Cyclic AMP is an intracellular messenger that regulates numerous processes in various types of cells. In pancreatic β-cells, cAMP potentiates the secretion of insulin by promoting Ca2+ signals and by amplifying Ca2+-triggered exocytosis. Whereas Ca2+ signals have been extensively characterized, little is known about the kinetics of cAMP signals. To enable measurements of the cAMP concentration beneath the plasma membrane ([cAMP]pm) of individual cells, a translocation biosensor was created based on fluorescent-protein-tagged subunits of protein kinase A (PKA). Evanescent wave microscopy imaging of biosensor-expressing clonal β-cells revealed that the insulinotropic hormones glucagon and GLP-1 triggered pronounced oscillations in [cAMP]pm. Simultaneous measurements of the intracellular Ca2+ concentration showed that cAMP and Ca2+ oscillations were synchronized and interdependent. [cAMP]pm oscillations were also triggered in clonal and primary mouse β-cells by an elevation of the glucose concentration from 3 to 11 mM. These oscillations were preceded and enhanced by elevations of Ca2+. However, conditions raising cytoplasmic ATP could trigger cAMP elevations also without accompanying Ca2+ changes, indicating that adenylyl cyclase activity may be directly controlled by the substrate concentration. Experiments with 3-isobutylmethylxanthine (IBMX) and various family-selective phosphodiesterase (PDE) inhibitors indicated that [cAMP]pm oscillations are generated by periodic formation of the messenger by adenylyl cyclases. PDE1 and PDE3 as well as IBMX-insensitive mechanisms shape [cAMP]pm, but no single PDE isoform was required for glucose generation of [cAMP]pm oscillations. Recordings of single-cell insulin secretion kinetics with a fluorescent biosensor that reports formation of the phospholipid PIP3 in the plasma membrane in response to autocrine insulin receptor activation showed that [cAMP]pm oscillations were paralleled by pulsatile insulin release. Whereas adenylyl cyclase inhibition suppressed both [cAMP]pm oscillations and pulsatile insulin release, elevation of [cAMP]pm enhanced secretion. Investigation of the effects of different temporal patterns of [cAMP]pm showed that brief [cAMP]pm elevation is sufficient to trigger cytoplasmic responses, whereas sustained elevation is required to induce translocation of the PKA catalytic subunit into the nucleus. In conclusion, these studies demonstrate for the first time in mammalian cells that [cAMP]pm oscillates in response to physiological stimuli. The glucose-induced [cAMP]pm oscillations are generated by periodic cAMP production mediated by interplay between ATP and Ca2+ in the sub-membrane space, and may contribute to both triggering and amplifying pathways of insulin secretion. Apart from regulating the precise kinetics of insulin exocytosis, temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.

Background and aims: Type 2 diabetes mellitus results from complex interplay between genetic predisposition and environmental factors that together promote impairment of insulin-producing β-cells. Elevated levels of glucose, fatty acid palmitate and apolipoprotein CIII (apoCIII) are implicated in this process. To delineate effects of these factors, the role of enhanced carnitine palmitoyltransferase 1 (CPT1) expression in glucolipotoxic cells, glucose-dependency of the unfolded protein response (UPR) in palmitate-induced apoptosis and activation of mitogen activated protein kinases (MAPKs) in apoCIII-induced apoptosis were evaluated. In addition, protein profiles of β-cell exposed to elevated levels of glucose or palmitate were generated to identify proteins regulated by these nutrients. Methodology: INS-1E cells were cultured at different glucose concentrations in the absence or presence of palmitate or apoCIII for up to 48 hours. CPT1 was over-expressed with a Tet-ON regulated adenovirus. In cells exposed to apoCIII, inhibitors of MAPKs p38 or ERK1/2 were included during culture. After culture, apoptosis, insulin secretion, expression of UPR-markers and MAPKs and protein profiles were determined. Results: INS-1E cells exposed to elevated levels of glucose and palmitate showed deranged insulin secretion with increased insulin secretion at non-stimulatory glucose level, enhanced apoptosis and induced expression of UPR-markers. Over-expression of CPT1 reduced basal insulin secretion and attenuated apoptosis. Palmitate-induced apoptosis was accentuated by increasing the culture glucose concentration. Markers of UPR were not modulated by the glucose concentration in INS-1E cell exposed to palmitate, however. ApoCIII-induced apoptosis in INS-1E cells was accompanied by activation of p38 and ERK1/2. Protein profiling of INS-1E cells exposed to elevated levels of glucose or palmitate revealed changes in expression of multiple β-cell proteins implicated in glucose metabolism, defence against reactive oxygen species, protein translation/folding/degradation and insulin granular trafficking. Conclusions: Over-expression of CPT1 counteracts β-cell glucolipotoxicity. Activation of UPR is not a major determinant for palmitate-induced β-cell apoptosis. ApoCIII-induced β-cell apoptosis involves activation of MAPKs. The identified differentially expressed proteins indicate a central role of altered glucose metabolism and protein synthesis in gluco- and lipotoxic β-cells and may provide specific molecular mechanisms offering new ways of treating the disease.

The adaptor protein Shb and the non-receptor tyrosine kinase Frk have been implicated in intracellular signalling in insulin-producing beta cells. In this thesis, knockout mice are used to further elucidate the role of Shb and Frk for beta cell number, cytokine-induced cell death, and glucose homeostasis. In addition, the effect of Shb deficiency upon tumour growth is studied in a mouse model of endogenous tumourigenesis. Previously, overexpression of Frk has been associated with increased beta cell replication, and increased susceptibility to cytokine induced beta cell destruction. To test whether Frk has a non-redundant role in regulating beta cell mass, beta cell number in Frk-/- mice was assessed at different stages of life. The results showed that Frk is involved in regulating beta cell number during embryonal and early postnatal life, but is probably redundant in the adult. An earlier study had suggested that Shb participates in cytokine-induced beta cell death, a model of autoimmune diabetes. To test this further, Shb-/- islets were exposed to cytokines, or to an ER-stress inducing agent. Shb knockout islets exhibited decreased cell death, and this effect appeared to be independent of NO, JNK, p38 MAP kinase, FAK and c-Abl, but may involve an augmented induction of Hsp70. Furthermore, glucose homeostasis in Shb-/- mice was impaired, with elevated basal blood sugar concentration and reduced glucose-induced insulin secretion. Previously Shb deficient mice had showed an impaired ability to sustain growth of implanted tumour cells, due to reduced angiogenesis. Tumour growth and angiogenesis were here assessed in an inheritable tumour model. Shb deficient mice exhibited fewer tumours, and reduced vessel density in small tumours, indicating impaired angiogenesis. However, a few large tumours developed in Shb-/- mice, suggesting that tumours can escape the angiogenic restriction caused by the absence of Shb.

Type 1 diabetes mellitus is a severe life-long disease with a pronounced risk of developing secondary complications. One way to avoid the latter is to restore the fine tuning of blood glucose homeostasis by transplantation of pancreatic islets. However, isolated islets need to be properly engrafted and to re-establish a vascular network in order to regain function. Earlier studies have shown that pancreatic islets experimentally transplanted to e.g. the liver or the kidney become poorly revascularized. In the present thesis, mice deficient of the angiostatic factor thrombospondin-1 (TSP-1) were found to have an impaired beta-cell function. Development of this beta-cell dysfunction was prevented by treatment of TSP-1 deficient mice from birth with the TGFbeta-1 activating sequence of TSP-1. TSP-1 in islets was predominantly expressed in the endothelial cells. Isolated islet endothelial cells was observed to have a low proliferatory and migratory capacity towards angiogenic stimuli, but this could be reversed by neutralizing antibodies to the angiostatic factors alpha1-antitrypsin, endostatin or TSP-1. Transient downregulation of TSP-1 expression in mouse islet cells prior to transplantation improved graft revascularization, blood perfusion, oxygenation and function when evaluated one-month post-transplantation. The same result was achieved when islets or recipients of islets were pre-treated with the hormone prolactin one-month post-transplantation. The present study illustrates the importance of the angiostatic factor TSP-1 for islet beta-cell function and engraftment of islets following transplantation. Interference with TSP-1 can possibly be used to improve the results of clinical islet transplantation.

Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and translation are still largely obscure, the work presented in this thesis therefore aimed to further investigate the role of insulin mRNA binding proteins in the control of insulin mRNA break-down and utilization for insulin biosynthesis. To clarify how glucose regulates insulin mRNA stability and translation we studied the correlation between polypyrimidine tract binding protein (PTB) gene expression and insulin mRNA levels. It was found that an increase in PTB mRNA and protein levels is paralleled by an increase in insulin mRNA levels. It was also found that PTB binds to the 5’-untranslated region of the insulin mRNA and that insulin mRNA can be translated through a cap-independent mechanism in human islets of Langerhans, possibly due to the interaction with PTB. Further it was discovered that the suppressed insulin biosynthesis in human islets during glucotoxicity is partly due to an induction of the microRNA miR-133a. This induction leads to decreased levels of PTB and insulin biosynthesis rates in human islets. Finally, we were able to identify two proteins, hnRNP U and TIAR, that bind specifically to the insulin mRNA in vitro, and show that the stability and translation of insulin mRNA is oppositely affected by these proteins. In conclusion, insulin producing cells seem to be able to regulate insulin messenger stability and translation by a control mechanism in which the binding of specific proteins to the insulin messenger dictates the outcome. A better understanding of the events leading to insulin production may in the future aid in optimal diagnosis and treatment of type 2 diabetes.

The development of different organs and tissues along the gastrointestinal tract, including the pancreas, depends on signalling between the endoderm and the adjacent mesenchyme. The Nkx gene Bapx1 is involved in spatial control of organ-positioning in the spleno-pancreatic region, and deficiency in this gene results in unacceptable proximity of the splenic mesenchyme to the pancreas. This permits agitating signals from the splenic mesenchyme to induce an in vivo (and in vitro) transformation of pancreatic epithelium to a cystic structure with gut like features. Also, wild type splenic mesenchyme is competent to induce a similar transformation. These findings illustrate the importance for strict control of organ positioning during spleno-pancreatic development. Several growth factors and receptors involved in pancreatic development are activated by protease processing. Some of these growth factors have been implicated as substrates for members of the A Disintegrin And Metalloprotease (ADAM) family. The ADAMs 9, 10, and 17 are expressed during pancreatic development and in the adult pancreas, suggesting a possible role for these ADAMs in pancreatic development and function. Animal model systems are widely used to investigate gene function during development and disease. However, spatial, molecular, and quantitative phenotype screening in animals is a time consuming effort. Optical Projection Tomography is a 3-dimensional imaging technique that, in combination with improvements in sample preparation and computer processing, can be used to visualize and quantify characteristics of intact adult mouse organs such as the total β-cell content in the pancreas.

The body is under constant attack from pathogens trying to slip by our immune defence. If the barrier is breached, invading pathogens enter the tissues and cause inflammation. During this process neutrophils, constituting the first line of defence, leave the bloodstream and seek out and kill the invading pathogens. The mechanisms leading to activation of receptors on neutrophils must be closely orchestrated. Pro- and anti-inflammatory substances can influence the outcome of the inflammation process by affecting the involved players. If not well balanced, inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, can be the outcome. The aim of this thesis was to elucidate the effect of pro- (fMLP, Leukotriene B4, and Interleukin-8) and anti- (lipoxins, aspirin and statins) inflammatory substances on the β2 integrins, mediating adhesion of neutrophils both under “normal” conditions and during coronary artery disease. More specifically, the effect of these substances on the β2 integrins were studied in regard to: i) the activity (i.e. affinity and avidity) of β2 integrins, ii) the signalling capacity of β2 integrins (i.e. detected as release of arachidonic acid, and the production of reactive oxygen species, and iii) the signal transduction mediated by the β2 integrins (i.e. phosphorylation of Pyk2). The pro-inflammatory substances belong to the family of chemoattractants that induces transmigration and chemotaxis. A hierarchy exists between the different family members; the end-target chemoattractants (e.g. fMLP) being more potent than intermediary chemoattractants (e.g. IL-8 and LTB4). It was found that intermediary chemoattractants regulate β2 integrins by mainly affecting the avidity of β2 integrins. End-target chemoattractants on the other hand, affected the β2 integrins by increasing the avidity and the affinity, as well as their signalling capacity. The anti-inflammatory substances used in this study were the exogenous aspirin and statins, and the endogenous lipoxins. In the presence of aspirin, stable analogues of lipoxin (i.e. epi-lipoxins) are formed in a trans-cellular process. Lipoxin inhibited the signalling capacity of β2 integrins mediated by intermediary chemoattractants, as well as the signal transduction induced by end-target chemoattractants. Moreover, the signalling capacity of β2 integrins in neutrophils from patients suffering from coronary artery disease (CAD) was impaired. Arachidonic acid, the precursor for both pro- and anti-inflammatory eicosanoid, induced an increase in the β2 integrin activity (both affinity and avidity), but had no effect on the signal transduction. In conclusion, different “roles” were observed for end-target and intermediary chemoattractants in the regulation of β2 integrins. The inhibitory effects of the anti-inflammatory lipoxins support earlier studies suggesting that these agents function as “stop signals” in inflammation. This is also confirmed by our findings in CAD patients, who have elevated levels of epi-lipoxins due to aspirin treatment. Moreover, Pyk2 was identified as a possible target for the inhibitory effect of anti-inflammatory drugs.

The microfilament system consists of actin filaments as the major component and is regulated by a number of actin binding proteins. It is juxtaposed to the plasma membrane where it forms a dense cortical weave from where it pervades into the cell interior. This filament system has multiple roles and participates in virtually all motile processes where its dynamic activities depend on receptor mediated signaling leading to constant polymerizations and depolymerizations. These activities are now also known to affect gene regulation. This thesis discusses these dynamic reorganizations of the microfilament system and how components are supplied to support these motile processes. The focus is on profilin/profilin:actin, actin polymerization and the localization of the transcripts of these proteins. The localization of profilin mRNA was examined in relation to the distribution of β-actin mRNA using fluorescent in situ hybridization. It was concluded that both these mRNAs localize to sites of massive actin polymerization called dorsal ruffles albeit the data obtained suggests that this localization must be dependent on distinct mechanisms. Additionally signal transduction and cell motility was studied after depleting the two profilin isoforms 1 and 2. The activity of the transcription factor SRF is known to be coupled to microfilament system dynamics via the cofactor MAL which binds to actin monomers and is released upon receptor mediated actin polymerization. Depletion of profilin was seen to influence SRF dependent signaling, most likely because the lack of profilin enables more MAL to bind actin monomers thereby preventing SRF dependent transcription. Finally, it was also investigated how the synaptic vesicle protein synaptotagmin 1 which is involved in exocytosis, has a role in actin polymerization. This protein has previously been described to cause filopodia formation when ectopically expressed. A polybasic sequence motif was identified as the effector sequence for this activity and it was established that this sequence interacts with anionic lipids. It is also discussed how this sequence could have a role in neurotransmitter release and actin polymerization in the nerve synapse. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted.

Humans have never before lived as long and we have never had as many cures and treatments for diseases as now. But we still spend the last years of our lives sick with age-related diseases. Some scientists say it is time to grasp the problem by its roots – which in this case is aging itself. A lot of studies show that life can be prolonged in model organisms with the diabetes medication metformin. In recent years, metformin has proven to both expand lifespan and inhibit age-related diseases such as cancer and stroke. Metformin can do this by five different mechanisms; 1) inhibition of mitochondrial complex I, which is a part of the electron transport chain, 2) inhibition of the accumulation of reactive oxygen species, which in turn protects DNA from damage and mutations, 3) activation of AMPK, a kinase that is activated in the body when energy levels are low, 4) decrease of signaling of IGF-1, a growth factor that has been linked to the development of tumors, and 5) inhibition of mTOR, a regulator of cell metabolism, growth, proliferation, survival and autophagy. Studies are constantly proving metformin to be useful in both treatment of diseases and in inhibiting aging. New studies like TAME (Targeting Aging with Metformin), where older patients will try out metformin fors everal years, will shed more light on whether metformin might have the same effect in humans. Metformin could potentially provide a new future of health care where we not only treat diseases, but also prolong aging itself.

Hemocytes are an important part of a crayfish’s immune system in helping tackling both virus and bacterial infections. Dscam is a protein that can be found in hemocytes, as well as many other tissues like the brain. In the brain, Dscam is thought to be important in the establishment of neuronal connections. Previous studies have found that the neurons in the crayfish brain do not replenish themselves, but instead are replenished by hemocytes that enter through a vascular cavity that pass through the neurogenic niche. There might be a specific type of hemocyte that is drawn to the niche and because of the link between Dscam and establishment of neuronal connections, Dscam have been chosen as a potential factor for this attraction. Dscam could be upregulated at many places along the way from the HPT to the brain. In this study, antibodies have been used to view BrdU and Dscam presence in hemocytes from crayfish P. leniusculus to find out where Dscam is upregulated and in what cells they are located. It was found that Dscam is not present on newly synthesized cells but rather on more differentiated cells, suggesting that Dscam is upregulated in older HPT cells or in circulation. It was found that LPS injections are an efficient way to upregulate Dscam in hemocytes and that expression of extracellular Dscam is peaking 24 hours post LPS injection.