Bone marrow stem cells (BMSCs) are able to differentiate along multiple lineages including adipogenic and osteogenic lineages. The adenosine A1 receptor (A1R) modulates adipose tissue function causing an inhibition of lipolysis. We therefore studied A1R expression in BMSCs induced to differentiate along the adipogenic lineage.
BMSCs were isolated from rat femur (10 wk male Wistar), cultured in control medium (alphaMEM 10% FCS) and then plated into adipocyte differentiation medium (containing a PPARgamma agonist) for 37 days or osteoblast differentiation medium (containing ascorbate, dexamethasone, and beta-glycerphosphate) for 14 days as a differentiation process control. Parallel cultures were stained for lipid with oil red O, mineralisation with alizarin red and subjected to RNA extraction. The RNA was reverse transcribed and adenosine A1, A2a, A2b and A3 receptor expression analysed by QPCR (Sybr green) using a Stratagene Mx3000P. Additionally, beta-actin transcript levels were analysed and used as a housekeeping gene as we had previously confirmed that this was unaltered during differentiation. Results were normalised for beta-actin expression and expressed as fold increase compared to undifferentiated control cultures.
Oil red O staining was only detectable in the adipocyte differentiated cells and alizarin red only in osteoblast differentiated cells. A1R transcript levels were markedly increased in adipocyte differentiated cells showing an 836 fold and a 357 fold increases (P<0.05) in cells differentiated along the adipogenic lineage for 3 and 7 days respectively compared to undifferentiated cells. By contrast, cells subjected to osteoblast differentiation showed a much smaller, 16 fold increase (ns). Differentiation had no effect on levels of A2a, A2b or A3 receptor transcript levels.
In conclusion, the A1R shows a specific and very marked upregulation in adipocyte but not in osteoblast differentiated BMSCs suggesting that the A1R plays a role in regulating adipocyte differentiation and/or metabolism.