Endogenous ligand for GPR55
GPR55.

Despite activation of GPR55 by cannabinoids, it is well accepted that LPI (L-α-lysophosphatidylinositol) is its more potent endogenous ligand known up to date. Plasma LPI levels previously have been found to be increased in patients with ovarian cancer, thereby considering LPI as a biomarker for this disease. However, to our knowledge, no reports have studied the interaction between LPI levels and metabolism. Similar to GPR55 expression, we found that plasma LPI is increased in obesity, suggesting that the LPI/GPR55 system is overactive in obese states. [1]

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L-α-lysophosphatidylinositol (LPI) is the endogenous ligand of GPR55. It activates GPR55, a G protein-coupled receptor that is involved in various physiological processes. Plasma LPI levels are increased in patients with ovarian cancer and obesity, suggesting its potential as a biomarker.

Intra-striatal administration of LPI in 6-OHDA-lesioned rats increased time on the rotarod, decreased latency to remove the label, with no significant effect on slip steps, and locomotor activity. Intra-striatal administration of ML193 also increased time on the rotarod, decreased latency to remove the label and slip steps in 6-OHDA-lesioned rats mostly at the dose of 1 µg/rat. Conclusions: This study suggests that the striatal GPR55 is involved in the control of motor functions. However, considering the similar effects of GPR55 agonist and antagonist, it may be concluded that this receptor has a modulatory role in the control of motor deficits in an experimental model of Parkinson.[3]

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In vivo, intra-striatal administration of LPI in 6-hydroxydopamine (6-OHDA)-lesioned rats increased time on the rotarod and decreased latency to remove the label, with no significant effect on slip steps or locomotor activity. Plasma LPI levels are elevated in obesity, suggesting that the LPI/GPR55 system is overactive in obese states.

LPI analysis.[1]
Plasma samples for the measurement of LPI were obtained from 78 individuals of cohort 1 (Supplementary Table 1). Total LPI was calculated by combining 16:0, 18:0, and 20:4 LPI measurements as previously reported (30). Please see Supplementary information for details.

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Effect of LPI on [Ca2+]i in visceral and subcutaneous adipocytes.[1]
Human SVF cells were isolated from VAT and SAT from obese subjects undergoing open abdominal surgery (gastrointestinal bypass) as previously described Please see Supplementary information for details.

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In vitro receptor binding assays for LPI are performed to evaluate its affinity for GPR55. Radioligand binding studies using membrane preparations from cells expressing GPR55 are conducted. The compound's ability to activate GPR55 is measured in functional assays, such as measuring calcium mobilization or ERK phosphorylation.

Effects of LPI on [Ca2+]i in cultured differentiated human adipocytes.[1]
LPI has previously been shown to increase [Ca2+]i in HEK293 cells expressing GPR55 as well as in rat pheochromocytoma PC12 cells. Given that increases in [Ca2+]i have been associated with lipogenesis in adipocytes, we next analyzed whether LPI was capable of enhancing [Ca2+]i in cultured differentiated adipocytes obtained from SVF of VAT and SAT of obese patients. For this purpose, after a 9-day differentiation period, VAT and SAT adipocytes were loaded with the calcium sensitive probe Fura 2-AM, and [Ca2+]i was monitored over time (8–10 min) in the absence and presence of LPI. We found that exposure of cells to 5 μmol/L LPI induced a substantial rise in [Ca2+]i in 48.4% of differentiated VAT adipocytes (46 of 95 cells; n = 3 independent experiments) and in 24.4% of differentiated SAT cells (29 of 119 cells; n = 3 independent experiments) (Fig. 5A and C, respectively). In terms of response intensity, differentiated VAT adipocytes treated with 5 μmol/L LPI exhibited an increase in [Ca2+]i significantly higher than that observed in differentiated SAT adipocytes (43.18 ± 2.77% vs. 19.69 ± 1.93% above basal levels in VAT and SAT, respectively; P < 0.001) (Fig. 5B and D, respectively).[1]

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Effect of LPI on adipocyte differentiation of 3T3-L1 cells.[1]
When 3T3-L1 cells were treated with LPI (1 and 10 μmol/L) during 10 days, we found no alteration in the Oil Red O staining in comparison with control cells (Supplementary Fig. 3). Thus, these results indicate that LPI may act differently in human and rodent adipocytes.[1]

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To investigate the effects of GPR55 activation on hNSC proliferation, cells were plated on laminin‐coated 6‐well plates. Cells were allowed to adhere overnight and then treated with LPI (1 μM), the endogenous ligand for GPR55, or synthetic agonists, O‐1602 (1 μM) or ML184 (1 μM), in a reduced growth factor media (5% growth factor). Reduced growth factor medium was utilized to better mimic a less proliferative phenotype while still maintaining a ‘stemness’ state. Analysis by flow cytometry showed no significant reduction of nestin+ or Sox2+ populations after 48 h (data not shown). Cells treated with the selective GPR55 antagonist ML193 (5 μM) were pretreated for 30 min prior to addition of agonist. Vehicle‐treated cells received 0.1% DMSO in 5% growth factor media. For differentiation studies, cells were treated with either vehicle, ML184 (1 μM), ML193 (5 μM), or a combination of ML184 (1 μM) and ML193 (5 μM) in ReNcell medium that did not contain growth factors.[2]

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In vitro cell-based assays are conducted using cells expressing recombinant GPR55. The cells are treated with LPI, and receptor activation is measured by assessing downstream signaling events such as calcium mobilization, ERK phosphorylation, or other GPR55-mediated pathways.

Experimental Parkinson was induced by unilateral intra-striatal administration of 6-hydroxydopamine (6-OHDA, 10 µg/rat). L-α-lysophosphatidylinositol (LPI, 1 and 5 µg/rat), an endogenous GPR55 agonist, and ML193 (1 and 5 µg/rat), a selective GPR55 antagonist, were injected into the striatum of 6-OHDA-lesioned rats. Motor performance and balance skills were evaluated using the accelerating rotating rod and the ledged beam tests. The sensorimotor function of the forelimbs and locomotor activity were assessed by the adhesive removal and open field tests, respectively.[3]

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In vivo animal studies are conducted to evaluate the effects of LPI on GPR55-mediated processes. The compound is typically administered via intra-striatal injection in rodent models. Its effects on motor function and behavior are assessed in models of Parkinson's disease and other neurological conditions.

L-α-lysophosphatidylinositol is an endogenous metabolite and is not a drug. As a lysophospholipid, it is involved in cell signaling and membrane biology. Its levels in biological fluids can be measured to assess disease states.

No specific toxicity data are publicly available for L-α-lysophosphatidylinositol. As an endogenous compound, it is generally well-tolerated at physiological concentrations. Elevated levels have been associated with ovarian cancer and obesity.

[1]. The L-α-lysophosphatidylinositol/GPR55 system and its potential role in human obesity. Diabetes. 2012 Feb;61(2):281-91.

GPR55 is a putative endocannabinoid receptor, and L-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. We investigated: 1) whether GPR55 is expressed in adipose tissue and liver; 2) the correlation between GPR55 and LPI and various metabolic parameters; and 3) the effect of LPI on human adipocytes. We analyzed the expression of CB1, CB2, and GPR55 genes and circulating LPI levels in two independent groups of obese and non-obese subjects (including those with normal or impaired glucose tolerance and those with type 2 diabetes). We used in vitro experiments to measure intracellular calcium and lipid accumulation. Compared with non-obese subjects, obese subjects had elevated GPR55 levels in adipose tissue, while obese patients with type 2 diabetes had even higher GPR55 levels. GPR55 in visceral adipose tissue was positively correlated with body weight, BMI, and body fat percentage, especially in women. GPR55 gene expression in the liver was similar in obese and type 2 diabetic patients. Circulating LPI levels were elevated in obese patients and were positively correlated with body fat percentage and BMI in women. LPI increased the expression of adipogenesis genes in visceral adipose tissue explants and the intracellular calcium ion concentration in differentiated visceral adipocytes. These findings suggest that the LPI/GPR55 system is positively correlated with human obesity. [1]

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Background and Objectives: The cannabinoid system has a functional regulatory role in neural stem cell (NSC) proliferation and adult neurogenesis, but not all observed effects of cannabinoid compounds can be attributed to cannabinoid 1 (CB1) or CB2 receptors. The recently discovered GPR55 receptor has been shown to be activated by a variety of cannabinoid ligands, suggesting that GPR55 is a third cannabinoid receptor. This study investigated the role of GPR55 activation in neural stem cell (NSC) proliferation and early adult neurogenesis.
Experimental Methods: Flow cytometry was used to assess the effects of GPR55 agonists (LPI, O-1602, ML184) on the proliferation of cultured human (h) NSCs in vitro. The differentiation of human NSCs was detected by flow cytometry, qPCR and immunohistochemistry. The formation of immature neurons in the hippocampus of C57BL/6 and GPR55-/- mice was assessed by immunohistochemistry.
Main results: Activation of GPR55 significantly increased the proliferation rate of cultured hNSCs in vitro. The selective GPR55 antagonist ML193 attenuated this effect. Compared with the solvent control group, ML184 significantly promoted the differentiation of neurons in vitro, while ML193 reduced the differentiation rate. Continuous injection of O-1602 into the hippocampus via a cannula connected to an osmotic pump resulted in an increase in the number of Ki67+ cells in the dentate gyrus. Compared with the vector control group, O-1602 increased the generation of immature neurons, which could be assessed by the number of DCX+ and BrdU+ cells. The proliferation and neurogenesis in the hippocampus of GPR55-/- mice were reduced, while O-1602 had no effect on them. [2]

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L-α-lysophosphatidylinositol (LPI) is an endogenous lysophospholipid and endocannabinoid neurotransmitter that serves as the endogenous ligand for GPR55. It is used as a research tool to study the endocannabinoid system, GPR55 signaling, and its role in cancer, obesity, and inflammation. The compound is not a drug and is strictly for research purposes.

C25H48NAO12P

594.60

642.278

796963-91-2

146159779

White to off-white solid powder

6

12

22

39

671

0

CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(=O)([O-])OC1[C@@H]([C@H](C([C@H]([C@H]1O)O)O)O)O)O.[Na+]

CPTWBHNULFRGAT-UHFFFAOYSA-M

InChI=1S/C25H49O12P.Na/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-19(27)35-16-18(26)17-36-38(33,34)37-25-23(31)21(29)20(28)22(30)24(25)32;/h18,20-26,28-32H,2-17H2,1H3,(H,33,34);/q;+1/p-1

sodium;(3-hexadecanoyloxy-2-hydroxypropyl) (2,3,4,5,6-pentahydroxycyclohexyl) phosphate

796963-91-2; l-alpha-lysophosphatidylinositol (soy, sodium salt); L-alpha-lysophosphatidylinositol (Soy) (sodium salt); DA-55133; L-; A-lysophosphatidylinositol (Soy) (sodium salt); sodium;(3-hexadecanoyloxy-2-hydroxypropyl) (2,3,4,5,6-pentahydroxycyclohexyl) phosphate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. store under nitrogen), avoid exposure to light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)