<p>Upon an inflammation the immune system signals the brain by secreted cytokines to elicit central nervous responses such as fever, loss of appetite and secretion of stress hormones. Since the blood brain barrier, (BBB) protects the brain from unwanted material, molecules like cytokines are not allowed to cross the barrier and enter the brain. However, it is clear that they in some way can signal the brain upon an inflammation. Many suggestions concerning this signaling has been made, one being that cytokines bind to receptors on the endothelial cells of the blood vessels of the brain and trigger the production of prostaglandins that can cross the BBB. This conversion is catalyzed by the enzyme cyclooxygenase-2, (COX-2), which is induced by transcription factors like NF-κB in response to cytokines. One of the central nervous responses to inflammatory stimuli is activation of the HPA-axis whose main purpose is glucocorticoid production. Glucocorticoids inhibit the inflammatory response by suppressing gene transcription of pro-inflammatory genes including those producing prostaglandins through direct interference with transcription factors such as NF-κB or initiation of transcription of anti-inflammatory genes like IκB or IL-10. It has however not been clear if glucocorticoids can target the endothelial cells of the brain in order to provide negative feed-back on the immune-to-brain signaling, and in that way inhibit central nervous inflammatory symptoms. An anatomical prerequisite for such a mechanism would be that the induced prostaglandin production occurs in cells expressing GR. This has however never been demonstrated. Here I show that a majority of the brain endothelial cells expressing the prostaglandin synthesizing enzyme COX-2 in response to immune challenge also express the glucocorticoid receptor, (GR). This indicates that immune-to-brain signaling is a target for negative regulation of inflammatory signaling executed by glucocorticoids and identifies brain endothelial GR as a possible future drug target for treatment of central nervous responses to inflammation such as fever and pain.</p>

<p>Protein tyrosine kinases play an important role in the regulation of various cellular processes such as</p><p>growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27^KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.</p>

<p>Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium.</p><p>Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. </p><p>Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. N^ω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. </p><p>In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.</p>

<p>The kidney is the major organ regulating the extracellular fluid volume and thereby the arterial blood pressure. The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa cells. These cells are sensors for both renin release and the tubuloglomerular feedback mechanism (TGF), which is an important regulator of the glomerular filtration rate and afferent arteriole tone. The aim of this investigation was to elucidate the function of nNOS in the macula densa cells.</p><p>Acute nNOS inhibition in rats resulted in an increased TGF responsiveness and unchanged blood pressure while, after chronic inhibition, the TGF was normalised and the blood pressure was elevated. The plasma renin concentration was elevated in rats on long-term low salt diet, but was not significantly affected by chronic nNOS inhibition. On the other hand, nNOS inhibition for four days increased plasma renin concentration in rats treated with a low salt diet. The renal vasculature of rats exhibits a diminished renal blood flow and intracellular Ca2+ response to angiotensin II after one week blockade of nNOS while angiotensin II’s effect on the renal blood flow was abolished after four weeks treatment. Acute extracellular volume expansion diminish the TGF sensitivity thus assisting the elimination of excess fluid but after acute addition of nNOS inhibitor to volume expanded rats the TGF sensitivity restored.</p><p>In conclusion, the results from the present study suggest an important role for nNOS in the macula densa cells in the regulation of the arterial blood pressure and the modulation of the TGF response.</p>

<p>Formation of blood vessels occurs through two processes: vasculogenesis and angiogenesis, which are regulated by various growth factors such as vascular endothelial growth factor, fibroblast growth factor and platelet-derived growth factor. The present study was carried out in order to investigate the roles of the Shb and Cbl proteins in growth factor-mediated signal transduction and blood vessel formation. Shb was found to be involved in NGF-stimulated Rap1 signaling in PC12 cells by forming a complex with CrkII and a 130-135 kDa protein. The Rap1 signaling pathway contributed to NGF-dependent neurite outgrowth. In immortomouse brain endothelial (IBE) cells, Shb increased cell spreading, migration and cytoskeletal rearrangements. Such effects may partly be due to altered Rap1 activation in Shb overexpressing IBE cells. Shb was required for tubular morphogenesis in collagen gels in the presence of FGF-2. In embryoid bodies (EBs) derived from murine embryonic stem cells, Shb up-regulated both VEGFR2 and Tal1 expression at early stages of EB development and thus promoted blood vessel formation both in the absence and in the presence of growth factors. In IBE cells, Cbl positively regulated FGF-2 signaling and increased cell proliferation. Mutation of RING finger alone did not affect blood vessel formation in EBs. However, EBs overexpressing the oncogenic form Cbl 70Z, which had a deletion of the linker region and the first cysteine of the RING finger, exhibited intense CD31 positive sheet-like staining and blood vessel. The results suggested that Cbl had dual roles in endothelial cells: it promoted FGF-2-induced proliferation whereas down-regulated proliferation of endothelial progenitor cells.</p><p>The present work suggests that Shb and Cbl play a crucial role in cell differentiation and blood vessel formation.</p>

<p>The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation may impair islet function.</p><p>In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. <i>In vitro</i>, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. <i>In vivo</i>, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.</p><p>Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The <i>in vitro</i> data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE₂ production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.</p>

<p>Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca²⁺]_i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from <i>ob/ob</i>-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K⁺ channels (K_ATP channels), depolarization, and Ca²⁺ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca²⁺]_i. When [Ca²⁺]_i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca²⁺]_i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca²⁺]_i oscillations are present. By permanently opening or closing the K_ATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca²⁺]_i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca²⁺]_i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca²⁺]_i, acting in synergy.</p>

<p>The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function.</p><p>Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. </p><p>Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect.</p><p>Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. </p><p>IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet.</p><p>It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.</p>

<p>Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.</p>

<p>Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy.</p><p> The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain.</p><p> Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. </p><p> The urinary excretion of the lipid peroxidation marker 8-iso-PGF_2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF_2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.</p>