2-Linoleoylglycerol Is a Partial Agonist of the Human Cannabinoid Type 1 Receptor that Can Suppress 2-Arachidonolyglycerol and Anandamide Activity

Leanne Lu, Gareth Williams, and Patrick Doherty*

Cannabis and Cannabinoid Research, 2019

Doi : 10.1089/can.2019.0030

 

Abstract

Introduction : The cannabinoid type 1 (CB1) receptor and cannabinoid type 2 (CB2) receptor are widely expressed in the body and anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are their best characterized endogenous ligands. The diacylglycerol lipases (diacylglycerol lipase alpha and diacylglycerol lipase beta) not only synthesize essentially all the 2-AG in the body but also generate other monoacylglycerols, including 2- linoleoylglycerol (2-LG). This lipid has been proposed to modulate endocannabinoid (eCB) signaling by protecting 2-AG from hydrolysis. However, more recently, 2-LG has been reported to be a CB1 antagonist.

Methods : The effect of 2-LG on the human CB1 receptor activity was evaluated in vitro using a cell-based reporter assay that couples CB1 receptor activation to the expression of the b-lactamase enzyme. Receptor activity can then be measured by a b-lactamase enzymatic assay.

Results : When benchmarked against 2-AG, AEA, and arachidonoyl-2¢-chloroethylamide (a synthetic CB1 agonist), 2-LG functions as a partial agonist at the CB1 receptor. The 2-LG response was potentiated by JZL195, a drug that inhibits the hydrolysis of monoacylglycerols. The 2-LG response was also fully inhibited by the synthetic CB1 antagonist AM251 and by the natural plant derived antagonist cannabidiol. 2-LG did not potentiate, and only blunted, the activity of 2-AG and AEA.

Conclusions : These results support the hypothesis that 2-LG is a partial agonist at the human CB1 receptor and capable of modulating the activity of the established eCBs.

Keywords : 2-AG; 2-LG; ACEA; anandamide; cannabinoid receptors; endocannabinoid system

Introduction

The cannabinoid type 1 (CB1) receptor and cannabinoid type 2 (CB2) receptor are widely expressed throughout the body with a plethora of functions.^1–3 For example, in the brain, CB1 receptors are expressed by embryonic and mature neurons, playing a role in axonal growth during development and in retrograde synaptic transmission at excitatory and inhibitory synapses in the adult.^4,5 CB1 and CB2 receptors are also expressed on neural stem cells and are involved in regulating several aspects of adult neurogenesis.^6–8 While inhibiting endocannabinoid (eCB) tone with CB1 antagonists has therapeutic potential for conditions such as obesity,⁹ increasing tone with plant-derived cannabinoids and/or inhibitors of eCB hydrolysis has therapeutic potential for the treatment of many conditions that include multiple sclerosis and pain.^10,11

Anandamide (AEA) was the first endogenous molecule to be identified with agonist activity at the cannabinoid receptors, with 2-arachidonoylglycerol (2-AG) identified as a second putative eCB soon after.^12,13 2-AG is now regarded as the “workhorse” eCB based on experiments that show loss of all the major eCB responses in the brain when the enzymes responsible for the synthesis of 2-AG, diacylglycerol lipase alpha, and beta (DAGLα/DAGLβ) are knocked out.^7,14

2-linoleoylglycerol (2-LG) is also synthesized by the DAGLs¹⁵ and has previously been reported to potentiate 2-AG activation of CB1 receptors in behavioral assays.¹⁶ It was suggested that this might reflect an “entourage” effect with 2-LG limiting 2-AG breakdown by competing for binding to monoacylglycerol lipase (MAGL), the enzyme largely responsible for the hydrolysis of 2-AG.¹⁷ However, experiments with cultured autaptic mouse hippocampal neurons show that 2-LG does not potentiate the effect of 2-AG on CB1-dependent depolarization-induced suppression of excitation (DSE). On the contrary, 2-LG displayed an antagonistic effect on the 2-AG response in this study.¹⁸

In this study, we evaluated the effect of 2-LG on the human CB1 receptor activity using a sensitive and quantitative cell-based reporter assay that couples receptor activation to the expression of the β-lactamase enzyme.¹⁹ In this assay, the synthetic CB1 agonist arachidonoyl-2′-chloroethylamide (ACEA)²⁰ and the natural eCBs AEA and 2-AG serve as positive controls to benchmark 2-LG activity. The results clearly show that 2-LG exhibits the properties of a partial agonist at the CB1 receptor. Interestingly, the response was potentiated by JZL195, an inhibitor of the hydrolytic enzymes that limit eCB activity.²¹ The 2-LG response was also fully inhibited by the competitive CB1 antagonist AM251²² and by the noncompetitive allosteric antagonist cannabidiol (CBD).²³ 2-LG did not potentiate the effect of any concentration of 2-AG or AEA; in contrast, it substantially flattened the concentration–response curve of each of them. These results support the hypothesis that 2-LG is a partial agonist at the human CB1 receptor capable of modulating the activity of the established eCBs.

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