Molecular Mechanisms Through Which Cannabidiol May Affect Skeletal Muscle Metabolism,Inflammation,Tissue Regeneration,and Anabolism: A Narrative Review

Abstract

Background:

Cannabidiol (CBD), a nonintoxicating constituent of the cannabis plant, recently gained a lot of interest among athletes, since it is no longer considered as a prohibited substance by the World Anti-Doping Agency. The increasing prevalence of CBD use among athletes is driven by a perceived improvement in muscle recovery and a reduction in pain. However, compelling evidence from intervention studies is lacking and the precise mechanisms through which CBD may improve muscle recovery remain unknown. This highlights the need for more scientific studies and an evidence-based background. In the current review, the state-of-the-art knowledge on the effects of CBD on skeletal muscle tissue is summarized with special emphasis on the underlying mechanisms and molecular targets. More specifically, the large variety of receptor families that are believed to be involved in CBD's physiological effects are discussed. Furthermore, in vivo and in vitro studies that investigated the actual effects of CBD on skeletal muscle metabolism, inflammation, tissue regeneration, and anabolism are summarized, together with the functional effects of CBD supplementation on muscle recovery in human intervention trials. Overall, CBD was effective to increase the expression of metabolic regulators in muscle of obese mice (e.g., Akt, glycogen synthase kinase-3). CBD treatment in rodents reduced muscle inflammation following eccentric exercise (i.e., nuclear factor kappa B [NF-κB]), in a model of muscle dystrophy (e.g., interleukin-6, tumor necrosis factor alpha) and of obesity (e.g., COX-2, NF-κB). In addition, CBD did not affect in vitro or in vivo muscle anabolism, but improved satellite cell differentiation in dystrophic muscle. In humans, there are some indications that CBD supplementation improved muscle recovery (e.g., creatine kinase) and performance (e.g., squat performance). However, CBD doses were highly variable (between 16.7 and 150 mg) and there are some methodological concerns that should be considered.

Conclusion:

CBD has the prospective to become an adequate supplement that may improve muscle recovery. However, this research domain is still in its infancy and future studies addressing the molecular and functional effects of CBD in response to exercise are required to further elucidate the ergogenic potential of CBD.

Introduction

Cannabidiol (CBD) is a nonintoxicating constituent of the cannabis plant and gained a lot of attention in clinical populations because of its analgesic,¹ neuroprotective,^2,3 anti-inflammatory,^4–8 and antioxidative ^9,10 effects with a safe adverse event profile.^11,12 This has contributed to the recent approval of CBD to treat epilepsy and tuberous sclerosis.¹³ In addition to clinical populations, CBD's popularity also drastically increased among the general public over the past few years. An online survey indicated that the top four reasons for using CBD were improvements in self-perceived anxiety, sleep problems, stress, and general health and wellbeing.¹⁴ A specific population that seems to benefit from CBD use are athletes. In fact, since the World Anti-Doping Agency (WADA) no longer considers CBD as a prohibited substance,¹⁵ CBD use among athletes is emerging. A study in elite rugby players showed a prevalence of 26%,¹⁶ which is a high number driven by a perceived improvement in recovery and a reduction in pain.¹⁶

As athletes are often subjected to strenuous exercise sessions that induce microstructural damage to the skeletal muscle, that is, exercise-induced muscle damage (EIMD),^17,18 they are continuously searching for ergogenic aids to improve their recovery process. EIMD goes along with inflammatory responses and a sensation of delayed onset of muscle soreness (DOMS), and eventually leads to decreased muscle performance.^17,19–21 CBD is believed to alleviate these symptoms, through its anti-inflammatory^4–8 and muscle regenerative⁴ effects, which have been proposed in preclinical studies. However, compelling evidence from human intervention studies is lacking and the precise mechanisms through which CBD may improve muscle recovery remain unknown.

Considering the high prevalence of CBD use among athletes, together with the lack of knowledge on its ergogenic effects in muscle recovery, there is an urgent need for more evidence-based scientific studies. This review is a first step in which we aim to provide a fundamental background, giving an overview of the current knowledge on the effects of CBD on skeletal muscle tissue with special emphasis on the underlying mechanisms, molecular targets, and functional adaptations.

Molecular Pathways Through Which CBD May Affect Skeletal Muscle Metabolism, Inflammation, Tissue Regeneration, or Anabolism

In this section, the potential molecular pathways and mechanisms underlying the physiological actions of CBD are discussed (Fig. 1). Although, these molecular mechanisms are not fully elucidated, yet, it is currently suggested that CBD is able to directly interact with different receptors, thereby targeting multiple pathways and mechanisms of action. Receptors that are believed to be involved in CBD signaling include transient receptor potential vanilloid channel 1 (TRPV1); peroxisome proliferator-activated receptor gamma (PPARγ); and G protein-coupled receptors (GPCRs) such as G protein-coupled receptor 55 (GPR55), adenosine A2a receptor, serotonin 1A receptor (5-HT1A), and the cannabinoid receptors 1 and 2 (CB1 and CB2). Additionally, CBD may activate these receptors indirectly through an increased concentration of anandamide (AEA, an endocannabinoid), as it was shown that CBD inhibits fatty acid amide hydrolase (FAAH), the enzyme which is responsible for the hydrolysis of AEA.

FIG. 1.

Schematic representation of potential molecular pathways and mechanisms underlying the physiological actions of CBD. The physiological effects of CBD as a result of the direct interaction (solid lines) with its receptors is depicted in the boxes with corresponding colors. CBD might also indirectly interact (dotted lines) with these receptors through increases in AEA and in adenosine. Indeed, CBD was shown to increase AEA concentration by inhibiting its hydrolysis to arachidonic acid through FAAH, and to increase extracellular adenosine concentration by inhibiting its uptake through ENT. 5-HT1A, serotonin 1A receptor; AEA, anandamide; CaMKII, calcium/calmodulin-dependent protein kinase II; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CBD, cannabidiol; FAAH, fatty acid amide hydrolase; GPR55, G protein-coupled receptor 55; MPS, muscle protein synthesis; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator-1alpha; PPARγ, peroxisome proliferator-activated receptor gamma; ROS, reactive oxygen species; TRPV1, transient receptor potential vanilloid channel 1.

Transient receptor potential vanilloid channel 1

Classically, TRPV1 channels are considered to play an important role in transducing noxious stimuli, such as heat²² and inflammation.²³ Coherently, they are highly expressed in nociceptive neurons, but they can also be found in many other cell types, including muscle cells.²⁴ Upon receptor activation, intracellular calcium levels increase and calcium-dependent processes result in desensitization of the channels inducing a refractory period. During this period, TRPV1-expressing sensory neurons are not responsive to further stimulation, thereby relieving symptoms of nociceptive behavior. Through this mechanism, TRPV1 agonism shows to be a potent manner to induce analgesic effects. In vitro experiments show that CBD may act as a TRPV1 agonist, inducing TRPV1 channel desensitization with similar efficacy as its well-known agonist capsaicin.^25,26 These findings are in line with in vivo experiments reporting that the analgesic effects of CBD in models of neuropathic and inflammatory pain in rats were reversed when combined with TRPV1 antagonists.^27,28

These results show that CBD induces analgesic actions through desensitization of TRPV1 channels. Whether the analgesic effects of CBD can be translated to reduced sensations of inflammatory muscle pain in humans, remains to be determined. However, it is conceivable that CBD acts through this pathway to attenuate for example, muscle soreness in athletes with EIMD.

TRPV1 is also expressed in the sarcoplasmic reticulum of skeletal muscle cells,²⁹ where its activation results in increased calcium concentrations in the cytoplasm.^24,30,31 Elevated calcium concentrations were shown to increase peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) expression through the calcium/calmodulin-dependent protein kinase II (CaMKII), thereby stimulating mitochondrial biogenesis.³² Indeed, mice fed with capsaicin for 4 months exhibited higher PGC-1α expression levels, mitochondrial biogenesis, and oxidative fibers, as well as an enhanced exercise endurance capacity.³³

Additionally, TRPV1-mediated increases in intracellular calcium levels also activated the mammalian target of rapamycin (mTOR) axis,^34,35 which is the main signaling pathway for muscle anabolism. Indeed, daily administration of capsaicin increased phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1), a key factor downstream of the mTOR pathway, and increased muscle weight, fiber size, and muscle force in mice.³⁶

Since CBD can act as a TRPV1 agonist, it is possible that CBD affects muscle metabolism and/or anabolism through similar calcium-dependent pathways, that is, CaMKII/PGC-1α and mTOR, respectively.

Peroxisome proliferator-activated receptor gamma

PPARs are ligand-activated transcription factors that control the expression of various genes involved in the modulation of substrate metabolism, insulin sensitivity, and immune function.³⁷ There are three PPAR subtypes, that is, PPARα, PPARδ, and PPARγ. Activation of the latter one has been associated with anti-inflammatory effects through proteasomal degradation of the nuclear factor kappa B (NF-κB) p65 subunit, which terminates the NF-κB signaling pathway and ultimately inhibits the expression of proinflammatory mediators.³⁸ Only one study investigated the anti-inflammatory effects of PPARγ agonism in skeletal muscle tissue, using a lipopolysaccharide (LPS) model of endotoxemia in rats. Treatment with the PPARγ agonist rosiglitazone blunted the LPS-induced increase in tumor necrosis factor alpha (TNFα) and interleukin (IL)-6 mRNA expression.³⁹

Interestingly, studies using pathological animal models showed that the anti-inflammatory effects of CBD are also mediated through PPARγ activation. A study on β-amyloid-induced neurotoxicity in rats showed that treatment with CBD inhibited the increased expression of the proinflammatory mediators nitric oxide, IL-1β, and TNFα, which was reversed by PPARγ antagonism.⁴⁰ Furthermore, CBD treatment resulted in a decreased protein expression of p65NF-κB, suggesting that PPARγ activation indeed may have occurred upstream of CBD-mediated NF-κB inhibition.⁴⁰

Besides a direct stimulation of PPARγ by CBD, it is plausible that CBD also indirectly activated PPARγ through elevated levels of AEA, as these were shown to activate PPARγ⁴¹ and exert anti-inflammatory effects.⁴² While the results above were obtained in neuronal cell lines and brain tissue, CBD may also exert anti-inflammatory effects through PPARγ activation in skeletal muscle tissue, but this remains to be investigated.

PPARγ also plays an important role in lipid metabolism and insulin sensitivity within the skeletal muscle and liver. In fact, PPARγ activation was associated with increased glucose tolerance, and expression of genes involved in fat oxidation and mitochondrial function in skeletal muscle tissue.^43,44 Whether CBD elicits similar effects upon PPARγ activation remains to be determined. A pilot study investigating the effects of CBD on metabolic effects in patients with type 2 diabetes did not show any beneficial effects of CBD supplementation (200 mg/day for 13 weeks).⁴⁵ However, the authors mentioned that the selected dose of 200 mg CBD per day was unable to elevate plasma levels of AEA. Therefore, it could be speculated that the dose was insufficient to exert any metabolic actions.

G protein-coupled receptors

GPCRs form a large family of cell surface receptors that contain a protein complex existing of an α, β, and γ subunit. Once a ligand binds to the receptor, the G protein undergoes a conformational change in which the α-subunit dissociates from the βγ-subunit. This dissociation induces a cascade of reactions in which the activated α-subunit regulates the membrane-bound enzyme adenylyl cyclase that converts ATP to cyclic adenosine monophosphate (cAMP), a second messenger involved in numerous signal transduction pathways. One of cAMPs main effectors is protein kinase A (PKA), which in turn activates the mitogen-activated protein kinase family (MAPK) that is involved in energy metabolism, cell proliferation and differentiation, hypertrophy, apoptosis, and inflammation.⁴⁶ Depending on the ligand, the G protein α-subunit type (i.e., Gα_i, Gα_s, and Gα_q), the cell type and the receptor type, inhibition or activation of adenylyl cyclase may result in the activation of several downstream signaling pathways.

The following paragraphs elaborate on the specific GPCRs through which CBD is expected to exert its effects.

G protein-coupled receptor 55

GPR55 is involved in different biological processes such as inflammation and metabolism. GPR55 activation was shown to promote proinflammatory responses by increasing neutrophil migration,⁴⁷ while deletion of this receptor was associated with a reduced inflammatory status and an increased expression of anti-inflammatory cytokines, such as IL-4 and IL-10.⁴⁸

CBD is an antagonist of GPR55⁴⁹ and can as such attenuate GPR55-induced proinflammatory responses. More specifically, CBD's antagonistic action at GPR55 was able to improve inflammatory status in a rat model of septic ileus as indicated by a reduced expression of proinflammatory cytokines (IL-6 and TNFα), edema, and infiltration of neutrophils and macrophages in the intestinal wall.⁵⁰ Similar findings have been obtained in vitro as CBD treatment reversed the proinflammatory actions of GPR55 agonism in human macrophage-derived foam cells.⁵¹ The anti-inflammatory effects of GPR55 antagonism in other cell types (relevant for skeletal muscle inflammation) remain to be elucidated.

As mentioned above, GPR55 also exerts a role in the regulation of energy metabolism. While GPR55 deletion was associated with adiposity and insulin resistance,^52,53 GPR55 activation positively regulated insulin sensitivity and metabolic function in vivo.^52,54 As such, GPR55 antagonism through CBD may also be associated with negative alterations on glucose homeostasis, but this needs to be further investigated.

Adenosine A2a receptor

Adenosine receptors are responsive to the nucleoside adenosine and can be classified in four subtypes, that is, A1, A2a, A2b, and A3. Extracellular adenosine signaling plays an important role in anti-inflammatory processes and tissue protection.^55,56 More specifically, activation of adenosine A2a receptors increases intracellular cAMP, which in turn inhibits intracellular proinflammatory signaling pathways.⁵⁷ The protective effects of adenosine A2a receptor activation have been shown in skeletal muscle tissue using a mouse hindlimb ischemia–reperfusion model to induce muscle damage.⁵⁸ Mice pretreated with an adenosine A2a receptor agonist showed reduced indices of muscle damage and serum creatine kinase compared with vehicle-treated mice. This effect was abrogated when the treatment was combined with an adenosine A2a antagonist.

Interestingly, CBD is able to increase the extracellular adenosine content available for adenosine receptor activation through inhibition of the equilibrative nucleoside transporter (ENT). ENT is responsible for the uptake of extracellular adenosine and terminates adenosine signaling.⁵⁹ Therefore, it is suggested that CBD may exert anti-inflammatory and tissue-protective actions through enhanced adenosine signaling.^60,61 In fact, CBD was able to reduce TNFα production in LPS-treated mice by enhancing adenosine signaling.⁵⁹

This mechanism was later supported by other animal models of induced inflammation. For instance, CBD was able to induce anti-inflammatory effects in LPS-induced retinal inflammation in rats,⁶² in LPS-induced lung injury in mice,⁶³ and in hypoxic–ischemic immature murine brain.⁶⁴ In all studies, the anti-inflammatory effect of CBD was abrogated by an adenosine A2a antagonist and authors speculated increased adenosine signaling to be the mechanism of action, although, the possibility that CBD directly binds to the adenosine A2a receptor cannot be ruled out.

While these findings seem promising with regard to the anti-inflammatory and tissue-protective effects of CBD in retinal, brain, and lung tissue, the anti-inflammatory effects of increased adenosine signaling by CBD within skeletal muscle tissue needs further investigation. Interestingly, adenosine receptors are expressed abundantly within skeletal muscle tissue⁶⁵ raising the possibility for CBD to exert its anti-inflammatory effects through this pathway. In fact, adenosine receptors are suggested to exert numerous effects within the skeletal muscle, among others, the regulation of muscle blood flow⁶⁵ and carbohydrate metabolism.⁶⁶ Whether CBD is also able to mediate these effects through increased adenosine A2a receptor activation is yet to be determined.

Serotonin 1A receptor

The 5-HT1A plays an important role in the protection of membranes against lipid peroxidation. Lipid peroxidation is a process in which free radicals, like reactive oxygen species (ROS), interact with membrane lipids causing degradation of the membrane. The 5-HT1A is able to intercept ROS before the process of lipid peroxidation is initiated.⁶⁷

CBD showed to be a 5-HT1A agonist⁶⁸ and the antioxidative effects of CBD through 5-HT1A stimulation was studied in vivo using a hypoxic–ischemic brain injury model in newborn pigs.³ Treatment with CBD prevented the increases in excitotoxicity, oxidative stress, and the proinflammatory cytokine IL-1, that were induced by hypoxic–ischemic brain injury. Interestingly, the neuroprotective effects of CBD were reversed by coadministration of a 5-HT1A receptor antagonist, confirming the involvement of the 5-HT1A receptor in this mechanism. While these findings show a possible mechanism of action through which CBD may reduce oxidative stress in neuronal tissue, the question remains whether these findings can be translated to skeletal muscle tissue, where 5-HT1A is less abundantly expressed than in the central and peripheral nervous system.

Cannabinoid receptor 1

CB1 is expressed in the central nervous system and in metabolic active tissues, including adipose,^69–71 liver,^72,73 and skeletal muscle tissue.^74–78 Despite a lower CB1 expression in skeletal muscle compared with brain or other metabolic tissues,⁷⁹ genetic knockout, and pharmacologically induced CB1 (ant)agonism showed that CB1 plays a critical role in metabolic regulation.⁸⁰ For example, CB1 overexpression was associated with the development of metabolic diseases,^73,74 whereas CB1 deletion or antagonism improved metabolic status.^81,82

In addition, CB1 was shown to be involved in muscle plasticity. More specifically, CB1 expression was higher in catabolic conditions associated with muscle degeneration such as aging, fasting, and dystrophy.^75,83–85 On the other hand, CB1 antagonism promotes muscle regeneration as was shown by increased expression of markers of myogenesis (i.e., myogenin and troponinT-1) in primary human satellite cells and myoblasts isolated from Duchenne's muscular dystrophy patients upon exposure to the CB1 antagonist rimonabant (1 μM).⁸⁵ Furthermore, CB1 deletion or antagonism stimulated the Akt/mTOR pathway and concomitantly increased muscle protein synthesis in vitro^86,87 and in vivo (unpublished data from our laboratory).

Lastly, CB1 was also suggested to be involved in inflammatory responses. In fact, activation of CB1 enhanced proinflammatory responses of macrophages resulting in increased ROS generation and expression of proinflammatory cytokines such as TNFα.⁸⁸ Additionally, a recent study confirmed the proinflammatory effect of CB1 activation in L6 skeletal muscle cells.⁸⁹ Moreover, incubation with the CB1 agonist arachidonyl-2′-chloroethylamide (ACEA, 10 nM) increased the expression of IL-6. This response was blocked upon pretreatment with the CB1 antagonist rimonabant (100 nM), suggesting CB1 mediation. Accordingly, a 2-week treatment with rimonabant (0.5 mg/kg, three times a week) reduced transcription levels of IL-6R, TNFα, transforming growth factor beta (TGF-β), and inducible nitric oxide synthase (iNOS) in gastrocnemius and quadriceps muscle of dystrophic mice.⁸⁵ These findings indicate that CB1 antagonism may be a promising target in reducing inflammation.

Like other receptors, CB1 has orthosteric and allosteric binding sites. Whereas (ant)agonists bind to the orthosteric binding site to (in)activate CB1-dependent signaling, binding of allosteric modulators can alter the efficacy and potency of orthosteric ligands. The classification of CBD as a negative allosteric modulator of CB1 explains its antagonistic effects despite its low affinity for the CB1 orthosteric binding site.^25,90,91 Indeed, in vitro and in vivo studies showed that CBD can antagonize agonist-stimulated CB1 activation.^92,93 Whether the antagonistic actions of CBD at CB1 are able to improve skeletal muscle metabolism, anabolism, and inflammation requires further investigation.

Cannabinoid receptor 2

CB2 is highly expressed in immune cells, including skeletal muscle resident macrophages and plays a pivotal role in their polarization and in skeletal muscle regeneration.^94,95 Depending on the immune microenvironment, macrophages can polarize into the M1 phenotype, which is responsible for proinflammatory actions and pathogen elimination, or into the M2 phenotype, which is responsible for anti-inflammatory functions and tissue repair. CB2 agonism was shown to inhibit M1^96–98 and to stimulate M2 macrophage polarization,^97,99 thereby inducing anti-inflammatory effects. Indeed, CB2^−/− knockout mice subjected to ischemic–reperfusion injury showed hampered skeletal muscle regeneration which was accompanied by increased M1 macrophage infiltration and decreased M2 macrophage infiltration.¹⁰⁰ Accordingly, CB2 activation improved skeletal muscle regeneration in mice subjected to ischemic–reperfusion injury by reducing oxidative damage and promoting early myogenesis.¹⁰¹

Some studies show that CBD acts as CB2 agonist,^3,64 raising the possibility that CBD may promote skeletal muscle regeneration via CB2 activation. However, in general, CBD showed to have very low affinity for CB2,²⁵ which questions the role of CBD at this receptor.

As previously mentioned, CBD is also able to indirectly interfere with receptor activity by increasing AEA levels. Since AEA also acts as an agonist of CB1 and (to a lesser extend) of CB2,¹⁰² CBD may indirectly activate the cannabinoid receptors through this mechanism. Nevertheless, CBD is believed to exert its actions mainly through alternative receptors such as TRPV1, PPARγ, GPR55, Adenosine A2a, and 5-HT1A that have been discussed above.

(Pre)clinical Studies on Skeletal Muscle Metabolism, Inflammation, Regeneration, and Anabolism

In the section above, we summarized the potential receptors through which CBD may exert its effects on skeletal muscle metabolism, inflammation, tissue regeneration, and anabolism. These speculations were based on studies that investigated the molecular effects of CBD on distinct tissues and cell lines. In the following section, studies investigating the actual effects of CBD on skeletal muscle tissue or skeletal muscle cells are discussed.

Muscle metabolism

As mentioned above, CBD may exert beneficial effects on energy metabolism in skeletal muscle through TRPV1 and PPARγ agonism, and through CB1 antagonism. Yet, the antagonistic properties of CBD at GPR55 may have a negative impact on metabolic functioning in skeletal muscle tissue. Therefore, CBD may affect muscle metabolism through different pathways, but the relative contribution and the relevance for metabolic control of each of these pathways remain to be determined.

To our knowledge, only one study investigated the effect of CBD on glucose metabolism in skeletal muscle tissue.¹⁰³ CBD treatment (10 mg/kg for 2 weeks) in rats fed with a high-fat diet (HFD) showed reduced de novo ceramide synthesis and thereby improved intramuscular insulin signaling, as indicated by enhanced phosphorylation of Akt and glycogen synthase kinase-3 (GSK-3) and an increased expression of the oxidative enzyme pyruvate dehydrogenase (PDH). These molecular adaptations might have contributed to a restored glycogen depletion in CBD-treated rats.¹⁰³ In addition, the authors also showed that HFD-induced obesity increased the expression of CB1, TRPV1, and 5-HT1A receptors in rats, while CBD administration reduced the expression of these receptors. This indicates that CBD is not only able to (de)activate these receptors but can also interfere with their expression.

Muscle inflammation

As described above, CBD may be able to exert anti-inflammatory effects through activation of several receptors. However, the effect of CBD on skeletal muscle inflammation is not fully understood.

The anti-inflammatory effects of CBD have been investigated in C₂C₁₂ cells. Incubation with the proinflammatory cytokine TNFα increased p65NF-κB phosphorylation, and treatment with CBD (1–5 μM) was unable to attenuate this effect.¹⁰⁴ The authors explained this observation by the low abundance of CB1 in muscle cells. However, as mentioned above, CBD has a low affinity for CB1 and is believed to express its effects through activation of alternative receptors.¹⁰⁵

In contrast to this in vitro study, in vivo studies investigated the anti-inflammatory effect of CBD in skeletal muscle tissue. Mdx mice (a murine model of Duchenne's muscular dystrophy) treated with CBD (60 mg/kg for 2 weeks) showed lower expression levels of different proinflammatory markers (i.e., IL-6, TNFα, TGF-β1, iNOS) in gastrocnemius and diaphragm muscles compared with vehicle-treated mdx mice.⁴ Additionally, CBD administration (10 mg/kg for 2 weeks) ameliorated the imbalance in n-6/n-3 PUFA ratio induced by HFD in rats and shifted the balance in favor of the anti-inflammatory n-3 PUFAs.⁸ Accordingly, the increase of HFD-induced proinflammatory markers, such as cyclooxygenase 1 and 2 (COX1 and COX2), 5-lipoxygenase (5-LOX), NF-κB, and IL-6 in the gastrocnemius muscle, were blunted by CBD administration.

It should be noted that inflammation was induced through pathological or dietary interventions in these rodent studies, which might have elicited different inflammatory responses compared with acute exercise. Interestingly, a recent study investigated the effect of acute CBD administration (100 mg/kg) on skeletal muscle molecular signaling following eccentric contractions in rats, and showed that CBD reduced phosphorylation of the major proinflammatory transcription factor NF-κB.¹⁰⁶ However, more studies need to be done to investigate whether the anti-inflammatory capacity of CBD also has implications for muscle functionality.

Muscle regeneration and anabolism

In section 2, a few potential molecular mechanisms through which CBD may stimulate muscle regeneration and anabolism have been proposed. However, there is still some conflicting evidence within the existing literature.

For instance, CBD does not seem to affect anabolic signaling through the mTOR pathway in vitro. More specifically, C₂C₁₂ cells that were incubated with CBD (1–5 μM) did not show any effects on downstream signaling proteins of the mTOR axis (i.e., S6K1, ribosomal protein S6 [rpS6], and eukaryotic translation initiation factor 4E-binding protein 1 [4E-BP1]) or on total protein synthesis.¹⁰⁴

On the other hand, promising effects of CBD on skeletal muscle differentiation were shown in vitro. Iannotti et al found that C₂C₁₂ cells incubated with CBD (1 μM) showed an increased expression of skeletal muscle differentiation markers, myogenin and troponinT-1, and increased levels of myosin heavy chain, a marker of myotube formation.⁴ Additionally, these authors showed that C₂C₁₂ and human satellite cell differentiation was promoted by CBD through increased calcium concentrations through TRPV(A)1 activation. These findings are very interesting, since satellite cell differentiation plays a role in muscle regeneration.

Iannotti et al also investigated the effects of CBD on muscle regeneration in vivo using mdx mice.⁴ Following 2 weeks of CBD treatment (60 mg/kg, three times a week) the mdx-related impairments in locomotor activity and muscle strength were ameliorated. Accordingly, histological analysis showed that CBD prevented tissue degradation in mdx mice as indicated by a lower number of centralized nuclei in muscle fibers of CBD treated mdx mice.

While these data show the effect of CBD in muscle recovery in a model of Duchenne's muscular dystrophy, its relevance in EIMD remains to be clarified as the severity of tissue damage is considerably lower in the latter. Interestingly, a recent study investigated the effect of acute CBD administration (100 mg/kg) on electrically stimulated eccentric contractions in the tibialis anterior muscle in rats.¹⁰⁶ CBD administration did not affect signaling intermediates of the anabolic mTOR axis.¹⁰⁶ Furthermore, there were no differences between the CBD and vehicle-treated animals based on histological signs of muscle damage, that is, central nuclei, necrosis, and nerve damage. However, it is important to note that these parameters also remained unaffected by the eccentric contractions. Therefore, it could be speculated that the stimulation was insufficient to induce a substantial level of muscle damage.

Studies in Healthy Subjects and Athletes

While the abovementioned effects of CBD on skeletal muscle metabolism, inflammation, regeneration, and anabolism highlight the potential beneficial effects of CBD in animal models, caution should be taken when extrapolating these findings to humans. Very few studies investigated the effect of CBD on muscle recovery and strength after strenuous exercise, and none of them looked at the muscle molecular signature upon CBD supplementation.

In a first study, trained participants were subjected to an exercise bout consisting of a warm-up followed by 4 sets of 10 back squats at 80% of the participants' one repetition maximum (1RM) to induce EIMD. Upon completion of the exercise bout, participants were randomly assigned to receive either CBD (16.67 mg solved in MCT oil), placebo (MCT oil only), or no supplement (null-group).¹⁰⁷ The CBD group reported lower muscle soreness 24 h postexercise compared with immediately postexercise and 72 h postexercise compared with 48 h postexercise.

However, these results were based on repeated t-tests at each time point, which are not suitable for this study design and might have led to false positive results. Besides, baseline 1RM back squat values were significantly different between the conditions, that is, participants in the CBD group had a higher 1RM back squat (102.8±45.2 kg) compared with the placebo (82.4±34.4 kg) and null group (65.9±16.2 kg). Differences in training level might have influenced the susceptibility to EIMD or the subjective experience of DOMS. Lastly, the results were only based on measures of perceived muscle soreness (through a visual analog scale) instead of analyzing markers of muscle damage and inflammation.

A second study investigated the effects of acute CBD supplementation (60 mg) on muscle recovery directly after a fatiguing series of back squats (3×12 at 70% 1RM) and drop-down jumps (3×15) in trained participants.¹⁰⁸ CBD restored the loss in maximal squat performance following the fatiguing exercise bout to a larger extent compared with placebo. CBD also attenuated the increase in plasma creatine kinase and myoglobin, which accompanied the loss in maximal squat performance.¹⁰⁸ Even though differences between CBD and placebo were significant, they were relatively small and only became apparent after 72 h.

It should be noted that both studies supplemented participants with a single, relatively small dose of CBD. Since the bioavailability of CBD is low (±6%)¹⁰⁹ a single dose of 16.67 or 60 mg might not have been sufficient to induce substantial effects in skeletal muscle. Additionally, participants of both studies received their supplement postexercise. Previous studies have shown that peak CBD plasma levels are reached between 0 and 4 h after oral supplement intake.¹¹⁰ Therefore, it could be suggested that postexercise CBD supplementation is less effective to attenuate EIMD. Unfortunately, in neither of these studies CBD plasma levels were measured.

In a third study, untrained men performed an eccentric elbow flexor exercise protocol and were supplemented with 150 mg CBD or placebo 2, 24, and 48 h following exercise.¹¹¹ Even though the dose was higher and the supplementation period was longer compared with the two previous studies, the authors were unable to detect any effects of CBD on perceived muscle soreness, arm circumference, hanging joint angle, and peak torque after the exercise session. However, the eccentric exercise load might have been insufficient to induce muscle damage, since indices of peak torque were unaffected by the eccentric exercise.

Overall, there is no clear consensus on the effects of CBD on functional responses upon strenuous exercise. The three studies mentioned above are highly variable in exercise protocol, dosing regimen, and measurement techniques, which makes it hard to draw any conclusions. More importantly, none of these studies collected muscle biopsies to analyze biochemical or histological signs of muscle damage. Therefore, the molecular effects of CBD in response to exercise in human muscle tissue remain unknown.

Conclusion and Future Directions

Because of the high prevalence of CBD use among athletes and the lack of knowledge on the effects of CBD supplementation on a muscular level, we aimed to summarize the current knowledge on the metabolic, anti-inflammatory, regenerative, and anabolic effects of CBD in skeletal muscle tissue and provided an overview of the possible molecular pathways and mechanisms through which CBD may exert these actions.

Several mechanisms were proposed to explain CBD's capacity to improve muscle metabolism, including TRPV1, PPARγ, and adenosine A2a receptor stimulation, and negative allosteric modulation of CB1. Possible negative effects of CBD on metabolism were suggested to occur through GPR55 antagonism. To our knowledge only one study investigated the effect of CBD on muscle metabolism, in which enhanced insulin signaling resulted in positive effects on glucose metabolism and glycogen storage in obese rats.¹⁰³ However, the improved insulin signaling upon CBD administration was attributed to reduced de novo ceramide synthesis. Therefore, it may be premature to translate these results to non-HFD conditions and more studies are required to elucidate the effects of CBD on fuel metabolism in healthy skeletal muscle tissue. Besides, the effect of CBD on other indices of metabolic processes such as mitochondrial activity and biogenesis, especially in response to exercise, should be explored in future research.

Next, a large variety of mechanisms through which CBD may exert anti-inflammatory actions were addressed in this review. For instance, activation of PPARγ by CBD may directly inhibit NF-κB signaling, reducing the expression of proinflammatory mediators. Alternatively, CBD may reduce the infiltration of neutrophils and proinflammatory macrophages through its actions at GPR55 and CB2 receptors, or inhibit lipid peroxidation through 5-HT1A agonism. Indeed, in vivo studies showed reduced NF-κB activation and inhibition of proinflammatory mediators upon CBD treatment in rodent studies.^4,106 However, the effect of CBD on neutrophil and macrophage infiltration remains to be elucidated. Since neutrophil and macrophage infiltration (and polarization) play a major role in EIMD and tissue repair,¹¹² its modulation by CBD (through GPR55 or CB2) is of great interest and should be further investigated.

Furthermore, the potential mechanisms through which CBD may influence muscle regeneration and anabolism were described. We speculated that CBD may induce muscle anabolism by stimulating the mTOR axis through increased calcium levels upon TRPV1 activation or by its negative allosteric actions at CB1. However, the amount of literature that is currently available on the anabolic and muscle regenerative effects of CBD is limited and inconsistent.

While chronic CBD treatment clearly improved muscle quality and regeneration in mdx mice,⁴ acute CBD treatment was unable to enhance anabolic signaling in response to eccentric contractions in healthy rats.¹⁰⁶ Among others, a different level of muscle damage induced in these interventions may have attributed to these contradictory findings, since skeletal muscle tissue of mdx mice showed significant levels of muscle degeneration, while the electrically stimulated eccentric muscle contractions were insufficient to induce clear histological signs of muscle damage. Besides, the outcomes of both studies, that is, satellite cell activation and regeneration versus mTOR activation and muscle protein synthesis, were also different. It is conceivable that CBD is only able to improve muscle regeneration once a certain level of muscle damage is induced.

To clarify the role of CBD on muscle regeneration in response to exercise, there is a need for more studies using adequate protocols to induce EIMD in healthy skeletal muscle tissue. Additionally, appropriate dosing schedules should be developed and applied to increase plasma CBD levels to physiologically relevant levels.

Lastly, the metabolic, anti-inflammatory, regenerative, and anabolic effects of CBD in human skeletal muscle tissue were addressed. Very limited information is available since the muscle molecular effects of CBD have not been investigated in humans and there were only three studies that examined the functional responses to acute or short-term CBD supplementation following exercise. More high-quality intervention trials using adequate training protocols and dosing schedules are needed to clarify the role of CBD in recovery from strenuous exercise. In addition, human intervention trials that include muscle biopsy sampling are required to investigate whether CBD can affect the molecular changes that underlie metabolic, inflammatory, regenerative, or anabolic adaptations. Finally, the long-term effects of CBD use on muscle functioning should be addressed in future research to determine whether the proposed effects of CBD on muscle recovery result in performance gains on the long term.

In conclusion, CBD has the prospective to become an adequate supplement that improves muscle regeneration and recovery and is thereby considered a potential ergogenic aid for athletes. However, to date, the knowledge about the effects of CBD on skeletal muscle tissue is still in its infancy and there is currently too little information available to properly advise athletes on any potential beneficial effects of CBD supplementation.

Footnotes

Authors' Contributions

M.S., S.D., and K.K. contributed to the conceptualization of the article. M.S. wrote the original draft and S.D. and K.K. reviewed and edited the article. All authors read and approved the final article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

SD has received a postdoctoral fellowship (grant no. 12Z8622N) from Research Foundation Flanders (FWO).

Abbreviations Used

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