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

Hormone-Sensitive Lipase and Protein Kinase B; Molecular characterization in testis, adipose tissue and pancreatic beta cells.

Hormone-sensitive lipase (HSL) and protein kinase B (PKB) are enzymes that in different ways are associated with lipid metabolism. While HSL is the well-known lipolytic enzyme responsible for hydrolysis of stored triacylglycerols in adipose tissue, PKB is a recently identified serine/threonine protein kinase, ubiquitously expressed and involved in signal transduction pathways induced by insulin and growth factors. Here, with focus on lipid-containing tissues from which information on their presence was previously scarce or lacking, expression of HSL in testis and expression/regulation of PKB in adipocytes and pancreatic beta cells are reported. A testicular isoform of HSL (HSLtes) is formed through addition of a unique N-terminal domain, comprising 300 amino acids, to the 768/775 amino acids, respectively, of rat and human HSLadi, i.e., the adipocyte form. The testis-specific part is more hydrophilic than HSLadi. In man, this addition occurs through alternative splicing of a testis-specific exon located 16 kb upstream of the first exon encoding HSLadi. HSLtes expression occurs primarily in spermatozoa and maturing sperm and not in the endocrine Leydig cells. HSLtes appears to exert a lipase/cholesterol esterase activity similar to that of HSLadi but its exact role is yet unknown. PKB isoforms (alfa, beta and gamma) are expressed in rat adipocytes and beta cells of pancreatic islets. Insulin stimulation of rat adipocytes leads to rapid and reversible phosphorylation and activation of PKB. Activation of PKB by the insulin-mimetic compound peroxovanadate is associated with translocation of PKB from the cytosol to the membrane fraction of rat adipocytes. In clonal beta cells, PKB is phosphorylated and activated upon stimulation with IGF-I. In both cells, the activation of PKB is abolished through inhibition of phosphatidylinositol-3 kinase (PI3K), suggesting a position for PKB down-stream of PI3K in signalling by insulin/IGF-I in these cells

Research and education form competing activity systems in externally funded doctoral education

ABSTRACTSeveral authors have described how the formalization of recent decades has steered doctoral education towards structured curricula, more managerial control and new models for supervision. Largely absent from these accounts, however, is if and how doctoral education has been affected by the concurrent changes in research governance, in particular by the ‘projectification’ of research. For this study, we were interested in the convergence of educational formalization with research projectification around doctoral education in the context of highly competitive, externally funded research in medicine and health sciences in Sweden. Using Cultural-historical activity theory and constructing activity systems for education and research, respectively, we were able to identify several contradictions and tensions, both within and between systems, that were consequences of adaptations to the abovementioned formalization and research policy changes. The contradictions were manifested in the tying of doctoral students, and their education, to their supervisors’ research projects, grants and future prospects, and in students being deprived of opportunities for learning and developing independence. Supervisors were torn between supervision and project management while doctoral students had to balance being students and project members. Our analysis provides a system level explanation to previously reported pedagogical and ethical challenges in STEM doctoral education

beta-cell PDE3B regulates Ca(2+)-stimulated exocytosis of insulin.

cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-I (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K+ for 5 min, was significantly reduced (similar to 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K+ was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca2+-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca2+, plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells. (c) 2007 Elsevier Inc. All rights reserved

Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B

To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells

The hormone-sensitive lipase (LIPE) gene located on chromosome 19q13.1-->13.2 is not duplicated on 19p13.3

The existence of a DNA polymorphism at the hormone-sensitive lipase locus could be of great interest for genetic analysis of obesity and related disorders since hormone-sensitive lipase is the rate-limiting enzyme of adipose tissue lipolysis and therefore plays a key role in energy metabolism. The polymorphic dinucleotide repeat D19S120 was identified within a human genomic clone selected with a rat hormone-sensitive lipase cDNA. This marker was subsequently localized to the short arm of chromosome 19 (p13.3) whereas human hormone-sensitive lipase (LIPE) had been mapped to the long arm of chromosome 19 (q13.1-->13.2). A duplication of the hormone-sensitive lipase gene or the presence of a pseudogene could explain the discrepancy. Cosmids from the two regions were analyzed in Southern blot experiments. A human adipose tissue hormone-sensitive lipase full-length cDNA probe hybridized only to cosmids from the 19q13.1-->13.2 region whereas the D19S120 amplicon probe hybridized only to cosmids from the p13.3 region. These data show that the occurrence of gene duplication or the presence of a pseudogene on the short arm of chromosome 19 is very unlikely and that D19S120 is unrelated to the hormone-sensitive lipase gene