Plant-Derived Trans-β-Caryophyllene Boosts Glucose Metabolism and ATP Synthesis in Skeletal Muscle Cells through Cannabinoid Type 2 Receptor Stimulation

Abstract

Skeletal muscle plays a pivotal role in whole-body glucose metabolism, accounting for the highest percentage of glucose uptake and utilization in healthy subjects. Impairment of these key functions occurs in several conditions including sedentary lifestyle and aging, driving toward hyperglycemia and metabolic chronic diseases. Therefore, strategies pointed to improve metabolic health by targeting skeletal muscle biochemical pathways are extremely attractive. Among them, we focused on the natural sesquiterpene and cannabinoid type 2 (CB2) receptor agonist Trans-β-caryophyllene (BCP) by analyzing its role in enhancing glucose metabolism in skeletal muscle cells. Experiments were performed on C2C12 myotubes. CB2 receptor membrane localization in myotubes was assessed by immunofluorescence. Within glucose metabolism, we evaluated glucose uptake (by the fluorescent glucose analog 2-NBDG), key enzymes of both glycolytic and oxidative pathways (by spectrophotometric assays and metabolic radiolabeling) and ATP production (by chemiluminescence-based assays). In all experiments, CB2 receptor involvement was tested with the CB2 antagonists AM630 and SR144528. Our results show that in myotubes, BCP significantly enhances glucose uptake, glycolytic and oxidative pathways, and ATP synthesis through a CB2-dependent mechanism. Giving these outcomes, CB2 receptor stimulation by BCP could represent an appealing tool to improve skeletal muscle glucose metabolism, both in physiological and pathological conditions.

Keywords: cannabinoid type 2 receptor; glucose metabolism; glucose uptake; trans-β-caryophyllene.

Figure 1

Qualitative detection with immunofluorescence staining of CB2 receptor in C2C12 skeletal muscle cells. Confocal image of a representative XY acquisition (60× magnification) of (A) differentiated cells incubated with primary CB2 receptor antibody, (B) differentiated cells incubated with CB2 receptor antibody and its blocking peptide (1/1 ratio), and (C) differentiated cells incubated with secondary antibody alone. Scale bar: 36 μm. Secondary antibody, anti-rabbit Alexa 200 Fluor 568, 1:1000. Images are presented in pseudocolor (LUT = fire) to better show the fluorescent intensity variations in the range 0–2552.

Figure 2

Trans-β-Caryophyllene (BCP) stimulates glucose uptake by CB2 receptor stimulation. (A) Representative confocal images of C2C12 incubated with the fluorescent glucose analog (2-NBDG) for 30 min in the dark. Images are presented in pseudocolor (LUT = fire) to better show the fluorescent intensity variations in the range 0–1031. Insulin was used as a positive control. Scale bar: 36 μm. (B) Bar graph summarizing glucose uptake experiments. CTRL: 103.66 ± 2.23, n cells = 129; Insulin: 124.98 ± 3.19, n cells = 133; BCP: 123.47 ± 2.76, n cells = 134; BCP + AM630: 99.43 ± 3.08, n cells = 86; AM630: 109.85 ± 3.54, n cells = 94. Data are represented as mean ± SEM (n = 6 independent experiments). * p < 0.05; *** p < 0.001.

Figure 3

BCP enhances glucose uptake by CB2 receptor stimulation. (A) Representative confocal images of C2C12 incubated with the fluorescent glucose analog (2-NBDG) for 30 min in the dark. Images are presented in pseudocolor (LUT = fire) to better show the fluorescent intensity variations in the range 0–1031. Insulin was used as a positive control. Scale bar: 36 μm. (B) Bar graph summarizing glucose uptake experiments. CTRL: 100.00 ± 3.15, n cells = 66; Insulin: 141.06 ± 4.17, n cells = 60; BCP: 130.30 ± 3.16, n cells = 67; BCP + SR144528: 109.04 ± 3.64, n cells = 65; SR144528: 139.45 ± 4.26, n cells = 44. Data are represented as mean ± SEM (n = 4 independent experiments). *** p < 0.001.

Figure 4

Effects of trans-β-carophyllene on glycolysis. C2C12 cells were left untreated (CTRL) or treated with insulin (25 nM for 30 min; INS), BCP (10 nM for 30 min), AM630 (5 µM for 2 h) or SR144528 (5 µM for 2 h), alone or in combination with 10 nM BCP, added in the last 30 min. The enzymatic activities of (A) phospho-fructokinase 1 (PFK1), (B) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (C) enolase, and (D) pyruvate kinase (PK) were measured spectrophotometrically in the whole cell lysates in triplicate. Data are expressed as means ± SEM (n = 3). *** p < 0.001.

Figure 5

Effects of BCP on mitochondria metabolism. C2C12 cells were left untreated (CTRL) or treated with insulin (25 nM for 30 min; INS), BCP (10 nM for 30 min), AM630 (5 µM for 2 h), or SR144528 (5 µM for 2 h), alone or in combination with 10 nM BCP, added in the last 30 min. The enzymatic activities of (A) pyruvate dehydrogenase (PDH), (B) citrate synthase, (C) aconitase, (D) isocitrate dehydrogenase, (E) α-ketoglutarate (αKG) dehydrogenase, and (F) succinate dehydrogenase were measured spectrophotometrically in mitochondrial extracts in triplicate. Data are expressed as means ± SEM (n = 3). * p < 0.1, ** p < 0.01, *** p < 0.001.

Figure 6

Effects of BCP on mitochondria metabolism. C2C12 cells were left untreated (CTRL) or treated with insulin (25 nM for 30 min; INS), BCP (10 nM for 30 min), AM630 (5 µM for 2 h) or SR144528 (5 µM for 2 h), alone or in combination with 10 nM BCP, added in the last 30 min. The electron flux from complex I to complex III (A) and the levels of ATP (B) were measured spectrophotometrically in mitochondrial extracts in triplicate. Data are expressed as means ± SEM (n = 3). *** p < 0.001.