In this study, we investigated the effect of CBD on anabolic and inflammatory signaling in C2C12 myotubes. We looked at the effect of CBD on mTORC1 signaling under fasted conditions and after addition of amino acids and IGF-1. We found that a range of 1–5 μM of CBD had no effect on anabolic signaling through the mTORC1-axis. However, we did detect a small increase in global protein synthesis via puromycin after IGF-1 treatment for the highest CBD dose. Since there were no baseline differences, the effect of CBD (5 μM) on puromycin after IGF-1 was small, and the technical replicates were limited (n=3; Fig. 2), we decided to repeat the experiment, double the technical replicates, and omit the fasted condition. In our follow-up experiment with increased statistical power (Fig. 3), the observed tendency of higher protein levels of mTORC1 signaling and global protein synthesis with increasing dosages of CBD disappeared.

To investigate the anti-inflammatory properties of CBD in muscle cell culture, we tested NF-κB signaling in the presence or absence of TNFα, together with CBD or a natural product inhibitor of NF-κB activation dioscin. Dioscin was identified as a potent inhibitor of TNFα using an NF-κB reporter cell line to screen 146 natural products. Compared with dioscin, CBD had little to no effect on inflammatory signaling through the NF-κB axis in muscle cells. Although dioscin decreased the phosphorylation and activation of p65 (Ser536), p105, and tended to decrease NF-κB p50, CBD had no effect on NF-κB signaling.

To determine whether the inability of CBD to affect anabolism or inflammatory signaling in C2C12 myotubes was the result of low receptor number, the primary CBD receptor CB1 was determined in C2C12 cells and skeletal muscle of rodents and compared with the cortex and hippocampus. Neither myotubes nor muscle demonstrated CB1 protein, and this likely explains the observation that CBD did not affect myotubes in cell culture and had modest effects on muscle in vivo. Previous studies on CB1 in muscle were able to detect CB1 in skeletal muscle cell culture and in rodent muscle samples.^10–12 However, these studies used immunoprecipitation of specific subfractions to enrich CB1 in an effort to detect CB1 protein levels. It is possible that using these methods we would be able to detect CB1 in our samples. However, the current work clearly demonstrates that the cellular abundance of CB1 is orders of magnitude lower in muscle cells than in brain tissue. This observation is in line with earlier findings on the subject.^13,14

A significant issue with any cell culture experiment is dosing and the translatability of findings to the in vivo setting. In our previous experiments in rats, we combined eccentric exercise with the injection of 100 mg CBD per kg bodyweight (intraperitoneal).⁵ Deiana et al. have measured plasma CBD levels after intraperitoneal injection of 120 mg per kg bodyweight in mice.¹⁵ Plasma levels peaked at 14 μg/mL at 120 min after injection, equivalent to about 45 mM. Since the dosage we used in our cell culture experiments is considerably lower (1–5 μM), it is possible that CBD would have started to exhibit effects at higher concentrations.

However, in a recent systematic review on CBD in cell culture, the authors concluded that CBD starts to negatively impact cell viability at doses higher than 2 μM and induces apoptosis at dosages higher than 10 μM.¹⁶ Therefore, we chose 5 μM as the upper limit of our dose–response studies. Indeed, we performed a subset of our experiments with 10 μM without seeing any beneficial effects compared with 5 μM. Similarly, going out to 24 h with the CBD treatment concomitantly with TNFα or before the addition of IGF-1 had no effect on NF-κB or mTORC1 signaling, respectively (data not shown).

Furthermore, it is important to remember that the effects of CBD on anabolic and inflammatory signaling in our in vivo experiments were small.⁵ This was the case despite a relatively high dose and an efficient route of administration, which could be associated with toxicity in chronic settings.^17–20 Given this context, it appears unlikely that there is a direct physiological role for CBD in anabolic and inflammatory signaling within skeletal muscle tissue.

Even though our data indicate that direct effects of CBD on anabolic or inflammatory signaling in skeletal muscle in vitro are small, it is still possible that CBD could exert indirect effects in vivo through modulation of immune cells that interact with skeletal muscle or by causing systemic changes to circulating cytokines and hormones that affect the central nervous system, immune system, and other endocrinologically active tissues. Future research needs to determine whether such indirect effects could be clinically relevant and whether they can be elicited without requiring toxic dosages of CBD. Additionally, future projects will need to investigate in how far the effects of CBD on skeletal muscle may differ from other cannabinoids, endocannabinoids and analogs.