Inhibition of proteasome activity blocks the ability of TNFα to down-regulate G_i proteins and stimulate lipolysis

Prolonged treatment of rat adipocytes with TNFα increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (G_i). Separately, down-regulation of G_i by prolonged treatment with an A₁-adenosine receptor agonist, N⁶-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNFα and PIA-mediated G_i down-regulation and stimulation of lipolysis, we used the protease inhibitors lactacystin (proteasome inhibitor) and calpeptin (calpain inhibitor). Rat adipocytes were preincubated for 1 h with lactacystin (10 μM) or calpeptin (50 μM), before 30-h treatment with either TNFα (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of α-subunits of G_i1 and G_i2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNFα and PIA stimulated lipolysis approximately 2-fold and caused G_i down-regulation. Although neither lactacystin nor calpeptin affected basal lipolysis, lactacystin completely inhibited both TNFα and PIA-stimulated lipolysis (the 50% inhibitory concentration was ∼2 μM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNFα-induced G_i down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNFα and PIA is secondary to G_i down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.