Activation Of Phosphodiesterase 3B Research Articles

Salicylate Sodium Suppresses Monocyte Chemoattractant Protein-1 Production by Directly Inhibiting Phosphodiesterase 3B in TNF-α-Stimulated Adipocytes.

As a worldwide health issue, obesity is associated with the infiltration of monocytes/macrophages into the adipose tissue causing unresolved inflammation. Monocyte chemoattractant protein-1 (MCP-1) exerts a crucial effect on obesity-related monocytes/macrophages infiltration. Clinically, aspirin and salsalate are beneficial for the treatment of metabolic diseases in which adipose tissue inflammation plays an essential role. Herein, we investigated the effect and precise mechanism of their active metabolite salicylate on TNF-α-elevated MCP-1 in adipocytes. The results indicated that salicylate sodium (SAS) could lower the level of MCP-1 in TNF-α-stimulated adipocytes, which resulted from a previously unrecognized target phosphodiesterase (PDE), 3B (PDE3B), rather than its known targets IKKβ and AMPK. The SAS directly bound to the PDE3B to inactivate it, thus elevating the intracellular cAMP level and activating PKA. Subsequently, the expression of MKP-1 was increased, which led to the decrease in p-EKR and p-p38. Both PDE3B silencing and the pharmacological inhibition of cAMP/PKA compromised the suppressive effect of SAS on MCP-1. In addition to PDE3B, the PDE3A and PDE4B activity was also inhibited by SAS. Our findings identify a previously unrecognized pathway through which SAS is capable of attenuating the inflammation of adipocytes.

Expression of Recombinant Phosphodiesterases 3A and 3B Using Baculovirus Expression System.

Background Phosphodiesterase 3A (PDE3A) and phosphodiesterase 3B (PDE3B) play a critical role in the regulation of intracellular level of adenosine 3´,5´-cyclic monophosphate (cyclic AMP, cAMP) and guanosine 3´,5´-cyclic monophosphate (cyclic GMP, cGMP). Subsequently PDE3 inhibitors have shown to relax vascular and inhibit platelet aggregation in cardiovascular disease. Objectives In this study, our aim was to establish a method of expression for the recombinant human PDE3A and PDE3B proteins in insect cells using baculovirus expression system in order to investigate the activity of the expressed PDE3A and PDE3B proteins. Materials and Methods The full length human PDE3A and PDE3B cDNA were cloned into recombinant baculovirus and transfected into the SF9 insect cells. Recombinant proteins were collected at 48 h, 60 h, 72 h, and 84 h post transfection. Transfection of recombinant baculovirus was verified by the morphological changes of the SF9 cells. Expression of human PDE3A and PDE3B was detected by using RT-PCR and western blot, respectively. The 125I RIA method was used to determine the level of adenosine 3´,5´-cyclic monophosphate cAMP and cGMP, correspondingly. The activity of the expressed PDE3A and PDE3B proteins were investigated by cAMP and cGMP dsgradation with or without addition of milrinone, a potent and selective PDE inhibitor. Results Recombinant human PDE3A and PDE3B proteins were stably expressed in SF9 cells and could be detected by distinct morphological changes in the SF9 cells, RT-PCR, and western blot at 48 h post-transfection. The molecular weights of the recombinant PDE3A and PDE3B molecular weights proteins were about 120 KDa and 135 KDa, respectively. Results of 125I RIA assay showed that the levels of cAMP and cGMP were significantly decreased after incubation with the expressed PDE3A and PDE3B proteins. Furthermore, degradation of cAMP and cGMP through the activity of PDE3A and PDE3B was suppressed following to the addition of milrinone. Conclusions Recombinant human PDE3A and PDE3B could be expressed in SF9 cells using baculovirus expression system, and thus provides the basic material for studying human PDE3A and PDE3B activity.

RNA-binding protein CUGBP1 regulates insulin secretion via activation of phosphodiesterase 3B in mice.

CUG-binding protein 1 (CUGBP1) is a multifunctional RNA-binding protein that regulates RNA processing at several stages including translation, deadenylation and alternative splicing, as well as RNA stability. Recent studies indicate that CUGBP1 may play a role in metabolic disorders. Our objective was to examine its role in endocrine pancreas function through gain- and loss-of-function experiments and to further decipher the underlying molecular mechanisms. A mouse model in which type 2 diabetes was induced by a high-fat diet (HFD; 60% energy from fat) and mice on a standard chow diet (10% energy from fat) were compared. Pancreas-specific CUGBP1 overexpression and knockdown mice were generated. Different lengths of the phosphodiesterase subtype 3B (PDE3B) 3' untranslated region (UTR) were cloned for luciferase reporter analysis. Purified CUGBP1 protein was used for gel shift experiments. CUGBP1 is present in rodent islets and in beta cell lines; it is overexpressed in the islets of diabetic mice. Compared with control mice, the plasma insulin level after a glucose load was significantly lower and glucose clearance was greatly delayed in mice with pancreas-specific CUGBP1 overexpression; the opposite results were obtained upon pancreas-specific CUGBP1 knockdown. Glucose- and glucagon-like peptide1 (GLP-1)-stimulated insulin secretion was significantly attenuated in mouse islets upon CUGBP1 overexpression. This was associated with a strong decrease in intracellular cAMP levels, pointing to a potential role for cAMP PDEs. CUGBP1 overexpression had no effect on the mRNA levels of PDE1A, 1C, 2A, 3A, 4A, 4B, 4D, 7A and 8B subtypes, but resulted in increased PDE3B expression. CUGBP1 was found to directly bind to a specific ATTTGTT sequence residing in the 3' UTR of PDE3B and stabilised PDE3B mRNA. In the presence of the PDE3 inhibitor cilostamide, glucose- and GLP-1-stimulated insulin secretion was no longer reduced by CUGBP1 overexpression. Similar to CUGBP1, PDE3B was overexpressed in the islets of diabetic mice. We conclude that CUGBP1 is a critical regulator of insulin secretion via activating PDE3B. Repressing this protein might provide a potential strategy for treating type 2 diabetes.

Hypothalamic Phosphodiesterase 3B Pathway Mediates Anorectic and Body Weight-Reducing Effects of Insulin in Male Mice

Background: Insulin action in the hypothalamus plays a critical role in the regulation of energy homeostasis, yet the intracellular signaling mechanisms mediating insulin action are incompletely understood. Although phosphodiesterase 3B (PDE3B) mediates insulin action in the adipose tissue and it is highly expressed in the hypothalamic areas implicated in energy homeostasis, its role, if any, in mediating insulin action in the hypothalamus is unknown. We tested the hypothesis that insulin action in the hypothalamus is mediated by PDE3B. Methods: Using enzymatic assay, we examined the effects of peripheral or central administration of insulin on hypothalamic PDE3B activity in adult mice. Western blotting and immunohistochemistry also examined p-Akt and p-STAT3 levels in the hypothalamus. Effects of leptin on these parameters were also compared. We injected cilostamide, a PDE3 inhibitor, prior to central injection of insulin and examined the 12- to 24-hour food intake and 24-hour body weight. Finally, we examined the effect of cilostamide on insulin-induced proopiomelanocortin (Pomc), neurotensin (Nt), neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression in the hypothalamus by qPCR. Results: Peripheral or central injection of insulin significantly increased PDE3B activity in the hypothalamus in association with increased p-Akt levels but without any change in p-STAT3 levels. However, leptin-induced increase in PDE3B activity was associated with an increase in both p-Akt and p-STAT3 levels in the hypothalamus. Prior administration of cilostamide reversed the anorectic and body weight-reducing effects as well as stimulatory effect of insulin on hypothalamic Pomc mRNA levels. Insulin did not alter Nt, Npy and Agrp mRNA levels. Conclusion: Insulin induction of hypothalamic PDE3B activity and the reversal of the anorectic and body weight-reducing effects and stimulatory effect of insulin on hypothalamic Pomc gene expression by cilostamide suggest that activation of PDE3B is a novel mechanism of insulin signaling in the hypothalamus.

Leptin receptor expressing neurons express phosphodiesterase-3B (PDE3B) and leptin induces STAT3 activation in PDE3B neurons in the mouse hypothalamus

Leptin signaling in the hypothalamus is critical for normal food intake and body weight regulation. Cumulative evidence suggests that besides the signal transducer and activator of transcription-3 (STAT3) pathway, several non-STAT3 pathways including the phosphodiesterase-3B (PDE3B) pathway mediate leptin signaling in the hypothalamus. We have shown that PDE3B is localized in various hypothalamic sites implicated in the regulation of energy homeostasis and that the anorectic and body weight reducing effects of leptin are mediated by the activation of PDE3B. It is still unknown if PDE3B is expressed in the long form of the leptin-receptor (ObRb)-expressing neurons in the hypothalamus and whether leptin induces STAT3 activation in PDE3B-expressing neurons. In this study, we examined co-localization of PDE3B with ObRb neurons in various hypothalamic nuclei in ObRb-GFP mice that were treated with leptin (5mg/kg, ip) for 2h. Results showed that most of the ObRb neurons in the arcuate nucleus (ARC, 93%), ventromedial nucleus (VMN, 94%), dorsomedial nucleus (DMN, 95%), ventral premammillary nucleus (PMv, 97%) and lateral hypothalamus (LH, 97%) co-expressed PDE3B. We next examined co-localization of p-STAT3 and PDE3B in the hypothalamus in C57BL6 mice that were treated with leptin (5mg/kg, ip) for 1h. The results showed that almost all p-STAT3 positive neurons in different hypothalamic nuclei including ARC, VMN, DMN, LH and PMv areas expressed PDE3B. These results suggest the possibility for a direct role for the PDE3B pathway in mediating leptin action in the hypothalamus.

Phosphodiesterase-3B-cAMP pathway of leptin signalling in the hypothalamus is impaired during the development of diet-induced obesity in FVB/N mice.

The phosphodiesterase-3B (PDE3B)-cAMP pathway plays an important role in transducing the action of leptin in the hypothalamus. Obesity is usually associated with hyperleptinaemia and resistance to anorectic and body weight-reducing effects of leptin. To determine whether the hypothalamic PDE3B-cAMP pathway of leptin signalling is impaired during the development of diet-induced obesity (DIO), we fed male FVB/N mice a high-fat diet (HFD: 58% kcal as fat) or low-fat diet (LFD: 6% kcal as fat) for 4 weeks. HFD fed mice developed DIO in association with hyperphagia, hyperleptinaemia and hyperinsulinaemia. Leptin (i.p.) significantly increased hypothalamic PDE3B activity and phosphorylated (p)-Akt levels in LFD-fed but not in HFD-fed mice. However, basal p-Akt levels in hypothalamus were increased in DIO mice. Additionally, amongst six-microdissected brain nuclei examined, leptin selectively decreased cAMP levels in the arcuate nucleus (ARC) of LFD-fed mice but failed to do so in HFD-fed mice. We next tested whether both the PBE3B and Akt pathways of leptin signalling remained impaired in DIO mice on the HFD for 12 weeks (long-term). DIO mice were hyperinsulinaemic and hyperleptinaemic in association with impaired glucose and insulin tolerance. Although, in LFD-fed mice, leptin significantly increased PDE3B activity and p-Akt levels in the hypothalamus, it failed to do so in HFD-fed mice. Also, basal p-Akt levels in the hypothalamus were increased in DIO mice and leptin had no further effect. Similarly, immunocytochemistry showed that leptin increased the number of p-Akt-positive cells in the ARC of LFD-fed but not in HFD-fed mice, and there was an increased basal number of p-Akt positive cells in the ARC of DIO mice. These results suggest that the PDE3B-cAMP- and Akt-pathways of leptin signalling in the hypothalamus are impaired during the development of DIO. Thus, a defect in the regulation by leptin of the hypothalamic PDE3B-cAMP pathway and Akt signalling may be one of the mechanisms of central leptin resistance and the development of DIO.

Phosphatidylinositol 3‐kinase is an Upstream Regulator of the Phosphodiesterase 3B Pathway of Leptin Signalling that may not Involve Activation of Akt in the Rat Hypothalamus

Leptin, the product of the obese gene, regulates energy homeostasis by acting primarily at the level of the hypothalamus. Leptin action through its receptor involves various pathways, including the signal transducer and activator of transcription (STAT)3, phosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B (PDE3B)-cAMP signalling in the central nervous system and peripheral tissues. In the hypothalamus, leptin stimulates STAT3 activation, and induces PI3K and PDE3B activities, among others. We have previously demonstrated that PDE3B activation in the hypothalamus is critical for transducing the anorectic and body weight reducing effects of leptin. Similarly, PI3K has been implicated to play a critical role in leptin signalling in the hypothalamus. Although, in the insulin signalling pathway, PI3K is known to be an upstream regulator of PDE3B in non-neuronal tissues, it is still unknown whether this is also the case for leptin signalling in the hypothalamus. To address this possibility, the effect of wortmannin, a specific PI3K inhibitor, was examined on leptin-induced PDE3B activity in the hypothalamus of male rats. Intracerebroventricular injection of leptin (4 μg) significantly increased PDE3B activity by two-fold in the hypothalamus as expected. However, previous administration of wortmannin completely reversed the stimulatory effect of leptin on PDE3B activity in the hypothalamus. To investigate whether leptin stimulates phospho (p)-Akt levels and that there might be a possible upstream regulator of PDE3B, we examined the effects of i.c.v. leptin on p-Akt levels in the hypothalamus and compared them with the known stimulatory effect of insulin on p-Akt. We observed that insulin increased p-Akt levels but leptin failed to do so, although it increased p-STAT3 levels, in the rat hypothalamus. Immunocytochemistry confirmed the biochemical findings in that leptin failed but insulin increased the number of p-Akt positive cells in various hypothalamic nuclei. Taken together, these results implicate PI3K but not Akt as an upstream regulator of the PDE3B pathway of leptin signalling in the rat hypothalamus.

Differential regulation of adipocyte PDE3B in distinct membrane compartments by insulin and the β3-adrenergic receptor agonist CL316243: effects of caveolin-1 knockdown on formation/maintenance of macromolecular signalling complexes

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.

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB

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.

Insulin‐induced formation of macromolecular complexes involved in activation of Phosphodiesterase3B (PDE3B) in 3T3‐L1 adipocytes

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.

Identification of the macromolecular complex responsible for PI3Kγ-dependent regulation of cAMP levels

PI3Kgamma is a phosphoinositide 3-kinase characterized by both lipid and protein kinase activity. It is activated by G-protein-coupled receptors and is predominantly expressed in leucocytes; in addition, recent work showed its presence in the heart and its involvement in regulating cardiac functions. In this tissue, PI3Kgamma acts as a negative modulator of contractility, by decreasing cAMP concentration through a kinase-independent mechanism. Indeed, whereas PI3Kgamma-deficient mice show an abnormal cAMP elevation, cAMP levels in knock-in mouse mutants, expressing a kinase-dead PI3Kgamma, are comparable with wild-type controls. PI3Kgamma regulates cardiac cAMP homoeostasis by forming a macromolecular complex containing PDE3B (phosphodiesterase 3B). In this complex, PI3Kgamma could regulate PDE3B activity through protein kinase A, a PDE activator.

Plasma membrane cyclic nucleotide phosphodiesterase 3B (PDE3B) is associated with caveolae in primary adipocytes

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.

Signaling Mechanism for the Insulin-like Effects of Growth Hormone - Another Example of a Classical Hormonal Negative Feedback Loop

Growth hormone (GH) exerts many effects in addition to its ability to stimulate growth. The metabolic effects are either chronic diabetogenic or acute insulin-like. The latter effects are only seen in cells that have been deprived of the hormone for a few hours. After exposure to GH the ability of the cells to respond with insulin-like effects disappears within a couple of hours, a negative feedback loop, which is a part of the chronic effects of the hormone. The insulin-like effects are mediated by the cytosolic tyrosine kinase Janus kinase 2 (JAK2) upon GH-GH receptor interaction, resulting in tyrosine phosphorylation of downstream targets including the GH receptor itself and insulin receptor substrate-1 (IRS-1) and IRS-2. Analogous to the post-receptor events for insulin this results in recruitment of phosphatidylinositol-3 kinase (PI3-kinase) to the IRS-proteins. Downstream PI3-kinase protein kinase B/Akt participates in the activation of glucose transporters (GLUT4) and increased glucose uptake as well as activation of phosphodiesterase 3B and hydrolysis of cAMP leading to a net dephosphorylation of the hormone sensitive lipase and inhibition of lipolysis. Simultaneously, JAK2 phosphorylates STAT-family transcription factors that move into the nucleus and activate the transcription of, among others, genes coding for negatively regulatory proteins called Suppressors of cytokine signalling (SOCS). The turnover of SOCS is rapid and in their presence JAK2 will still activate STAT-proteins (and the diabetogenic effects), but no longer phosphorylate the IRS-proteins (and induce insulin-like effects), closing the loop of yet another classical hormonal negative feedback loop.

Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.

HSL (hormone-sensitive lipase) is a key enzyme in the mobilization of fatty acids from acylglycerols in adipocytes as well as non-adipocytes. In adipocytes, catecholamines stimulate lipolysis mainly through PKA (protein kinase A)-mediated phosphorylation of HSL and perilipin, a protein coating the lipid droplet. The anti-lipolytic action of insulin is mediated mainly via lowered cAMP levels, accomplished through activation of phosphodiesterase 3B. Phosphorylation of HSL by PKA occurs at three sites, the serines 563, 659 and 660, both in vitro and in primary rat adipocytes. Phosphorylation of Ser-659 and -660 is required for in vitro activation as well as translocation from the cytosol to the lipid droplet, whereas the role of the third PKA site remains elusive. Adipocytes isolated from homozygous HSL-null mice, generated in our laboratory, exhibit completely blunted catecholamine-induced glycerol release and reduced fatty acid release, suggesting the presence of additional, although not necessarily hormone-activatable, triacylglycerol lipase(s). Basal hyperinsulinaemia, release of exaggerated amounts of insulin during glucose challenges and retarded glucose disposal during insulin tolerance tests suggest that HSL-null mice are insulin resistant. Liver, adipose tissue and skeletal muscle appear all to be sites of impaired insulin sensitivity in HSL-null mice.

Systematic search for single nucleotide polymorphisms in the 5 ′ flanking region of the human phosphodiesterase 3B gene: absence of evidence for major effects of identified polymorphisms on susceptibility to Japanese type 2 diabetes

The activation of phosphodiesterase 3B (PDE3B) reduces free fatty acid output from adipocytes. A reduced PDE3B gene expression could lead to insulin resistance. To determine whether there are polymorphisms associated with type 2 diabetes in PDE3B gene promoter, this 5 ′ flanking region was isolated. The transcription initiation site was located 206 bp upstream from the translation start site. Sequences of 2 kb of the 5 ′ flanking region for 24 type 2 diabetic Japanese subjects were initially analyzed using PCR direct sequencing, and the regions including the identified polymorphisms were then examined. In 98 controls and 98 type 2 diabetic subjects, −1947T > C, −567G > A, −465G > T, −458T > C, and −1727_−1726insTCAATT were found. Only −465G > T and this insertion had more than 5% frequencies. Since a complete linkage disequilibrium existed between them, −465G > T was further analyzed, along with a previously identified +1389G > A in the coding region, in a total of 200 controls and 207 type 2 diabetic subjects. These allele frequencies were not significantly different between these two groups (controls vs. cases; −465G > T, 12.0% vs. 10.1%, P=0.435; +1389G > A, 30.3% vs. 33.3%, P=0.408). These genotype distributions were not significantly different between these two groups. The T/T genotype at −465 was rare although this frequency could be higher in type 2 diabetes (4/207 subjects) than controls (0/200 subjects). The linkage disequilibrium existed between −465G > T and +1389G > A, and the estimated haplotype frequencies defined by these SNPs were not significantly different between the cases and controls. Thus, the identified polymorphisms are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation.

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.

Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells.

Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba-, F/Bb-, F/Bc-) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B- cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities approximately 2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approximately 10-fold and PDE3B phosphorylation and activity ( approximately 4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased ( approximately 10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B- cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.

Leptin inhibits insulin secretion by activation of phosphodiesterase 3B.

The molecular signaling events by which leptin exerts its functions in vivo are not well delineated. Here, we show a novel leptin signaling mechanism that requires phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and subsequent suppression of cAMP levels. In pancreatic beta cells, leptin causes the activation of PDE3B, which leads to marked inhibition of glucagon-like peptide-1-stimulated insulin secretion. The effect of leptin is abolished when insulin secretion is induced with cAMP analogues that cannot be hydrolyzed by PDE3B. Selective inhibitors of PDE3B and PI 3-kinase completely prevent the leptin effect on insulin secretion and cAMP accumulation. The results demonstrate that one of the physiological effects of leptin, suppression of insulin secretion, is mediated through activation of PDE3B and suggest PDE3B as a mediator of leptin action in other tissues.

Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B.

Both insulin and insulin-like growth factor 1 (IGF-1) are known to reduce glucose-dependent insulin secretion from the beta cells of pancreatic islets. In this paper we show that the mechanism by which IGF-1 mediates this effect is in large part through activation of a specific cyclic nucleotide phosphodiesterase, phosphodiesterase 3B (PDE3B). More specifically, in both isolated pancreatic islets and insulin-secreting HIT-T15 cells, IGF-1 inhibits insulin secretion that has been increased by glucose and glucagonlike peptide 1 (GLP-1). Moreover, IGF-1 decreases cAMP levels in parallel to the reduction of insulin secretion. Insulin secretion stimulated by cAMP analogs that activate protein kinase A and also are substrates for PDE3B is also inhibited by IGF-1. However, IGF-1 does not inhibit insulin secretion stimulated by nonhydrolyzable cAMP analogs. In addition, selective inhibitors of PDE3B completely block the ability of IGF-1 to inhibit insulin secretion. Finally, PDE3B activity measured in vitro after immunoprecipitation from cells treated with IGF-1 is higher than the activity from control cells. Taken together with the fact that pancreatic beta cells express little or no insulin receptor but large amounts of IGF-1 receptor, these data strongly suggest a new regulatory feedback loop model for the control of insulin secretion. In this model, increased insulin secretion in vivo will stimulate IGF-1 synthesis by the liver, and the secreted IGF-1 in turn feedback inhibits insulin secretion from the beta cells through an IGF-1 receptor-mediated pathway. This pathway is likely to be particularly important when levels of both glucose and secretagogues such as GLP-1 are elevated.