Studies on signal transduction mechanisms for adrenaline-driven lipolysis in white and brown adipocytes.
White and brown rat adipocytes have been permeabilised by repeated exposure of the cells in suspension to high voltage electrical discharges. The resulting preparations were permeable to low molecular weight materials, e.g., cyclic AMP, propidium iodide, and were stable in suspension with little evidence of rapid resealing, or of gross damage to the cell membrane. Leakage of lactate dehydrogenase was not markedly enhanced except at voltages in excess of 2 kV cm-1 for brown adipocytes. Exogenously-added cyclic AMP stimulated lipolysis (measured as glycerol release) in the electropermeabilised adipocytes far more effectively than in intact adipocytes. In brown, but not in white, adipocytes this effect was enhanced by addition of millimolar ATP. The EC50 for stimulation of glycerol release by cyclic AMP was 0.2 microM in electropermeabilised brown adipocytes, and 2 microM and 40 microM in electropermeabilised white adipocytes obtained from weanling and adult rats respectively. The effect of cyclic AMP on lipolysis was enhanced by addition of an inhibitor of cyclic AMP phosphodiesterases and was reduced by addition of 5'-AMP, adenosine or inosine (in brown adipocytes). Addition of adenosine deaminase caused a small, but significant, enhancement of cyclic AMP-driven lipolysis. Catecholamine-driven lipolysis was observed in electropermeabilised brown and white adipocytes, especially in the presence of GTP. Adrenaline-, and to a lesser extent cyclic AMP-, driven lipolysis in electropermeabilised white adipocytes was inhibited by insulin. This effect of insulin was not enhanced by addition of GTP or of a metabolically stable GTP analogue. The results obtained establish the electropermeabilised preparation as suitable for analysis of signal transduction pathways in white and brown adipocytes.
Kokkaliari
M
M
Division of Life Sciences, King's College, London, UK.
Chew
E
E
Scrutton
M C
MC
Perry
M C
MC
D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects*; D002001:Adipose Tissue, Brown / Q000187:drug effects; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004558:Electric Stimulation; D004837:Epinephrine / Q000494:pharmacology*; D005260:Female; D005990:Glycerol / Q000032:analysis; D007328:Insulin / Q000494:pharmacology; D008066:Lipolysis / Q000187:drug effects*; D008297:Male; D009638:Norepinephrine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
D007328:Insulin; D000242:Cyclic AMP; D005990:Glycerol; D009638:Norepinephrine; D004837:Epinephrine