Ghrelin and Cannabinoid Functional Interactions Mediated by Ghrelin/CB1 Receptor Heteromers That Are Upregulated in the Striatum From Offspring of Mice Under a High-Fat Diet.

Alejandro Lillo,Jaume Lillo,Cristina Miralpeix,Iu Raïch,Rafael Franco,Gemma Navarro,Francesc Dosrius

doi:10.3389/fncel.2021.786597

Abstract

There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits. The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors. In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes. Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling. On the one hand, just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling. On the other hand, cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist, arachidonyl-2’-chloroethylamide (ACEA). The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays. Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet. Surprisingly, the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM). These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain.

Highlights

Cell surface cannabinoid receptors were identified as targets of natural compounds present in Cannabis sativa, specially of ∆9tetrahydrocannabinol (∆9-THC; (6aR, 10aR)-6,6,9-trimethyl3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol; CAS registry number: #1972-08-3)
Immunocytochemical assays in HEK-293T cells transfected with the cDNA of the GHS-R1a fused to Renilla luciferase (Rluc) and/or the cDNA for the CB1 receptor (CB1R) fused to the Yellow Fluorescent Protein (YFP) led to detect the receptors at the plasma membrane level with a marked colocalization when coexpressed (Figure 1A)
As colocalization may be found for proteins that are close but may not be directly interacting, a Bioluminescence Resonance Energy Transfer (BRET) assay was performed in HEK-293T cells cotransfected with a constant amount of the cDNA for GHS-R1a-Renilla luciferase protein (Rluc) and increasing amounts of cDNA for CB1R-YFP

Summary

Introduction

Cell surface cannabinoid receptors were identified as targets of natural compounds present in Cannabis sativa, specially of ∆9tetrahydrocannabinol (∆9-THC; (6aR, 10aR)-6,6,9-trimethyl3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol; CAS registry number: #1972-08-3). Two cannabinoid receptors have been cloned and pharmacologically characterized, the CB1 and the CB2 receptors. They belong to class A rhodopsin-like G-protein coupled receptors (GPCRs) and both have Gi as the canonical heterotrimeric G protein to which they couple (Alexander et al, 2021). Subsequent to the discovery of cannabinoid receptors, the main compounds that act as endogenous agonists were identified, 2-arachidonoylglycerol (2AG) and anandamide (N-arachidonoylethanolamine). Cannabis smoking leads to psychotropic events that are due to ∆9-THC acting on the CB1 receptor (CB1R), which is reportedly the most abundant GPCR in the central nervous system, being expressed in almost any region of the brain and both in neurons and glia (Elphick and Egertová, 2001; Mackie, 2005). It is well established that cannabis use has orexigenic properties (Pagotto et al, 2006)

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