title: Plasma membrane cyclic nucleotide phosphodiesterase 3B (PDE3B) is associated with caveolae in primary adipocytes.
abstract: Caveolae, plasma membrane invaginations particularly abundant in adipocytes, have been suggested to be important in organizing insulin signalling. Insulin-induced activation of the membrane bound cAMP degrading enzyme, phosphodiesterase 3B (PDE3B) is a key step in insulin-mediated inhibition of lipolysis and is also involved in the regulation of insulin-mediated glucose uptake and lipogenesis in adipocytes. The aim of this work was to evaluate whether PDE3B is associated with caveolae. Subcellular fractionation of primary rat and mouse adipocytes demonstrated the presence of PDE3B in endoplasmic reticulum and plasma membrane fractions. The plasma membrane PDE3B was further analyzed by detergent treatment at 4 degrees C, which did not solubilize PDE3B, indicating an association of PDE3B with lipid rafts. Detergent-treated plasma membranes were studied using Superose-6 chromatography which demonstrated co-elution of PDE3B with caveolae and lipid raft markers (caveolin-1, flotillin-1 and cholesterol) at a Mw of >4000 kDa. On sucrose density gradient centrifugation of sonicated plasma membranes, a method known to enrich caveolae, PDE3B co-migrated with the caveolae markers. Immunoprecipitation of caveolin-1 using anti caveolin-1 antibodies co-immunoprecipitated PDE3B and immunoprecipitation of flag-PDE3B from adipocytes infected with a flag-PDE3B adenovirus resulted in co-immunoprecipitation of caveolin-1. Studies on adipocytes with disrupted caveolae, using either caveolin-1 deficient mice or treatment of adipocytes with methyl-beta-cyclodextrin, reduced the membrane associated PDE3B activity. Furthermore, inhibition of PDE3 in primary rat adipocytes resulted in reduced insulin stimulated glucose transporter-4 translocation to caveolae, isolated by immunoprecipitation using caveolin-1 antibodies. Thus, PDE3B, a key enzyme in insulin signalling, appears to be associated with caveolae in adipocytes and this localization seems to be functionally important.
authors.affiliation: Department of Experimental Medical Science, Lund University, BMC C11, 221 84 Lund, Sweden. rebecka.nilsson@med.lu.se
authors.forename: Rebecka
authors.initials: R
authors.lastname: Nilsson
authors.forename: Faiyaz
authors.initials: F
authors.lastname: Ahmad
authors.forename: Karl
authors.initials: K
authors.lastname: Swärd
authors.forename: Ulrika
authors.initials: U
authors.lastname: Andersson
authors.forename: Marie
authors.initials: M
authors.lastname: Weston
authors.forename: Vincent
authors.initials: V
authors.lastname: Manganiello
authors.forename: Eva
authors.initials: E
authors.lastname: Degerman
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D003902:Detergents / Q000494:pharmacology; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D051242:Caveolin 1; D003902:Detergents; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; C106354:flotillins; D002784:Cholesterol; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517293:Pde3b protein, mouse; C517294:Pde3b protein, rat