Endocrine Abstracts

Background

Cannabinoid (CB) receptors are expressed in adipocytes. CB1 antagonism may have potential in the metabolic syndrome, with effects mediated through central orexigenic mechanisms and via peripheral action on white adipose tissue (WAT). Equivalent studies in brown adipose tissue (BAT) are lacking.

Objective

We aimed to compare CB1 and CB2 receptor expression during WAT and BAT adipogenesis and to investigate the effects of CB1 activation/inactivation on proliferation.

Method

3T3-L1 (murine WAT) and PAZ-6 (human BAT) preadipocytes were cultured in differentiation medium containing 1 μM pioglitazone. mRNA was extracted at various time points and reverse transcribed. Transcripts for CB1, CB2, Pref-1, C/EBPβ, PPARγ, and GPDH were measured using SYBR Green and the Stratagene MX3000 QPCR System. Copy numbers were quantified by comparison to dilutions of relevant plasmid DNA. Both populations were exposed to varying concentrations of CB1 agonists (anandamide and ACEA) or antagonist (AM251) and proliferation assessed by direct cell counting (Coulter).

Results

Standard curves were obtained with a linear range (correlation coefficient >0.97, amplification efficiency >99%) from 10⁵ to 10¹ copies for each gene. In 3T3-L1 CB1 expression increased around day 6 of adipogenesis achieving levels up to 50-fold higher (10⁴ copies) than at the outset, which coincided with the upregulation of PPARγ. CB2 was expressed at low levels (10²) but with no significant change. In PAZ6, both CB1 and CB2 were detected but at low copy numbers (<10¹).

CB1 agonists caused a concentration dependent stimulation of proliferation of 3T3-L1 (peak effect 10⁻⁸ M P<0.001). The effect was inhibited by AM251, which was without effect alone. The agents had no effect on PAZ6.

Conclusion

These results suggest that while CB1 modulation provides therapeutic benefit in manipulation of WAT, given the low expression levels its role may be more limited in BAT. However this, and the role of CB2 modulation, requires further investigation.