Orexin Signalling in Brown Adipose Tissue Precursor Cells
2013
Hunter, Kerri | Helsingin yliopisto, Maatalous-metsätieteellinen tiedekunta, Maataloustieteiden laitos | University of Helsinki, Faculty of Agriculture and Forestry, Department of Agricultural Sciences | Helsingfors universitet, Agrikultur- och forstvetenskapliga fakulteten, Institutionen för lantsbruksvetenskaper
Orexins are neuropeptides for which signalling effects have been noted in multiple functions of the central nervous system, and also potentially in the periphery of the body. Orexin receptors couple to a number of different proteins eliciting cellular responses such as activation/inhibition of ion channels, kinase activation, and second messenger generation, and downstream effects such as neuronal excitation, synaptic plasticity, and cell death. The majority of knowledge on orexin signalling has been obtained from recombinant expression systems, thus studies of signalling responses in specific cell or tissue types are desired. The goal of this master’s thesis project was to investigate orexin signalling in two mouse brown adipocyte precursor cell lines (C3H10T1/2 and HIB1b) with native OX1 receptor expression. p38 mitogen-activated protein kinase (MAPK) activation was assessed by western blot analysis, while phospholipase D (PLD) activity, arachidonic acid (AA) and 2-arachidonoylglycerol (2-AG) release, and adenylyl cyclase activity were assessed by radioactive prelabelling, extraction and separation of the molecular species, and quantification of radioactivity. p38 was activated by orexin in C3H10T1/2 cells, but not HIB1b cells; PLD, AA, and 2-AG showed no response to orexin; and adenylyl cyclase appears to be both stimulated and inhibited by orexin, at different concentrations. The results indicate that orexin signalling in these cell lines significantly differs from recombinantly expressed orexin receptors. This raises interesting questions regarding the variability of responses to orexin in different tissue types.
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