Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the β3-adrenergic receptor agonist CL316243: Effects of caveolin-1 knockdown on formation/ maintenance of macromolecular signalling complexes

Faiyaz Ahmad, Rebecka Lindh, Yan Tang, Iida Ruishalme, Anita Ost, Bobby Sahachartsiri, Peter Strålfors, Eva Degerman, Vincent C. Manganiello

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the β3-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KD of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained β3-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KD. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.

Original languageEnglish (US)
Pages (from-to)399-410
Number of pages12
JournalBiochemical Journal
Volume424
Issue number3
DOIs
StatePublished - Dec 15 2009
Externally publishedYes

Keywords

  • Adipocyte
  • Caveolin-1
  • Insulin
  • Phosphodiesterase 3 (PDE3)
  • Protein kinase A (PKA)
  • β-adrenergic receptor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the β<sub>3</sub>-adrenergic receptor agonist CL316243: Effects of caveolin-1 knockdown on formation/ maintenance of macromolecular signalling complexes'. Together they form a unique fingerprint.

  • Cite this