Phosphodiesterase 3B Is Localized in Caveolae and Smooth ER in Mouse Hepatocytes and Is Important in the Regulation of Glucose and Lipid Metabolism

Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes

Expression and Regulation of Cyclic Nucleotide Phosphodiesterases in Human and Rat Pancreatic Islets

As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic β-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for β-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets

The role of phosphodiesterase 3B in energy metabolism

Energy metabolism at the level of any particular tissue is tightly regulated by hormones, neurotransmitters and cytokines in order to maintain overall energy homeostasis. Any dysfunctions in energy metabolism may lead to more or less severe consequences, with obesity and type 2 diabetes being the most prevalent ones. Type 2 diabetes is characterized by defective insulin secretion (?-cell dysfunction) and action (insulin resistance). The cAMP-degrading enzyme, PDE3B mediates antilipolytic insulin action in adipocytes and antiglycogenolytic insulin action in hepatocytes. The aim of this work was to evaluate the role of PDE3B in overall energy homeostasis as well as in insulin-induced glucose uptake and lipogenesis in adipocytes. Furthermore, we investigated mechanisms whereby PDE3B could regulate energy metabolism as well as long-term regulation of the enzyme by insulin and catecholamines. Used models included PDE3B KO mice generated for the first time, primary adipocytes and cell lines. The results indicate that PDE3B plays unique roles in adipocytes, liver and pancreatic ?-cells, and is crucial for the regulation of overall energy metabolism. Furthermore, in adipocyes, PDE3B is involved in the regulation of insulin-induced glucose uptake and lipogenesis, presumably via regulation of cAMP/Epac signalling which has an impact on phosphorylation and activation of PKB and ACC. At last, in adipocytes PDE3B and PDE4 are regulated by insulin and catecholamines also on a long-term basis. Altered expression of adipocyte PDEs by insulin and catecholamines may contribute to altered regulation of metabolism in obesity and diabetic states

Novel mechanisms of the regulation of Protein kinase B in adipocytes; implications for Protein kinase A, Epac, Phosphodiesterases 3 and 4.

Crosstalk between insulin and cAMP signalling pathways has a great impact on adipocyte metabolism. Whilst Protein kinase B (PKB) is a pivotal mediator of insulin action, in some cells regulation of PKB by cAMP has also been demonstrated. Here we provide evidence that, in a phosphatidyl inositol 3-kinase dependent manner, beta 3-adrenergic stimulation (using CL316243) in adipocytes induces PKB phosphorylation in the absence of insulin and also potentiates insulin-induced phosphorylation of PKB. Interestingly, insulin- and CL316243-induced PKB phosphorylation was found to be inhibited by pools of cAMP controlled by PDE3B and PDE4 (mainly in the context of insulin), whereas a cAMP pool controlling protein kinase A appeared to mediate stimulation of PKB phosphorylation (mainly in the context of CL316243). Furthermore, an Epac (exchange protein directly activated by cAMP) agonist (8-pCPT-2'-O-Me-cAMP) mimicked the effect of the PDE inhibitors, giving evidence that Epac has an inhibitory effect on PKB phosphorylation in adipocytes. Further, we put the results obtained at the level of PKB in the context of possible downstream signalling components in the regulation of adipocyte metabolism. Thus, we found that overexpression of PKB induced lipogenesis in a PDE3B-dependent manner. Furthermore, overexpression or inhibition of PDE3B was associated with reduced or increased phosphorylation of the key lipogenic enzyme acetyl-CoA carboxylase (ACC), respectively. These PDE3B-dependent effects on ACC correlated with changes in lipogenesis. The Epac agonist, 8-pCPT-2'-O-Me-cAMP, mimicked the effect of PDE3B inhibition on ACC phosphorylation and lipogenesis. (c) 2006 Elsevier Inc. All rights reserved

Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocytes.

In adipocytes, phosphorylation and activation of PDE3B is a key event in the antilipolytic action of insulin. The role of PDE4, another PDE present in adipocytes, is not yet known. In this work we investigate the role of PDE3B and PDE4 in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis. Inhibition of PDE3 (OPC3911, milrinone) but not PDE4 (RO 20-1724) lowered insulin-induced glucose uptake and lipogenesis, especially in the presence of isoproterenol (a general beta-adrenergic agonist), CL316243, a selective beta 3-adrenergic agonist, and pituitary adenylate cyclase-activating peptide. The inhibitory effect of OPC3911 was associated with reduced translocation of GLUT-4 from the cytosol to the plasma membrane. Both OPC3911 and RO 20-1724 increased protein kinase A (PKA) activity and lipolysis. H89, a PKA inhibitor, did not affect OPC3911-mediated inhibition of insulin-induced glucose uptake and lipogenesis, whereas 8-pCPT-2'-O-Me-cAMP, an Epac agonist which mediates PKA independent cAMP signaling events, mimicked all the effects of OPC3911. Insulin-mediated activation of protein kinase B, a kinase involved in insulin-induced glucose uptake, was apparently not altered by OPC3911. In summary, our data suggest that PDE3B, but not PDE4, contributes to the regulation of insulin-induced glucose uptake, GLUT-4 translocation, and lipogenesis presumably by regulation of a cAMP/Epac signalling mechanisms. (c) 2005 Elsevier Inc. All rights reserved

Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocytes.

Phosphodiesterase 3B (PDE3B), is known to play an important role in acute insulin and cAMP-mediated regulation of lipid metabolism, and PDE4 are the main PDE types expressed in adipocytes. Here, we show that members of all PDE4 isoforms are expressed in 3T3-L1 and primary mouse adipocytes. Long-term treatment of 3T3-L1 adipocytes with insulin induced up-regulation of PDE3B and PDE4D in a phosphatidylinositol 3-kinase-dependent manner whereas long-term treatment with P-adrenergic agonists induced down-regulation of PDE3B and up-regulation of PDE4D. Thus, PDE3B and PDE4D can be added to the list of genes regulated by insulin and cAMP-increasing hormones. Altered expression of PDE3B and PDE4D in response to long-term treatment with insulin and catecholamines may contribute to altered regulation of metabolism in diabetes. (c) 2006 Elsevier Inc. All rights reserved

Regulation of AMP-activated protein kinase by cAMP in adipocytes: Roles for phosphodiesterases, protein kinase B, protein kinase A, Epac and lipolysis.

AMP-activated protein kinase (AMPK) is an important regulator of cellular energy status. In adipocytes, stimuli that increase intracellular cyclic AMP (cAMP) have also been shown to increase the activity of AMPK. The precise molecular mechanisms responsible for cAMP-induced AMPK activation are not clear. Phosphodiesterase 3B (PDE3B) is a critical regulator of cAMP signaling in adipocytes. Here we investigated the roles of PDE3B, PDE4, protein kinase B (PKB) and the exchange protein activated by cAMP 1 (Epac1), as well as lipolysis, in the regulation of AMPK in primary rat adipocytes. We demonstrate that the increase in phosphorylation of AMPK at T172 induced by the adrenergic agonist isoproterenol can be diminished by co-incubation with insulin. The diminishing effect of insulin on AMPK activation was reversed upon treatment with the PDE3B specific inhibitor OPC3911 but not with the PDE4 inhibitor Rolipram. Adenovirus-mediated overexpression of PDE3B and constitutively active PKB both resulted in greatly reduced isoproterenol-induced phosphorylation of AMPK at T172. Co-incubation of adipocytes with isoproterenol and the PKA inhibitor H89 resulted in a total ablation of lipolysis and a reduction in AMPK phosphorylation/activation. Stimulation of adipocytes with the Epac1 agonist 8-pCPT-2'O-Me-cAMP led to increased phosphorylation of AMPK at T172. The general lipase inhibitor Orlistat decreased isoproterenol-induced phosphorylation of AMPK at T172. This decrease corresponded to a reduction of lipolysis from adipocytes. Taken together, these data suggest that PDE3B and PDE4 regulate cAMP pools that affect the activation/phosphorylation state of AMPK and that the effects of cyclic AMP on AMPK involve Epac1, PKA and lipolysis

PDE3B is present in caveolae-enriched fractions of hepatocyte plasma membranes.

<p>Plasma membranes isolated from mouse hepatocytes (50–120×10⁶) were sonicated in NaCO₃ and subjected to sucrose gradient fractionation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004671#s2" target="_blank">Materials and Methods</a>. Fractions of 0.8 ml were collected starting from the top. The fractions were analyzed for PDE3 activity and cholesterol (a), proteins (b) and subjected to immunoblot analysis (90 µl of each fraction) using antibodies against PDE3B, caveolin-1 (Cav-1) and flotillin-1 (Flot-1) (c). One representative experiment is shown (n = 4).</p

Transmission electron micrographs of mouse hepatocytes; PDE3B is localized to the smooth ER.

<p>Sections of lowicryl embedded mouse livers with immunogold labeling for PDE3B (A and C). B and D: negative control with omission of the primary antibody. C and D are higher magnifications of the indicated areas in A and B, respectively. Arrows show examples of gold labeling. Representative micrographs are shown (n = 4). G; Glycogen storage M; Mitochondria, Nucl; nucleus, rER; rough endoplasmic reticulum, sER; smooth ER.</p