Inhibition of proteasome activity blocks the ability of TNF alpha to down-regulate G(i) proteins and stimulate lipolysis.
Prolonged treatment of rat adipocytes with TNF alpha 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 A(1)-adenosine receptor agonist, N(6)-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNF alpha 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 microM) or calpeptin (50 microM), before 30-h treatment with either TNF alpha (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of alpha-subunits of G(i)1 and G(i)2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF alpha 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 alpha and PIA-stimulated lipolysis (the 50% inhibitory concentration was approximately 2 microM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNF alpha-induced G(i) down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF alpha and PIA is secondary to G(i) down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.
Botion
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Depto de Fisiologia e Biofísica-Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 31270-901.
Brasier
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Tian
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Udupi
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Green
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D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D015536:Down-Regulation / Q000502:physiology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008066:Lipolysis / Q000187:drug effects / Q000502:physiology*; D008297:Male; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000502:physiology*; D010660:Phenylisopropyladenosine / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D009097:Multienzyme Complexes; D014409:Tumor Necrosis Factor-alpha; C067713:lactacystin; C071482:calpeptin; D010660:Phenylisopropyladenosine; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000111:Acetylcysteine