Expression, Structure/function, Regulation, and Roles of PDE3 Isoforms

肥胖是2型糖尿病和心血管疾病的主要危险因素。白色脂肪组织（WAT）是一个高度调控和动态的分泌器官，通过甘油三酯的储存和转化/水解影响身体脂肪和能量的利用。此外，通过产生内分泌因子、脂肪细胞因子和脂质，WAT调节和整合重要的生理通路，包括饱腹感、能量利用、葡萄糖敏感性、胰岛素敏感性和炎症。然而，WAT也有助于代谢失调，这是胰岛素抵抗和肥胖相关的代谢和心血管并发症的特征。在PDE3B KO小鼠的白色附睾脂肪组织（EWAT）中，cAMP/PKA-和amp激活的蛋白激酶（AMPK）信号通路被激活，其整合导致KO EWAT具有棕色脂肪组织（BAT）的表型特征，包括形态学改变和基因表达增加，如PDRM16， LRP130和PGC-1，这些基因在BAT和线粒体生物发生的分化中很重要。在KO EWAT中，能量耗散和脂肪酸氧化所需的基因、转录调节因子和线粒体蛋白的表达存在协调调节，如PPAR、UCP-1、CIDEA和其他参与选举转运和脂肪酸氧化的线粒体蛋白。UCP-1是褐色脂肪组织（BAT）的标记物，通常在EWAT中不存在，但在KO EWAT中明显升高。这些发现有助于PDE3B KO小鼠的几个表型特征，包括对高脂肪饮食的体重增加较小，性腺脂肪沉积和脂肪细胞减少，不偶联的EWAT线粒体呼吸，对β -3肾上腺素能受体激动剂刺激的体内耗氧量增加，分离的BAT和EWAT片段的耗氧量增加，PDE3B KO脂肪细胞的脂肪酸氧化增加以及跑步机耐力增加。在培养的3T3 L1脂肪细胞中，西洛胺（特异性PDE3抑制剂）激活PKA和AMPK，西洛胺或siRNA敲低PDE3B可显著增强β -3肾上腺素能受体激动剂对UCP-1的诱导。这些结果表明，PDE3B可能调节cAMP对WAT/BAT表型转换的敏感开关，调节cAMP对cAMP/PKA-和ampk信号传导、线粒体生物发生和功能以及能量消耗的下游影响。了解KO EWAT这些变化的机制很重要，因为将脂肪储存的EWAT转化为脂肪燃烧的BAT代表了治疗肥胖和糖尿病的潜在策略。

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.