Impact of circadian rhythmicity and sleep restriction on circulating endocannabinoid (eCB) N-arachidonoylethanolamine (anandamide)

Abstract

Objective

The endocannabinoid (eCB) system is involved in diverse aspects of human physiology and behavior but little is known about the impact of circadian rhythmicity on the system. The two most studied endocannabinoids, AEA (ananamide) and 2-AG (2-arachidonoylglycerol), can be measured in peripheral blood however the functional relevance of peripheral eCB levels is not clear. Having previously detailed the 24-h profile of serum 2-AG, here we report the 24-h serum profile of AEA to determine if these two endocannabinoids vary in parallel across the biological day including a nocturnal 8.5-h sleep period. Further, we assessed and compared the effect of a physiological challenge, in the form of sleep restriction to 4.5-h, on these two profiles.

Methods

In this randomized crossover study, we examined serum concentrations of AEA across a 24-h period in fourteen young adults. Congeners of AEA, the structural analogs oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were simultaneously assayed. Prior to 24-h blood sampling, each participant was exposed to two nights of normal (8.5 h) or restricted sleep (4.5 h). The two sleep conditions were separated by at least one month. In both sleep conditions, during the period of blood sampling, each individual ate the same high-carbohydrate meal at 0900, 1400, and 1900.

Results

Mean 24-h concentrations of AEA were 0.697 ± 0.11 pmol/ml. A reproducible biphasic 24-h profile of AEA was observed with a first peak occurring during early sleep (0200) and a second peak in the mid-afternoon (1500) while a nadir was detected in the mid-morning (1000). The 24-h profiles for both OEA and PEA followed a similar pattern to that observed for AEA. AEA, OEA, and PEA levels were not affected by sleep restriction at any time of day, contrasting with the elevation of early afternoon levels previously observed for 2-AG.

Conclusions

The 24-h rhythm of AEA is markedly different from that of 2-AG, being of lesser amplitude and biphasic, rather than monophasic. These observations suggest distinct regulatory pathways of the two eCB and indicate that time of day needs to be carefully controlled in studies attempting to delineate their relative roles. Moreover, unlike 2-AG, AEA is not altered by sleep restriction, suggesting that physiological perturbations may affect AEA and 2-AG differently. Similar 24-h profiles were observed for OEA and PEA following normal and restricted sleep, further corroborating the validity of the wave-shape and lack of response to sleep loss observed for the AEA profile. Therapeutic approaches involving agonism or antagonism of peripheral eCB signaling will likely need to be tailored according to time of day.

Introduction

Humans have long been using Cannabis sativa with the noted effects of euphoria, decreased stress, increased hunger, and potential alterations in anxiety. The active component of cannabis, Δ⁹-tetra-hydrocannabinol (THC) was isolated in 1964 (Gaoni and Mechoulam, 1964) but the major components of the endogenous cannabinoid (eCB) system were not identified until the early 1990′s. It is now well established that the endocannabinoid system is comprised of the cannabinoid receptors (CBR), the endogenous ligands of these receptors, including 2 – arachidonoylglycerol (2-AG) and N – arachidonylethanolamine (AEA or anandamide), and the enzymes responsible for the biosynthesis and degradation of the eCBs (Devane et al., 1992; Gerard et al., 1991; Mechoulam et al., 1995; Munro et al., 1993; Sugiura et al., 1995). Components of the eCB system are located both centrally and peripherally and the ubiquity of the system lends itself to involvement in multiple aspects of human physiology (Mazier et al., 2015). A large body of literature has documented that the eCB system is not only involved in mediating feeding behavior, reward, stress and anxiety but also in glucose metabolism, pain, immune response, neurological disorders, and depression (Hillard, 2018; Iannotti et al., 2016; Turcotte et al., 2015). Endocannabinoids can be measured in blood from lipid extracts of plasma or serum, although the specific origin of peripheral concentrations of serum eCBs remains unclear (Hillard, 2018). Some data suggests that circulating eCBs are derived from the multiple tissues in which the enzymatic machinery to synthesize eCBs are located (Hillard, 2018). These tissues include, but are not limited to brain, gut, muscle, pancreas, and adipose tissue (Mazier et al., 2015). The eCB like compounds N-acyl ethanolamines (NAEs), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), are structurally similar to AEA but do not bind cannabinoid receptors. These lipids are produced by similar enzymatic machinery as for AEA and can also be measured in circulation (Hillard, 2018). They are frequently measured in conjunction with AEA as they may produce similar physiological effects as AEA, without binding eCB receptors (Lam et al., 2010). Whether these eCBs and NAEs are purposefully released into the circulation as a physiological signal with explicit function or are simply a marker of tissue endocannabinoid tone remains to be seen.

Much attention has been paid in recent years to the ability of the eCB system to control feeding, body weight, and peripheral metabolism in diet-induced or genetically obese animals (Chen and Pang, 2013; Fong et al., 2007; Jbilo et al., 2005; Poirier et al., 2005; Ravinet Trillou et al., 2003); thus this system has been the target of efforts to develop new anti-obesity drugs (Chen and Pang, 2013; Despres et al., 2005; Kipnes et al., 2010; Proietto et al., 2010; Scheen et al., 2006; Van Gaal et al., 2008). Most notably, rimonabant, a selective CB1 receptor blocker was approved in Europe as an appetite-suppressant in 2006 and was shown to have beneficial metabolic effects beyond those mediated by weight loss (Despres et al., 2005; Scheen et al., 2006; Van Gaal et al., 2005, 2008). The drug had to be withdrawn in 2008 due to serious psychiatric adverse effects (Sam et al., 2011). These notable side-effects have sidelined drug development, but the role of the eCB system in the regulation of energy balance, glucose and lipid metabolism, and food intake remains nonetheless a putative target of the pharmacological treatment of obesity. The eCB system is indeed involved in modulating not only homeostatic (energy balance) pathways but also hedonic and reward mechanisms that govern food intake (Coccurello and Maccarrone, 2018). The endocannabinoid system is also known to affect other reward-related behaviors as well as reinforcement, mood, anxiety, and cognition (Wenzel and Cheer, 2018). There is also evidence that the eCB system is involved in the inflammatory response; eCBs can alter macrophage migration, and macrophages along with T and B cells have the capacity to release eCBs (Cabral and Griffin-Thomas, 2009; Di Marzo et al., 1999; Sido et al., 2016). Moreover, reports suggest that the eCB system can mediate stress responses and, alternatively, can be altered by stress (Hillard, 2014), suggesting their role as regulators of endocrine response to stress (Hillard et al., 2016). Surprisingly, the vast literature on the functions of the eCB system and the efforts to target the eCB system in drug development have largely ignored the role of the circadian system and of sleep, both major modulators of mammalian metabolism, mood and behavior.

Indeed, only a limited number of reports have examined circadian fluctuations in the eCB system or regulation of circadian rhythms by the eCB system (reviewed in (Prospero-Garcia et al., 2016)). An early study by Perron and colleagues (Perron et al., 2001), revealed that discontinuation of treatment with an exogenous cannabinoid (THC), inverted the circadian rhythm of brain temperature suggesting a role for the eCB system in modulating brain temperature rhythm. More recent studies have shown diurnal variation in CB1 receptor and protein levels, as well as in the CBR ligands AEA and 2-AG, in rat brain (Martinez-Vargas et al., 2013, 2003; Rueda-Orozco et al., 2008; Valenti et al., 2004), and in rat liver (Bazwinsky-Wutschke et al., 2017). However, to date, only a few studies have examined the rhythm of circulating endocannabinoid levels in humans (Hanlon et al., 2015; Vaughn et al., 2010). Whether the impact of the circadian system and sleep may differ for the two major eCB ligands, AEA and 2-AG, has not been examined. Delineating the 24-h variation in the activity of the eCB system may help unravel the links between eCBs and disturbances of circadian and sleep regulation, and their behavioral and physiological implications.

We therefore examined the 24-h profile of serum AEA under normal sleep conditions and during sleep restriction. We contrast the findings with our previously published characterization of the 24-h profile of 2-AG (Hanlon et al., 2016).