The Role of Phosphodiesterase 3B in the Regulation of Insulin Secretion

Pancreatic beta-cell dysfunction and insulin resistance are the two hallmarks of type 2 diabetes. An early sign of beta-cell dysfunction is impaired nutrient-induced insulin release. Several insulin secretagogues act by increasing the formation of intracellular cAMP. Thus, accurate regulation of cAMP is of vital importance for the ability of the beta-cell to respond properly to these stimuli. The level of cAMP is defined by the activities of adenylyl cyclases and cAMP-degrading phosphodiesterases (PDEs). The aim of this thesis was to study the role of PDE3B in the regulation of insulin secretory processes in pancreatic beta-cells and in the regulation of overall energy homeostasis. Results in this thesis demonstrate that mice with a specific increase in beta-cell PDE3B activity (RIP-PDE3B mice) have, in comparison to control mice, a reduced insulin response to glucose as well as to glucose in combination with GLP-1, glucose intolerance and altered islet morphology. Moreover, when metabolically challenged RIP-PDE3B mice develop severe obesity and insulin resistance. The insulin secretory capacity of isolated islets from RIP-PDE3B mice was studied and a specific reduction in the first phase of glucose-stimulated insulin release was identified. An important role of beta-cell PDE3B for exocytosis and release of insulin was further demonstrated by overexpression of PDE3B and by selective inhibition of the enzyme in both insulinoma cell lines and rat pancreatic islets. Of specific interest was the marked decrease of glucose-stimulated cAMP levels and concomitant decrease in insulin release observed in cells overexpressing PDE3B. In summary, this thesis has contributed to an increased understanding for the role of beta-cell PDE3B in the regulation of insulin secretory processes. Results suggest that PDE3B regulates cAMP pools important for exocytosis of insulin-containing granules responsible for the first phase of insulin release. Also, these studies bring forward the role of cAMP in nutrient-induced insulin release. Finally, the work in this thesis demonstrates for the first time a functional role for beta-cell PDE3B in the maintenance of whole body energy homeostasis in mice

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

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

A Chloroplast Localized Small Heat Shock Protein, Hsp21. Importance of Hsp21 in stress protection of transgenic Arabidopsis thaliana, recombinant expression and characterization of oxidation-dependent conformational changes.

A specific group of heat shock proteins, the so called small heat shock proteins (sHsps), exists in all organisms but are especially abundant in plants. The sHsps prevent aggregation of other proteins (substrate proteins) during transient heat and oxidative stress. Molten globule forms of the substrate proteins are kept in a re-folding competent state by binding onto the outer surface of the sHsp. Members of the superfamily of sHsps and a-crystallins share a conserved C-terminal domain, the a-crystallin domain. This thesis is concerned with a chloroplast localized sHsp, Hsp21. In addition to the a-crystallin domain Hsp21 contains a conserved N-terminal domain predicted to form an amphipathic a-helix with highly conserved methionine residues on the hydrophobic side. Using transgenic Arabidopsis thaliana plants that overexpress Hsp21 I found that Hsp21 improves the plant performance during concomitant heat and light stress and that Hsp21 undergoes conformational changes during stress. To characterize the different conformations, recombinant Hsp21 protein was used and different biochemical and biophysical methods as circular dichroism, fluorescence spectroscopy, mass spectrometry and site-directed mutagenesis. A similar conformational change could be induced in the recombinant Hsp21 protein by oxidation with hydrogen peroxide. In the oxidized Hsp21 protein the highly conserved methionine residues was found to be in methionine sulfoxide form. The oxidized Hsp21 showed loss in a-helical secondary structure and chaperone activity. An enzyme, peptide methionine sulfoxide reductase, was able to reduce the methionine sulfoxides and recover the chaperone activity. The present data indicate that the methionine-rich amphipathic a-helix, which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone activity very dependent on the chloroplast redox state

Earnings management under coronapandemin : En kvantitativ studie av svenska börsnoterade företag

Denna uppsats undersöker förekomsten av earnings management under coronakrisen i svenska börsnoterade företag samt ämnar att undersöka huruvida förekomsten skiljer sig branscher emellan. För att undersöka detta har den modifierade Jones model för beräkningen av diskretionära periodiseringar tillämpats. De svenska börsnoterade företagens diskretionära periodiseringar och absoluta diskretionära periodiseringar undersöktes för perioden innan pandemin, 2018-2019, samt under pandemins första år, 2020. Statistiska tester upprättades, vilka visade att det förelåg en signifikant skillnad mellan perioden innan pandemin och pandemiåret gällande förekomsten av negativ earnings management. Vidare undersöktes huruvida förekomsten av earnings management minskat under pandemin, men ingen statistisk signifikans kunde fastställas för detta samband. Slutligen kunde ej en signifikant skillnad mellan nivån av earnings management mellan olika branscher fastställas. Varken före eller under pandemin

Earnings management under coronapandemin : En kvantitativ studie av svenska börsnoterade företag

Denna uppsats undersöker förekomsten av earnings management under coronakrisen i svenska börsnoterade företag samt ämnar att undersöka huruvida förekomsten skiljer sig branscher emellan. För att undersöka detta har den modifierade Jones model för beräkningen av diskretionära periodiseringar tillämpats. De svenska börsnoterade företagens diskretionära periodiseringar och absoluta diskretionära periodiseringar undersöktes för perioden innan pandemin, 2018-2019, samt under pandemins första år, 2020. Statistiska tester upprättades, vilka visade att det förelåg en signifikant skillnad mellan perioden innan pandemin och pandemiåret gällande förekomsten av negativ earnings management. Vidare undersöktes huruvida förekomsten av earnings management minskat under pandemin, men ingen statistisk signifikans kunde fastställas för detta samband. Slutligen kunde ej en signifikant skillnad mellan nivån av earnings management mellan olika branscher fastställas. Varken före eller under pandemin

What symptoms does the patient experience before heart transplantation and one year after? A quantitative study

Bakgrund: Hjärttransplantation är idag den mest effektiva behandlingen vid terminal hjärtsvikt. Under de senaste decennierna har utvecklingen av den mediciniska behandlingen efter hjärttransplantation gjort stora framsteg. Omvårdnaden till patienterna behöver därmed förbättras och vidareutvecklas. För att öka kunskaperna om hjärttransplanterade patienters symtom krävs studier som inriktar sig mot patientperspektivet. Syfte: Att undersöka samt jämföra personers skattade välbefinnande och upplevda symtom före hjärttransplantation och ett år efter. Metod: Patienter som genomgått hjärttransplantation vid Sahlgrenska universitetssjukhuset i Göteborg under åren 2013 till 2019 inkluderades och skattade sina symtom före- och ett år efter hjärttransplantation med enkäten Organ Transplant Symptom and Wellbeing Instrument (OTSWI). Instrumentet OTSWI är utvecklat i Sverige och har genomgått psykometrisk testning. Data analyserade med statistikprogrammet SPSS. Statistiska analyser som gjorts var deskriptiv- samt korrelationsanalys för att undersöka eventuella samband. Resultat: De vanligaste symtomen patienterna upplevde innan hjärttransplantation var: sover dåligt, har ingen energi samt är slö och håglös (95%). Symtom som andfåddhet, måste vila på grund av andfåddhet, har svårt att somna och fysisk trötthet var symtom som också var vanligt förekommande innan hjärttransplantationen. Majoriteten av de vanligaste symtomen efter hjärttransplantation var kopplade till fysisk hälsa, såsom darrningar i händer, fysisk trötthet, andfåddhet och ont i leder. Vid jämförelse av symtom så var det en signifikant förbättring vad gäller sömnen, fysisk trötthet, har ingen energi samt känner mig slö och håglös efter hjärttransplantation. Sexlusten hade ökat signifikant vid mätningen ett år efter hjärttransplantationen. Slutsats: Innan hjärttransplantation var större delen av upplevda symtom kopplade till psykisk hälsa i jämförelse med efter hjärttransplantation, då fysisk hälsa stod för majoriteten av upplevda symtom. För att ge en mer personcentrerad vård är det viktigt att tydliggöra individens specifikt upplevda symtom – och utifrån dessa utveckla vårdplaner, här kan OTSWI vara ett bra instrument

The Chloroplast Small Heat Shock Protein-Purification and Characterization of Pea Recombinant Protein

We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using anEscherichia coliexpression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5.0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment. This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens @a-crystallin. The crystallins are known from both crystallographic and spectroscopic data to be all-@b proteins. In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% @a-helical secondary structure