ATM in focus:a damage sensor and cancer target

The ability of a cell to conserve and maintain its native DNA sequence is fundamental for the survival and normal functioning of the whole organism and protection from cancer development. Here we review recently obtained results and current topics concerning the role of the ataxia-telangiectasia mutated (ATM) protein kinase as a damage sensor and its potential as therapeutic target for treating cancer. This monograph discusses DNA repair mechanisms activated after DNA double-strand breaks (DSBs), i.e. non-homologous end joining, homologous recombination and single strand annealing and the role of ATM in the above types of repair. In addition to DNA repair, ATM participates in a diverse set of physiological processes involving metabolic regulation, oxidative stress, transcriptional modulation, protein degradation and cell proliferation. Full understanding of the complexity of ATM functions and the design of therapeutics that modulate its activity to combat diseases such as cancer necessitates parallel theoretical and experimental efforts. This could be best addressed by employing a systems biology approach, involving mathematical modelling of cell signalling pathways

The association between metabolic health status and smell perception in obesity: behavioral and brain anatomical correlates

Obesity is a major health concern that is accompanied by a high risk for several disorders such as type 2 diabetes, cardiovascular disease and certain forms of cancer (Stevens et al., 2012; Lahey and Khan, 2018). Since the prevalence of obesity has nearly tripled within the last 45 years and is still on the rise (WHO, 2018), it is imperative to understand mechanisms that might underlie the emergence and maintenance of obesity to develop new prevention- and intervention strategies. The high availability of energy-rich food is one of the main contributing factors for the increasing prevalence of obesity. Hence, the mechanisms underlying eating without physiological needs come into focus. In that regard, the olfactory system plays a major role: it is equally involved in homeostatic signaling of hunger and hedonic eating (Palouzier-Paulignan et al., 2012). Given that the sense of smell is altered in obesity (for an overview see Peng et al., 2019), the overall goal of this thesis is to contribute to further understanding of the mechanisms that might underlie this phenomenon. It has been previously shown that people with obesity evaluate food odors as more pleasant (Stafford and Whittle, 2015) and show higher reactivity towards them (Proserpio et al., 2019), however, they persistently have lower olfactory function. This low function is most evident in olfactory sensitivity, i.e. picking up odors from the environment, and is therefore related to appetite and food search. Odor sensitivity evaluation is usually a lengthy procedure and is standardly performed with non-food odors. However, Stafford and Whittle (2015), revealed a different result for sensitivity to food odors: obese outperformed normal-weight participants for chocolate odor. Strikingly, further scrutiny reveals that metabolic and endocrine health factors could provide a possible explanation for divergent results of olfactory sensitivity to food and non-food odors in obesity: whereas the chocolate-study included only class 1 obese participants (BMI 30-35 kg/m2), all other studies included class 2-3 obese participants (BMI > 35 kg/m2). It is likely that obese class 2-3 participants are more affected by hormonal changes, such as higher insulin resistance and higher leptin levels than their less obese counterparts. On a recent note, it has been shown that the olfactory and endocrine systems are closely linked (Palouzier-Paulignan et al., 2012). As such there are many receptors for hunger-related hormones located in brain structures that are highly relevant for odor processing as well as for the regulation of homeostatic needs (Baly et al., 2007; Lacroix et al., 2008; Henkin, 2010). Especially, the olfactory bulbs, where olfactory information is firstly processed in the brain, have a high density of insulin and leptin receptors (Baskin et al., 1983; Thanarajah et al., 2019; Havrankova et al., 1981; Marks et al., 1990). Further, animal studies have reliably demonstrated that obesity leads to structural and functional changes in the olfactory system (Thiebaud et al., 2014; Fadool et al.2011; Riviére et al., 2016). However, brain anatomical changes in the olfactory system of humans have not been studied yet. To conclude, we firstly aimed to develop an olfactory test that is easy to administer and of short duration to apply in a complex research design, because available tests are time consuming and highly variable in duration (10-25 min). Secondly, in order to elucidate the potential link between olfactory impairments in obesity and metabolic health factors, we investigated food and non-food odor sensitivity in a wide body weight range and related it to metabolic and endocrine factors such as insulin resistance. Third, we aimed to investigate the possible relationship between obesity and brain anatomical changes in the olfactory bulbs. Study 1: In our first study, we measured olfactory sensitivity in a within-subject repeated-measures design in 20 young and healthy participants. Using the odor detection threshold subtest from the “Sniffin’ Sticks” test battery, we applied three different presentation methods: (1) gold standard, (2) shorter single staircase method and (3) ascending procedure. Compared to the gold-standard, the shorter single staircase procedure was 26% and the ascending procedure was 51% shorter in duration. Both short procedure thresholds correlated highly with the gold standard threshold. All three tests showed similar test-retest reliability. To conclude, we have developed a test that takes on average 5-7 minutes less time and is as reliable as the gold standard. Study 2: Within the second study, we focused on metabolic health parameters that might explain the relationship between odor sensitivity and obesity. We investigated food and non-food odor sensitivity in the hungry and sated state in 75 young healthy participants with normal weight, overweight and obesity in a within-subject, repeated-measures design. We assessed metabolic health status with BMI, WHR, pre- and postprandial levels of insulin, leptin, glucose, and ghrelin. We showed that odor sensitivity did not directly depend on body weight status or BMI. However, we found a strong negative mediating effect of insulin resistance as assessed by HOMA-IR score on the relationship between BMI and olfactory sensitivity for the food odor. Post-hoc regression models revealed that insulin resistance rather than obesity is responsible for this effect. To conclude, our findings indicate a strong negative association between insulin resistance and sensitivity to food odors. Study 3: In the third study, we examined neuroanatomical correlates of smell perception in obesity and its relationship with metabolic health factors. Olfactory bulb volume was assessed with magnetic resonance imaging in 67 healthy normal weight, overweight and obese participants. To examine recently proposed mechanistic explanatory models of altered smell perception in obesity, we collected parameters that are associated with metabolic health in obesity, such as insulin resistance, leptin, body fat percentage and fat mass index. We showed that in our sample, people with obesity had significantly lower olfactory bulb volume when compared to people with normal weight. Further, we found that olfactory bulb volume was negatively associated with other measures of metabolic health, especially insulin resistance, leptin, and body fat percentage. Our results imply that, similar to other diseases such as depression and Parkinson’s disease, obesity also involves a neuroanatomical change in the olfactory bulbs compared to healthy participants with normal weight. Hence, our study provides first indications that obesity is associated with brain anatomical changes in the olfactory bulbs. Conclusion The overall aim of this thesis was to shed light on the complex relationship between obesity and olfaction. Study 1 provides two easy-to-use odor threshold test procedures for clinical use or for complex research designs with limited time frames. Importantly, this thesis emphasizes the major role of metabolic health status and especially insulin resistance in the altered smell perception in obesity. Most notably, poor metabolic health mediates the relationship between obesity and olfactory sensitivity (study 2). Metabolic health parameters rather than obesity per se might be responsible for low olfactory function and should be further scrutinized in future studies. In particular, a group-design with elevated vs. normal HOMA-IR participants instead of BMI groups could provide more insights. Intriguingly, a high BMI and related metabolic health factors, such as high insulin resistance and high body fat percentage are associated with neuroanatomical changes in the olfactory system, i.e., lower olfactory bulb volume (study 3). These findings contribute to a further understanding of explanatory models introduced by Peng et al. (2019). In accordance with this metabolic and hormonal model our results support the theoretical framework that metabolic and hormonal shifts in obesity might be crucial for changes in olfactory perception. Thereby, these results provide a deeper understanding of the pathophysiological mechanisms underlying altered olfactory function in obesity. Subsequently, olfaction might represent a new target for prevention or therapy.:LIST OF ABBREVIATIONS I LIST OF FIGURES II LIST OF TABLES III I. INTRODUCTION 1 1. THE OBESITY PANDEMIC 1 2. HORMONES INVOLVED IN OBESITY AND OLFACTION 4 2.1 HORMONES IN THE REGULATION OF EATING BEHAVIOR AND OBESITY 4 2.2 HORMONES IN THE CONTEXT OF SMELL PERCEPTION 7 3. THE OLFACTORY SYSTEM 9 3.1 ANATOMY AND PHYSIOLOGY 9 3.2 MEASURING SMELL ABILITY: THREE DIMENSIONS OF OLFACTORY FUNCTION 13 3.3 THE ROLE OF OLFACTION IN THE CONTROL OF EATING BEHAVIOR 14 3.4 SMELL PERCEPTION IN OBESITY 15 4. THE LINK: WHY TARGET THE OLFACTORY SYSTEM IN OBESITY? 19 II. RATIONALE OF THE EXPERIMENTAL WORK 20 III. EXPERIMENTAL WORK 21 STUDY 1: SHORT PROCEDURE TO ASSESS ODOR DETECTION THRESHOLDS 21 STUDY 2: ODOR SENSITIVITY FOR FOOD AND NON-FOOD ODORS IN OBESITY 30 STUDY 3: BRAIN ANATOMICAL CORRELATES OF SMELL PERCEPTION IN OBESITY 47 IV. SUMMARY 60 V. REFERENCES 65 VI. APPENDIX 75 A. DECLARATION OF AUTHENTICITY 75 B. AUTHOR CONTRIBUTIONS 76 C. CURRICULUM VITAE 80 D. ACKNOWLEDGEMENTS 8

Pancreatic beta-cell insulin signaling in genetic and dietary models of obesity and insulin resistance

Type 2 Diabetes Mellitus (T2DM) is a heterogeneous metabolic disease characterized by elevated blood glucose levels that has reached pandemic proportions. Genome-wide association studies have linked T2DM to the function of the insulin-producing pancreatic βcell residing in the micro-organ islet of Langerhans. An individual´s risk to develop T2DM depends on genetic predisposition and environmental factors, e.g. life style. Central for disease development is the interplay between insulin resistance in insulin target tissues like muscle, liver and fat and deficient β-cell insulin secretion. Since the β-cell is an insulin target itself, βcell insulin resistance can contribute to β-cell dysfunction and the development of T2DM. This was shown in several genetic (knockout) mouse models, however the dynamics of β-cell insulin resistance and its relevance in a diet-induced context has so far not been explored. Furthermore the consequences of diet-induced β-cell insulin resistance for β-cell function remain to be understood. The difficulty to study β-cell insulin resistance in vivo has partly been due to the lack of a technique to monitor β-cell insulin resistance non-invasively and longitudinally in the living organism. In my thesis I employed the anterior chamber of the eye of mice as a transplantation site for biosensor-expressing reporter islets and the cornea as a natural body window to monitor β-cell insulin resistance non-invasively and longitudinally by microscopic imaging. The β-cell insulin resistance biosensor is based on GFP-labeled FoxO1, that changes its intracellular localization from cytoplasmic (insulin responsive) to nuclear (insulin resistant). With this technique we investigated β-cell insulin resistance dynamics in ob/ob and NZO mice and demonstrated that β-cell insulin resistance dynamics vary in animal models of insulin resistance and obesity. Furthermore, we showed that β-cell insulin resistance developed in the presence of whole-body insulin resistance, impaired glucose tolerance and increased body weight, but independently from liver insulin resistance. To study the relevance of β-cell insulin resistance in diet-induced T2DM development, we treated diabetes-prone male C57BL/6J mice with different combinations of solid high fat diet and drinking water containing either sucrose or fructose. Employing our new monitoring technique we showed that only mice that were fed a High-FatHigh-Sucrose-Diet developed β-cell insulin resistance. This demonstrated the importance of βcell insulin resistance in a model of diet-induced obesity and insulin resistance and highlighted the importance of diet composition for the development of T2DM. The β-cell insulin resistance was accompanied by a decreased functional β-cell mass and impaired insulin secretion downstream of glucose-stimulated Ca2+ influx, due to a reduction of syntaxin-1A. We were also able to show that β-cell insulin resistance in one insulin signaling cascade can re-route the insulin signal, thus allowing the co-existence of reduced and increased insulin response in the same cell. In conclusion, my in vivo studies of diet-induced β-cell insulin resistance and its consequences on β-cell function and survival contribute to better understanding of the development of T2DM

Interindividual variability in perceived appetite and appetite-related hormone responses to eating and exercise in humans

As obesity rates continue to rise worldwide, scientific interest in the area of appetite regulation has increased in an attempt to identify strategies that can prevent energy overconsumption and body weight gain. Appetite regulation is complex and involves many different physiological and psychological factors, allowing for great interindividual variability. Recently, some studies assessing appetite and energy intake responses to meal or exercise interventions have shifted the focus on presenting findings exclusively as group means to assessing individual responses and exploring interindividual variability. However, important methodological limitations may have impaired the detection of true interindividual variability, and gold standard study design and statistical approaches that address these limitations have been recently suggested. Therefore, this thesis aimed to assess the reproducibility and quantify the interindividual variability in appetite responses to acute exercise and to a standardised meal, and to explore the influence of genetic, physiological and behavioural characteristics on fasting and postprandial appetite-related outcomes. To achieve this, a total of 145 healthy men and women were recruited into four experimental studies.The first experimental study (Chapter 4) demonstrated, using a replicated crossover design, that young men exhibited reproducible appetite responses after 60-min of fasted treadmill running at 70% peak oxygen uptake. True interindividual variability was observed in acylated ghrelin, total peptide YY (PYY) and perceived appetite responses over and above any random within-subject variability and measurement error, even after adjustment for individual baseline measurements. In the second experimental study (Chapter 5), the fat mass and obesity- associated gene (FTO) was not significantly associated with fasting or postprandial perceived appetite, acylated ghrelin, total PYY, insulin, glucose and leptin in healthy men and women, with or without the addition of physiological and behavioural covariates in the statistical models. While fasting leptin, glucose and insulin and postprandial insulin concentrations were associated with adiposity outcomes, the associations between fasting and postprandial acylated ghrelin, total PYY and general or abdominal adiposity were small. The third experimental study (Chapter 6) employed a replicated crossover design to demonstrate that the reproducibility of appetite responses to a standardised meal (5025 kJ) is generally good in healthy men. True interindividual variability was present in perceived appetite, acylated ghrelin, total PYY, insulin and glucose responses to the meal beyond any random within-isubject variation over time, but the magnitude of change in postprandial appetite responses was not influenced by the FTO gene. The final experimental study (Chapter 7) consisted of a pilot study which showed no significant association between brown adipose tissue activity assessed with thermal imaging, FTO genotype and fasting and postprandial acylated ghrelin, total PYY, insulin and glucose in healthy males.Collectively, these studies demonstrate that appetite responses to acute exercise and to eating are reproducible in healthy men, and true interindividual variability exist in these responses. However, the FTO genotype was not significantly associated with fasting and postprandial perceived appetite and appetite-related hormones, and further studies are warranted to investigate other individual characteristics that may moderate the observed interindividual variability. These findings highlight the importance of exploring individual differences in appetite responses in the context of the prevention and/or management of obesity.</div

Analysis of Indirect Biomarkers of Effect after Exposure to Low Doses of Bisphenol A in a Study of Successive Generations of Mice

Bisphenol A (BPA) is considered as being an emerging pollutant, to which both animal and human populations are continuously and inadvertently exposed. The identification of indirect biomarkers of effect could be a key factor in determining early adverse outcomes from exposure to low doses of BPA. Thus, this study on mice aims to evaluate and identify indirect biomarkers of effect through the analysis of their blood biochemistry, and of certain reproduction parameters after exposure to different BPA concentrations (0.5, 2, 4, 50, and 100 µg/kg BW/day) in drinking water over generations. Our results showed that there were no modifications in the reproductive parameters evaluated, like estrous cycle duration, litter size, or the percentage of the young alive at reaching the weaning stage, at the exposure levels evaluated. However, there were modifications in the biochemical parameters, e.g., alterations in the glucose levels, that increased significantly (p < 0.05) in the breeders at the higher exposure doses (50 and 100 µg/kg BW/day in F1; 50 µg/kg BW/day in F2 and 100 µg/kg BW/day in F3), that would suggest that the BPA could induce hyperglycemia and its complications in adult animals, probably due to some damage in the pancreas cells; albumin, that increased in the breeders exposed to the highest dose in F1 and F3, inferring possible hepatic alterations. Further, total proteins showed a diminution in their values in F1 and F2, except the group exposed to 100 µg/kg BW/day, whereas in F3 the values of this parameter increased with respect to the control group, this aspect likely being related to a possible hepatic and renal alteration. Based on these results, glucose, albumin, and total proteins could initially be considered as early indicators of indirect effect after prolonged exposure to low BPA doses over generations

TMEM27: A Cleaved and Shed Plasma Membrane Protein That Stimulates Pancreatic Beta Cell Proliferation

The signals and the molecular mechanisms that regulate the replication of terminally differentiated β cells are unclear. In this thesis I report the identification of a gene encoding transmembrane protein 27 (TMEM27) in pancreatic β cells. Expression of Tmem27 is reduced in Tcf1–/– mice, which exhibit defects in proliferation, and is increased in islets of ob/ob, db/db and aP2- Srebp-1c transgenic mice with marked hypertrophy of the endocrine pancreas. Tmem27 is expressed in hormone positive cells at early stages of pancreas development and becomes restricted to pancreatic β cells in the mature pancreas. Biochemical characterization revealed that the Tmem27 exists as a dimer and that its extracellular domain is glycosylated, cleaved and shed from the plasma membrane. The cleavage process of Tmem27 is β cell-specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, in MIN6 cells leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic β cells exhibit increased β cell mass compared to their control littermates. The following results identify a novel pancreatic β cell-shed protein that regulates cell growth of pancreatic islets

Roles for the Type 2 diabetes-associated genes C2CD4A and C2CD4B in the control of insulin secretion

Genome-wide Association Studies (GWAS) have identified several SNPs in human chromosome 15 at the C2CD4A/C2CD4B/VPS13C locus associated with increased proinsulin levels and type 2 diabetes (T2D) risk. A recent in vivo study has shown that murine Vpc13c has a minor role in glucose homeostasis. Therefore, in this study, I sought to investigate the roles of C2CD4A and C2CD4B in glucose homeostasis and insulin secretion. C2CD4A and C2CD4B have been predicted to encode nucleus-localised calcium-binding proteins in endothelial cells. To investigate the role of these genes in pancreatic β-cells, we first addressed their subcellular localisation. Our results suggested a novel role for these genes products since they showed localisation at the plasma membrane in addition to the nucleus as shown previously in endothelial cells. We also found that C2CD4A translocates from the cytoplasm to the plasma membrane in response to an elevation in intracellular free calcium. This suggests that the C2 domain of this protein binds to calcium and membrane phospholipids. We also studied the role of C2cd4b in vivo. Our data showed that a lack of C2cd4b in female mice leads to impaired glucose tolerance, caused by a significantly decreased plasma insulin level. In these mice, we also observed a significant reduction of follicle-stimulating hormone (FSH). In contrast to the latter data, C2cd4a null mice did not present any glucose intolerance phenotype, suggesting a minor role for this gene in glucose homeostasis. However, in humans, deletion of this gene from human pancreas-derived β-cells caused an impairment in insulin secretion. Taken together, our data demonstrate a novel role for these genes in the control of insulin secretion and glucose homeostasis. Further studies investigating the structures, their specific functions and interacting partners may demonstrate the mechanisms of action of the nearby T2D variants and open new avenues for the treatment of T2D.Open Acces

3D in vitro cancer models for drug screening: A study of glucose metabolism and drug response in 2D and 3D culture models

Current drug screening protocols use in vitro cancer cell panels grown in 2D to evaluate drug response and select the most promising candidates for further in vivo testing. Most drug candidates fail at this stage, not showing the same efficacy in vivo as seen in vitro. An improved first screening that is more translatable to the in vivo tumor situation could aid in reducing both time and cost of cancer drug development. 3D cell cultures are an emerging standard for in vitro cancer cell models, being more representative of in vivo tumour conditions. To overcome the translational challenges with 2D cell cultures, 3D systems better model the more complex cell-to-cell contact and nutrient levels present in a tumour, improving our understanding of cancer complexity. Furthermore, cancer cells exhibit altered metabolism, a phenomenon described a century ago by Otto Warburg, and possibly related to changes in nutrient access. However, there are few reports on how 3D cultures differ metabolically from 2D cultures, especially when grown in physiological glucose conditions. Along with this, metabolic drug targeting is considered an underutilized and poorly understood area of cancer therapy. Therefore, the aim of this work was to investigate the effect of culture conditions on response to metabolic drugs and study the metabolism of 3D spheroid cultures in detail. To achieve this, multiple cancer cell lines were studied in high and low glucose concentrations and in 2D and 3D cultures. We found that glucose concentration is important at a basic level for growth properties of cell lines with different metabolic phenotypes and it affects sensitivity to metformin. Furthermore, metformin is able to shift metabolic phenotype away from OXPHOS dependency. There are significant differences in glucose metabolism of 3D cultures compared to 2D cultures, both related to glycolysis and oxidative phosphorylation. Spheroids have higher ATP-linked respiration in standard nutrient conditions and higher non-aerobic ATP production in the absence of supplemented glucose. Multi-round treatment of spheroids is able to show more robust response than standard 2D drug screening, including resistance to therapy. Results from 2D cultures both over and underestimate drug response at different concentrations of 5-fluorouracil (5-FU). A higher maximum effect of 5-FU is seen in models with lower OCR/ECAR ratios, an indication of a more glycolytic metabolic phenotype. In conclusion, both culture method and nutrient conditions are important consideration for in vitro cancer models. There is good reason to not maintain in vitro cultures in artificially high glucose conditions. It can have downstream affects on drug response and likely other important metrics. If possible, assays should also be implemented in 3D. If not in everyday assays, at least as a required increase in complexity to validate 2D results. Finally, metabolism even in the small scope presented here, is complex in terms of phenotypic variation. This shows the importance of metabolic screening in vitro to better understand the effects of these small changes and to model how a specific tumor may behave based on its complex metabolism

HEPATOCYTE GROWTH FACTOR REGULATES INFLAMMATORY MEDIATED DISEASES BY SUPPRESSION OF IL-6: IMPLICATIONS FOR TYPE 2 DIABETES

The generation of the pro-inflammatory cytokines IL-6 and TNF-α by macrophages recruited to adipose tissue facilitates obesity-induced inflammation resulting in insulin resistance and type 2 diabetes (T2D). Increased adipose tissue is associated with inflammation and expression of acute phase response (APR) proteins secreted by the liver. Proper homeostasis of the liver is regulated by IL-6-depdendent expression of Hepatocyte Growth Factor (HGF) upon cleavage to its active form (aHGF) by the urokinase-type plasminogen activator (uPA). Plasminogen Activator Inhibitor Type-1 (PAI-1) is a pro-thrombotic APR protein known to inhibit the function of uPA; however, since HGF's activation, interaction and signaling through its receptor, MET are dependent upon uPA, PAI-1 is also capable of regulating the function of hepatic HGF. In vitro data demonstrates that aHGF significantly suppressed IL-6 production by macrophages stimulated with LPS via an increase in phosphorylation of GSK3β, rendering it inactive. Phosphorylated GSK3β correlated with increased retention of the phosphorylated NF-κB p65 subunit in the cytoplasm and an enhanced interaction between CBP and phosphorylated CREB resulting in IL-10 cytokine production. These changes were a direct result of signaling through MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice.It is known that obese T2D patients present with an accumulation of PAI-1, which we hypothesize, results in the inactivation of HGF. The loss of HGF-MET signaling results in increased active GSK3β and the progression to unchecked inflammation and disease progression. In vivo studies using male, C57BL6 mice on a high fat diet alongside control fed mice demonstrates move severe hepatic steatosis in obese mice at 44 weeks compared to control. Steatosis coincided with the decrease in aHGF and elevated levels of PAI-1 protein. These results demonstrate that elevated levels of PAI-1 inhibit aHGF, leading to unresolved chronic inflammation in obesity and T2D