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 Citations (Scopus)

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

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Type 3 Cyclic Nucleotide Phosphodiesterases
Macromolecular Substances
Caveolin 1
Adrenergic Agonists
Adipocytes
Maintenance
Insulin
Membranes
Chemical activation
14-3-3 Proteins
Sterol Esterase
Small Interfering RNA
Cholesterol
disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate
Insulin Receptor Substrate Proteins
Caveolae
Protein Phosphatase 2
Phosphorylation
Molecules
Molecular Chaperones

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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

In: Biochemical Journal, Vol. 424, No. 3, 15.12.2009, p. 399-410.

Research output: Contribution to journalArticle

Ahmad, Faiyaz ; Lindh, Rebecka ; Tang, Yan ; Ruishalme, Iida ; Ost, Anita ; Sahachartsiri, Bobby ; Strålfors, Peter ; Degerman, Eva ; Manganiello, Vincent C. / 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. In: Biochemical Journal. 2009 ; Vol. 424, No. 3. pp. 399-410.
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