GPR39 Knockout Research Articles

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.

Loss of GPR40 in LDL receptor-deficient mice exacerbates high-fat diet-induced hyperlipidemia and nonalcoholic steatohepatitis.

GPR40, a G protein-coupled receptor for free fatty acids (FFAs), is considered as a therapeutic target for type 2 diabetes mellitus (T2DM) since GPR40 activation in pancreatic beta cells enhances glucose-stimulated insulin secretion. Nonalcoholic fatty liver disease (NAFLD) is a common complication of T2DM or metabolic syndrome (MetS). However, the role of GPR40 in NAFLD associated with T2DM or MetS has not been well established. Given that it is known that cholesterol and FFAs are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH) and LDL receptor (LDLR)-deficient mice are a good animal model for human hyperlipidemia including high cholesterol and FFAs, we generated GPR40 and LDLR double knockout (KO) mice in this study to determine the effect of GPR40 KO on hyperlipidemia-promoted NASH. We showed that GPR40 KO increased plasma levels of cholesterol and FFAs in high-fat diet (HFD)-fed LDLR-deficient mice. We also showed that GPR40 KO exacerbated HFD-induced hepatic steatosis, inflammation and fibrosis. Further study demonstrated that GPR40 KO led to upregulation of hepatic CD36 and genes involved in lipogenesis, fatty acid oxidation, fibrosis and inflammation. Finally, our in vitro mechanistic studies showed that while CD36 was involved in upregulation of proinflammatory molecules in macrophages by palmitic acid (PA) and lipopolysaccharide (LPS), GPR40 activation in macrophages exerts anti-inflammatory effects. Taken together, this study demonstrated for the first time that loss of GPR40 in LDLR-deficient mice exacerbated HFD-induced hyperlipidemia, hepatic steatosis, inflammation and fibrosis potentially through a CD36-dependent mechanism, suggesting that GPR40 may play a beneficial role in hyperlipidemia-associated NASH in LDLR-deficient mice.

GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24h or brain atrophy at 3weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1week post-stroke, though functional deficit disappeared in both groups by 3weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

The Zinc-sensing Receptor (GPR39) Modulates Declarative Memory and Age-related Hippocampal Gene Expression in Male Mice

As a neuromodulator, zinc regulates synaptic plasticity, learning and memory. Synaptic zinc is also a crucial factor in the development of toxic forms of amyloid beta protein and, subsequently, of Alzheimer’s dementia (AD). Therefore, efforts to pinpoint mechanisms underlying zinc-dependent cognitive functions might aid AD research, by providing potential novel targets for drugs. One of the most understudied proteins in this regard is a zinc-sensing metabotropic receptor: GPR39. In this study we investigated the impact of GPR39 knock-out (KO) on age-related memory decline in mice of both sexes, by comparing them to age-matched wild-type (WT) littermates. We also tested the effects of a GPR39 agonist (TC-G 1008) on declarative memory of old animals, and its disruption in adult mice. We observed episodic-like memory (ELM) and spatial memory (SM) deficits in male GPR39 KO mice, as well as intact procedural memory in GPR39 KO mice regardless of age and sex. ELM was also absent in old WT male mice, and all female mice regardless of their genotype. Acute application of TC-G 1008 (10 mg/kg) reversed a deficit in two of three ELM components in old WT male mice, and had no promnesic effect on consolidation interference of ELM in adult WT mice. We discuss the possible neurobiological mechanisms and the translational value of these results for potential add-on pharmacotherapy of AD aimed at the zinc-sensing receptor.

Dysfunction of a peripheral lipid sensor GPR120 causes PGD<sub>2</sub>-microglia-provoked neuroinflammation and neurodegeneration in the mouse hippocampus

Neuroinflammation is a key pathological component of neurodegenerative disease and is characterized by microglial activation and the secretion of proinflammatory mediators. We previously reported that a surge in prostaglandin D2 (PGD2) production and PGD2-induced microglial activation could provoke neuroinflammation. We also reported that a lipid sensor GPR120, which is expressed in intestine, could be activated by polyunsaturated fatty acids, thereby mediating secretion of glucagon-like peptide-1 (GLP-1). To reveal the relationship between PGD2-microglia-provoked neuroinflammation and intestinal PUFA/GPR120 signaling, we investigated neuroinflammation and neuronal function in GPR120 knockout (KO) mice.

A peripheral lipid sensor GPR120 remotely contributes to suppression of PGD2-microglia-provoked neuroinflammation and neurodegeneration in the mouse hippocampus

BackgroundNeuroinflammation is a key pathological component of neurodegenerative disease and is characterized by microglial activation and the secretion of proinflammatory mediators. We previously reported that a surge in prostaglandin D2 (PGD2) production and PGD2-induced microglial activation could provoke neuroinflammation. We also reported that a lipid sensor GPR120 (free fatty acid receptor 4), which is expressed in intestine, could be activated by polyunsaturated fatty acids (PUFA), thereby mediating secretion of glucagon-like peptide-1 (GLP-1). Dysfunction of GPR120 results in obesity in both mice and humans.MethodsTo reveal the relationship between PGD2-microglia-provoked neuroinflammation and intestinal PUFA/GPR120 signaling, we investigated neuroinflammation and neuronal function with gene and protein expression, histological, and behavioral analysis in GPR120 knockout (KO) mice.ResultsIn the current study, we discovered notable neuroinflammation (increased PGD2 production and microglial activation) and neurodegeneration (declines in neurogenesis, hippocampal volume, and cognitive function) in GPR120 KO mice. We also found that Hematopoietic–prostaglandin D synthase (H-PGDS) was expressed in microglia, microglia were activated by PGD2, H-PGDS expression was upregulated in GPR120 KO hippocampus, and inhibition of PGD2 production attenuated this neuroinflammation. GPR120 KO mice exhibited reduced intestinal, plasma, and intracerebral GLP-1 contents. Peripheral administration of a GLP-1 analogue, liraglutide, reduced PGD2-microglia-provoked neuroinflammation and further neurodegeneration in GPR120 KO mice.ConclusionsOur results suggest that neurological phenotypes in GPR120 KO mice are probably caused by dysfunction of intestinal GPR120. These observations raise the possibility that intestinal GLP-1 secretion, stimulated by intestinal GPR120, may remotely contributed to suppress PGD2-microglia-provoked neuroinflammation in the hippocampus.

A novel GPR119 agonist DA-1241 preserves pancreatic function via the suppression of ER stress and increased PDX1 expression

DA-1241 is a novel small molecule G protein-coupled receptor 119 (GPR119) agonist in early clinical development for type 2 diabetic patients. This study aimed to elucidate the pharmacological characteristics of DA-1241 for its hypoglycemic action. DA-1241 potently and selectively activated GPR119 with enhanced maximum efficacy. DA-1241 increased intracellular cAMP in HIT-T15 insulinoma cells (EC50, 14.7 nM) and increased insulin secretion (EC50, 22.3 nM) in association with enhanced human insulin promoter activity. Accordingly, postprandial plasma insulin levels were increased in mice after single oral administration of DA-1241. Postprandial glucose excursion was significantly reduced by single oral administration of DA-1241 in wild-type mice but not in GPR119 knockout mice. GLP-1 secretion was increased by DA-1241 treatment in mice. Thus, upon combined sitagliptin and DA-1241 treatment in high-fat diet/streptozotocin (HFD/STZ)-induced diabetic mice, plasma active GLP-1 levels were synergistically increased. Accordingly, blood glucose and triglyceride levels were significantly lowered both by DA-1241 and sitagliptin alone and in combination. Immunohistochemical analysis revealed that β-cell mass with reduced PDX1 levels in the islets from HFD/STZ diabetic mice was significantly preserved by DA-1241, whereas increased glucagon and BiP levels were significantly suppressed. In HIT-T15 insulinoma cells subjected to ER stress, decreased cell viability was significantly rescued by treatment with DA-1241. Additionally, increased apoptosis was largely attenuated by DA-1241 by inhibiting BiP and CHOP expression through suppression of p38 MAPK. In conclusion, these studies provide evidence that DA-1241 can be a promising antidiabetic drug by potentially preserving pancreatic functions through suppressing ER stress and increasing PDX1 expression.

Neuronal correlates underlying the role of the zinc sensing receptor (GPR39) in passive-coping behaviour

The Zn2+ receptor GPR39 is proposed to be involved in the pathophysiology of depression. GPR39 knockout (KO) animals show depressive- and anxiety-like behaviour, and resistance to conventional monoamine-based antidepressants. However, it is unclear as to which brain regions are involved in the pro-depressive phenotype of GPR39KO mice and the resistance to monoamine-targeting antidepressant treatment. Our current study confirmed previous results, showing that mice lacking GPR39 display enhanced passive coping-like behaviour compared with their wild-type controls. Furthermore, this study shows for the first time that GPR39KO displayed aberrant challenge-induced neuronal activity in key brain regions associated with passive coping behaviour. Imipramine induced only a marginal reduction in the enhanced passive coping behaviour in GPR39KO mice, which was associated with attenuation of the hyperactive prefrontal cortex. Similarly, the aberrant activity within the amygdalar subregions was normalized following imipramine treatment in the GPR39KO mice, indicating that imipramine mediates these effects independently of GPR39 in the prefrontal cortex and amygdala. However, imipramine failed to modulate the aberrant brain activity in other brain regions, such as the anterior CA3 and the dentate gyrus, in GPR39KO mice. Normalization of aberrant activity in these areas has been shown previously to accompany successful behavioural effects of antidepressants. Taken together, our data suggest that monoamine-based antidepressants such as imipramine exert their action via GPR39-dependent and -independent pathways. Failure to modulate passive-coping related aberrant activity in important brain areas of the depression circuitry is proposed to mediate/contribute to the greatly reduced antidepressant action of monoamine-based antidepressants in GPR39KO mice.

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.

1840-P: GPR40 Deficiency Is Associated with Steatosis, Hepatic Inflammation, and CD36 Upregulation in C57BL/6 Mice

GPR40 is a G-protein coupled receptor for medium and long-chain fatty acids. Since GPR40 is highly expressed in pancreatic islets and GPR40 activation increases glucose-stimulated insulin secretion from pancreatic beta cells, GPR40 is considered as a potential target for treatment of type 2 diabetes mellitus (T2DM). However, although nonalcoholic fatty liver disease (NAFLD) is a common complication of T2DM and GPR40 is also expressed in hepatocytes and macrophages, the role of GPR40 in high-fat diet (HFD)-induced NAFLD in animal models remains unknown. In this study, we fed wild type or GPR40 knockout (KO) C57BL/6 mice low-fat diet (LFD) or HFD for 20 weeks and then analyzed the effect of GPR40 KO on HFD-induced NAFLD. Metabolic assays showed that HFD increased bodyweight, glucose, insulin, insulin resistance, cholesterol and alanine aminotransferase (ALT) as expected, but GPR40 KO did not further change these parameters in HFD-fed mice. Interestingly, in LFD-fed mice, GPR40 KO increased bodyweight, insulin, insulin resistance, cholesterol and ALT. Histological analysis and Oil Red O staining showed that HFD induced steatosis while GPR40 KO only slightly reduced HFD-induced steatosis. Surprisingly, GPR40 KO in LFD-fed mice induced steatosis. Furthermore, study showed that while HFD increased immunostaining of F4/80, a biomarker for macrophages, GPR40 KO also increased F4/80 immunostaining in LFD-fed mice. Finally, we found that while HFD increased mRNA expression of CD36, a scavenger receptor that also imports long-chain fatty acids into cells, by 5-fold in liver, GPR40 KO increased it by 9-fold in LFD-fed mice. Taken together, this study showed that GPR40 deficiency was associated with insulin resistance, dyslipidemia, steatosis and hepatic inflammation in LFD-fed mice. This study also showed that GPR40-deficiency led to a robust upregulation of CD36 expression in liver, which is likely involved in GPR40 deficiency-associated steatosis and hepatic inflammation. Disclosure Z. Lu: None. Y. Li: None. M.F. Lopes-Virella: None. Y. Huang: None. Funding National Institutes of Health (DE027070); U.S. Department of Veterans Affairs (BX000854)

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.

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.

Omega-3 fatty acids are protective in hepatic ischemia reperfusion injury in the absence of GPR120 signaling

BackgroundA single dose of IV fish oil (FO) before hepatic ischemia reperfusion injury (HIRI) increases hepatocyte proliferation and reduces necrosis in wild type (WT) mice. It has been suggested that the GPR120 receptor on Kupffer cells mediates FO's ability to reduce HIRI. The purpose of this study was to determine whether GPR120 is required for FO to reduce HIRI. MethodsSixty-four (n = 8/group) adult male WT (C57BL/6) and GPR120 knockout (KO) mice received IV FO (1 g/kg) or saline 1 h prior to HIRI or sham operation. Mice were euthanized 24 h postoperatively for analysis of hepatic histology, NFκB activity, and serum alanine transaminase (ALT) levels. ResultsFO pretreated livers had less necrosis after HIRI than saline pretreated livers in both WT (mean ± SEM 25.9 ± 7.3% less, P = 0.007) and KO (36.6 ± 7.3% less, P < 0.0001) mice. There was no significant difference in percent necrosis between WT-FO and KO-FO groups. Sham groups demonstrated minimal necrosis (0–1.9%). Mean [95% CI] ALT after HIRI was significantly higher (P = 0.04) in WT-Saline mice (1604 U/L [751–3427]) compared to WT-FO (321 U/L [150–686]) but was not significantly higher in KO-Saline mice compared to KO-FO. There were no differences in ALT between WT-FO and KO-FO mice who underwent HIRI or between groups who underwent sham surgery. There were no differences in NFκB or IKKβ activation among groups as measured by Western blot analysis. ConclusionsIV FO pretreatment was able to reduce HIRI in GPR120 KO mice, suggesting the hepatoprotective effects of FO are not mediated by GPR120 alone.

Role of Host GPR120 in Mediating Dietary Omega-3 Fatty Acid Inhibition of Prostate Cancer.

GPR120, a G protein-coupled receptor for long-chain polyunsaturated fatty acids (FAs), mediates the anti-inflammatory effects of omega-3 (ω-3) FAs. We investigated whether host or tumor GPR120 plays a role in the anti-prostate cancer effects of ω-3 FAs. MycCap prostate cancer allografts were grown in immunocompetent wild-type (WT) and GPR120 knockout (KO) mice fed ω-3 (fish oil) or ω-6 (corn oil) diets. Immune cell infiltration was quantified by flow cytometry, and gene expression of immune cell markers in isolated tumor-associated macrophages (TAMs) was quantified by quantitative real-time polymerase chain reaction. Archived tissue from a fish oil intervention trial was used to correlate gene expression of GPR120 with cell cycle progression (CCP) genes and Ki67 index (n = 11-15 per group). All statistical tests were two-sided. In WT mice (n = 7 per group), dietary ω-3 FAs decreased MycCap allograft tumor growth (mean [SD] final tumor volume ω-6 = 491 [437] mm3 vs ω-3 = 127 [77] mm3, P = .04), whereas in global GPR120KO mice (n = 7 per group) ω-3 FAs had no anticancer effects. Dietary ω-3 FAs inhibited GPR120KO-MycCaP allografts grown in WT mice (n = 8 per group; mean [SD] final tumor volume ω-6 = 776 [767] mm3 vs ω-3 = 36 [34] mm3, P = .02). Omega-3 FA treatment decreased the number of M2-like TAMs in tumor tissue and gene expression of M2 markers in isolated TAMs compared with ω-6 controls in WT (n = 7 per group) but not in GPR120KO mice (n = 7 per group). In human tissue, higher expression of stromal GPR120 correlated with greater reduction in expression of CCP genes in men with prostate cancer on a high-ω-3 diet (r = -.57, P = .04). Host GPR120 plays a central role in the anti-prostate cancer effects of dietary ω-3 FAs. Future studies are required to determine if the anticancer effects of ω-3 FAs are mediated through inhibition of M2-like macrophages and if host GPR120 status predicts anticancer effects of dietary ω-3 FAs in men with prostate cancer.

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 &amp; Development. T. Martin: Employee; Self; Janssen Research &amp; Development. Stock/Shareholder; Self; Janssen Research &amp; Development. B. Rady: Employee; Self; Janssen Research &amp; Development. S. Meegalla: None. H. Huang: None. M.P. Winters: None. L.D. Norquay: Employee; Self; Janssen Research &amp; Development. J. Qi: None. F. Du: None. A.R. Nawrocki: Employee; Self; Janssen Pharmaceuticals, Inc.. M. Player: None. A. Pocai: None.

GPR119 Agonism Increases Glucagon Secretion During Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia in diabetes is associated with impaired glucagon secretion. In this study, we tested whether stimulation of GPR119, a G-protein-coupled receptor expressed in pancreatic islet as well as enteroendocrine cells and previously shown to stimulate insulin and incretin secretion, might enhance glucagon secretion during hypoglycemia. In the study, GPR119 agonists were applied to isolated islets or perfused pancreata to assess insulin and glucagon secretion during hypoglycemic or hyperglycemic conditions. Insulin infusion hypoglycemic clamps were performed with or without GPR119 agonist pretreatment to assess glucagon counterregulation in healthy and streptozotocin (STZ)-induced diabetic rats, including those exposed to recurrent bouts of insulin-induced hypoglycemia that leads to suppression of hypoglycemia-induced glucagon release. Hypoglycemic clamp studies were also conducted in GPR119 knockout (KO) mice to evaluate whether the pharmacological stimulatory actions of GPR119 agonists on glucagon secretion during hypoglycemia were an on-target effect. The results revealed that GPR119 agonist-treated pancreata or cultured islets had increased glucagon secretion during low glucose perfusion. In vivo, GPR119 agonists also significantly increased glucagon secretion during hypoglycemia in healthy and STZ-diabetic rats, a response that was absent in GPR119 KO mice. In addition, impaired glucagon counterregulatory responses were restored by a GPR119 agonist in STZ-diabetic rats that were exposed to antecedent bouts of hypoglycemia. Thus, GPR119 agonists have the ability to pharmacologically augment glucagon secretion, specifically in response to hypoglycemia in diabetic rodents. Whether this effect might serve to diminish the occurrence and severity of iatrogenic hypoglycemia during intensive insulin therapy in patients with diabetes remains to be established.