A cannabinoid receptor agonist N-arachidonoyl dopamine inhibits adipocyte differentiation in human mesenchymal stem cells

Abstract

Endocannabinoids can affect multiple cellular targets, such as cannabinoid (CB) receptors, transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and peroxisome proliferator-activated receptor γ (PPARγ). The stimuli to induce adipocyte differentiation in hBM-MSCs increase the gene transcription of the CB1 receptor, TRPV1 and PPARγ. In this study, the effects of three endocannabinoids, N-arachidonoyl ethanolamine (AEA), N-arachidonoyl dopamine (NADA) and 2-arachidonoyl glycerol (2-AG), on adipogenesis in hBM-MSCs were evaluated. The adipocyte differentiation was promoted by AEA whereas inhibited by NADA. No change was observed by the treatment of non-cytotoxic concentrations of 2-AG. The difference between AEA and NADA in the regulation of adipogenesis is associated with their effects on PPARγ transactivation. AEA can directly activate PPARγ. The effect of AEA on PPARγ in hBM-MSCs may prevail over that on the CB1 receptor mediated signal transduction, giving rise to the AEA-induced promotion of adipogenesis. In contrast, NADA had no effect on the PPARγ activity in the PPARγ transactivation assay. The inhibitory effect of NADA on adipogenesis in hBM-MSCs was reversed not by capsazepine, a TRPV1 antagonist, but by rimonabant, a CB1 antagonist/inverse agonist. Rimonabant by itself promoted adipogenesis in hBM-MSCs, which may be interpreted as the result of the inverse agonism of the CB1 receptor. This result suggests that the constantly active CB1 receptor may contribute to suppress the adipocyte differentiation of hBM-MSCs. Therefore, the selective CB1 agonists that are unable to affect cellular PPARγ activity inhibit adipogenesis in hBM-MSCs.

Keywords: Adipogenesis; Cannbinoid type 1 (CB1) receptor; Endocannabinoids; Human mesenchymal stem cells; Rimonabant.

Fig. 1.

Transcriptional expression profile of CNR1, CNR2 and TRPV1 during adipogenesis in hBM-MSCs. Adipocyte differentiation was induced in hBM-MSCs by exchanging culture media supplemented with 1 μg/ml insulin, 0.1 μM dexamethasone and 0.5 mM isobutylmethylxanthine (IDX). At the seventh day after the induction of adipogenesis, total RNA was extracted and Q-RT-PCR analysis was performed for (A) CNR1 and (B) TRPV1. Values represent the mean expression ± SE of the mRNA of the various genes relative to human GAPDH expression (n=3), *p≤0.05, **p≤0.01.

Fig. 2.

Effects of endocannabinoids on adipogenesis in hBM-MSCs. hBM-MSCs were cultured in 24 well plates. When confluent, the cell viability effects of endocannabinoids were evaluated in hBM-MSCs. NADA (A), AEA (B) and 2-AG (C), were treated for 72 hours in hBM-MSC culture. The cell viability was determined with a detecting reagent, 10 μM of WST-1. Adipogenesis was induced in hBM-MSCs under the presence of the IDX adipocyte differentiation inducing medium. After treating NADA, AEA and 2-AG, in every two or three day, media were exchanged. At the 7^th days in culture, lipid droplets in differentiated adipocytes were stained with Oil Red O (ORO) (D). After dissolving the ORO in isopropyl alcohol, the level of staining was quantified at 500 nm using a spectrometer. Data were normalized by setting the control as 1 (E). Results are the mean ± standard deviation (SD) of three measurements using independent hBM-MSCs from three different donors (n=3). *p≤0.05 and **p≤0.01.

Fig. 3.

Effects of endocannabinoids on adipocyte differentiation marker expression during adipogenesis in hBM-MSCs. When hBM-MSCs were in confluent state, adipogenesis was induced by exchanging media with the IDX adipogenic cocktail. At the 7^th days in culture, total RNA samples were extracted and Q-RT-PCR was performed for PPARγ (A), FABP4 (B) and adiponectin (C). GAPDH was used as an internal control for Q-RT-PCR standardization. Values represent the mean expression ± standard deviation (SD) (n=3). *p≤0.05 and **p≤0.01.

Fig. 4.

Effects of endocannabinoids on adiponectin production during adipogenesis in hBM-MSCs. When hBM-MSCs were in confluent state, adipogenesis was induced by exchanging media with the IDX adipogenic cocktail. ELISA was performed to measure the concentration of adiponectin (A) accumulated in cell culture supernatants for 48 hours after the last medium exchange. The concentration-dependent effect of arachidonyl dopamine (NADA) on the inhibition of adipogenesis was evaluated (B). Values represent the mean expression ± standard deviation (SD) (n=3). *p≤0.05 and **p≤0.01.

Fig. 5.

Effects of rimonabant and capsazepine on the NADA-dependent inhibition of adipogenesis in hBM-MSCs. hBM-MSCs were cultured in 24 well plates. When confluent, the cell viability effects were evaluated for rimonabant (A) and capsazepine (B), both of which were treated for 72 hours in hBM-MSC culture. The cell viability was determined by WST-1 assay. For testing the effects of capsazepine (C) and rimonabant (D) on the NADA-induced inhibition on the adipogenesis in hBM-MSCs, cell culture supernatants were harvested for the measurement of adiponectin by ELISA at the 7^th days after exchanging media containing with IDX. Values represent the mean expression ± standard deviation (SD) (n=3). *p ≤0.05 and **p≤0.01.

Fig. 6.

The effects of rimonabant on adipogenesis in hBM-MSCs and PPARγ transactivation. (A) The concentration-dependent effect of rimonabant on adipogenesis in hBM-MSCs was evaluated in 24 well plates. The effect of rimonabant was compared with those of aspirin, glibenclamide and troglitazone. Cell culture supernatants were harvested for the measurement of adiponectin by ELISA at the 7^th days after exchanging media containing with IDX. (B) CV-1 cells were transiently cotransfected with the PPARγ expression vector and the PPRE-TK-Luciferase reporter, and then treated with vehicle, rimonabant (RIMO), AEA, NADA and 2-AG (1, 3, and 10 μM) or troglitazone (TRO, 1 μM). Values represent the mean expression ± standard deviation (SD) (n=3). *p≤0.05 and **p≤0.01.