Endocannabinoid Anandamide Mediates the Effect of Skeletal Muscle Sphingomyelins on Human Energy Expenditure

Abstract

Context: Skeletal muscle endocannabinoids and sphingolipids (particularly sphingomyelins) are inversely associated with sleeping energy expenditure (SLEEP) in humans. The endocannabinoid system may increase sphingolipid synthesis via cannabinoid receptor-1.

Objective: To investigate in human skeletal muscle whether endocannabinoids are responsible for the effect of sphingomyelins on SLEEP.

Design: Muscle endocannabinoid [anandamide (AEA), 2-arachidonoylglycerol (2-AG)], endocannabinoid congeners [oleoylethanolamide (OEA), palmitoylethanolamide (PEA)], and sphingomyelin content were measured with liquid chromatography/mass spectrometry. SLEEP was assessed in a whole-room indirect calorimeter. Mediation analyses tested whether the inverse associations between sphingomyelins and SLEEP depended on endocannabinoids and endocannabinoid-related OEA and PEA.

Setting: Inpatient study.

Participants: Fifty-three Native Americans who are overweight.

Main outcome measure: SLEEP.

Results: AEA (r = 0.45, P = 0.001), 2-AG (r = 0.47, P = 0.0004), OEA (r = 0.27, P = 0.05), and PEA (r = 0.53, P < 0.0001) concentrations were associated with the total sphingomyelin content. AEA, OEA, and PEA correlated with specific sphingomyelins (SM18:1/23:0, SM18:1/23:1, and SM18:1/26:1) previously reported to be determinants of SLEEP in Native Americans (all r > 0.31, all P < 0.03). Up to half of the negative effect of these specific sphingomyelins on SLEEP was accounted for by AEA (all P < 0.04), rendering the direct effect by sphingomyelins per se on SLEEP negligible (P > 0.05).

Conclusions: In skeletal muscle, AEA is responsible for the sphingomyelin effect on SLEEP, indicating that endocannabinoids and sphingomyelins may jointly reduce human whole-body energy metabolism.

Trial registration: ClinicalTrials.gov NCT00340132.

Figures

Figure 1.

Relationships between (A) AEA, (B) 2-AG, (C) OEA, and (D) PEA and the overall sphingomyelin content in skeletal muscle (SMcont), calculated as the average of individual sphingomyelin z scores reported in Table 2. For PEA, n = 52 because one subject had highly leveraged values for these measures (association between SMcont and PEA r = 0.54, P < 0.0001 upon inclusion). (E) The positive relationship between SPTLC3 and CNR1 gene expression levels in skeletal muscle. Gene expression levels were batch- and sex-standardized and reported in SD units. Pearson partial correlations are reported.

Figure 2.

Correlation of the average (A) sphingomyelin content (SMcont) in skeletal muscle, (B) AEA, (C) 2-AG, and (D) PEA, with sleeping EE adjusted for FFM (residual SLEEP). For correlation of SMcont with residual SLEEP, 29 subjects were included because of incomplete assessment of sphingomyelins. For correlation of PEA with residual SLEEP, 29 subjects were included because of one subject with highly leveraged PEA measurement. As previously shown (4), the association of AEA and residual SLEEP was still significant upon exclusion of two subjects with lower AEA content (all P < 0.05). Pearson correlations and P values are reported.

Figure 3.

(A) Example of the mediation analysis of the sphingomyelin effect on residual SLEEP accounted for by AEA. Residual SLEEP was calculated after adjustment for FFM via linear regression analysis. Path coefficients (±SE) are shown for the associations between SM18:1/23:1 (independent variable) and AEA (mediator), SM18:1/23:1 and SLEEP (dependent variable), and AEA and residual SLEEP, and were derived by linear regression analysis. The endocannabinoid-specific effect of SM18:1/23:1 on residual SLEEP exerted by AEA was calculated by multiplication of the path coefficients for the associations between SM18:1/23:1 and AEA and between AEA and residual SLEEP, respectively. The Sobel test was used to test the significance of the endocannabinoid-specific effect. Mediation analysis was performed in 29 subjects with complete SLEEP, endocannabinoid, and sphingolipid measurements. (B) Percentage of the total effect of SM18:1/23:1 on residual SLEEP as accounted by the endocannabinoid-specific (i.e., via AEA) vs direct (i.e., SM18:1/23:1 only, independent of AEA) effect.