Endogenous ligand for 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]

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]

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.

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]

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]

[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 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.[1]

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Background and purpose: The cannabinoid system exerts functional regulation of neural stem cell (NSC) proliferation and adult neurogenesis, yet not all effects of cannabinoid-like compounds seen can be attributed to the cannabinoid 1 (CB1 ) or CB2 receptor. The recently de-orphaned GPR55 has been shown to be activated by numerous cannabinoid ligands suggesting that GPR55 is a third cannabinoid receptor. Here, we examined the role of GPR55 activation in NSC proliferation and early adult neurogenesis.
Experimental approach: The effects of GPR55 agonists (LPI, O-1602, ML184) on human (h) NSC proliferation in vitro were assessed by flow cytometry. Human NSC differentiation was determined by flow cytometry, qPCR and immunohistochemistry. Immature neuron formation in the hippocampus of C57BL/6 and GPR55-/- mice was evaluated by immunohistochemistry.
Key results: Activation of GPR55 significantly increased proliferation rates of hNSCs in vitro. These effects were attenuated by ML193, a selective GPR55 antagonist. ML184 significantly promoted neuronal differentiation in vitro while ML193 reduced differentiation rates as compared to vehicle treatment. Continuous administration of O-1602 into the hippocampus via a cannula connected to an osmotic pump resulted in increased Ki67+ cells within the dentate gyrus. O-1602 increased immature neuron generation, as assessed by DCX+ and BrdU+ cells, as compared to vehicle-treated animals. GPR55-/- animals displayed reduced rates of proliferation and neurogenesis within the hippocampus while O-1602 had no effect as compared to vehicle controls.[2]

C25H48NAO12P

594.60

642.278

796963-91-2

146159779

White to off-white solid powder

6

12

22

39

671

0

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.)