G(I) DOWN-REGULATION AND HETEROLOGOUS DESENSITIZATION IN ADIPOCYTES AFTER TREATMENT WITH THE ALPHA(2)-AGONIST UK-14304

Prolonged treatment of rat adipocytes with the A(1)-adenosine receptor agonist [-]N-6-phenylisopropyl adenosine (PIA) or prostaglandin E(1) down-regulates G(i) and induces heterologous desensitization. alpha(2)-Adrenergic receptors also inhibit adenylyl cyclase through G(i), but whether alpha 2-receptors are present on rat adipocytes has been controversial. We have investigated the effects of the highly specific alpha 2-adrenergic agonist UK 14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) on rat adipocytes. In previous studies on young rats, we were unable to demonstrate an effect of the alpha 2-agonist. We now report that, in cells isolated from older, more obese rats (300-400 g), UK 14304 inhibited lipolysis (measured as the rate of glycerol release) by similar to 40% (EC(50) similar to 40 nM). To determine whether UK 14304 would induce heterologous desensitization, we incubated adipocytes with or without 1 mu M UK 14304 for 4 days in primary culture. The cells were then washed, and the rate of lipolysis was determined during a 30-min incubation in the presence of various concentrations of PIA. The concentration-response curve for PIA-induced inhibition of lipolysis was shifted to the right, with the EC(50) for UK 14304-treated cells about 2-fold higher than in the control cells. This finding demonstrates that the alpha 2-agonist can desensitize the response to PIA and indicates heterologous desensitization. To investigate the mechanism of this phenomenon, we isolated crude membrane fractions from the cells and analyzed them on Western blots using antibodies against G(i) alpha 1, 2 and 3. In cells treated with UK 14304 for 4 days, G(i)1 alpha and G(i)2 alpha were down-regulated to about 15% of the control level, and G(i)3 alpha was decreased to 30% of control. We conclude that prolonged treatment of adipocytes with the alpha 2-agonist induces heterologous desensitization of lipolysis and causes down-regulation of G(i). The findings suggest that G-protein down-regulation is a mechanism for heterologous desensitization.