Cardiovascular diseases are the leading cause of death worldwide, with atherosclerosis as most common underlying pathology. Atherosclerosis is characterized by arterial narrowing due to cholesterol... Show moreCardiovascular diseases are the leading cause of death worldwide, with atherosclerosis as most common underlying pathology. Atherosclerosis is characterized by arterial narrowing due to cholesterol and lipid accumulation. Despite available effective cholesterol lowering medication, considerable risk for recurrent vascular events remains. This residual risk is at least in part explained by high blood lipid levels. The research described in this thesis revealed novel therapeutic strategies that improve lipid metabolism and reduce atherosclerosis development in mice. Inhibition of the endocannabinoid system was found to be an effective strategy, as well as concomitant activation of two incretin hormone receptors, namely those for GIP and GLP1. For combined GIP/GLP1 receptor agonism we additionally showed strongly attenuated hepatic steatosis. We were also able to identify additional targets to attenuate hyperlipidemia by studying the mechanisms underlying the strong day-night rhythm of brown adipose tissue, which is a lipid combusting tissue. In this thesis, I also stress the importance of the choice in animal model when studying lipid-modifying interventions, and describe the development of the software tool RandoMice which can be used to improve the quality of preclinical studies by creating well-balanced experimental groups. Show less
Background Migraine is a highly prevalent disorder with significant economical and personal burden. Despite the development of effective therapeutics, the causes which precipitate migraine attacks... Show moreBackground Migraine is a highly prevalent disorder with significant economical and personal burden. Despite the development of effective therapeutics, the causes which precipitate migraine attacks remain elusive. Clinical studies have highlighted altered metabolic flux and mitochondrial function in patients. In vivo animal experiments can allude to the metabolic mechanisms which may underlie migraine susceptibility. Understanding the translational relevance of these studies are important to identifying triggers, biomarkers and therapeutic targets in migraine. Main body Functional imaging studies have suggested that migraineurs feature metabolic syndrome, exhibiting hallmark features including upregulated oxidative phosphorylation yet depleted available free energy. Glucose hypometabolism is also evident in migraine patients and can lead to altered neuronal hyperexcitability such as the incidence of cortical spreading depression (CSD). The association between obesity and increased risk, frequency and worse prognosis of migraine also highlights lipid dysregulation in migraine pathology. Calcitonin gene related peptide (CGRP) has demonstrated an important role in sensitisation and nociception in headache, however its role in metabolic regulation in connection with migraine has not been thoroughly explored. Whether impaired metabolic function leads to increased release of peptides such as CGRP or excessive nociception leads to altered flux is yet unknown. Conclusion Migraine susceptibility may be underpinned by impaired metabolism resulting in depleted energy stores and altered neuronal function. This review discusses both clinical and in vivo studies which provide evidence of altered metabolic flux which contribute toward pathophysiology. It also reviews the translational relevance of animal studies in identifying targets of biomarker or therapeutic development. Show less
Fuchs, S.; Helden, R.W.J. van; Wiendels, M.; Graaf, M.N.S. de; Orlova, V.V.; Mummery, C.L.; ... ; Mayr, T. 2022
Recent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard... Show moreRecent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard plastic tissue culture plates. Microfluidic devices, also called Organs-on-Chip (OoC), allow complex engineering of the cellular compartment, yielding designs in which microfluidic flow can be precisely controlled. However, it is important that cellular physiology is not only controlled but can also be monitored in these devices. Here, we integrated oxygen and pH sensors into microfluidics, allowing close monitoring of the extracellular flux from the cells, enabling constant assessment of features such as glycolysis and mitochondrial oxidative phosphorylation in situ. Using human -induced pluripotent stem cells (hiPSCs) as an exemplar of a highly metabolic and relatively challenging cell type to maintain, we showed that monitoring the extracellular environment allowed rapid optimization of the seeding protocol. Based on the measurements, we implemented earlier and more frequent media refreshment to counteract the rapid acidification and depletion of oxygen. The integrated sensors showed that hiPSCs in the devices exhibited mitochondrial and glycolytic capacity similar to that measured with the Seahorse extracellular flux system, the most widely used standard for these types of assays in conventional cell culture. Under both conditions, hiPSCs showed greater reliance on glycolysis than mitochondrial OXPHOS and the absolute values obtained were similar. These results thus pave the way for the assessment of cell metabolism in situ under con-ditions of fluidic flow with the same precision and relevance as current standard static cell cultures. Show less
Eggelbusch, M.; Shi, A.D.; Broeksma, B.C.; Vazquez-Cruz, M.; Soares, M.N.; Wit, G.M.J. de; ... ; Wust, R.C.I. 2022
Background Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain... Show moreBackground Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement.Methods Differentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100-200 ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3-induced IL-1 ss production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function.Results NLRP3 gene expression and protein concentrations increased in a time-dependent and dose-dependent manner. Intracellular IL-1 ss concentration significantly increased (P < 0.0001), but significantly less with MCC950 (P = 0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24 h (P = 0.03), and myotubes remained smaller up to 72 h (P = 0.0009). Exposure of myotubes to IL-1 ss caused similar alterations in cell morphology, and MCC950 mitigated these LPS-induced differences in cell diameter. NLRP3 appeared to co-localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P = 0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation.Conclusions LPS- induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association. Show less
Aims/hypothesis: Numerous genome-wide association studies have been performed to understand the influence of genetic variation on type 2 diabetes etiology. Many identified risk variants are located... Show moreAims/hypothesis: Numerous genome-wide association studies have been performed to understand the influence of genetic variation on type 2 diabetes etiology. Many identified risk variants are located in non-coding and intergenic regions, which complicates understanding of how genes and their downstream pathways are influenced. An integrative data approach will help to understand the mechanism and consequences of identified risk variants. Methods: In the current study we use our previously developed method CONQUER to overlap 403 type 2 diabetes risk variants with regulatory, expression and protein data to identify tissue-shared disease-relevant mechanisms. Results: One SNP rs474513 was found to be an expression-, protein- and metabolite QTL. Rs474513 influenced LPA mRNA and protein levels in the pancreas and plasma, respectively. On the pathway level, in investigated tissues most SNPs linked to metabolism. However, in eleven of the twelve tissues investigated nine SNPs were linked to differential expression of the ribosome pathway. Furthermore, seven SNPs were linked to altered expression of genes linked to the immune system. Among them, rs601945 was found to influence multiple HLA genes, including HLA-DQA2, in all twelve tissues investigated. Conclusion: Our results show that in addition to the classical metabolism pathways, other pathways may be important to type 2 diabetes that show a potential overlap with type 1 diabetes. Show less
The research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and... Show moreThe research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and glucocorticoids.Leptin plays an important role during TB infection and has a huge impact on insulin sensitivity in zebrafish larvae. Similarly to what has been observed in the murine model, leptin deficiency in zebrafish increased the bacterial burden and mortality during the infection, leading to hyperglycemia and the development of insulin resistance. In addition, a novel SHP-1/SHP-2 inhibitor, NSC-87877, was shown to represent a promising anti-diabetic drug that can be used for further DM2 research, as it is able to rescue the phenotype of the leptin-deficient zebrafish and to restore glucose transport to the tissues. In contrast to metformin, NSC-87877 can act at very early developmental stages and inhibits the function of SHP-1 and factors that underlay impaired glucose metabolism, whereas metformin is mostly known to improve insulin sensitivity. Additionally, treatment with the glucocorticoid beclomethasone attenuates the metabolic changes associated with the infection, and transcriptional alterations induced by beclomethasone treatment suggest that genes involved in glucose metabolism, insulin and leptin signaling all play an important role in the modulation of the metabolism.Our data show that zebrafish larvae represent an interesting model system to investigate the complex pathology of TB, and the studies described in this thesis in which this model has been used have provided novel insights into the molecular mechanisms underlying wasting syndrome and the possibilities for adjunctive glucocorticoid therapy to alleviate this metabolic state. Show less
Dendritic cells are the canonical professional antigen-presenting cell and are therefore crucial in the generation of efficient adaptive T cell responses. It is now well described that immune cells... Show moreDendritic cells are the canonical professional antigen-presenting cell and are therefore crucial in the generation of efficient adaptive T cell responses. It is now well described that immune cells – including dendritic cells – make drastic changes to their biology to transition between different life stages and to deal efficiently with the threat of infection. However, an unanswered question was if DCs with different T cell polarizing properties - that is to say they preferentially skew T cells towards a specific specialization (for example T helper 1 cells over T helper 2 cells) - rely on distinct metabolic characteristics for their T cell polarizing ability. This thesis tries to address that question by studying the metabolism of dendritic cells after in vitro stimulation with antigens or immunomodulatory compounds that are known to prime either T helper 1 cells, T helper 2 cells, T helper 17 cells or regulatory T cells. In addition, we interrogate the role of liver kinase B1 (LKB1) and mechanistic target of rapamycin complex 1 (mTORC1) in DC biology. Show less
This thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome... Show moreThis thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome scale metabolic reconstruction for zebrafish, that we used to study zebrafish metabolism upon infection with Mycobacterium marinum integrating gene expression data from control and infected zebrafish larvae. The chapters focusing on cell motility in response to the environment, revolve around the question of how the environmental inputs of cell-matrix interactions, cell-sized obstacles and cell-signalling upon wounding shape and guide cell motility. Show less
Metabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile... Show moreMetabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile technique for the elaboration of physiology and disease, studying metabolism using standard cell culture protocols is profoundly interfered by the Crabtree effect. This phenomenon refers to the adaptation of cultured cells to a glycolytic phenotype, away from oxidative phosphorylation in glucose-containing medium, and questions the applicability of cell culture in certain fields of research. In this systematic review we aim to provide a comprehensive overview and critical appraisal of strategies reported to circumvent the Crabtree effect. Show less
Objective: Carbonyl reductase 1 (Cbr1), a recently discovered contributor to tissue glucocorticoid metabolism converting corticosterone to 2013dihydrocorticosterone (2013-DHB), is upregulated in... Show moreObjective: Carbonyl reductase 1 (Cbr1), a recently discovered contributor to tissue glucocorticoid metabolism converting corticosterone to 2013dihydrocorticosterone (2013-DHB), is upregulated in adipose tissue of obese humans and mice and may contribute to cardiometabolic complications of obesity. This study tested the hypothesis that Cbr1-mediated glucocorticoid metabolism influences glucocorticoid and mineralocorticoid receptor activation in adipose tissue and impacts glucose homeostasis in lean and obese states. Methods: The actions of 2013-DHB on corticosteroid receptors in adipose tissue were investigated first using a combination of in silico, in vitro, and transcriptomic techniques and then in vivo administration in combination with receptor antagonists. Mice lacking one Cbr1 allele and mice overexpressing Cbr1 in their adipose tissue underwent metabolic phenotyping before and after induction of obesity with high-fat feeding. Results: 2013-DHB activated both the glucocorticoid and mineralocorticoid receptor in adipose tissue and systemic administration to wild-type mice induced glucose intolerance, an effect that was ameliorated by both glucocorticoid and mineralocorticoid receptor antagonism. Cbr1 haploinsufficient lean male mice had lower fasting glucose and improved glucose tolerance compared with littermate controls, a difference that was abolished by administration of 2013-DHB and absent in female mice with higher baseline adipose 2013-DHB concentrations than male mice. Conversely, overexpression of Cbr1 in adipose tissue resulted in worsened glucose tolerance and higher fasting glucose in lean male and female mice. However, neither Cbr1 haploinsfficiency nor adipose overexpression affected glucose dyshomeostasis induced by high-fat feeding. Conclusions: Carbonyl reductase 1 is a novel regulator of glucocorticoid and mineralocorticoid receptor activation in adipose tissue that influences glucose homeostasis in lean mice. (c) 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
In recent years there have been major advances in our understanding of the role of free fatty acids (FAs) and their metabolism in shaping the functional properties of macrophages and DCs. This... Show moreIn recent years there have been major advances in our understanding of the role of free fatty acids (FAs) and their metabolism in shaping the functional properties of macrophages and DCs. This review presents the most recent insights into how cell intrinsic FA metabolism controls DC and macrophage function, as well as the current evidence of the importance of various exogenous FAs (such as polyunsaturated FAs and their oxidation products-prostaglandins, leukotrienes, and proresolving lipid mediators) in affecting DC and macrophage biology, by modulating their metabolic properties. Finally, we explore whether targeted modulation of FA metabolism of myeloid cells to steer their function could hold promise in therapeutic settings. Show less
Metabolic reprogramming of cancer cells generates a tumour microenvironment (TME) characterised by nutrient restriction, hypoxia, acidity and oxidative stress. While these conditions are... Show moreMetabolic reprogramming of cancer cells generates a tumour microenvironment (TME) characterised by nutrient restriction, hypoxia, acidity and oxidative stress. While these conditions are unfavourable for infiltrating effector T cells, accumulating evidence suggests that regulatory T cells (Tregs) continue to exert their immune-suppressive functions within the TME. The advantages of Tregs within the TME stem from their metabolic profile. Tregs rely on oxidative phosphorylation for their functions, which can be fuelled by a variety of substrates. Even though Tregs are an attractive target to augment anti-tumour immune responses, it remains a challenge to specifically target intra-tumoral Tregs. We provide a comprehensive review of distinct mechanistic links and pathways involved in regulation of Treg metabolism under the prevailing conditions within the tumour. We also describe how these Tregs differ from the ones in the periphery, and from conventional T cells in the tumour. Targeting pathways responsible for adaptation of Tregs in the tumour microenvironment improves anti-tumour immunity in preclinical models. This may provide alternative therapies aiming at reducing immune suppression in the tumour. Show less
Kok, M.J.C. de; Schaapherder, A.F.; Wüst, R.C.I.; Zuiderwijk, M.; Bakker, J.A.; Lindeman, J.H.N.; Le Dévédec S.E. 2021
Metabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile... Show moreMetabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile technique for the elaboration of physiology and disease, studying metabolism using standard cell culture protocols is profoundly interfered by the Crabtree effect. This phenomenon refers to the adaptation of cultured cells to a glycolytic phenotype, away from aoxidative phosphorylation in glucose-containing medium, and questions the applicability of cell culture in certain fields of research. In this systematic review we aim to provide a comprehensive overview and critical appraisal of strategies reported to circumvent the Crabtree effect. Show less
Throughout evolution, humans have lived in synchrony with the natural light-dark cycle. Our bodies were used to going to sleep a few hours after dark, and waking up just before dawn. However, in... Show moreThroughout evolution, humans have lived in synchrony with the natural light-dark cycle. Our bodies were used to going to sleep a few hours after dark, and waking up just before dawn. However, in modern society the unambiguous availability of artificial light has desynchronized our biological clock from the naturally occurring day and night, with large consequences for metabolic health. This thesis sheds light on the negative health consequences of a disturbed biological clock, and elucidates novel approaches to prevent disease associated with chronic rhythm disruption, as occurs in shift work. We have identified important mechanisms through which rhythm disruption contributes to (cardio)metabolic disease, namely by exacerbating vascular inflammation and by deregulating rhythm in glucocorticoid hormone, thereby affecting the metabolic activity of tissues such as brown fat and bone. We continued by investigating two main approaches to prevent diseases associated with circadian disturbances: (1) by limiting disruption of the circadian timing system, and (2) by directly targeting the affected tissues. We found that timed feeding (1) and stimulation of the metabolic activity of brown fat (2) are both promising strategies to prevent and/or reduce (cardio)metabolic disease risk in the ever-increasing population of individuals who suffer from circadian disturbances. Show less
MHC class I antigen-presentation plays a pivotal role in anti-tumor immunity. High surface expression of MHC-I molecules is generally correlated with high CD8 T cell infiltrate and improved overall... Show moreMHC class I antigen-presentation plays a pivotal role in anti-tumor immunity. High surface expression of MHC-I molecules is generally correlated with high CD8 T cell infiltrate and improved overall survival in many cancers. In contrast, partial or complete loss of MHC-I surface expression is associated with reduced survival and primary-resistance to immunotherapy in cancers. Expression of additional molecules in the tumor microenvironment (TME), such as PD-L1 and HLA-E, further shape immune responses. The presence of immune cells and the expression of immune-related genes together determine the ‘immune landscape’ of cancers, while the local production of interferons strongly impacts this environment. Although MHC-I and PD-L1 are both regulated by the IFN pathway, an in-depth study on immune escape of NSCLC showed that the expression of co-inhibitory markers and the loss of MHC-I expression are two independent mechanisms of immune evasion. This classifies tumors into different “types” depending on their MHC-I and PD-L1 expression. The differential expression of MHC-I and PD-L1 suggests that immune-escape of cancer cells occurs through a multitude of distinct “hard-wired” and “soft-wired” modifications and knowing which of the mechanisms underlie immune escape determines which immunotherapeutic strategy has the most potential for clinical success. Show less
Venetoclax is an oral BCL2 inhibitor undergoing investigation for use in relapsed or refractory multiple myeloma (RRMM), particularly in combination with proteasome inhibitors (VPI)[1,2]. An... Show moreVenetoclax is an oral BCL2 inhibitor undergoing investigation for use in relapsed or refractory multiple myeloma (RRMM), particularly in combination with proteasome inhibitors (VPI)[1,2]. An interim analysis of a current phase 2 trial of venetoclax with carfilzomib in RRMM demonstrated an overall response rate of 78% with a very good partial response rate of 56%[3,4]. However, a separate ongoing phase 3 trial of venetoclax with bortezomib found a decrease in overall survival due to increased fatal infections in the venetoclax arm compared to placebo. Better describing these infections may give insight into the pathophysiology and prove useful in mitigating strategies for use with VPI therapy in RRMM. Show less
Introduction: Disturbances in onset and resolution of inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. Dietary polyunsaturated fatty acids (PUFAs) can be... Show moreIntroduction: Disturbances in onset and resolution of inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. Dietary polyunsaturated fatty acids (PUFAs) can be converted into lipid mediators here collectively named oxylipins. These include classical eicosanoids, but also pro-resolving mediators. A balanced production of pro-inflammatory and pro-resolving oxylipins is of importance for adequate inflammatory responses and subsequent return to homeostasis.Objectives: Here we investigated if PUFA metabolism is disturbed in COPD patients.Methods: Free PUFA and oxylipin levels were measured in induced sputum samples from the Bergen COPD cohort and COPD exacerbation study using liquid chromatography-mass spectrometry. Additionally, effects of whole cigarette smoke on PUFA metabolism in air-liquid interface cultures of primary bronchial epithelial cells were assessed.Results: Significantly lower levels of free alpha-linolenic acid, linoleic acid and eicosapentaenoic acid (EPA) were detected in sputum from stable COPD patients compared to controls. During acute exacerbation (AE), levels of free arachidonic acid and docosapentaenoic acid were higher than in stable COPD patients. Furthermore, levels of omega-3 EPA- and docosahexaenoic acid-derived oxylipins were lower in sputum from stable COPD patients compared to controls. Cyclooxygenase-2-converted mediators were mostly increased during AE. In vitro studies additionally showed that cigarette smoke exposure may also directly contribute to altered epithelial PUFA metabolism, and indirectly by causing airway epithelial remodelling.Conclusions: Our findings show significant differences in PUFA metabolism in COPD patients compared to controls, further changed during AE. Airway epithelial remodelling may contribute to these changes. These findings provide new insight in impaired inflammatory resolution in COPD. Show less
Atherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the vessel wall. Over time, this accumulation of lipids and immune cells induce morphological abnormalities in the vessel wall which cause the vessel lumen to narrow. This narrowing of the lumen (stenosis) causes ischemia in the downstream tissue. Prolonged ischemia causes myocardial ischemia and/or stroke. The research described in my thesis examines a well-recognized risk factor of atherosclerosis, being dyslipidemia, from an entirely new perspective. More specifically, it describes how dyslipidemia affects intrinsic metabolic processes in T cells, the conductors of the immune response characterizing atherosclerosis, and how this affects their function. My research has contributed to knowledge on the pathophysiology of atherosclerosis and might one day pave the way for the development of novel therapeutic approaches to treat cardiovascular disease. Show less
