Prolonged treatment of rat adipocytes with TNFα increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (Gi). Separately, down-regulation of Gi 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 Gi 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 Gi1 and Gi2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNFα and PIA stimulated lipolysis approximately 2-fold and caused Gi 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 Gi down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNFα and PIA is secondary to Gi down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.
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