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

Leida M. Botion, Allan R. Brasier, Bing Tian, Vidya Udupi, Allan Green

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5069-5075
Number of pages7
JournalEndocrinology
Volume142
Issue number12
DOIs
StatePublished - 2001

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Lipolysis
Proteasome Endopeptidase Complex
Adenosine
Down-Regulation
Proteins
Adipocytes
Adenosine A1 Receptor Agonists
Gi-Go GTP-Binding Protein alpha Subunits
Proteasome Inhibitors
Protease Inhibitors
Glycerol
Proteolysis
Inhibitory Concentration 50
Peptide Hydrolases
Western Blotting
Cell Membrane
lactacystin
calpeptin

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Inhibition of proteasome activity blocks the ability of TNFα to down-regulate Gi proteins and stimulate lipolysis. / Botion, Leida M.; Brasier, Allan R.; Tian, Bing; Udupi, Vidya; Green, Allan.

In: Endocrinology, Vol. 142, No. 12, 2001, p. 5069-5075.

Research output: Contribution to journalArticle

Botion, Leida M. ; Brasier, Allan R. ; Tian, Bing ; Udupi, Vidya ; Green, Allan. / Inhibition of proteasome activity blocks the ability of TNFα to down-regulate Gi proteins and stimulate lipolysis. In: Endocrinology. 2001 ; Vol. 142, No. 12. pp. 5069-5075.
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