GPR56 Functions Together with α3β1 Integrin in Regulating Cerebral Cortical Development

Loss of function mutations in GPR56, which encodes a G protein-coupled receptor, cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Studies from BFPP postmortem brain tissue and Gpr56 knockout mice have previously showed that GPR56 deletion leads to breaches in the pial basement membrane (BM) and neuronal ectopias during cerebral cortical development. Since α3β1 integrin also plays a role in pial BM assembly and maintenance, we evaluated whether it functions together with GPR56 in regulating the same developmental process. We reveal that loss of α3 integrin enhances the cortical phenotype associated with Gpr56 deletion, and that neuronal overmigration through a breached pial BM occurs earlier in double knockout than in Gpr56 single knockout mice. These observations provide compelling evidence of the synergism of GPR56 and α3β1 integrin in regulating the development of cerebral cortex.     

The Beneficial Effects of n-3 Polyunsaturated Fatty Acids on Diet Induced Obesity and Impaired Glucose Control Do Not Require Gpr120

GPR120 (Ffar4) has been postulated to represent an important receptor mediating the improved metabolic profile seen upon ingestion of a diet enriched in polyunsaturated fatty acids (PUFAs). GPR120 is highly expressed in the digestive system, adipose tissue, lung and macrophages and also present in the endocrine pancreas. A new Gpr120 deficient mouse model on pure C57bl/6N background was developed to investigate the importance of the receptor for long-term feeding with a diet enriched with fish oil. Male Gpr120 deficient mice were fed two different high fat diets (HFDs) for 18 weeks. The diets contained lipids that were mainly saturated (SAT) or mainly n-3 polyunsaturated fatty acids (PUFA). Body composition, as well as glucose, lipid and energy metabolism, was studied. As expected, wild type mice fed the PUFA HFD gained less body weight and had lower body fat mass, hepatic lipid levels, plasma cholesterol and insulin levels and better glucose tolerance as compared to those fed the SAT HFD. Gpr120 deficient mice showed a similar improvement on the PUFA HFD as was observed for wild type mice. If anything, the Gpr120 deficient mice responded better to the PUFA HFD as compared to wild type mice with respect to liver fat content, plasma glucose levels and islet morphology. Gpr120 deficient animals were found to have similar energy, glucose and lipid metabolism when fed HFD PUFA compared to wild type mice. Therefore, GPR120 appears to be dispensable for the improved metabolic profile associated with intake of a diet enriched in n-3 PUFA fatty acids.     

Sex- and age-dependent effects of Gpr30 genetic deletion on the metabolic and cardiovascular profiles of diet-induced obese mice

The G protein-coupled receptor 30 (GPR30) has been claimed as an estrogen receptor. However, the literature reports controversial findings and the physiological function of GPR30 is not fully understood yet. Consistent with studies assigning a role of GPR30 in the cardiovascular and metabolic systems, GPR30 expression has been reported in small arterial vessels, pancreas and chief gastric cells of the stomach. Therefore, we hypothesized a role of GPR30 in the onset and progression of cardiovascular and metabolic diseases. In order to test our hypothesis, we investigated the effects of a high-fat diet on the metabolic and cardiovascular profiles of Gpr30-deficient mice (GPR30-lacZ mice). We found that GPR30-lacZ female, rather than male, mice had significant lower levels of HDL along with an increase in fat liver accumulation as compared to control mice. However, two indicators of cardiac performance assessed by echocardiography, ejection fraction and fractional shortening were both decreased in an age-dependent manner only in Gpr30-lacZ male mice. Collectively our results point to a potential role of Gpr30 in preserving lipid metabolism and cardiac function in a sex- and age-dependent fashion.     

Cloning,molecular characterization,and spatial and developmental expression analysis of GPR41 and GPR43 genes in New Zealand rabbits

Short-chain fatty acids (SCFAs) play a regulatory role in various physiological processes in mammals and act as endogenous ligands for the G protein-coupled receptors (GPR) 41 and 43. The role of GPR41 and GPR43 in mediating SCFA signaling in the rabbit remains unclear. The present study was to investigate the sequence of the GPR41 and GPR43 messenger RNA (mRNA) and their expression pattern in different tissues and developmental stages in New Zealand rabbit. Comparison of genomic sequences in GenBank using the Basic Local Alignment Search Tool program suggested that the New Zealand rabbit GPR41 mRNA has high similarities with the human (84%), bovine (84%) and Capra hircus (84%) genes. Similarly, GPR43 mRNA has high similarity with the rat (84%) and mouse (84%) genes. Real-time PCR results indicated that GPR41 and GPR43 mRNA were expressed throughout rabbit’s whole development and were expressed in several tissues. G protein-coupled receptor 41 and GPR43 mRNA were most highly expressed in pancreas (P<0.05) and s.c. adipose tissue (P<0.05), respectively. The expression levels of GPR41 mRNA was down-regulated in duodenum, cecum (P<0.05) and pancreas and up-regulated in jejunum, ileum, adipose tissue and spleen during growth. G protein-coupled receptor 43GPR43 mRNA was highly expressed in the duodenum, jejunum, ileum, colon, cecum and lung at 15^th day (P<0.05), whereas the expression levels in the pancreas and spleen increased later after birth, with the highest expression at 60^th day (P<0.05).     

Impaired working memory,cognitive flexibility and reward processing in mice genetically lacking Gpr88: Evidence for a key role for Gpr88 in multiple cortico-striatal-thalamic circuits

The GPR88 orphan G protein-coupled receptor is expressed throughout the striatum, being preferentially localised in medium spiny neurons. It is also present in lower densities in frontal cortex and thalamus. Rare mutations in humans suggest a role in cognition and motor function, while common variants are associated with psychosis. Here we evaluate the influence of genetic deletion of GPR88 upon performance in translational tasks interrogating motivation, reward evaluation and cognitive function. In an automated radial arm maze ‘N-back’ working memory task, Gpr88 KO mice showed impaired correct responding, suggesting a role for GPR88 receptors in working memory circuitry. Associative learning performance was similar to wild-type controls in a touchscreen task but performance was impaired at the reversal learning stage, suggesting cognitive inflexibility. Gpr88 KO mice showed higher breakpoints, reduced latencies and lengthened session time in a progressive ratio task consistent with enhanced motivation. Simultaneously, locomotor hyperactivity was apparent in this task, supporting previous findings of actions of GPR88 in a cortico-striatal-thalamic motor loop. Evidence for a role of GPR88 in reward processing was demonstrated in a touchscreen-based equivalent of the Iowa gambling task. Although both Gpr88 KO and wild-type mice showed a preference for an optimum contingency choice, Gpr88 KO mice selected more risky choices at the expense of more advantageous lower risk options. Together these novel data suggest that striatal GPR88 receptors influence activity in a range of procedures integrated by prefrontal, orbitofrontal and anterior cingulate cortico-striatal-thalamic loops leading to altered cognitive, motivational and reward evaluation processes.     

The Orphan G Protein-Coupled Receptor Gene GPR178 Is Evolutionary Conserved and Altered in Response to Acute Changes in Food Intake

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12–48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward.     

Analyzing human knockouts to validate GPR151 as a therapeutic target for reduction of body mass index

Novel drug targets for sustained reduction in body mass index (BMI) are needed to curb the epidemic of obesity, which affects 650 million individuals worldwide and is a causal driver of cardiovascular and metabolic disease and mortality. Previous studies reported that the Arg95Ter nonsense variant of GPR151, an orphan G protein-coupled receptor, is associated with reduced BMI and reduced risk of Type 2 Diabetes (T2D). Here, we further investigate GPR151 with the Pakistan Genome Resource (PGR), which is one of the largest exome biobanks of human homozygous loss-of-function carriers (knockouts) in the world. Among PGR participants, we identify eleven GPR151 putative loss-of-function (plof) variants, three of which are present at homozygosity (Arg95Ter, Tyr99Ter, and Phe175LeufsTer7), with a cumulative allele frequency of 2.2%. We confirm these alleles in vitro as loss-of-function. We test if GPR151 plof is associated with BMI, T2D, or other metabolic traits and find that GPR151 deficiency in complete human knockouts is not associated with clinically significant differences in these traits. Relative to Gpr151^+/+ mice, Gpr151^-/- animals exhibit no difference in body weight on normal chow and higher body weight on a high-fat diet. Together, our findings indicate that GPR151 antagonism is not a compelling therapeutic approach to treatment of obesity.     

The regulation of adipogenesis through GPR120

Recently, it has been found that long-chain fatty acids activate the G protein-coupled receptors (GPRs), GPR120 and GPR40. However, there have been no reports to date on the possible physiological roles of these GPRs in adipose tissue development and adipocyte differentiation. GPR120 mRNA was highly expressed in the four different adipose tissues, and the amount of mRNA was elevated in adipose tissues of mice fed a high fat diet. However, GPR40 mRNA was not detected in any of the adipose tissues. The expression of GPR120 mRNA was higher in adipocytes compared to stromal-vascular (S-V) cells. The level of GPR120 mRNA increased during adipocyte differentiation in 3T3-L1 cells. Similar results were observed in human adipose tissue, human preadipocytes, and cultured adipocytes. Moreover, use of a small interference RNA (siRNA) to down-regulate GPR120 expression resulted in inhibition of adipocyte differentiation. Our results suggest that GPR120 regulates adipogenic processes such as adipocyte development and differentiation.     

Inulin-type fructans with prebiotic properties counteract GPR43 overexpression and PPARγ-related adipogenesis in the white adipose tissue of high-fat diet-fed mice

Inulin-type fructans (ITF) are nondigestible/fermentable carbohydrates which are able — through the modification of the gut microbiota — to counteract high-fat (HF) diet-induced obesity, endotoxemia and related-metabolic alterations. However, their influence on adipose tissue metabolism has been poorly studied until now. The aim of this study was to assess the influence of ITF supplementation on adipose tissue metabolism, by focusing on a G protein-coupled receptor (GPR), GPR43, as a potential link between gut fermentation processes and white adipose tissue development. Male C57bl6/J mice were fed a standard diet or an HF diet without or with ITF (0.2 g/day per mouse) during 4 weeks. The HF diet induced an accumulation of large adipocytes, promoted peroxisome proliferator activated receptor gamma (PPARγ)-activated differentiation factors and led to a huge increase in GPR43 expression in the subcutaneous adipose tissue. All those effects were blunted by ITF treatment, which modulated the gut microbiota in favor of bifidobacteria at the expense of Roseburia spp. and of Clostridium cluster XIVa. The dietary modulation of GPR43 expression seems independent of endotoxemia, in view of data obtained in vivo (acute and chronic lipopolysaccharides treatment). In conclusion, ITF, which promote gut fermentation, paradoxically counteract GPR43 overexpression induced in the adipose tissue by an HF diet, a phenomenon that correlates with a beneficial effect on adiposity and with potential decrease in PPARγ-activated processes.     

猪脂肪组织和原代脂肪细胞中GPR43基因的转录表达规律

根据GenBank已发表的人、小鼠及大鼠GPR43(G protein-coupled receptor 43)基因序列, 设计并合成一对引物, RT-PCR扩增获得猪GPR43基因cDNA, 并利用PCR技术检测该基因在不同猪种、不同发育阶段、不同部位脂肪组织及原代脂肪细胞中的转录表达规律。结果显示, 成功克隆猪GPR43 cDNA片段, 长度为486 bp (GenBank登陆号为EU122439); 同源性分析发现, 猪GPR43与人、小鼠和大鼠同源性达83%以上; GPR43 mRNA表达量在脂肪型猪种上显著高于瘦肉型猪种, 随月龄增长表达量逐渐上升, 且皮下脂肪表达量较内脏脂肪高; 在猪前体脂肪细胞诱导分化过程中, GPR43 mRNA表达量呈时间依赖性升高。揭示GPR43 mRNA表达与猪肥胖程度、年龄、脂肪沉积部位以及脂肪细胞分化程度密切相关。     

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes

In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity.     

Phytonutrient genistein is a survival factor for pancreatic β-cells via GPR30-mediated mechanism

We previously discovered that phytonutrient genistein rapidly activates cAMP signaling in β-cells and improves islet mass in diabetic mice. However, the mechanism underlying these actions of genistein is still unclear. Here, we show that pharmacological or molecular inhibition of Gαs blocked genistein-stimulated adenylate cyclase activity in plasma membrane and intracellular cAMP production in INS1 cells and islets. Further, genistein stimulation of cAMP generation was abolished in islets exposed to a specific GPR30 inhibitor G15 or islets from GPR30 deficient (GPR30−/−) mice. In vivo, dietary provision of genistein (0.5 g/kg diet) significantly mitigated streptozotocin-induced hyperglycemia in male WT mice, which was associated with improved blood insulin levels and pancreatic islet mass and survival, whereas these effects were absent in Gpr30−/− mice. Genistein treatment promoted survival of INS1 cells and human islets chronically exposed to palmitate and high glucose. At molecular level, genistein activated CREB phosphorylation and subsequently induced Bcl-2 expression, and knockdown of CREB diminished the protective effect of genistein on β-cells induced by lipoglucotoxicity. Finally, deletion of GPR30 in β-cells or islets ablated genistein-induced CREB phosphorylation and its cytoprotective effect. These findings demonstrate that genistein is a survival factor for β-cells via GPR30-initiated, Gαs-mediated activation of CREB.     

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Gα_i. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gα_i signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gα_i signalling mediated by GPR43 in SCFA-stimulated leptin secretion.     

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein G(s) and an orphan member of the G protein-coupled receptor family, GPR3. To determine whether GPR3 activates G(s), the localization of Galpha(s) in follicle-enclosed oocytes from Gpr3(+/+) and Gpr3(-/-) mice was compared by using immunofluorescence and Galpha(s)GFP. GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte. Both of these properties indicate that GPR3 activates G(s). The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.     

GPR30 does not mediate estrogenic responses in reproductive organs in mice

The G protein-coupled receptor Gpr30 (Gper) was recently claimed to bind to estradiol and to activate cytoplasmic signal transduction pathways in response to estradiol. However, there are conflicting data regarding the role of Gpr30 as an estrogen receptor (ER): several laboratories were unable to demonstrate estradiol binding to GPR30 or estradiol-activated signal transduction in Gpr30-expressing cells. To clarify the potential role of Gpr30 as an ER, we generated Gpr30-deficient mice. Although Gpr30 was expressed in all reproductive organs, histopathological analysis did not reveal any abnormalities in these organs in Gpr30-deficient mice. Mutant male and female mice were as fertile as their wild-type littermates, indicating normal function of the hypothalamic-pituitary-gonadal axis. Moreover, we analyzed estrogenic responses in two major estradiol target organs, the uterus and the mammary gland. For that purpose, we examined different readout paradigms such as morphological measures, cellular proliferation, and target gene expression. Our data demonstrate that in vivo Gpr30 is dispensable for the mediation of estradiol effects in reproductive organs. These results are in clear contrast to the phenotype of mice lacking the classic ER alpha (Esr1) or aromatase (Cyp19a1). We conclude that the perception of Gpr30 (based on homology related to peptide receptors) as an ER might be premature and has to be reconsidered.     

Increasing Adipocyte Lipoprotein Lipase Improves Glucose Metabolism in High Fat Diet-induced Obesity

Lipid accumulation in liver and skeletal muscle contributes to co-morbidities associated with diabetes and obesity. We made a transgenic mouse in which the adiponectin (Adipoq) promoter drives expression of lipoprotein lipase (LPL) in adipocytes to potentially increase adipose tissue lipid storage. These mice (Adipoq-LPL) have improved glucose and insulin tolerance as well as increased energy expenditure when challenged with a high fat diet (HFD). To identify the mechanism(s) involved, we determined whether the Adipoq-LPL mice diverted dietary lipid to adipose tissue to reduce peripheral lipotoxicity, but we found no evidence for this. Instead, characterization of the adipose tissue of the male mice after HFD challenge revealed that the mRNA levels of peroxisome proliferator-activated receptor-γ (PPARγ) and a number of PPARγ-regulated genes were higher in the epididymal fat pads of Adipoq-LPL mice than control mice. This included adiponectin, whose mRNA levels were increased, leading to increased adiponectin serum levels in the Adipoq-LPL mice. In many respects, the adipose phenotype of these animals resembles thiazolidinedione treatment except for one important difference, the Adipoq-LPL mice did not gain more fat mass on HFD than control mice and did not have increased expression of genes in adipose such as glycerol kinase, which are induced by high affinity PPAR agonists. Rather, there was selective induction of PPARγ-regulated genes such as adiponectin in the adipose of the Adipoq-LPL mice, suggesting that increasing adipose tissue LPL improves glucose metabolism in diet-induced obesity by improving the adipose tissue phenotype. Adipoq-LPL mice also have increased energy expenditure.     

Generation of mouse oocytes defective in cAMP synthesis and degradation: endogenous cyclic AMP is essential for meiotic arrest

Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a^−/− mice with Gpr3^−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3^−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3^−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.     

Indomethacin Treatment Prevents High Fat Diet-induced Obesity and Insulin Resistance but Not Glucose Intolerance in C57BL/6J Mice

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.