The Impact of CB1 Receptor on Nuclear Receptors in Skeletal Muscle Cells

Abstract

Cannabinoids are abundant signaling compounds; their influence predominantly arises via engagement with the principal two G-protein-coupled cannabinoid receptors, CB1 and CB2. One suggested theory is that cannabinoids regulate a variety of physiological processes within the cells of skeletal muscle. Earlier publications have indicated that expression of CB1 receptor mRNA and protein has been recognized within myotubes and tissues of skeletal muscle from both murines and humans, thus representing a potentially significant pathway which plays a role in the control of skeletal muscular activities. The part played by CB1 receptor activation or inhibition with respect to these functions and relevant to targets in the periphery, especially skeletal muscle, is not fully delineated. Thus, the aim of the current research was to explore the influence of CB1 receptor stimulation and inhibition on downstream signaling of the nuclear receptor, NR4A, which regulates the immediate impacts of arachidonyl-2'-chloroethylamide (ACEA) and/or rimonabant in the cells of skeletal muscle. Murine L6 skeletal muscle cells were used in order to clarify additional possible molecular signaling pathways which contribute to alterations in the CB1 receptor. Skeletal muscle cells have often been used; it is well-documented that they express cannabinoid receptors. Quantitative real-time probe-based polymerase chain reaction (qRT-PCR) assays are deployed in order to assess the gene expression characteristics of CB1 receptor signaling. In the current work, it is demonstrated that skeletal muscle cells exhibit functional expression of CB1 receptors. This can be deduced from the qRT-PCR assays; triggering CB1 receptors amplifies both NR4A1 and NR4A3 mRNA gene expression. The impact of ACEA is inhibited by the selective CB1 receptor antagonist, rimonabant. The present research demonstrated that 10 nM of ACEA notably amplified mRNA gene expression of NR4A1 and NR4A3; this effect was suppressed by the addition of 100 nM rimonabant. Furthermore, the CB1 receptor antagonist led to the downregulation of mRNA gene expression of NR4A1, NR4A2 and NR4A3. In conclusion, in skeletal muscle, CB1 receptors were recognized to be important moderators of NR4A1 and NR4A3 mRNA gene expression; these actions may have possible clinical benefits. Thus, in skeletal muscle cells, a possible physiological expression of CB1 receptors was identified. It is as yet unknown whether these CB1 receptors contribute to pathways underlying skeletal muscle biological function and disease processes. Further research is required to fully delineate their role(s).

Keywords: ACEA; NR4A; cannabinoid CB1 receptors; rimonabant; skeletal muscle cells.

Figure 1

Images depicting L6 skeletal muscle myoblasts from rats, differentiated into L6 skeletal muscle myotubes (passage 7). (a) Myoblasts obtained on day 3 of the tissue culture in 10% FBS Ham F-10 media (10×). (b) Cells obtained on day 4 of the tissue culture in 6% horse serum Ham F-10 media (10×). (c) Myotubes obtained on day 5 of the tissue culture in 2% horse serum Ham F-10 media (10×). (d) Myotubes obtained after 4 h of tissue culture in 2% delipidated serum Ham F-10 media (10×). (e) Myotubes obtained after one hour of cell starvation in only Ham F-10 media (10×). (f) Myotubes obtained after 19 h of cell starvation in only Ham F-10 media (10×).

Figure 1

Images depicting L6 skeletal muscle myoblasts from rats, differentiated into L6 skeletal muscle myotubes (passage 7). (a) Myoblasts obtained on day 3 of the tissue culture in 10% FBS Ham F-10 media (10×). (b) Cells obtained on day 4 of the tissue culture in 6% horse serum Ham F-10 media (10×). (c) Myotubes obtained on day 5 of the tissue culture in 2% horse serum Ham F-10 media (10×). (d) Myotubes obtained after 4 h of tissue culture in 2% delipidated serum Ham F-10 media (10×). (e) Myotubes obtained after one hour of cell starvation in only Ham F-10 media (10×). (f) Myotubes obtained after 19 h of cell starvation in only Ham F-10 media (10×).

Figure 2

NR4A1 muscle myotubes—ACEA, rimonabant, ACEA and rimonabant, and insulin affect NR4A1 gene expression; myotubes fed with delipidated serum. The stimulation time covered 3 to 24 h, and NR4A1 mRNA levels, relative to TATA-Box, was evaluated by quantitative real-time PCR (qRT-PCR) (100 nM). The following scenarios explain the stimulation process conducted: (a) Stimulation was done for 1 h. (b) Stimulation was done for 3 h. (c) Stimulation was applied for up to 5 h. (d) Stimulation was applied for up to 24 h. The data were reported as the mean ± SEM of three separate groups. (n = 3; * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001). Data were investigated by conducting one-way ANOVA test and Tukey test. NR4A1; nuclear receptor subfamily 4, group A, member 1.

Figure 3

NR4A2 muscle myotubes—ACEA, rimonabant, ACEA and rimonabant, and insulin affect NR4A2 gene expression; myotubes fed with delipidated serum. The stimulation time covered 3 to 24 h, and NR4A2 mRNA levels, relative to TATA-Box, was evaluated by quantitative real-time PCR (qRT-PCR) (100 nM). The following scenarios explain the stimulation process conducted: (a) Stimulation was done for 1 h. (b) Stimulation was done for 3 h. (c) Stimulation was applied for up to 5 h. (d) Stimulation was applied for up to 24 h. The data were reported as the mean ± SEM of three separate groups. (n = 3; * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001). Data were investigated by conducting one-way ANOVA test and Tukey test. NR4A2; nuclear receptor subfamily 4, group A, member 2.

Figure 4

NR4A3 muscle myotubes—ACEA, rimonabant, ACEA and rimonabant, and insulin affect NR4A3 gene expression; myotubes fed with delipidated serum. The stimulation time covered 3 to 24 h, and NR4A3 mRNA levels, relative to TATA-Box, was evaluated by quantitative real-time PCR (qRT-PCR) (100 nM). The following scenarios explain the stimulation process conducted: (a) Stimulation was done for 1 h. (b) Stimulation was done for 3 h. (c) Stimulation was applied for up to 5 h. (d) Stimulation was applied for up to 24 h. The data were reported as the mean ± SEM of three separate groups. (n = 3; * denotes p < 0.05, and ** denotes p < 0.01). Data were investigated by conducting one-way ANOVA test and Tukey test. NR4A3; nuclear receptor subfamily 4, group A, member 1.