Objective: Orexin-A and orexin-B and their G-protein coupled receptors (orexin receptor-1 & -2: OX1R, OX2R) have divergent effects on physiological behaviour, cardiovascular regulation, glucocorticoid and insulin release. Furthermore, orexins have been shown to affect both brown adipose tissue energy expenditure and thermogenesis through stimulation of sympathetic nerve activity. Despite in vivo studies demonstrating a role for orexins acting centrally on adipose tissue, there are no data on the expression of orexin receptors in brown adipose tissue. We therefore analyzed the expression and localization of OX1R and OX2R in mouse brown adipocytes and in the T37i brown adipocyte cell line. Furthermore, the effects of exposure to orexin-A and orexin-B were measured on the expression of key genes involved in thermoregulation and insulin sensitivity; leptin, uncoupling protein-1 (UCP-1), adipocyte-specific fatty acid binding protein-2 (AP2) and PPARγ.
Methods: Quantitative real time RT-PCR was performed using a Roche Light Cycler system, and genes of interest were standardised against the housekeeping gene β-actin. OX1R and OX2R were detected in differentiated T37i brown adipocytes using immunocytochemistry and confocal microscopy.
Results: mRNA expression was detected for OX1R and OX2R in mouse mature interscapular brown adipocytes, as well as in differentiated T37i brown adipocytes in vitro. Furthermore, mRNA expression of both receptors increased as a function of the degree of differentiation. Confocal analysis revealed intense localised staining for OX1R around intracellular lipid droplets, whereas more membrane-localised staining was observed for OX2R. Treatment with orexin-A and -B (100 nM, 4 h), resulted in a significant increase in PPARγ and AP2 mRNA (n=6, P<0.05). Whereas only orexin B treatment resulted in an increase in leptin and UCP-1 mRNA (n=6, P<0.05).
Conclusions: These novel findings indicate a direct role for orexins in brown adipocyte tissue metabolism and thermogenesis and the potential to affect insulin-sensitivity. Furthermore, the differing cellular receptor localisation suggests divergent roles for orexins in brown adipocytes.