2-Linoleoylglycerol Is a Partial Agonist of the Human
Cannabinoid Type 1 Receptor that Can Suppress
2-Arachidonolyglycerol and Anandamide Activity
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 diacyl-glycerol 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 β-lactamase enzyme. Receptor activity can then be measured by a β-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
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.