors. To gain system-level understanding of their alcohol endophenotype, we also analyzed whole-brain functional connectivity in naïve mice using resting-state functional magnetic resonance imaging. ResultsGpr88 knockout mice showed increased voluntary alcohol drinking at both moderate and excessive levels, with intact alcohol sedation and metabolism. Mutant mice also showed increased operant responding and motivation for alcohol, while food and chocolate operant self-administration were unchanged. Alcohol place conditioning and alcohol-induced dopamine release in the nucleus accumbens were decreased, suggesting reduced alcohol reward in mutant mice that may partly explain enhanced alcohol drinking. Seed-based voxelwise functional connectivity analysis revealed significant remodeling of mesocorticolimbic centers, whose hallmark was predominant weakening of prefrontal cortex, ventral tegmental area, and amygdala connectional patterns. Also, effective connectivity from the ventral tegmental area to the nucleus accumbens and amygdala was reduced. ConclusionsGpr88 deletion disrupts executive, reward, and emotional networks in a configuration that reduces alcohol reward and promotes alcohol seeking and drinking. The functional connectivity signature is reminiscent of alterations observed in individuals at risk for AUD. The Gpr88 gene, therefore, may represent a vulnerability/resilience factor for AUD, and a potential drug target for AUD treatment.

Mapping GPR88-Venus illuminates a novel role for GPR88 in sensory processing.

GPR88 is an orphan G-protein coupled receptor originally characterized as a striatal-enriched transcript and is a potential target for neuropsychiatric disorders. At present, gene knockout studies in the mouse have essentially focused on striatal-related functions and a comprehensive knowledge of GPR88 protein distribution and function in the brain is still lacking. Here, we first created Gpr88-Venus knock-in mice expressing a functional fluorescent receptor to fine-map GPR88 localization in the brain. The receptor protein was detected in neuronal soma, fibers and primary cilia depending on the brain region, and remarkably, whole-brain mapping revealed a yet unreported layer-4 cortical lamination pattern specifically in sensory processing areas. The unique GPR88 barrel pattern in L4 of the somatosensory cortex appeared 3 days after birth and persisted into adulthood, suggesting a potential function for GPR88 in sensory integration. We next examined Gpr88 knockout mice for cortical structure and behavioral responses in sensory tasks. Magnetic resonance imaging of live mice revealed abnormally high fractional anisotropy, predominant in somatosensory cortex and caudate putamen, indicating significant microstructural alterations in these GPR88-enriched areas. Further, behavioral analysis showed delayed responses in somatosensory-, visual- and olfactory-dependent tasks, demonstrating a role for GPR88 in the integration rather than perception of sensory stimuli. In conclusion, our data show for the first time a prominent role for GPR88 in multisensory processing. Because sensory integration is disrupted in many psychiatric diseases, our study definitely positions GPR88 as a target to treat mental disorders perhaps via activity on cortical sensory networks.

G protein-coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1.

The putative cannabinoid receptor GPR55 has been shown to play a tumor-promoting role in various cancers, and is involved in many physiological and pathological processes of the gastrointestinal (GI) tract. While the cannabinoid receptor 1 (CB1 ) has been reported to suppress intestinal tumor growth, the role of GPR55 in the development of GI cancers is unclear. We, therefore, aimed at elucidating the role of GPR55 in colorectal cancer (CRC), the third most common cancer worldwide. Using azoxymethane (AOM)- and dextran sulfate sodium (DSS)-driven CRC mouse models, we found that GPR55 plays a tumor-promoting role that involves alterations of leukocyte populations, i.e. myeloid-derived suppressor cells and T lymphocytes, within the tumor tissues. Concomitantly, expression levels of COX-2 and STAT3 were reduced in tumor tissue of GPR55 knockout mice, indicating reduced presence of tumor-promoting factors. By employing the experimental CRC models to CB1 knockout and CB1 /GPR55 double knockout mice, we can further show that GPR55 plays an opposing role to CB1 . We report that GPR55 and CB1 mRNA expression are differentially regulated in the experimental models and in a cohort of 86 CRC patients. Epigenetic methylation of CNR1 and GPR55 was also differentially regulated in human CRC tissue compared to control samples. Collectively, our data suggest that GPR55 and CB1 play differential roles in colon carcinogenesis where the former seems to act as oncogene and the latter as tumor suppressor.

GPR88 in A2A receptor-expressing neurons modulates locomotor response to dopamine agonists but not sensorimotor gating.

The orphan receptor, GPR88, is emerging as a key player in the pathophysiology of several neuropsychiatric diseases, including psychotic disorders. Knockout (KO) mice lacking GPR88 throughout the brain exhibit many abnormalities relevant to schizophrenia including locomotor hyperactivity, behavioural hypersensitivity to dopaminergic psychostimulants and deficient sensorimotor gating. Here, we used conditional knockout (cKO) mice lacking GPR88 selectively in striatal medium spiny neurons expressing A2A receptor to determine neuronal circuits underlying these phenotypes. We first studied locomotor responses of A2A R-Gpr88 KO mice and their control littermates to psychotomimetic, amphetamine, and to selective D1 and D2 receptor agonists, SKF-81297 and quinpirole, respectively. To assess sensorimotor gating performance, mice were submitted to acoustic and visual prepulse inhibition (PPI) paradigms. Total knockout GPR88 mice were also studied for comparison. Like total GPR88 KO mice, A2A R-Gpr88 KO mice displayed a heightened sensitivity to locomotor stimulant effects of amphetamine and SKF-81297. They also exhibited enhanced locomotor activity to quinpirole, which tended to suppress locomotion in control mice. By contrast, they had normal acoustic and visual PPI, unlike total GPR88 KO mice that show impairments across different sensory modalities. Finally, none of the genetic manipulations altered central auditory temporal processing assessed by gap-PPI. Together, these findings support the role of GPR88 in the pathophysiology of schizophrenia and show that GPR88 in A2A receptor-expressing neurons modulates psychomotor behaviour but not sensorimotor gating.

Inflammatory and Neuropathic Nociception is Preserved in GPR55 Knockout Mice

The G-protein coupled receptor GPR55 has been postulated to serve as a novel cannabinoid receptor. A previous report indicated that GPR55 knockout mice fail to develop mechanical hyperalgesia, suggesting a pro-nociceptive role for GPR55 in the control of nociceptive responding. However, GPR55 knockout mice remain incompletely characterized in models of pathological pain. Here we provide a comprehensive assessment of responses of GPR55 knockout and wild-type mice to mechanical and thermal (heat, cold) stimulation in multiple, mechanistically distinct models of inflammatory and neuropathic pain. Inflammatory sensitization was produced by intraplantar administration of capsaicin, formalin or complete Freund’s adjuvant. No differences in responding were detected between GPR55 knockout and wild-type mice in any model of inflammatory nociception assessed. Neuropathic pain was induced by partial sciatic nerve ligation (which induces hypersensitivity to mechanical, cold and heat stimulation) or by treatment with the chemotherapeutic agent paclitaxel (which induces hypersensitivity to mechanical and cold stimulation only). No differences were observed between GPR55 knockout and wild type mice in either development or maintenance of neuropathic nociception in either neuropathic pain model. In conclusion, genetic deletion of GPR55 did not alter the development of pathological pain in adult mice in any chronic pain model evaluated.