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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

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Inhibition of proteasome activity blocks the ability of TNF alpha to down-regulate G(i) proteins and stimulate

L M Botion1, A R Brasier, B Tian

  • 1Depto de Fisiologia e Biofísica-Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 31270-901.

Endocrinology
|November 20, 2001
PubMed

Insights

Tumor necrosis factor alpha (TNF alpha) and N(6)-phenylisopropyl adenosine (PIA) increase fat breakdown (lipolysis) by reducing inhibitory G proteins (G(i)). Proteasome inhibition blocks this effect, indicating proteolysis is key to TNF alpha and PIA-induced lipolysis.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Pharmacology

Background:

  • Tumor necrosis factor alpha (TNF alpha) and N(6)-phenylisopropyl adenosine (PIA) promote lipolysis in adipocytes.
  • This lipolytic effect is partly mediated by the down-regulation of inhibitory G proteins (G(i)).
  • The precise mechanism of G(i) down-regulation and its role in lipolysis requires further investigation, particularly concerning proteolysis.

Purpose of the Study:

  • To investigate the role of proteolysis in TNF alpha and PIA-induced G(i) down-regulation.
  • To determine if proteasome or calpain pathways are involved in the lipolytic effects of TNF alpha and PIA.
  • To elucidate the mechanism by which TNF alpha and PIA stimulate lipolysis.

Main Methods:

  • Rat adipocytes were treated with TNF alpha or PIA following preincubation with proteasome inhibitor lactacystin or calpain inhibitor calpeptin.
  • Lipolysis was quantified by measuring glycerol release.
  • G(i) protein alpha-subunit abundance was assessed using Western blotting, and protease activities were measured via fluorogenic assays.

Main Results:

  • Both TNF alpha and PIA significantly increased lipolysis and down-regulated G(i) protein levels.
  • Lactacystin completely inhibited TNF alpha and PIA-stimulated lipolysis and G(i) down-regulation.
  • Calpeptin had no significant effect on basal or stimulated lipolysis or G(i) down-regulation.

Conclusions:

  • The lipolytic effects of TNF alpha and PIA are dependent on the down-regulation of inhibitory G proteins (G(i)).
  • Proteolytic degradation, specifically via the proteasome pathway, mediates the down-regulation of G(i) in response to TNF alpha and PIA.
  • These findings highlight the proteasome pathway as a critical component in regulating adipocyte lipolysis.

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