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Inhibition of Proteasome Activity Blocks the Ability of TNF to Down-Regulate G_i Proteins and Stimulate Lipolysis

Depto de Fisiologia e Biofísica–Instituto de Ciências Biológicas (L.M.B.), Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 31270-901; Department of Internal Medicine (A.R.B., B.T.), University of Texas Medical Branch, Galveston, Texas 77555; and the Bassett Research Institute (V.U., A.G.), Mary Imogene Bassett Hospital, Cooperstown, New York 13326

Address all correspondence and requests for reprints to: Allan Green, Ph.D., Basset Research Institute, Mary Imogene Bassett Hospital, One Atwell Road, Cooperstown, New York 13326.

Prolonged treatment of rat adipocytes with TNF 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 TNF 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 TNF (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of -subunits of G_i1 and G_i2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF 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 TNF 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 TNF-induced G_i down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF and PIA is secondary to G_i down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.

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