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Academic collection --- Theses

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The obesity rates continue to rise worldwide and are associated with adverse health problems, including increased risk of type 2 diabetes. Excessive weight gain is often considered to be the result of excessive food intake and/or insufficient physical activity. In addition, the food landscape has shifted dramatically over the past several decades and the increased consumption of soft drinks and other sugar-sweetened beverages is considered as a major contributor to the obesity epidemic. Not surprisingly, non-caloric sweeteners have increased in popularity over the years as a mean to facilitate weight loss by reducing the sugar content of meals without affecting its taste. Next to these non-caloric sweeteners, low-caloric prebiotic sweeteners (oligofructose; OFS) have been proposed as functional food ingredients that could improve lipid metabolism and body weight through beneficial effects ascribed to their fermentation products, the short-chain fatty acids (SCFAs).^ When dietary changes are insufficient, pharmacotherapy can be added, although the risks and modest nature of weight loss that can be achieved with these anti-obesity drugs highlights the need for new treatment strategies. The gastrointestinal tract is an obvious target for new anti-obesity treatment strategies as it coordinates the release of gut hormones, such as the ‘hunger hormone’ ghrelin and satiety hormones GLP-1 and PYY, to regulate energy uptake and utilization. Restoring postprandial gut hormone levels (ghrelin, GLP-1, PYY), known to be dysregulated in obesity, may play a role in the metabolic improvements after bariatric surgery such as Roux-en-Y gastric bypass (RYGB) surgery. RYGB surgery is still an invasive technique but mimicking the sustained and enhanced release of GLP-1 and PYY through combination therapy might be a valuable, non-invasive alternative for bariatric surgery. The magnitude of postprandial gut hormone release depends on the meal composition.^ Several taste receptors (sweet, umami, bitter, fatty acid) and the taste receptor coupled G-protein, α-gustducin, are not only present on taste buds of the tongue but also on entero-endocrine cells (EECs) and may tune gut hormone release according to the macronutrient composition of the meal. The physiological role of taste receptors on EECs has not been fully elucidated yet.In this thesis, we aimed to unravel whether α-gustducin coupled sweet taste receptors (TAS1R2-TAS1R3) play a role in the sensing of carbohydrates and sweeteners by the ghrelin cell. In addition, we elucidated whether intragastric supplementation of artificial sweeteners (sucralose) or prebiotic sweeteners (OFS) can prevent the deleterious effects of a high-fat diet in mice by altering gut hormone release through gustducin-mediated taste receptor activation.^ As a last aim, we investigated the effect of nutrient rerouting during RYGB surgery on the gustducin-mediated signaling pathways that contribute to the metabolic improvements and physiological adaptations along the gut after RYGB surgery.In the first part of this thesis we investigated if α-gustducin mediated sweet taste receptor signaling is involved in the sensing of sweeteners by the ghrelin cell in three different experimental models (a ghrelinoma cell line, ex vivo intestinal segments, in vivo experiments).The carbohydrate D-glucose and prebiotic sweetener OFS decreased ghrelin release from a gastric ghrelinoma cell line at concentrations physiological to the postprandial luminal fluid. In contrast, the artificial sweetener sucralose increased ghrelin release in vitro at a supraphysiological (200mM) concentration.^ Furthermore, by using pharmacological inhibitors we showed that neither sweet taste receptor activation, nor glucose transport (SGLT-1, GLUT family) played a role in the effect of D-glucose, OFS or sucralose on ghrelin release from the ghrelinoma cell line. Ghrelin release from gastric (only containing the TAS1R3 subunit) and jejunal (containing the TAS1R2 and TAS1R3 subunit) segments from WT and α-gust-/-mice, mimicked the in vitro effects of the sweeteners in the ghrelinoma cell line to a similar extent in both genotypes. These findings indicate that the effect of D-glucose, OFS and sucralose on ghrelin release is neither α-gustducin nor region-dependent and thus does not involve the α-gustducin coupled TAS1R2-TAS1R3 heterodimer. Intragastric, but not intravenous administration of D-glucose decreased plasma octanoyl ghrelin levels in WT and α-gust-/- mice. These results indicate that the sensing of D-glucose is polarized and occurs via the luminal side of the X/A cell.^ In contrast, neither OFS nor sucralose at “equisweet” concentrations affected octanoyl ghrelin release after an intragastric administration in WT or α-gust-/- mice. In conclusion, our findings indicate that α-gustducin-mediated sweet taste receptor signaling does not play a functional role in the effect of sweeteners on ghrelin release. In contrast to the in vitro findings, only acute intragastric administration of D-glucose but not OFS or sucralose affected ghrelin release.In the second part of this thesis we studied whether daily intragastric administration of equisweet concentrations of an artificial sweetener (sucralose) or a prebiotic sweetener (OFS) for 8 weeks can prevent high-fat diet induced body weight gain, glucose intolerance and impairment of gut permeability, via activation of taste receptors coupled to α-gustducin, using WT and α-gust-/- mice.Sucralose administration did not modulate gut hormone release nor did it prevent body weight gain or glucose intolerance.^ Instead we provided evidence that OFS (300 mg/day) administration decreased HFD-induced body weight gain with about 20% without improving glucose homeostasis. This effect was not accompanied by a reduced food intake. Furthermore, OFS induced a similar but delayed decrease in body weight gain in α-gust-/- mice, indicating that the α-gustducin mediated signaling pathway did not play a major role in this effect. OFS administration did not affect plasma levels of ‘the hunger hormone’ ghrelin and ‘satiety hormone’ PYY, but decreased plasma levels of ‘the satiety hormone’ GLP-1 in WT mice. These changes in gut hormone levels cannot explain the beneficial effects on body weight. Neither OFS, nor sucralose administration altered the mRNA expression levels of the TAS1R2 or TAS1R3 subunit of the sweet taste receptor in the gastro-intestinal tract.^ However, OFS supplementation decreased cecal acetate and butyrate levels, downregulated colonic short chain fatty acid receptor (FFAR2/3) mRNA levels and upregulated FFAR2 in peripheral adipose tissue. These findings suggest that, not sweet taste receptor activation, but enhanced uptake of SCFAs produced by the fermentation of OFS interacting with FFAR2 in peripheral adipose tissue may reduce adipogenesis and lead to the decrease (60%) in fat mass. Moreover, OFS improved the increased colonic permeability which results in metabolic complications in obesity, independent from taste receptors coupled to α-gustducin. In conclusion, this study provided evidence that despite the controversy in the field, artificial sweeteners are metabolically inert. Furthermore, neither OFS nor sucralose affected TAS1R2 or TAS1R3 mRNA levels, while OFS supplementation altered FFAR2/3 expression levels in the gastrointestinal tract and on adipose tissue.^ Therefore, not sucralose but OFS and especially the produced SCFAs, are interesting metabolites that could beneficially affect body weight gain.In the third part we studied the role of gustducin-mediated signaling in the metabolic improvements and intestinal adaptations along the gut after RYGB surgery in obese WT and α-gust-/- mice.We showed that RYGB surgery decreased body weight in WT and a-gust-/- mice. Furthermore, pair-feeding to the RYGB group induced similar blood glucose and plasma insulin profiles during an oral glucose tolerance test compared to RYGB surgery, indicating that the reduced food intake after RYGB surgery was responsible for the improved glucose homeostasis. Moreover, a-gust-/- mice were partially protected from the diabetogenic properties of a western style diet, highlighting the importance of the gustatory signaling pathway in glucose homeostasis.^ After RYGB surgery plasma GLP1 levels were increased in both genotypes, plasma PYY levels were increased in α-gust-/-mice and plasma octanoyl ghrelin levels were not affected. The mechanism behind the postsurgical changes in gut hormone levels seemed to differ between WT and a-gust-/- mice.In WT mice, nutrients act via α-gustducin to increase L-cell differentiation (in the Roux limb which comes in contact with more undigested nutrients) and L-cell number (Roux limb and colon) after RYGB surgery, in a region-dependent manner. However, this nutrient rerouting did not alter the mRNA expression levels of nutrient sensors in the Roux Limb, nor did it alter bacterial fermentation in the caecum of WT mice. In contrast, a-gust-/- mice did not display an altered L-cell number or L-cell differentiation in the Roux limb or colon. However, a-gust-/- mice did show increased mRNA expression levels of the glucose transporters (SGLT1 and GLUT2) and the protein sensor (LPAR5) in the Roux limb.^ Furthermore, RYGB surgery changed bacterial fermentation in the caecum of a-gust-/- mice, which showed increased butyrate and propionate levels compared to WT mice. This resulted in decreased colonic FFAR2/3 mRNA levels in a-gust-/- mice. These results suggest that a changed L-cell number and differentiation will be responsible for the increased plasma GLP-1 levels in WT mice. In contrast, alterations in nutrient signaling in the foregut, and altered bacterial fermentation and short-chain fatty acid sensing in the distal gut of a-gust-/- mice could explain the increased plasma GLP-1 and PYY levels in this genotype.Finally, signaling via α-gustducin plays a role in t

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Academic collection --- Theses

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In T1D patients, auto-Ags of pancreatic insulin-producing ß-cells are targeted by auto-reactive T cells. The underlying events triggering this excessive auto-reactivity remain unknown, but clearly involve genetic predisposition, allowing inappropriate interaction of environmental factors with the immune system. When diagnosed with T1D, patients are limited to exogenous insulin administrations to control their aberrant glucose metabolism. However, this does not fully prevent the occurrence of complications at later stages in the disease, making it difficult to pursue normal life quality and quantity. Moreover, this does not address the underlying Ag-specific autoimmunity. Various auto-Ags a.o. PINS, GAD65 and IA-2 are involved in the pathogenesis of T1D. Numerous studies in preclinical models have highlighted the potential of Ag-specific approaches to restore tolerance in a highly specific and safe manner. Oral auto-Ag administration uses the tolerogenic nature of the gut-associated immune system to induce Ag-specific tolerance, a phenomenon referred to as oral tolerance induction. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant L. lactis have proven to be effective and safe biological carriers for gastrointestinal delivery of therapeutic products. Preclinical successes with systemic broad immune modulating or Ag-specific therapies have repeatedly failed to demonstrate prolonged efficacy in the clinic, urging the need to explore new therapeutic strategies, such as combinatorial immune approaches, targeting multiple molecular pathways. Previously, recombinant L. lactis secreting PINS and IL-10 have demonstrated to induce diabetes remission in a well-defined mouse model for T1D, when combined with low-doses anti-CD3 mAbs. Here, we wanted to explore (i) whether targeting other prominent auto-Ags GAD65 and IA-2 could enhance the therapeutic success of this combination strategy and (ii) whether co-administration of systemic immunomodulators are essential for therapeutic efficacy. First, we generated recombinant L. lactis secreting human GAD65 or IA-2, two major diabetes-related auto-Ags, by themselves and combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering Ag secretion were resolved by trimming the full size proteins. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis while maintaining sufficient bacterial growth. Our data demonstrated that secretion efficiencies could be influenced by mere physico-chemical properties, such as hydrophobic regions, but emphasized that engineering strategies based on functional epitope-distribution could provide a more relevant secretion product in terms of epitope density and secretion efficiency. To explore the tolerogenic potential of mucosally delivered GAD65 and IA-2, recent-onset diabetic NOD mice received intragastric inoculations of recombinant L. lactis secreting GAD65370-575 or IA-2635-979 and IL-10. In combination with short-course low-dose anti-CD3, L. lactis secreting GAD65370-575 and IL-10 stabilized insulitis, preserved functional ß-cell mass and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but significantly decreased the inflammatory profile of islet cell infiltrates locally in the pancreas. Moreover, this combi-GAD therapy increased the presence of functional CD4+Foxp3+CD25+ Tregs in the PLN. In addition, we show, for the first time, that also IA-2 enholds the potential to normalize hyperglycemia in recent-onset NOD mice. Regardless of IL-10 co-delivery, recombinant L. lactis secreting IA-2635-979 induced diabetes remission when combined with low-doses anti-CD3 mAbs. Moreover, efficacy for both auto-Ags was similar in mice with mild and severe hyperglycemia at time of diagnosis. The combined delivery of the three prominent auto-Ags PINS, GAD65370-575 and IA-2635-979 in our combination setting did not enhance efficacy. The comparable success rates suggest that, at least in part, Treg-mediated bystander suppression could account for the tolerogenic effects, as was partly demonstrated in cured combi-GAD-treated mice. These data further suggest that inherent features of the NOD mouse model, such as insufficient ß-cell mass or fullblown inflammation levels in a subgroup of diagnosed mice, could account for the limited efficacy. We further explored an innovative Ag-specific combination strategy that could open up new therapeutic possibilities for T1D. As a general conclusion, we can say that recombinant L. lactis are an exquisite and flexible biological delivery platform that could be applied for oral tolerance induction in the emerging field of autoimmune diseases. In the NOD mouse model, we have demonstrated therapeutic efficacy with a combinatory approach whereby systemic immunomodulation installs the tolerogenic conditions needed for Ag-specific immune regulation to stably restore tolerance. Small Ag-steered differences in therapeutic efficacy favor the exploration of a patient-tailored clinical validation. In order to ensure efficient clinical translation of combination therapies, a substantially validated choice of monotherapies, defined target groups and relevant endpoints are key and need to be carefully considered. Taken together, our data point to a clear opportunity of combinatory strategies, where recombinant L. lactis could make the difference in medicine.

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It is well established that oxygen and nutrients are crucial for cellular functioning, including survival, proliferation and differentiation. As simple oxygen diffusion is limited to about 200 µm, cells will become hypoxic in tissues beyond a certain size. Cells have however developed mechanisms to resist tissue hypoxia and these are mediated by hypoxia inducible factors (HIFs) that are considered as the executors of the cellular response to low oxygen together with HIF prolyl hydroxylases (PHDs) that regulate HIFs in an oxygen-dependent manner. In general, activation of the HIF pathway will ultimately result in (i) stimulation of angiogenesis to resupply oxygen and (ii) adaptations in cell metabolism to ensure adequate energy generation and redox balance.Despite the remarkable healing potential of bone tissue, about 10% of all fractures results in delayed healing or non-union. Bone tissue engineering combines biological basic research and the principles of engineering in order to repair damaged bone using skeletal progenitor or stem cells and/or appropriate signaling molecules seeded on a biocompatible scaffold. The clinical impact of cell-based constructs is however limited, which urges for a better understanding of the cellular and molecular mechanisms of bone regeneration. Upon transplantation, grafted cells encounter an injured tissue that is poorly perfused, turning them in a hypoxic state. In a first part of this PhD project, we investigated the importance of endogenous HIF-1a signaling for bone regeneration. To this end, we specifically deleted the HIF­1a isoform in periosteal progenitor cells and show that activation of HIF-1a signaling in these cells is critical for bone repair by modulating angiogenic and metabolic processes. Activation of HIF‑1a is not only crucial for blood vessel invasion, by enhancing angiogenic growth factor production, but also for periosteal cell survival early after implantation, when blood vessels have not yet invaded the construct. Mechanistically, HIF-1a signaling limits oxygen consumption in hypoxia to avoid accumulation of harmful reactive oxygen species and thereby preserves redox balance, and additionally induces a switch to glycolysis to prevent energetic distress.Although activation of endogenous HIF-1a signaling prevents massive apoptosis, the majority of the grafted cells do not survive. Therefore, we preconditioned periosteal cells to the hypoxic environment of the bone defect site through inhibition of PHD2, the most important PHD isoform in skeletal cells. This strategy increased post-implantation cell survival and improved bone regeneration, an effect that was mediated by HIF-1a. The enhanced cell viability was angiogenesis-independent, but relied on simultaneous and combined changes in glutamine and glycogen metabolism. HIF­1a stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. At the same time, HIF­1a signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Lastly, we showed that pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and thus the beneficial effects on cell survival, supporting further investigations to develop novel treatment modalities for bone regeneration.Throughout life, bone is being constantly remodeled through the coupled action of bone-forming osteoblasts and bone-resorbing osteoclasts, which is controlled among others by matrix-embedded osteocytes. This observation suggests that an adequate supply of oxygen and nutrients is needed to meet the high metabolic demand of these processes. Intriguingly, the bone microenvironment during development, adult life and several pathologies is characterized by low oxygen tensions and several reports have demonstrated a functional role for HIF signaling in osteoprogenitors and osteoblasts. However, the role of the hypoxia signaling pathway in osteocytes, the key regulators of bone mass during homeostasis and pathology, remains elusive.The aim of the last part of this PhD study was therefore to investigate the role of PHD2 in osteocytes, which are considered to be crucial regulators of postnatal bone mass. Transgenic mice lacking PHD2 in osteocytes displayed a high bone mass phenotype, caused by increased bone formation and decreased resorption, processes that were associated with enhanced angiogenesis. Mechanistically, stabilization of HIF-1a in PHD2-deficient osteocytes resulted in a Sirtuin 1-dependent decrease in the WNT/b-catenin inhibitor sclerostin, which stimulates osteogenesis while limiting osteoclast activity. The enhanced angiogenic response was mediated by a HIF-1a-dependent increase in vascular endothelial growth factor. Lastly, we show that genetic ablation of PHD2 was sufficient to protect mice from osteoporotic bone loss, which was potentially mediated by sustained WNT/b-catenin signaling.In this PhD thesis, we have determined the pleiotropic role of PHD2/HIF signaling during bone regeneration and homeostasis. Moreover, our data suggest that targeting PHD2 might be an interesting strategy to improve bone regeneration and, potentially, to limit bone loss during osteoporosis.