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Tumor necrosis factor-α (TNF-α) stimulates lipolysis in human adipocytes. However, the mechanisms regulating this process are largely unknown. We demonstrate that TNF-α increases lipolysis in differentiated human adipocytes by activation of mitogen-activated protein kinase (MEK), extracellular signal-related kinase (ERK), and elevation of intracellular cAMP. TNF-α activated ERK and increased lipolysis; these effects were inhibited by two specific MEK inhibitors, PD98059 and U0126. TNF-α treatment caused an electrophoretic shift of perilipin from 65 to 67 kDa, consistent with perilipin hyperphosphorylation by activated cAMP dependent protein kinase A (PKA). Coincubation with TNF-α and MEK inhibitors caused perilipin to migrate as a single 65-kDa band. Consistent with the hypothesis that TNF-α induces perilipin hyperphosphorylation by activating PKA, TNF-α increased intracellular cAMP ∼l.7-fold, and the increase was abrogated by PD98059. Furthermore, H89, a specific PKA inhibitor, blocked TNF-α-induced lipolysis and the electrophoretic shift of perilipin, suggesting a role for PKA in TNF-α-induced lipolysis. Finally, TNF-α decreased the expression of cyclic-nucleotide phosphodiesterase 3B (PDE3B) by ∼50%, delineating a mechanism by which TNF-α could increase intracellular cAMP. Cotreatment with PD98059 restored PDE3B expression. These studies suggest that in human adipocytes, TNF-α stimulates lipolysis through activation of MEK-ERK and subsequent increase in intracellular cAMP. [ABSTRACT FROM AUTHOR]

PDE3B is an important regulator of several insulin- and cAMP-dependent signaling pathways. Fractionation of membranes from adipocytes revealed that PDE3B was associated with plasma membrane (PM) and internal membrane (IM) fractions, and that insulin-induced phosphorylation/activation of PDE3B was greater in IM than PM fractions. Insulin increased tyrosine phosphorylation of IRS-1, and activated IRS-1-associated PI3-K and PKB in both membrane and cytosolic fractions. Insulin also induced formation of large macromolecular complexes (LMCs), separated during gel filtration (Superose 6) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and several signaling molecules potentially involved in its activation by insulin, e.g. IRS-1, PI3-K, PKB, HSP-90, and 14-3-3. Expression of full length recombinant Flag-tagged murine (M)PDE3B (M3B) and M3BD604(M3B lacking N-terminal 604 amino acids) indicated that N-terminal region of M3B (which contains sites phosphorylated by insulin) was necessary for insulin-induced activation and recruitment of PDE3B into LMCs. M3BD604 was not activated by insulin, was not recruited into LMCs, and did not interfere with recruitment of endogenous PDE3B. siRNA knockdown of PDE3B indicated that PDE3B was not required for formation of insulin-induced LMCs. Wortmannin inhibited insulin-induced LMCs, as well as insulin-induced phosphorylation/activation of PKB and PDE3B, their co-immunoprecipitation, and interactions during Superose 6 chromatography. Insulin-stimulated complex formation of PDE3B and other signaling molecules may be critical for down stream events regulating adipocyte metabolism. [ABSTRACT FROM AUTHOR]

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic β cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The β3-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance. [ABSTRACT FROM AUTHOR]