C2C12 myoblasts were cultured in growth media (high glucose Dulbecco's modified Eagle medium [DMEM], 10% fetal bovine serum, 0.1% penicillin) until they reached 95–100% confluence at which time, they were shifted into differentiation media (DM; high glucose DMEM, 2% horse serum, 0.1% penicillin) to promote the differentiation and fusion of cells to form myotubes. All experiments were conducted on fully formed myotubes 5 days after the introduction of DM.

The first dose–response relationship experiment for CBD and anabolic signaling was assessed using a previously published protocol with modifications (Fig. 1).⁸ Myotubes plated in six-well plates were fasted for 15 min by replacing DM with starvation media (SM; phosphate-buffered saline [PBS], 2% horse serum, and increasing dosages of CBD [0, 1, 2.5 and 5 μM]). The CBD product (Amber Metric, Santa Rosa, CA) was laboratory tested and hemp derived (99.8% purity). After the 15 min fasting period, SM in half of the wells was replaced with DM containing insulin-like growth factor 1 (IGF-1) (5 nM; PeproTech, NJ) and increasing dosages of CBD for a duration of 30 min before the cells were collected.

To improve statistical power and based on the absence of baseline differences in the first experiment, we grew the cells on 24-well plates and omitted the starved condition from the second dose–response experiment (Fig. 2). Instead, DM in all wells was replaced with DM + IGF-1 (5 nM) with increasing dosages of CBD (0, 1, 2.5, and 5 μM) for 60 min before collection.

To identify natural products that could decrease the activity of NF-κB in response to tumor necrosis factor α (TNFα), NF-κB reporter (luc)-HEK293 cells (BPS Bioscience, San Diego, CA) containing the firefly luciferase gene driven by four copies of NF-κB response element were treated with 10 ng/mL TNFα. Following treatment for 6 h, cells were collected in passive lysis buffer and luciferase activity was determined using a GloMax(R) 20/20 Luminometer System w/dual injectors (Promega, Madison, WI). To find NF-κB inhibitors, 146 structurally diverse, bioactive, and cell permeable natural products (Selleck Chemicals, Houston, TX) were added together with TNFα at a final concentration of 10 μM. Of the six natural products that decreased the NF-κB activity by more than 85%, the effect of increasing doses was determined. Data are presented for one of these natural products (dioscin).

For the experiment on CBD and acute inflammatory signaling, C2C12 cells were differentiated into myotubes in 24-well plates. On the day of the experiment, existing media in the wells was replaced with fresh DM that contained no additional compounds, DM + TNFα (2.5 ng/mL; PeproTech), DM + TNFα + CBD (2.5 μM), or DM + TNFα + the phytochemical dioscin (10 μM; Selleck Chemicals) as a positive control. Cells were treated for 1 h before collection (Fig. 3).

To compare protein levels of cannabinoid receptor type 1 (CB1) between skeletal muscle in vitro and in vivo with a positive control (brain tissue), we used cell culture samples from the experiments above and analyzed them alongside of rat tibialis anterior tissue from previous CBD experiments⁵ and murine cortex and hippocampus samples (Fig. 4).

For assessment of global muscle protein synthesis via the SUnSET method, 50 μL of puromycin (45 μM; Research Products International, IL) was added to every cell culture well 5 min before collection. Upon collection, all wells were placed on ice and washed three times with ice-cold PBS. For six-well plates, 100 μL of ice-cold sucrose lysis buffer was added to each well after the last wash, as described previously.⁸ Cells were then scraped off the plate using a spatula and collected in an Eppendorf tube. After centrifuging them at 10,000 g for 5 min at 4°C, 80 μL of the supernatant was transferred to a fresh tube and 20 μL of 4×Laemmli sample buffer (LSB) was added. The samples were then vortexed, heated at 100°C for 5 min, and finally stored at −30°C until Western blot analysis was completed. For the 24-well plate experiments, the collection protocol was identical except for two modifications: after the last washing step with ice-cold PBS, 75 μL of 1×LSB was directly added to each well before the samples were transferred to a fresh tube. The samples were then briefly sonicated before boiling and storage at −30°C until further analysis.

Western blotting was carried out as described previously.⁸ Briefly, for the cell culture experiments, 10 μL of sample was loaded per lane, run for 40 min at 200 V, and transferred onto polyvinylidene fluoride or nitrocellulose membrane in an ice-cold transfer buffer at 100 V for 30 min. For the rodent skeletal muscle and brain samples, 10 μg of protein per lane was loaded. Membranes were blocked in 1% fish skin gelatin dissolved in Tris-buffered saline with 0.1% Tween-20 for 30 min and probed with the primary antibody overnight. The following antibodies were used: Cell Signaling (Cell Signaling Technology, Danvers, MA): S6K1 (Thr389) (No. 9205; lot 16), ribosomal protein S6 (Ser240/244) (No. 5364), 4E-BP1 (Thr37/46) (No. 2855), NF-kB p65 (Ser536) (No. 3033; lot14), NF-kB p105/p50 (No. 13586), CB1 receptor (No. 93815); Millipore Sigma (Merck Group): IRS1 (No. 06-248; lot 2465193), puromycin (No. MABE343). Levels of each protein were normalized to total protein content per lane as assessed via Ponceau S staining of the membrane or a fluorescent gel.⁹

Data analysis was carried out using GraphPad Prism 9 (GraphPad Software, San Diego, CA). One- or two-way analysis of variance combined with Dunnett's (one-way analysis of variance [ANOVA]; Fig. 2) or Tukey's (one-way ANOVA and two-way ANOVA; Figs. 1, 3, and 4) multiple comparison test to allocate differences post hoc were applied depending on the number of groups and variables. The alpha level was set at p=0.05; p-values <0.05 were deemed statistically significant, and p-values between 0.05 and 0.1 are described as trends.

IRS1 protein showed a distinct upwards shift in the gel indicating post-translational modification. The absolute amount of protein tended to increase with IGF-1 treatment (p=0.06) and CBD did not affect this process (CBD: p=0.65 and interaction: p=0.99; Fig. 1A). Phosphorylated S6K1 (Thr389) levels increased significantly with IGF-1 treatment (p<0.0001) without significant effect of CBD (p=0.27) or an interaction effect (p=0.3). No differences at baseline or after IGF-1 treatment between the different CBD conditions could be detected via post hoc testing (Fig. 1B). Similarly, phosphorylated rpS6 (Ser240/244) increased with IGF-1 treatment (p<0.0001) without an effect of CBD (p=0.76) or an interaction effect (p=0.91; Fig. 1C). Finally, global protein synthesis as assessed via puromycin increased significantly with IGF-1 treatment (p<0.0001) and showed a trend for an effect of CBD (p=0.06) but no interaction effect (p=0.18). Post hoc testing determined a difference between the IGF-1 control condition and the IGF-1 CBD (5 μM) condition (p<0.05; Fig. 1D).

To increase technical replicates in each trial, the starved conditions were omitted and the effect of CBD on the IGF-1 response was determined. Total IRS1 protein levels did not differ significantly (Fig. 2A). Similarly, S6K1 (Thr389), rpS6 (Ser240/244), 4E-BP1 (Thr37/46), and puromycin failed to show a main effect of CBD (p=0.27, p=0.81, p=0.87, and p=0.26, respectively) or post hoc group differences (Fig. 2B–E).

To investigate NF-κB signaling, natural product inhibitors of NF-κB transcription were initially identified in HEK293 NF-κB reporter cells. Of 146 structurally diverse natural products, 6 decreased the NF-κB activity >85%. Of these factors, we focused of dioscin, a natural steroidal saponin that inhibited NF-κB activation by TNFα in a dose-dependent manner (Fig. 3A, B). C2C12 cells were then treated with 2.5 ng/mL TNFα in the presence or absence of 2.5 μM CBD or 10 μM dioscin (Fig. 3). We found a main effect for p105 protein levels (p<0.01) with group differences between the control condition and cells that were treated with TNFα and dioscin (p<0.05) as well as a difference between the TNFα condition and the TNFα plus dioscin condition (p<0.05; Fig. 3D), whereas CBD had no effect. Phosphorylated p65 (Ser536) also showed a main effect (p<0.001) with various group differences. TNFα treatment increased phosphorylated-p65 compared with the control condition (p<0.001). Similarly, TNFα in conjunction with CBD increased phosphorylated-p65 levels significantly (p<0.01), whereas phosphorylated-p65 did not increase with TNFα and dioscin treatment. Accordingly, phosphorylated-p65 levels with TNFα or TNFα plus CBD were significantly higher than with TNFα and dioscin (p<0.01 and p<0.05, respectively; Fig. 3E). There was no significant effect of any treatment on p50 levels, but a trend toward a main effect (p=0.07) and a trend toward a group difference between TNFα and TNFα plus dioscin (p=0.05; Fig. 3F).

Since CBD had modest effects on skeletal muscle in vitro and in vivo, levels of the main cannabinoid receptor (CB1) were determined in C2C12 cells and rat tibialis anterior muscle, each with and without CBD treatment. As a positive control, murine brain tissue from either the cortex or hippocampus where CB1 receptor is abundant was run in parallel. The predicted molecular weight of CB1 is 52.8 kDa, but the protein tends to run at around 60 kDa. CB1 was highest in the murine cortex, significantly less CB1 was observed in the hippocampus (p<0.01), and the protein was not detected in either the C2C12 myotubes or the rat TA muscle (Fig. 4).