Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1

Int J Obes Relat Metab Disord. 2004 Feb;28(2):191-8. doi: 10.1038/sj.ijo.0802554.

Abstract

Objective: Insulin-responsive adipogenic signaling molecules include insulin receptor substrates (IRS)-1 and -2, phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB; also known as Akt). Mammalian target of rapamycin (mTOR) is a PKB substrate, and regulates p70 S6 kinase (p70 S6K). Since p70 S6K is an insulin-responsive kinase downstream of PI3K and PKB, its potential role in adipogenic insulin signaling was investigated.

Design: We measured the effect of rapamycin, a specific inhibitor of mTOR, on insulin-induced 3T3-L1 adipogenesis and on insulin-stimulated p70 S6K activation.

Results: Rapamycin partially reduced differentiation, measured by Oil Red O staining, triacylglycerol accumulation (by up to 46%), and peroxisome proliferator-activated receptor gamma protein expression (by 50%). In contrast, rapamycin completely inhibited insulin-stimulated p70 S6K activation, assessed by phosphorylation of p70 S6K and its substrate, S6. Expression of a constitutively activated form of p70 S6K did not promote 3T3-L1 adipogenesis. The considerable residual differentiation in the presence of rapamycin, despite the complete blockade of p70 S6K activation, prompted us to measure the phosphorylation of another rapamycin-sensitive protein, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Insulin-stimulated 4E-BP1 phosphorylation in 3T3-L1 preadipocytes was only partially affected by rapamycin, consistent with the differentiation data. Phosphorylation of eIF4E itself, an expected consequence of 4E-BP1 phosphorylation, was also only partially inhibited.

Conclusion: Our data suggest that adipogenic mTOR signaling occurs via the 4E-BP1/eIF4E pathway, rather than through p70 S6K.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3-L1 Cells
  • Adaptor Proteins, Signal Transducing
  • Adipocytes / cytology
  • Adipocytes / drug effects*
  • Adipocytes / metabolism
  • Animals
  • Carrier Proteins / metabolism*
  • Cell Cycle Proteins
  • Cell Differentiation / drug effects
  • Eukaryotic Initiation Factors
  • Insulin / pharmacology*
  • Insulin Antagonists / pharmacology*
  • Mice
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors
  • Protein Kinases*
  • Ribosomal Protein S6 Kinases, 70-kDa / metabolism*
  • Signal Transduction / drug effects
  • Sirolimus / pharmacology*
  • TOR Serine-Threonine Kinases

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Cell Cycle Proteins
  • Eif4ebp1 protein, mouse
  • Eukaryotic Initiation Factors
  • Insulin
  • Insulin Antagonists
  • Phosphoproteins
  • Protein Kinase Inhibitors
  • Protein Kinases
  • TOR Serine-Threonine Kinases
  • mTOR protein, mouse
  • Ribosomal Protein S6 Kinases, 70-kDa
  • Sirolimus