Obesity is a major risk factor for type 2 diabetes and cardiovascular disease. White adipose tissue (WAT), a highly regulated and dynamic secretory organ, affects body fat and energy utilization through storage and turnover/hydrolysis of triglycerides. In addition, via production of endocrine factors, adipocytokines, and lipids, WAT regulates and integrates important physiological pathways, including satiety, energy utilization, glucose sensitivity, insulin sensitivity, and inflammation. WAT, however, also contributes to metabolic dysregulation that characterizes insulin resistance and obesity-related metabolic and cardiovascular complications. In white epididymal adipose tissue (EWAT) of PDE3B KO mice, cAMP/PKA- and AMP-activated protein kinase (AMPK)-signaling pathways are activated, the integration of which results in KO EWAT assuming phenotypic characteristics of brown adipose tissue (BAT), including alterations in morphology and increased expression of genes, such as PDRM16, LRP130, and PGC-1, that are important in differentiation of BAT and mitochondrial biogenesis. In KO EWAT, there is coordinate regulation of expression of genes, transcriptional regulators, and mitochondrial proteins required for energy dissipation and fatty acid oxidation, such as PPAR, UCP-1, CIDEA, and other mitochondrial proteins involved in election transport and fatty acid -oxidation. UCP-1, a marker for brown adipose tissue (BAT) usually not present in EWAT, is markedly elevated in KO EWAT. These findings contribute to several phenotypic characteristics of PDE3B KO mice, including a smaller increase in body weight in response to high fat diets, smaller gonadal fat deposits and adipocytes, uncoupled EWAT mitochondrial respiration, increased oxygen consumption in vivo response to Beta-3 adrenergic receptor agonist stimulation, increased oxygen consumption in isolated BAT and EWAT fragments, increased fatty acid oxidation in PDE3B KO adipocytes and increased treadmill endurance. In cultured 3T3 L1 adipocytes, cilostamide (specific PDE3 inhibitor) activated PKA and AMPK, and cilostamide or siRNA knockdown of PDE3B markedly potentiated induction of UCP-1 by a Beta-3 adrenergic receptor agonist. These results suggest that PDE3B may regulate a cAMP-sensitive switch for WAT/BAT phenotypic conversion, regulating downstream effects of cAMP on cAMP/PKA- and AMPK-signaling, mitochondrial biogenesis and function, and energy dissipation. Understanding mechanisms for these changes in KO EWAT is important, since conversion of fat-storing EWAT to fat-burning BAT represents a potential strategy in treatment of obesity and diabetes. Apolipoprotein E knockout (apoE-/-) and low density lipoprotein receptor (LDL-R-/-) mice develop hypocholesterolmia and atherosclerosis, either spontaneously or under a high cholesterol diet, respectively. Inflammatory infiltrates in the atherosclerotic plaques contain cholesterol-laden macrophages (foam cells) and T lymphocytes. These and other inflammation-related cells are presumably responsible for the increased circulating levels of proinflammatory cytokines, interferon-gamma (INF-gamma), and tumor necrosis factor-α(TNF-α), as well as macrophage-derived interleukin (IL)-12 and IL-18, in apoE-/- mice.Interestingly, we found that targeted disruption of PDE3B was associated with decreased macrophage markers in epididymal white adipose tissue (EWAT). Moreover, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein-1 (MCP-1) and its receptor CCR2, which play an important role in macrophage chemotaxis, were less highly expressed in EWAT of PDE3B-/- mice than WT mice. In addition, after lipopolysaccaride (LPS) injection, plasma levels of TNF-α, IL-12 and CCL2/MCP-1 were lower in PDE3B-/- mice than WT mice. To examine the possible effects of PDE3B on macrophage infiltration and atherosclerotic plaque formation, apoE-/-/PDE3B-/-, as well as LDL-R-/-/PDE3B--/- mice were generated. Compared to apoE-/- and LDL-R-/- mice, in the aorta of apoE-/-/PDE3B-/- (normal diet) and LDL-R-/-/PDE 3B-/- (Western diet high in fat for 5 months) mice, plaque formation was significantly reduced, respectively, suggesting a role for PDE3B in modulating the inflammatory response and suggesting that PDE3B signaling pathways might provide possible therapeutic targets to moderate atherosclerosis. In animal models, caloric restriction (CR) decreases the incidence of age-associated disorders such as cardiovascular disease, diabetes, and cancer. SIRT1 activators, i.e., resveratrol and SRT1720 (1000 fold more potent than resveratrol), mimic effects of CR in lower organisms and mice. In 3T3-L1 adipocytes, resveratrol and SRT1720 inhibited PDE activities in membrane (IC50, 40 and 7.5 uM respectively), and cytosolic fractions (IC50, 90 and 10 uM, respectively). Cilostamide (PDE3 inhibitor, 10 uM), resveratrol (12.5 uM), SRT-1720 (2 uM), and rolipram (PDE4 inhibitor, 30 uM), increased phosphorylation of Ser133-CREB, Ser431-LKB, Thr172-AMPK, Ser79-ACC as well as other unidentified PKA substrates. Rp-8-Br-cAMPs, a PKA inhibitor, blocked phosphorylation of these signaling molecules by cilostamide and rolipram. Rolipram had a relatively stronger effect on phosphorylation of PKA substrates and CREB than cilostamide, resveratrol and SRT1720, but had smaller effect on the phosphorylation of LKB, AMPK, and ACC, suggesting the presence of a distinct cAMP pool involved in activation of AMPK. In adipocytes incubated for 90 min with CL-316243 (B3-agonist), 10 uM resveratrol or 2 uM SRT1720 increased phosphorylation of Ser563 of hormone-sensitive lipase (S563-HSL), resulting in a significant increase in lipolysis (glycerol release). These and other results provide evidence that at least some effects of resveratrol and SRT-1720 may be related to their inhibition of PDEs and, thereby, alteration of intracellular cAMP concentrations. Mammalian PDE3 is known to play an important role in insulin signaling pathways and in platelets, cardiovascular tissues, adipocytes, and oocytes.Caenorhabditis elegans represents a unique model for genetic manipulation and thus can facilitate the identification of regulatory genes and characterization of their functions. We report here studies of expression and characterization of the C.elegans Phosphodiesterase3 (CEPDE3) gene, a homolog of the mammalian PDE3 family. The nematode PDE3 gene is present on chromosome II, spaning about 22.2 Kb, and encodes two different CEPDE3 isoforms. The CEPDE3 long form (LF) consists of 11 exons and codes for a 63.5 kDa protein;the short form (SF) has 8 exons and codes for a 54.2 kDa protein. Both CEPDE3 isoforms have the characteristic mammalian PDE Pfam and phosphodiester domains, and also contain the HD metal binding motif, which is unique for the PDE superfamily. Multiple sequence homology alignments of CEPDE3 with that of the human PDE families shows that C.elegans PDE3 is close to mammalian PDE3 on the PDE family tree. The predicted sequences of CEPDE3LF and SF isoforms show an overall 97 % homology between each other,with identical catalytic domains. PDE activity assays indicated that recombinant CEPDE3 long and short forms are markedly inhibited by cilostamide (a specific inhibitor of mammalian PDE3 ), but not by rolipram (a specific inhibitor of PDE4). The IC50 values for cilostamide and rolipram inhibition are similar for recombinant CEPDE3 long form and recombinant mammalian PDE3. The MS/MS sequence of purified recombinant CEPDE3LF contained several predicted phosphorylation sites.
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