Imaging of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of PET Tracers for Vascular Inflammation

Atherosclerosis is a vascular inflammatory disease causing coronary artery disease, myocardial infarct and stroke, the leading causes of death in Finland and in many other countries. The development of atherosclerotic plaques starts already in childhood and is an ongoing process throughout life. Rupture of a plaque and the following occlusion of the vessel is the main reason for myocardial infarct and stroke, but despite extensive research, the prediction of rupture remains a major clinical problem. Inflammation is considered a key factor in the vulnerability of plaques to rupture. Measuring the inflammation in plaques non-invasively is one potential approach for identification of vulnerable plaques. The aim of this study was to evaluate tracers for positron emission tomography (PET) imaging of vascular inflammation. The studies were performed with a mouse model of atherosclerosis by using ex vivo biodistribution, autoradiography and in vivo PET and computed tomography (CT). Several tracers for inflammation activity were tested and compared with the morphology of the plaques. Inflammation in the atherosclerotic plaques was evaluated as expression of active macrophages. Systematic analysis revealed that the uptake of 18F-FDG and 11C-choline, tracers for metabolic activity in inflammatory cells, was more prominent in the atherosclerotic plaques than in the surrounding healthy vessel wall. The tracer for αvβ3 integrin, 18Fgalacto- RGD, was also found to have high potential for imaging inflammation in the plaques. While 11C-PK11195, a tracer targeted to receptors in active macrophages, was shown to accumulate in active plaques, the target-to-background ratio was not found to be ideal for in vivo imaging purposes. In conclusion, tracers for the imaging of inflammation in atherosclerotic plaques can be tested in experimental pre-clinical settings to select potential imaging agents for further clinical testing. 18F-FDG, 18F-galacto-RGD and 11C-choline choline have good properties, and further studies to clarify their applicability for atherosclerosis imaging in humans are warranted.

Imaging of Tumour Microenvironment for the Planning of Oncological Therapies Using Positron Emission Tomography

Tumour cells differ from normal tissue cells in several important ways. These differences, like for example changed energy metabolism, result in altered microenvironment of malignant tumours. Non-invasive imaging of tumour microenvironment has been at the centre of intense research recently due to the important role that this changed environement plays in the development of malignant tumours and due to the role it plays in the treatment of these tumours. In this respect, perhaps the most important characteristics of the tumour microenvironment from this point of view are the lack of oxygen or hypoxia and changes in blood flow (BF). The purpose of this thesis was to investigate the processes of energy metabolism, BF and oxygenation in head and neck cancer and pancreatic tumours and to explore the possibilities of improving the methods for their quantification using positron emission tomography (PET). To this end [18F]EF5, a new PET tracer for detection of tumour hypoxia was investigated. Favourable uptake properties of the tracer were observed. In addition, it was established that the uptake of this tracer does not correlate with the uptake of existing tracers for the imaging of energy metabolism and BF, so the information about the presence of tissue hypoxia cannot therefore be obtained using tracers such as [18F]FDG or [15O]H2O. These results were complemented by the results of the follow-up study in which it was shown that the uptake of [18F]EF5 in head and neck tumours prior to treatment is also associated with the overall survival of the patients, indicating that tumour hypoxia is a negative prognostic factor and might be associated with therapeutic resistance. The influences of energy metabolism and BF on the survival of patients with pancreatic cancer were investigated in the second study. The results indicate that the best predictor of survival of patients with pancreatic cancer is the relationship between energy metabolism and BF. These results suggest that the cells with high metabolic activity in a hypoperfused tissue have the most aggressive phenotype.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species’ adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-γ or IL-1β have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens’ ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine’s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1β produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.

Transgenic mice overexpressing neuropeptide Y: An experimental model of metabolic and cardiovascular diseases

Neuropeptide Y (NPY) is an abundant neurotransmitter in the brain and sympathetic nervous system (SNS). Hypothalamic NPY is known to be a key player in food intake and energy expenditure. NPY’s role in cardiovascular regulation has also been shown. In humans, a Leucine 7 to Proline 7 single nucleotide polymorphism (p.L7P) in the signal peptide of the NPY gene has been associated with traits of metabolic syndrome. The p.L7P subjects also show increased stress-related release of NPY, which suggests that more NPY is produced and released from SNS. The main objective of this study was to create a novel mouse model with noradrenergic cell-targeted overexpression of NPY, and to characterize the metabolic and vascular phenotype of this model. The mouse model was named OE-NPY^DBH mouse. Overexpression of NPY in SNS and brain noradrenergic neurons led to increased adiposity without significant weight gain or increased food intake. The mice showed lipid accumulation in the liver at young age, which together with adiposity led to impaired glucose tolerance and hyperinsulinemia with age. The mice displayed stress-related increased mean arterial blood pressure, increased plasma levels of catecholamines and enhanced SNS activity measured by GDP binding activity to brown adipose tissue mitochondria. Sexual dimorphism in NPY secretion pattern in response to stress was also seen. In an experimental model of vascular injury, the OE-NPY^DBH mice developed more pronounced neointima formation compared with wildtype controls. These results together with the clinical data indicate that NPY in noradrenergic cells plays an important role in the pathogenesis of metabolic syndrome and related diseases. Furthermore, new insights on the role of the extrahypothalamic NPY in the process have been obtained. The OE-NPY^DBH model provides an important tool for further stress and metabolic syndrome-related studies.

Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.

Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

Early nutritional determinants in cardio-metabolic programming – A prospective randomized controlled dietary intervention study

Western societies have been faced with the fact that overweight, impaired glucose regulation and elevated blood pressure are already prevalent in pediatric populations. This will inevitably mean an increase in later manifestations of cardio-metabolic diseases. The dilemma has been suggested to stem from fetal life and it is surmised that the early nutritional environment plays an important role in the process called programming. The aim of the present study was to characterize early nutritional determinants associating with cardio-metabolic risk factors in fetuses, infants and children. Further, the study was designated to establish whether dietary counseling initiated in early pregnancy can modify this cascade. Healthy mother-child pairs (n=256) participating in a dietary intervention study were followed from early pregnancy to childhood. The intervention included detailed dietary counseling by a nutritionist targeting saturated fat intake in excess of recommendations and fiber consumption below recommendations. Cardio-metabolic programming was studied by characterizing the offspring’s cardio-metabolic risk factors such as over-activation of the autonomic nervous system, elevated blood pressure and adverse metabolic status (e.g. serum high split proinsulin concentration). Fetal cardiac sympathovagal activation was measured during labor. Postnatally, children’s blood pressure was measured at six-month and four-year follow-up visits. Further, infants’ metabolic status was assessed by means of growth and serum biomarkers (32-33 split proinsulin, leptin and adiponectin) at the age of six months. This study proved that fetal cardiac sympathovagal activity was positively associated with maternal pre-pregnancy body mass index indicating adverse cardio-metabolic programming in the offspring. Further, a reduced risk of high split proinsulin in infancy and lower blood pressure in childhood were found in those offspring whose mothers’ weight gain and amount and type of fats in the diet during pregnancy were as recommended. Of note, maternal dietary counseling from early pregnancy onwards could ameliorate the offspring’s metabolic status by reducing the risk of high split proinsulin concentration, although it had no effect on the other cardio-metabolic markers in the offspring. At postnatal period breastfeeding proved to entail benefits in cardio-metabolic programming. Finally, the recommended dietary protein and total fat content in the child’s diet were important nutritional determinants reducing blood pressure at the age of four years. The intrauterine and immediate postnatal period comprise a window of opportunity for interventions aiming to reduce the risk of cardio-metabolic disorders and brings the prospect of achieving health benefits over one generation.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Oxidized LDL and physical fitness in healthy young men: associations with body composition, smoking, metabolic syndrome and androgen status

Background: In the past, oxidized low density lipoprotein (ox-LDL) has been associated with an unbeneficial lipid profile. This atherogenic lipid profile increases the risk of atherosclerotic cardiovascular diseases. Physical fitness has substantial effect on serum lipoprotein concentration as well as body composition and humoral responses, however interrelationships between ox-LDL and physical fitness have not been widely examined in a nationally representative sample. Aims: This thesis evaluates how cardiorespiratory and muscular fitness associate with ox-LDL lipids and how the other known risk factors of atherosclerosis might alter these associations. Subjects and Methods: The study cohort consisted of 846 healthy young males (mean age 25.1, SD 4.6) who were gathered by voluntary nationwide recruitment. Each participant conducted a series of physical fitness tests (cardiorespiratory and muscular fitness) and answered a detailed questionnaire that included lifestyle habits (i.e. smoking and leisuretime physical activity). Venous blood samples including ox-LDL and serum lipids were also collected. Results: Higher levels of ox-LDL were found in overweight and obese men, however, high cardiorespiratory fitness seemed to protect the overweight from high levels of ox-LDL. Young men who smoked and had poor cardiorespiratory or muscular fitness possessed a higher concentration of ox-LDL lipids when compared to comparable levels of cardiorespiratory or muscular fitness non-smoking young men. Metabolic syndrome was associated with increased levels of ox-LDL and high levels of ox-LDL combined with poor cardiorespiratory and abdominal muscle fitness seems to predict metabolic syndrome in young men. Also, participants with poor cardiorespiratory fitness and low levels of testosterone had higher levels of ox-LDL when compared to participants with high cardiorespiratory fitness / low testosterone as well as those with poor cardiorespiratory fitness / high testosterone. Conclusions: Good cardiorespiratory and muscular fitness protects young men from increased levels of ox-LDL lipids. This association was discovered in young men who were categorized as being overweight, smokers, metabolic syndrome or with low levels of testosterone. Being fit seems to prevent higher levels of ox-LDL, even in young healthy

Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds.

Alpha- and gamma-melanocyte stimulating hormones in obesity. A study of the key central areas of metabolic regulation

The melanocortin system is an important regulator of feeding, energy metabolism,and cardiovascular function and it consists of the pro-opiomelanocortin (POMC) derived melanocyte stimulating hormones (α-, β- and γ-MSH) and their endogenous melanocortin receptors, MC1R to MC5R. In the hypothalamus, α-MSH reduces food intake, and increases energy expenditure and sympathetic tone by binding to MC4R. Mutations affecting the MC4R gene lead to obesity in mammals. On the other hand, the metabolic effects of MC3R stimulation using agonists such as the endogenously expressed γ-MSH have been less extensively explored. The main objective of this study was to investigate the long-term effects of increased melanocortin tone in key areas of metabolic regulation in the central nervous system (CNS) in order to investigate the sitespecific roles of both α-MSH and γ-MSH. The aim was to stereotaxically induce local overexpression of single melanocortin peptides using lentiviral vectors expressing α-MSH (LVi-α-MSH-EGFP) and γ-MSH (LVi-γ-MSH-EGFP). The lentiviral vectors were shown to produce a long-term overexpression and biologically active peptides in cell-based assays. The LVi-α-MSHEGFP was targeted to the arcuate nucleus in the hypothalamus of diet induced obese mice where it reduced weight gain and adiposity independently of food intake. When the nucleus tractus solitarus in the brainstem was targeted, the LVi-α-MSH-EGFP treatment was shown to cause a small decrease in adiposity, which did not impact weight development. However, the α-MSH treatment increased heart rate, which was attenuated by adrenergic receptor blockade indicative of increased sympathetic activity. The LVi-γ-MSH-EGFP was targeted to the hypothalamus where it decreased fat mass in mice eating the standard diet, but the effect was abated if animals consumed a high-fat Western type diet. When the diet induced obese mice were subjected again to the standard diet, the LVi-γ-MSH-EGFP treated animals displayed increased weight loss and reduced adiposity. These results indicate that the long-term central anti-obesity effects of α-MSH are independent of food intake. In addition, overexpression of α-MSH in the brain stem efficiently blocked the development of adiposity, but increased sympathetic tone. The evidence presented in this thesis also indicates that selective MC3R agonists such as γ-MSH could be potential therapeutics in combination with low fat diets.

Bariatric and Post-Bariatric Surgery: from Metabolic Surgery to Plastic Surgery Indications

Background: Controversy exists concerning indications and outcomes of major bariatric surgery procedures. Massive weight loss after bariatric surgery leads to excess skin with functional and aesthetic impairments. The aim of this study was to investigate the major bariatric surgery procedures and their outcomes in two specific subgroups of morbidly obese patients, ≥55-year-olds and the superobese. Further aims were to evaluate whether the preoperative weight loss correlates with laparoscopic gastric bypass complications. The prevalence and impact of excess skin and the desire for body contouring after bariatric surgery were also studied. Patients and Methods: Data from patients who underwent Laparoscopic Adjustable Gastric Banding (LAGB) and Laparoscopic Roux-en-Y Gastric Bypass (LRYGB) at Vaasa Central Hospital were collected and postoperative outcomes were evaluated according to the BMI, age and preoperative weight loss. Patients who had undergone bariatric surgery procedures were asked to complete a questionnaire to estimate any impairment due to redundant skin and to analyse each patient’s desire for body contouring by area. Results: No significant difference was found in operative time, hospital stay, or overall early postoperative morbidity between LAGB and LRYGB. Mean excess weight loss percents (EWL%) at 6 and 12 months after LRYGB were significantly higher. A significant difference was found in operative time favouring patients <55 years. Intraoperative complications were significantly more frequent in the group aged >55 years. No significant difference was detected in overall postoperative morbidity rates. A significant difference was found in operative time and hospital stay favouring all patients who lost weight preoperatively. Most patients reported problems with redundant skin, especially on the abdomen, upper arms and rear/buttocks, which impaired daily physical activity in half of them. Excess skin was significantly associated with female gender, weight loss and ΔBMI. Patients with a WL >20 kg, ΔBMI ≥10 kg/m2 and an EWL % > 50 showed a significantly surplus skin discomfort (p < 0.001). Most patients desired body contouring surgery, with high or very high desire for waist/abdomen (62.2%), upper arm (37.6%), chest/breast (28.3%), and rear/buttock (35.6%) contouring. Conclusions: LRYGB is effective and safe in superobese (BMI >50) and elderly (>55 years) patients. A preoperative weight loss >5% is recommended to improve the outcomes and reduce complications. A WL >20 kg, ΔBMI ≥10 kg/m2 and an EWL % > 50 are associated with a higher functional discomfort due to redundant skin and to a stronger desire for body contouring plastic surgery.

Gut Microbiota and Metabolic Disorders

Obesity and its co-morbidities, such as metabolic syndrome (MetS), non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes, have increased over the last few decades like an epidemic. So far the mechanisms of many metabolic diseases are not known in detail and currently there are not enough effective means to prevent and treat them. Several recent studies have shown that the unbalanced gut microbiota composition (GMC) and activity have an influence on the fat accumulation in the body. Further, it seems that the GMC of obese individuals differs from the lean. The aim of this study was to investigate whether there are differences between the GMC of metabolically impaired overweight/obese (MetS group), metabolically healthy overweight/obese and normal-weight individuals. In addition, the mechanisms by which the gut bacteria as well as their specific structures, such as flagellin (FLG) that stimulates the Toll-like receptor 5 (TLR5) affect metabolism, were investigated both in vivo and in vitro in human adipocytes and hepatocytes. The results of this study show that the abundance of certain gram-positive bacteria belonging to the Clostridial cluster XIV was higher in the MetS group subjects compared to their metabolically healthy overweight/obese and lean counterparts. Metabolically impaired subjects tended to also have a greater abundance of potentionally inflammatory Enterobacteria in their gut and thus seemed to have aberrant GMC. In addition, it was found that subjects with a high hepatic fat content (HHFC group) had less Faecalibacterium prausnitzii in their gut than individuals with low hepatic fat content. Further gene expression analysis revealed that the HHFC group also had increased inflammation cascades in their adipose tissue. Additionally, metabolically impaired individuals displayed an increased expression of FLG-recognizing TLR5 in adipose tissue, and the TLR5 expression levels associated positively both with liver fat content and insulin resistance in humans. These changes in the adipose tissue may further contribute to the impaired metabolism observed, such as insulin resistance and dyslipidemia. In vitro -studies showed that the FLG-induced TLR5 activation in adipocytes enhanced the hepatic fat accumulation by decreasing insulin signaling and mitochondrial functions and increasing triglyceride synthesis due to increased glycerol secretion from adipocytes. In conclusion, the findings of this study suggest that it may be possible that the novel prevention and personalized treatment strategies based on GM modulation will succesfully be developed for obesity and metabolic disorders in the future.