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Inhibition of adipocyte lipolysis by insulin is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance and type 2 diabetes mellitus. The main target of the antilipolytic action of insulin is believed to be phosphodiesterase 3B (PDE3B), whose phosphorylation by Akt leads to accelerated degradation of the prolipolytic second messenger cyclic AMP (cAMP). To test this hypothesis genetically, brown adipocytes lacking PDE3B were examined for their regulation of lipolysis. In Pde3b knockout (KO) adipocytes, insulin was unable to suppress β-adrenergic receptor-stimulated glycerol release. Reexpressing wild-type PDE3B in KO adipocytes fully rescued the action of insulin against lipolysis. Surprisingly, a mutant form of PDE3B that ablates the major Akt phosphorylation site, murine S273, also restored the ability of insulin to suppress lipolysis. Taken together, these data suggest that phosphorylation of PDE3B by Akt is not required for insulin to suppress adipocyte lipolysis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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 beta3-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 beta3-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.

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.

Abstract: Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B. [Copyright &y& Elsevier]

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 °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-β-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. [Copyright &y& Elsevier]

Tests whether protein tyrosine phosphatases (PTPases) may play a role in insulin resistance of insulinopenic diabetes by assessing PTPase activity as well as the protein and mRNA abundance of candidate PTPases in subcellular fractions of liver and skeletal. Protein for leukocyte common antigen PTPase; Pretransnational PTPase regulation.