L-α-lysophosphatidylinositol Research Articles

The L-α-Lysophosphatidylinositol/G Protein-Coupled Receptor 55 System Induces the Development of Nonalcoholic Steatosis and Steatohepatitis.

Background and AimsG protein–coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l‐α‐lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown.Approach and ResultsWe measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up‐regulated in patients with NASH. LPI induced adenosine monophosphate–activated protein kinase activation of acetyl–coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing β‐oxidation. The inhibition of GPR55 and ACCα blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high‐fat diet and in mice fed a methionine‐choline–deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC.ConclusionsThe LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.

L-α-Lysophosphatidylinositol (LPI) aggravates myocardial ischemia/reperfusion injury via a GPR55/ROCK-dependent pathway.

The phospholipid l‐α‐lysophosphatidylinositol (LPI), an endogenous ligand for GPR55, is elevated in patients with acute coronary syndrome, and a GPR55 antagonist cannabidiol (CBD) reduces experimental ischemia/reperfusion (I/R) injury. While LPI activates multiple signaling pathways, little is known about which ones are important in cardiomyocytes. In this study we explored whether activation of the Rho kinase/ROCK/p38 MAPK pathway is responsible for LPI‐induced extension of I/R injury. Using a high‐throughput screening method (dynamic mass redistribution; DMR), mouse‐ and human‐induced pluripotent stem cell (iPSC) cardiomyocytes exposed to LPI were shown to exhibit a rapid, sustained, and concentration‐dependent (1 nmol L−1‐30 μmol L−1) cellular response. Y‐27632 (ROCK inhibitor; 10 & 50 μmol L−1) and CBD (1 μmol L−1) both abolished the DMR response to LPI (10 μmol L−1). In murine iPSC cardiomyocytes, LPI‐induced ROCK and p38 MAPK phosphorylation, both of which were prevented by Y‐27632 and CBD, but did not induce JNK activation or cleavage of caspase‐3. In hearts isolated from wild type (WT) mice subjected to 30 minutes global I/R, LPI (10 μmol L−1) administered via the coronary circulation increased infarct size when applied prior to ischemia onset, but not when given at the time of reperfusion. The exacerbation of tissue injury by LPI was not seen in hearts from GPR55−/− mice or in the presence of Y‐27632, confirming that injury is mediated via the GPR55/ROCK/p38 MAPK pathway. These findings suggest that raised levels of LPI in the vicinity of a developing infarct may worsen the outcome of AMI.

A novel GPR55-mediated satiety signal in the oval Bed Nucleus of the Stria Terminalis.

Nestled within feeding circuits, the oval (ov) region of the Bed Nucleus of the Stria Terminalis (BNST) may be critical for monitoring energy balance through changes in synaptic strength. Here we report that bidirectional plasticity at ovBNST GABA synapses was tightly linked to the caloric state of male rats, seesawing between long-term potentiation (iLTP, fed) and depression (iLTD, food restricted). L-α-lysophosphatidylinositol (LPI) acting on GPR55 receptors and 2-arachidonoylglycerol (2-AG) through CB1R were respectively responsible for fed (iLTP) and food restricted (iLTD) states. Thus, we have characterized a potential gating mechanism within the ovBNST that may signal metabolic state within the rat brain feeding circuitry.

GPR55 and the regulation of glucose homeostasis

Pathophysiological conditions such as obesity and type 2 diabetes (T2D) are reportedly associated to over-activation of the endocannabinoid system (ECS). Therefore, modulation of the ECS offers potential therapeutic benefits on those diseases. GPR55, the receptor for L-α-lysophosphatidylinositol (LPI) that has also affinity for various cannabinoid ligands, is distributed at the central and peripheral level and it is involved in several physiological processes. This review summarizes the localization and role of GPR55 in tissues that are crucial for the regulation of glucose metabolism, and provides an update on its contribution in obesity and insulin resistance. Finally, the therapeutic potential of targeting the GPR55 receptor is also discussed.

Identification of Crucial Amino Acid Residues Involved in Agonist Signaling at the GPR55 Receptor.

GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.

The GPR 55 agonist, L-α-lysophosphatidylinositol, mediates ovarian carcinoma cell-induced angiogenesis.

Background and PurposeHighly vascularized ovarian carcinoma secretes the putative endocannabinoid and GPR55 agonist, L-α-lysophosphatidylinositol (LPI), into the circulation. We aimed to assess the involvement of this agonist and its receptor in ovarian cancer angiogenesis.Experimental ApproachSecretion of LPI by three ovarian cancer cell lines (OVCAR-3, OVCAR-5 and COV-362) was tested by mass spectrometry. Involvement of cancer cell-derived LPI on angiogenesis was tested in the in vivo chicken chorioallantoic membrane (CAM) assay along with the assessment of the effect of LPI on proliferation, network formation, and migration of neonatal and adult human endothelial colony-forming cells (ECFCs). Engagement of GPR55 was verified by using its pharmacological inhibitor CID16020046 and diminution of GPR55 expression by four different target-specific siRNAs. To study underlying signal transduction, Western blot analysis was performed.Key ResultsOvarian carcinoma cell-derived LPI stimulated angiogenesis in the CAM assay. Applied LPI stimulated proliferation, network formation, and migration of neonatal ECFCs in vitro and angiogenesis in the in vivo CAM. The pharmacological GPR55 inhibitor CID16020046 inhibited LPI-stimulated ECFC proliferation, network formation and migration in vitro as well as ovarian carcinoma cell- and LPI-induced angiogenesis in vivo. Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis. These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.Conclusions and ImplicationsWe conclude that inhibiting the pro-angiogenic LPI/GPR55 pathway appears a promising target against angiogenesis in ovarian carcinoma.

A role for GPR55 in human placental venous endothelial cells.

Endocannabinoids and their G protein-coupled receptors have been suggested to play a key role in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labor. G protein-coupled receptor 55 (GPR55) was previously postulated to be another cannabinoid receptor, since specific cannabinoids were shown to act independently of the classical cannabinoid receptors CB1 or CB2. Current knowledge about GPR55 expression and function in human placenta is very limited and motivated us to evaluate human placental GPR55 expression in relation to other human peripheral tissues and to analyze spatiotemporal GPR55 expression in human placenta. Gene expression analysis revealed low GPR55 levels in human placenta, when compared to spleen and lung, the organs showing highest GPR55 expression. Moreover, expression analysis showed 5.8 fold increased placental GPR55 expression at term compared to first trimester. Immunohistochemistry located GPR55 solely at the fetal endothelium of first trimester and term placentas. qPCR and immunocytochemistry consistently confirmed GPR55 expression in isolated primary placental arterial and venous endothelial cells. Incubation with L-α-lysophosphatidylinositol (LPI), the specific and functional ligand for GPR55, at a concentration of 1 µM, significantly enhanced migration of venous, but not arterial endothelial cells. LPI-enhanced migration was inhibited by the GPR55 antagonist O-1918, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function.

Pharmacological Profiling of Lysophosphatidylinositol Species at GPR55 Using xCELLigence Cellular Impedance Analysis

L-α-lysophosphatidylinositol (LPI) is the endogenous ligand for GPR55, a Gα13-coupled GPCR. The most abundant endogenous LPI species are stearoyl-LPI (16:0 LPI), palmitoyl-LPI (18:0 LPI) and arachidonoyl-LPI (20:4 LPI). The pharmacology of ligands at GPR55 is contentious therefore further studies are required to elucidate the pharmacology of GPR55 in native cells. This study aimed to elucidate the signaling profiles of LPI species in stably expressing GPR55-HEK293 cells and human osteoclasts using xCELLigence cellular impedance in order to validate this approach as a screening tool for GPR55 ligands. In GPR55-HEK293 cells, all LPI species produced a dose dependent decrease in cellular impedance that was absent in HEK293 cells and antagonised by GPR55 antagonists CID1261822 and CID16020046. Immunocytochemical staining confirmed that the decrease in impedance was attributable to cellular retraction. GPR55 mediated signaling via Gαi, Gαs, Gαq/11 or Gβγ subunits was excluded due to lack of inhibition by pre-treatment with pertussis toxin, SQ22536, U73122 and gallein respectively. Responses to all species were completely inhibited by Y27632 and H89 suggesting that the main pathways contributing to the decrease in impedance are Rho and PKA. Interestingly, the response to 20:4 LPI was also significantly attenuated by the PKC inhibitor Bisindolylmaleimide II, indicative of biased agonism. Treatment with all LPI species in human osteoclasts produced a dose dependant decrease in cellular impedance attributed to Rho, ERK and PKC as well as Gi and Gβγ, reflective of more diverse signaling. These experiments validate use of xCELLigence technology to screen novel GPR55 ligands in native cells.

Identification of the GPR55 Antagonist Binding Site Using a Novel Set of High-Potency GPR55 Selective Ligands

GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ~300000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50 values in the range of 0.16-2.72 μM [Heynen-Genel, S., et al. (2010) Screening for Selective Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second-generation GPR55 selective antagonists.

The G Protein‐Coupled Receptor 55 Ligand l‐α‐Lysophosphatidylinositol Exerts Microglia‐Dependent Neuroprotection After Excitotoxic Lesion

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein-coupled cannabinoid receptor 1 (CB1 ) and microglial G protein-coupled cannabinoid receptor 2 (CB2 ). However, pharmacological experiments with CB1 /CB2 -deficient mice unraveled the existence of further, so-called non-CB1 /non-CB2 G protein-coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l-α-Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI-mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca(2+) and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI-mediated modulation of microglia function.

Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55

The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca(2+) signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca(2+) entry via L-type Ca(2+) channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca(2+) release. The latter signal is further amplified by Ca(2+)-induced Ca(2+) release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca(2+) release from acidic-like Ca(2+) stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca(2+)-induced Ca(2+) release via ryanodine receptors. Extracellularly applied LPI produces Ca(2+)-independent membrane depolarization, whereas the Ca(2+) signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.

Thel-α-Lysophosphatidylinositol/GPR55System and Its Potential Role in Human Obesity

GPR55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. We investigated 1) whether GPR55 is expressed in fat and liver; 2) the correlation of both GPR55 and LPI with several metabolic parameters; and 3) the actions of LPI on human adipocytes. We analyzed CB1, CB2, and GPR55 gene expression and circulating LPI levels in two independent cohorts of obese and lean subjects, with both normal or impaired glucose tolerance and type 2 diabetes. Ex vivo experiments were used to measure intracellular calcium and lipid accumulation. GPR55 levels were augmented in the adipose tissue of obese subjects and further so in obese patients with type 2 diabetes when compared with nonobese subjects. Visceral adipose tissue GPR55 correlated positively with weight, BMI, and percent fat mass, particularly in women. Hepatic GPR55 gene expression was similar in obese and type 2 diabetic subjects. Circulating LPI levels were increased in obese patients and correlated with fat percentage and BMI in women. LPI increased the expression of lipogenic genes in visceral adipose tissue explants and intracellular calcium in differentiated visceral adipocytes. These findings indicate that the LPI/GPR55 system is positively associated with obesity in humans.

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

Lipid bilayer molecular dynamics study of lipid-derived agonists of the putative cannabinoid receptor, GPR55

Both L-α-lysophosphatidylinositol (LPI) and 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) have been reported to activate the putative cannabinoid receptor, GPR55. Recent microsecond time-scale molecular dynamics (MD) simulations and isothiocyanate covalent labeling studies have suggested that a transmembrane helix 6/7 (TMH6/7) lipid pathway for ligand entry may be necessary for interaction with cannabinoid receptors. Because LPI and 2-AGPI are lipid-derived ligands, conformations that each assumes in the lipid bilayer are therefore likely important for their interaction with GPR55. We report here the results of 70ns NAMD molecular dynamics (MD) simulations of LPI and of 2-AGPI in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These simulations are compared with a 70ns simulation of the cannabinoid CB1 receptor endogenous ligand, N-arachidonoylethanolamine (anandamide, AEA) in a POPC bilayer. These simulations revealed that (1) LPI and 2-AGPI sit much higher in the bilayer than AEA, with inositol headgroups that can at times be solvated completely by water; (2) the behavior of the acyl chains of AEA and 2-AGPI are similar in their flexibilities in the bilayer, while the acyl chain of LPI has reduced flexibility; and (3) both 2-AGPI and LPI can adopt a tilted headgroup orientation by hydrogen bonding to the phospholipid phosphate/glycerol groups or via intramolecular hydrogen bonding. This tilted head group conformation (which represents over 40% of the conformer population of LPI (42.2±3.3%) and 2-AGPI (43.7±1.4%)) may provide a low enough profile in the lipid bilayer for LPI and 2-AGPI to enter GPR55 via the putative TMH6/7 entry port.