{"id":11444,"date":"2026-04-13T10:52:10","date_gmt":"2026-04-13T10:52:10","guid":{"rendered":"https:\/\/www.proefschriftmaken.nl\/portfolio\/wessel-theeuwes\/"},"modified":"2026-04-13T10:52:17","modified_gmt":"2026-04-13T10:52:17","slug":"wessel-theeuwes","status":"publish","type":"us_portfolio","link":"https:\/\/www.proefschriftmaken.nl\/en\/portfolio\/wessel-theeuwes\/","title":{"rendered":"Wessel Theeuwes"},"content":{"rendered":"","protected":false},"excerpt":{"rendered":"","protected":false},"author":8,"featured_media":11445,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"us_portfolio_category":[45],"class_list":["post-11444","us_portfolio","type-us_portfolio","status-publish","has-post-thumbnail","hentry","us_portfolio_category-new-template"],"acf":{"naam_van_het_proefschift":"Regulation of skeletal muscle mitochondrial biogenesis by GSK-3\u03b2","samenvatting":"Onze spieren bezitten een opmerkelijk vermogen om zich aan te passen aan veranderende omstandigheden. Dit betekent dat zowel spiermassa als spiermetabolisme zich normaliter goed kunnen aanpassen aan veranderingen in onder andere fysieke activiteit of spierbelasting. Weefsels, waaronder ook de skeletspieren, bestaan uit cellen. Cellen bevatten op hun beurt verschillende celorganellen waaronder mitochondri\u00ebn. Mitochondri\u00ebn zijn erg belangrijk voor energieproductie door middel van substraatoxidatie, ofwel de verbranding van voedingsstoffen met behulp van zuurstof ook wel energiemetabolisme genoemd. Hoofdstuk 1 introduceert de moleculaire processen die mitochondri\u00eble biogenese (de aanmaak van nieuwe mitochondri\u00ebn) reguleren. De peroxisome proliferator-activated receptor-\u03b3 co-activator-1 (PGC-1)\u03b1 signalering is essentieel voor dit proces en is onder andere betrokken bij het aansturen van de aanmaak van de zogenaamde oxidatieve fosforylering (OXPHOS) complexen. Deze OXPHOS-complexen zijn belangrijk voor de energieproductie van de cel en bevinden zich in de mitochondri\u00ebn. Dit hoofdstuk zet ook de betrokkenheid van het eiwit glycogeen synthase kinase (GSK)-3\u03b2 in de regulatie van PGC-1\u03b1 in andere celtypen uiteen en het daaruit voortvloeiende algemene doel van dit proefschrift: \u2018het onderzoeken van de rol van GSK-3\u03b2 in de regulatie van oxidatief energiemetabolisme en mitochondri\u00eble biogenese in de spier\u2019. De betrokkenheid van GSK-3\u03b2 in de moleculaire regulatie van spiermassa (eiwitaanmaak, -afbraak en postnatale myogenese (dat laatste is de vorming en regeneratie van spierweefsel)) wordt eveneens in Hoofdstuk 1 besproken. Daarnaast introduceren we de experimentele modellen die we gebruikt hebben om de rol van GSK-3\u03b2 in mitochondri\u00eble biogenese te onderzoeken. Fysieke (in)activiteit wordt daarbij gepositioneerd als belangrijke factor die energiemetabolisme in de spier kan be\u00efnvloeden.\n\nIn Hoofdstuk 2, hebben we vervolgens onderzocht of uitschakelen van GSK-3\u03b2 essentieel is voor de processen van myogenese, de aansturing van eiwitaanmaak en -afbraak, PGC-1\u03b1 signalering en de aanmaak van OXPHOS-complexen tijdens spierherstel after inactiviteit. We hebben dit onderzocht door gebruik te maken van transgene muizen waarbij een permanent actieve vorm van GSK-3 door middel van genetische modificatie is aangebracht (in het hoofdstuk aangeduid met \u2018C.A. GSK-3 KI\u2019 muizen). Deze muizen hebben vervolgens een protocol doorlopen waarbij fysieke inactiviteit werd gesimuleerd door 14 dagen de achterpoten te ontlasten door muizen op te hangen aan hun staart. Om spierherstel te stimuleren zijn deze muizen vervolgens teruggeplaatst op hun achterpoten. We vonden geen uitgesproken verschillen tussen de gewone en transgene muizen tijdens de herstelfase. Hieruit hebben we geconcludeerd dat uitschakelen van GSK-3 niet noodzakelijk is voor boven-genoemde processen tijdens spierherstel na een periode van fysieke inactiviteit. Daarentegen vonden we wel dat de aanmaak van onderdelen van de PGC-1\u03b1 signalering en OXPHOS-complexen verminderd is in de spieren van transgene muizen ten opzichte van de gewone dieren onder basale condities.\n\nDe bovenstaande bevindingen hebben geleid tot de volgende hypothese: \u2018het uitschakelen van GSK-3\u03b2 in de skeletspier verhoogt PGC-1\u03b1 signalering, hetgeen mitochondri\u00eble biogenese en verhoogde aanmaak van de OXPHOS-complexen tot gevolg heeft\u2019. Om deze hypothese te toetsen hebben we in Hoofdstuk 3 gebruik gemaakt van een celkweek model van volledig gedifferentieerde spiercellen. Om de rol van GSK-3\u03b2 in mitochondri\u00eble biogenese in de spiercel te onderzoeken hebben we de activiteit van GSK-3\u03b2 farmacologisch geremd of het GSK-3\u03b2 eiwit verwijderd. Ook hebben we transgene muizen gebruikt waarbij het GSK-3\u03b2 eiwit door middel van genetische modificatie is verwijderd uit de skeletspier (in het hoofdstuk aangeduid met \u2018GSK-3 KO\u2019) en deze spieren geanalyseerd v\u00f3\u00f3r en n\u00e1 14 dagen inactiviteit (zoals hierboven beschreven). Farmacologische remming en genetische verwijdering van GSK-3\u03b2 verhoogden de aanmaak van zowel PGC-1\u03b1 als de OXPHOS-complexen in deze spiercellen. Ook was de hoeveelheid mitochondrieel DNA (mtDNA; een veelgebruikte maat voor de hoeveelheid mitochondri\u00ebn in de cel) verhoogd. Deze verhoogde aanmaak van OXPHOS-complexen na het uitschakelen van GSK-3\u03b2 was afhankelijk van de aanwezigheid van PGC-1\u03b1. Tevens waren de dieren zonder het GSK-3\u03b2 eiwit in de spier beschermd tegen een door fysieke inactiviteit ge\u00efnduceerd verlies van belangrijke spelers in de PGC-1\u03b1 signalering en de OXPHOS-complexen. Hierdoor hebben we geconcludeerd dat uitschakelen van GSK-3\u03b2 leidt tot een verhoogde aanmaak van het PGC-1\u03b1 in de spier, hetgeen vervolgens mitochondri\u00eble biogenese en daarmee een vermeerdering van de hoeveelheid OXPHOS-complexen tot gevolg heeft.\n\nHet onderhouden van de spieren en spierherstel zijn beide belangrijk voor het intact houden van gezonde skeletspieren. Daarom hebben we in Hoofdstuk 4 onderzocht of het uitschakelen van GSK-3\u03b2 tijdens myogene differentiatie (als onderdeel van spierontwikkeling) en spierherstel na een periode van inactiviteit ook leidt tot verhoging van PGC-1\u03b1 signalering, mitochondri\u00eble biogenese en aanmaak OXPHOS-complexen. Hiervoor hebben we gebruik gemaakt van gekweekte spiercellen tijdens het proces van myogene differentiatie en hebben we de muizen zonder het GSK-3\u03b2 eiwit in de spier gebruikt die na 14 dagen inactiviteit teruggeplaatst zijn op hun achterpoten om spierherstel te stimuleren. Net als in de gedifferentieerde spiercellen leidde uitschakelen van GSK-3\u03b2 tijdens myogene differentiatie tot een verhoging van belangrijke spelers van de PGC-1\u03b1 signalering alsook de aanmaak van OXPHOS-complexen. Aan het einde van de myogene differentiatie resulteerde uitschakelen van GSK-3\u03b2 tot een verhoogd mitochondrieel zuurstofverbruik wat duidt op een verbeterde functie van de mitochondri\u00ebn. Tijdens de herstelfase (dus bij het weer belasten van de achterpoten) namen de PGC-1\u03b1 signalering en de aanmaak van OXPHOS-complexen sterker toe in de muizen zonder GSK-3\u03b2 in de spier vergeleken met de controledieren. Hierdoor concluderen we dat uitschakelen van GSK-3\u03b2 tijdens spierherstel leidt tot een verhoogde mitochondri\u00eble biogenese en een verbeterde mitochondri\u00eble functie.\n\nIn Hoofdstuk 5 hebben we getracht te ontrafelen hoe het uitschakelen van GSK-3\u03b2 nu precies leidt tot een verhoging van de aanmaak van PGC-1\u03b1 in de spiercellen. Hiervoor hebben we wederom gebruik gemaakt van volledig gedifferentieerde spiercellen. Naast een verhoogde aanmaak van PGC-1\u03b1 leidde het uitschakelen van GSK-3\u03b2 ook tot een verhoogde activiteit van de PGC-1\u03b1 promoter (een stukje DNA dat de aanmaak van PGC-1\u03b1 reguleert). Om dit verder te onderzoeken we de betrokkenheid van verschillende transcriptiefactoren, waarvan bekend was dat deze aan de PGC-1\u03b1 promoter kunnen binden en vervolgens diens aanmaak kunnen be\u00efnvloeden, onder de loep genomen. Dit is gedaan door middel van farmacologische remming en genetische verwijdering van deze transcriptiefactoren. Hierdoor hebben we de betrokkenheid van verschillende transcriptiefactoren (myocyte enhancer factor 2 (MEF2) en estrogen-related receptor (ERR)) uitgesloten. Het uitschakelen van GSK-3\u03b2 zorgt er wel voor dat een andere transcriptiefactor (transcriptie factor EB (TFEB)) geactiveerd wordt. Door het uitschakelen van GSK-3\u03b2 kan TFEB namelijk naar de celkern (de plaats in de cel waar de aanmaak van genen gereguleerd wordt) migreren. Hierdoor kan TFEB aan de PGC-1\u03b1 promoter binden, wat de aanmaak van PGC-1\u03b1 tot gevolg heeft. Hieruit concluderen we dat het uitschakelen van GSK-3\u03b2 resulteert in de translocatie van TFEB naar de celkern. Dit is noodzakelijk voor de activatie van de PGC-1\u03b1 promoter en de aanmaak van PGC-1\u03b1.\n\nIn Hoofdstuk 6 worden de resultaten van dit proefschrift besproken aan de hand van de meest recente wetenschappelijke literatuur en worden de resultaten in breder perspectief geplaatst. In dit hoofdstuk wordt GSK-3\u03b2 als sleutelspeler in de regulatie van spiermassa en spiermetabolisme gepresenteerd. Verschillende aspecten van chronische ziekten die mogelijk geassocieerd zijn met een verandering van de activiteit van GSK-3\u03b2 in de skeletspieren worden belicht. Vanuit een klinisch perspectief geeft dit het therapeutische potentieel aan van farmaceutica die GSK-3\u03b2 kunnen remmen of TFEB kunnen activeren om spierafwijkingen (zoals verlies van spiermassa en\/of -metabolisme) te verhelpen of te voorkomen bij pati\u00ebnten waarbij de spiermassa is verminderd en\/of de spierstofwisseling is aangedaan zoals pati\u00ebnten met een chronische longziekte, chronisch hartfalen en bij ouderen. De algemene conclusie van dit proefschrift is dat het uitschakelen van GSK-3\u03b2 leidt tot aanmaak van PGC-1\u03b1 via TFEB in spiercellen. De verhoogde aanmaak van PGC-1\u03b1 na het uitschakelen van GSK-3\u03b2 gaat gepaard met een verhoging van het oxidatieve energiemetabolisme en de mitochondri\u00eble biogenese.\n\nValorization\n\nSocietal relevance\n\nPrevalence and burden of skeletal muscle dysfunction\n\nAging as well as several chronic diseases, such as chronic obstructive pulmonary disease (COPD), chronic kidney disease, type II diabetes and chronic heart failure, are characterized by a decline in peripheral muscle mass and a reduced number and impaired functionality of skeletal muscle mitochondria, primarily in the lower limbs [1-4]. The latter is associated with a reduced capacity of the muscle to produce energy. These skeletal muscle abnormalities ultimately dramatically affect the quality of life of these patients and are associated with increased morbidity and even higher mortality [5-7]. Indeed, good physical performance and muscle health strongly correlate with the ability to perform daily life activities, which is positively associated with quality of life and represses healthcare costs [8]. Indicative of the colossal impact of the abovementioned muscle abnormalities, sarcopenia defined as the loss of muscle mass and muscle function as a result of ageing, is highly prevalent in healthy aged individuals (10% [9]). Alarmingly, sarcopenia is even more prevalent in patients suffering from chronic diseases, such as type II diabetes and COPD [10-12] and numbers are predicted to rise further in the near future [13]. Sarcopenia is thus highly prevalent and, as stated above, correlates with an inability to adequately perform daily life tasks which ultimately leads to a reduced social status and increased hospitalization [14]. In 2014, the costs of sarcopenia-related hospitalization in the United States was estimated to be $40.4 billion [15]. Although Bruy\u00e8re et al. could not make definitive conclusions regarding the economic burden of sarcopenia, because the studies used were heterogeneous in the assessment of sarcopenia and the type of costs evaluated, their systematic review gives a recent overview of several papers indicating elevated healthcare-cost in sarcopenic individuals. In addition, already in 2000, Janssen et al. estimated that a 10% reduction in the prevalence of sarcopenia in the United States alone would save $1.1 billion in healthcare-related expenses per year [16]. Treatment or even prevention of skeletal muscle impairments can therefore contribute significantly to increasing quality of life of large groups of patients and can help with decreasing the massive healthcare costs associated with (the management of) chronic diseases [17, 18]. Besides the high prevalence of skeletal muscle abnormalities, the above-mentioned diseases are also often associated with a high cardiometabolic risk. For example, COPD patients have an increased risk for the development of cardiovascular disease [19]. Interestingly, reduced skeletal muscle oxidative phenotype (OXPHEN) has been suggested as a major driver of increased cardiometabolic risk in COPD [20] and type II diabetes [21, 22]. This further stresses the need to minimize skeletal muscle abnormalities and thereby reduce cardiometabolic disease-related healthcare costs in these patient populations [23].\n\nFuture perspective for GSK-3\u03b2 and TFEB to alleviate muscle abnormalities\n\nIn this thesis, we describe a previously unknown role for glycogen synthase kinase (GSK)-3\u03b2 in the molecular regulation of mitochondrial biogenesis and oxidative energy metabolism in skeletal muscle. We unraveled that inactivation of GSK-3\u03b2 activated peroxisome proliferator-activated receptor-\u03b3 co-activator-1 (PGC-1)\u03b1 gene expression via transcription factor EB (TFEB), which resulted in enhanced mitochondrial biogenesis and oxidative substrate use in skeletal muscle. This indicates that both GSK-3\u03b2 as well as TFEB may serve as potential targets for therapeutic intervention to alleviate abnormalities at the level of the mitochondrion in skeletal muscle during ageing or in chronic disease. However, the relevance or efficacy of specific GSK-3\u03b2 inhibitors or TFEB agonists in this context remains to be explored in more detail. Moreover, it has been reported that the inhibitory phosphorylation of GSK-3\u03b2 and nuclear abundance of TFEB increase during physical activity. Combined with the data presented in this thesis, this indicates that improving muscle OXPHEN via modulation of the GSK-3\u03b2-TFEB-PGC-1\u03b1 pathway can be achieved by adopting a healthier and more active lifestyle. As a long-term perspective, the knowledge gained by research described in this thesis could aid in the development of GSK-3\u03b2 inhibitors, TFEB agonists or lifestyle interventions aiming to alleviate muscle abnormalities in the above-mentioned conditions.\n\nTreatment strategies for increasing muscle oxidative phenotype\n\nExercise and exercise mimetics\n\nPhysical exercise is the most common and effective method to increase skeletal muscle health. However, exercise training is not always feasible in humans due to disease-related symptoms or age-related frailty. In addition, lifestyle changes are particularly hard to accomplish in elderly [24]. Nonetheless, exercise training or increased loading of skeletal muscle are highly effective evidence-based intervention methods for improving skeletal muscle function in several chronic conditions, including COPD [25]. However, the anabolic and mitochondrial response to exercise training is often blunted in COPD patients [26] as well as during aging [27, 28]. Alterations in the abundance or activity of TFEB in these conditions might underlie this mal-adaptability, because TFEB knock-out mice have been shown to have a reduced ability to increase mitochondrial function and maintain the same exercise capacity compared to control animals. This makes them unable to fully benefit from exercise in terms mitochondrial regeneration [29], which speculatively could result in a blunted anabolic response. In this context, pharmacological activation of the pathways controlling muscle OXPHEN and muscle mass (so called \u2018exercise-mimetics\u2019) may prove beneficial to alleviate skeletal muscle abnormalities in the above-mentioned disorders and the aging population [30]. The mechanistic insight in the regulation of skeletal muscle PGC-1\u03b1 expression gained in this thesis suggests a potential role for specific GSK-3\u03b2 inhibitors or TFEB agonists as exercise-mimetics. Although the effects of inactivation of GSK-3\u03b2 on mitochondrial biogenesis and oxidative energy metabolism and the involvement of TFEB herein needs to be studied in humans, the promising potential of GSK-3\u03b2 inhibitors for clinical implication is clear.\n\nPharmacological potential of GSK-3\u03b2 inhibitors\n\nIn order to verify the importance of GSK-3\u03b2 in the (dys)regulation of skeletal muscle mitochondrial biogenesis and energy metabolism, it needs to be elucidated whether or not GSK-3\u03b2 abundance or activity is altered in skeletal muscle in the above-mentioned human conditions associated with loss of muscle OXPHEN. In this context, it has been shown that the abundance and activity of GSK-3\u03b2 is higher in skeletal muscle of obese type II diabetic patients compared with lean and weight-matched non-diabetic subjects [31]. In addition, GSK-3\u03b2 phosphorylation was found to be reduced (indicating increased GSK-3\u03b2 activity) in skeletal muscle of pregnancy-associated diabetes mellitus patients [32]. If GSK-3\u03b2 activity is also increased in skeletal muscle of COPD patients is unclear. Several studies reported contradicting evidence on the phosphorylation status of GSK-3\u03b2 in skeletal muscle of COPD patients. Indeed, unaltered [33, 34] and increased [35] levels of phosphorylated\/inactivated (Ser9) GSK-3\u03b2 have both been reported in skeletal muscle of COPD patients. Literature on the activity of GSK-3\u03b2 in skeletal muscle in chronic human diseases is thus scarce and contradictory and needs to be investigated in closer detail. Furthermore, it needs to be elucidated if structural absence of GSK-3\u03b2 (in ablation modalities) or short-term acute inactivation of GSK-3\u03b2 exert similar effects on TFEB nuclear translocation. To date, several experimental studies reveal that inactivation of GSK-3\u03b2 in skeletal muscle is sufficient for nuclear translocation and activation of TFEB. Besides the effect of inactivation of GSK-3\u03b2 on TFEB nuclear translocation in healthy conditions, these effects have not been verified in experimental models of chronic disease associated with reduced mitochondrial biogenesis. In order to fully exploit the benefits of exercise on muscle function, specific inhibition of GSK-3\u03b2 (or activation of TFEB) combined with an exercise protocol may be a potential novel treatment strategy [29]. Indicative of the therapeutic potential of GSK-3\u03b2 inhibition in augmenting muscle functionality, lithium chloride (LiCl; an inhibitor of GSK-3) improved muscle strength and muscle mass in a murine muscle dystrophy model [36]. However, whether or not pharmacological inhibition of GSK-3\u03b2 can rescue mitochondrial abnormalities in skeletal muscle remain to be investigated. Therefore, new studies should focus on the effect of exercise and pharmacological inhibition of GSK-3\u03b2 on TFEB nuclear translocation in skeletal muscle in health and disease. In order to accomplish this, placebo-controlled pilot experiments aiming to investigate the effect of GSK-3\u03b2 inhibitors in combination with or without exercise training on skeletal muscle mitochondrial biogenesis should reveal the clinical potential of GSK-3\u03b2 inhibitors. Interestingly, lithium is clinically approved and often used to treat bipolar disorder [37]. However, some minor side effects of lithium treatment have been reported [38]. The clinical use of lithium indicates that lithium as well as more specific GSK-3\u03b2 inhibitor are likely safe for human application. In this thesis, we report that GSK-3\u03b2 inactivation enhanced TFEB nuclear translocation. In addition, TFEB was required for the inactivation of GSK-3\u03b2-mediated induction of PGC-1\u03b1, hence TFEB agonist might also be a promising pharmacological intervention aiming to induce muscle PGC-1\u03b1 levels and thereby enhance muscle mitochondrial biogenesis.\n\nPharmacological potential for TFEB agonists\n\nThe role of TFEB in the regulation of PGC-1\u03b1 and the subsequent control over mitochondrial biogenesis and oxidative energy metabolism was only recently discovered [29]. Despite this, several studies using multiple cell-types now support that nuclear translocation of TFEB, or other microphthalmia\/TFE (MiT) family members, increases the expression of PGC-1\u03b1 and enhance mitochondrial biogenesis [29, 39, 40]. Interestingly, TFEB agonists that stimulate nuclear translocation are being developed and tested [41]. However, if pharmacological activation of TFEB can rescue mitochondrial abnormalities in skeletal muscle remains to be investigated. In order to accomplish this, placebo-controlled pilot experiments aiming to investigate the effect of these compounds on mitochondrial function can be the first step to elucidate potential pharmacological potential of TFEB agonist in humans. Interestingly, digoxin (chemical TFEB agonist) is clinically approved and thus found to be safe for human application. The above-mentioned trials could increase our understanding of mitochondrial abnormalities in skeletal muscle within human disease and ultimately could reverse aging- or chronic disease-associated skeletal muscle mitochondrial deficits.\n\nA potential role for GSK-3\u03b2 and TFEB in neurodegenerative disorders\n\nNext to skeletal muscle, mitochondria are highly important for the function of a myriad of other tissues, for example the brain. Growing lines of evidence suggest that mitochondrial dysfunction is involved in neurodegenerative diseases such as Parkinson\u2019s, Alzheimer\u2019s and Huntington\u2019s disease. Interestingly, it has been proposed that inactivation of GSK-3\u03b2, aiming to prevent mitochondrial dysfunction, is a potential treatment of neurodegenerative disorders. Pre-clinical data showed that pharmacological inhibition of GSK-3\u03b2 restored mitochondrial biogenesis in a mouse model of Parkinson\u2019s disease [42]. Interestingly, Alzheimer\u2019s disease is linked to increased activity of GSK-3\u03b2 in the cortex. Furthermore, reduced levels or decreased activity of PGC-1\u03b1 and TFEB have been associated with Alzheimer\u2019s disease progression [43, 44]. This suggest that GSK-3\u03b2 inhibitors or TFEB agonists might also be beneficial for patients with Alzheimer\u2019s disease. Intriguingly, pre-clinical data revealed that inhibition of GSK-3\u03b2 using LiCl reduced both \u03b2-amyloid as well as tau protein (the two major protein aggregates involved in Alzheimer\u2019s disease progression) formation [45, 46]. LiCl treatment was previously reported to reduce cognitive decline in Alzheimer\u2019s disease [47]. This indicates that inhibition of GSK-3\u03b2 indeed might be suitable to alleviate the burden of Alzheimer\u2019s disease [48].\n\nConclusion\n\nThe data in this thesis convincingly shows that inactivation of GSK-3\u03b2 in skeletal muscle enhances PGC-1\u03b1-mediated mitochondrial biogenesis and oxidative substrate metabolism via TFEB. This suggests that both GSK-3\u03b2 inhibitors as well as TFEB agonists are potential therapeutic strategies to treat or even prevent deficits in skeletal muscle OXPHEN associated with chronic diseases and aging. In addition, as outlined in the general discussion (Chapter 6), GSK-3\u03b2 is involved in both the regulation of muscle mass as well as the regulation of muscle mitochondrial biogenesis and oxidative energy metabolism. Thus, specific GSK-3\u03b2 inhibitors might be beneficial in alleviating both abnormalities in muscle mass and muscle OXPHEN that often coincide in chronic diseases. To date however it remains unclear if pharmacological inhibition of GSK-3\u03b2 or activation of TFEB can induce skeletal muscle mitochondrial biogenesis or enhance oxidative substrate metabolism in a clinical setting. In the future, new randomized placebo-controlled human trials should shed light on these remaining questions. In addition, particular care should be given to confirm safety of novel pharmaceuticals for human application. Furthermore, in order to minimize side effects, the possibility to use specific drug-delivery techniques should be explored to transport the medication to the targeted tissue, for example the skeletal muscle [49]. Moreover, the beneficial effects of inhibition of GSK-3\u03b2 on the brain, as discussed above, indicates a broader perspective for the pharmacological inhibition of GSK-3\u03b2 in human diseases. Although speculative in nature, the data presented in this thesis thus may be applied in several research disciplines beyond skeletal muscle pathology.","summary":"Skeletal muscle tissue has a remarkably high plasticity. This is reflected by its large capacity to adapt its mass and oxidative phenotype (OXPHEN; defined as the proportion of oxidative muscle fibers and mitochondrial oxidative capacity) in response to, amongst others, changes in physical activity or muscle loading. In Chapter 1, the molecular pathways regulating skeletal muscle OXPHEN are introduced. This primarily encompasses the peroxisome proliferator-activated receptor-\u03b3 co-activator-1 (PGC-1)\u03b1 signaling network which has been convincingly shown to be a pivotal pathway in the regulation of mitochondrial biogenesis not only in skeletal muscle but in a wide variety of cell types. In addition, this chapter introduces glycogen synthase kinase (GSK)-3\u03b2 and its role in the regulation of PGC-1\u03b1 in non-muscle cells. This led to the main objective of this thesis: \u2018To investigate the role of GSK-3\u03b2 in oxidative substrate metabolism and mitochondrial biogenesis in skeletal muscle\u2019. The models that were used to investigate this are presented and physical (in)activity is introduced as one of the most potent external triggers affecting muscle OXPHEN. Furthermore, Chapter 1 introduces the involvement of GSK-3\u03b2 in the regulation of skeletal muscle mass, e.g. muscle protein turnover and post-natal myogenesis.\n\nIn Chapter 2, we investigated if GSK-3\u03b2 inactivation during muscle reloading (after a period of muscle unloading) is essential for improvements in myogenesis, protein turnover signaling, PGC-1\u03b1 signaling and subsequent expression of oxidative phosphorylation (OXPHOS) sub-units. We address this hypothesis using whole-body constitutively active (C.A.) GSK\u20113\u03b1\/\u03b2 knock-in and wild-type mice and investigated the soleus muscle of the hind-limb under baseline conditions, directly after 14-days of hind-limb suspension (HLS; a disuse-induced atrophy model) and during reloading of the hind-limbs. No consistent or significant alterations in reloading-induced changes in muscle mass, protein turnover, post-natal myogenesis nor in the regulation of muscle OXPHEN were observed between the two genotypes. In this chapter, we conclude that GSK-3 inactivation is dispensable for the above-mentioned processes during muscle reloading after a period of inactivity. However, subtle but consistent differences are observed at baseline between the two genotypes suggesting suppression of protein turnover signaling, PGC-1\u03b1 signaling and mRNA expression of several OXPHOS sub-units resulting from constitutive activation of GSK-3.\n\nThese findings led to the hypothesis that inactivation of GSK-3\u03b2 increases gene expression of PGC-1\u03b1, which subsequently induces mitochondrial biogenesis and gene expression of OXPHOS sub-units in adult muscle (Chapter 3). To address this hypothesis, we used pharmacological and genetic approaches to inhibit GSK-3\u03b2 in fully differentiated C2C12 murine myotubes, as an in vitro analogue of myofibers. In addition, we used muscle-specific GSK-3\u03b2 knock-out (KO) mice at baseline and directly after 14-days of HLS. Largely in line with our findings in the C.A. GSK-3\u03b2 knock-in mice (Chapter 2), overexpression of GSK-3\u03b2 did not alter the expression of PGC-1\u03b1 or OXPHOS sub-units in C2C12 myotubes. However, pharmacologic and genetic inhibition of GSK-3\u03b2 potently increases mRNA and protein content of PGC-1\u03b1 and OXPHOS sub-units in C2C12 myotubes. This is accompanied by increased levels of mitochondrial (mt)DNA. Furthermore, we reveal that increased gene expression of OXPHOS sub-units mediated by inhibition of GSK-3\u03b2 requires PGC-1\u03b1 by deploying double knock-down experiments. In addition, muscle-specific GSK-3\u03b2 KO protects against unloading-induced loss of gene expression of components of the PGC-1\u03b1 signaling cascade and OXPHOS sub-units. In conclusion, in this chapter we identified that GSK-3\u03b2 inactivation increases PGC-1\u03b1 levels in muscle cells and subsequently induces mitochondrial biogenesis and expression of sub-units of OXPHOS complexes.\n\nSeveral molecular mechanisms control the oxidative capacity of the muscle during both maintenance as well as regeneration of adult skeletal muscle. Impairments in these regulatory mechanisms contribute to the development of skeletal muscle abnormalities. In Chapter 4, we therefore investigated if inactivation of GSK-3\u03b2 also potentiates the PGC-1\u03b1 signaling cascade during myogenic differentiation and during recovery of inactivity-induced muscle atrophy. We report that GSK-3\u03b2 inhibition increases the abundance of key constituents of the PGC-1\u03b1 signaling pathway and increases expression of OXPHOS sub-units during myogenic differentiation of C2C12 myoblast into myotubes, which is in line with the effects of inhibition of GSK-3\u03b2 in fully differentiated myotubes that we describe in Chapter 3. Ultimately, knock down of GSK-3\u03b2 during myogenic differentiation results in enhanced mitochondrial respiration at the end of the myogenic differentiation program. In addition to these findings, in vivo experiments reveals that muscle-specific GSK-3\u03b2 KO potentiates reloading-induced inductions in gene expression of components of the PGC-1\u03b1 signaling and OXPHOS sub-units after a period of inactivity. Overall, we conclude that inactivation of GSK-3\u03b2 potentiates the PGC-1\u03b1 signaling, resulting in mitochondrial biogenesis and mitochondrial respiration during myogenic differentiation and muscle recovery after a period of physical inactivity.\n\nIn Chapter 5, we aimed to elucidate the molecular mechanism by which inactivation of GSK-3\u03b2 increases Pgc-1\u03b1 mRNA abundance in skeletal muscle cells. We used fully differentiated C2C12 myotubes to fundamentally underpin this underlying molecular mechanism. PGC-1\u03b1 promoter activity enhances following inactivation of GSK-3\u03b2 while chromatin accessibility of the PGC-1\u03b1 promoter remained unaltered, indicating a transcriptionally-controlled mechanism. Subsequently, several transcription factors known to be involved in the transcriptional control of PGC-1\u03b1 were pharmacologically or genetically inhibited in C2C12 myotubes in order to investigate their possible involvement in transcriptional regulation of PGC-1\u03b1 mediated by inactivation of GSK-3\u03b2. Inhibition of GSK-3\u03b2 did not alter myocyte enhancer factor 2 (MEF2) transcriptional activity, indicating that MEF2 isoforms are likely not involved in enhanced Pgc-1\u03b1 transcription mediated by inactivation of GSK-3\u03b2. Furthermore, while knock-down of GSK-3\u03b2 increased estrogen-related receptor (ERR) expression levels and potentiated transcription of its down-stream targets, ERR transcription factors are not essential for increased Pgc-1\u03b1 levels mediated by inhibition of GSK-3\u03b2. Interestingly, inhibition of GSK-3 activity results in nuclear translocation of transcription factor EB (TFEB). Furthermore, increased Pgc-1\u03b1 gene expression and activation of the PGC-1\u03b1 promoter mediated by inactivation of GSK-3\u03b2 require TFEB. In addition, mutation of a TFEB binding sequence located on the proximal PGC-1\u03b1 promoter blocked PGC-1\u03b1 promoter activation induced by inactivation of GSK-3. Overall, we report that inactivation of GSK-3\u03b2 causes nuclear translocation of TFEB, which is required for induction of PGC-1\u03b1 promoter activation and subsequent transcription of the Pgc-1\u03b1 gene.\n\nFinally, in Chapter 6, the overall results of this thesis are discussed and placed in a broader perspective. We review the potential role of GSK-3\u03b2 as key node in the regulation of skeletal muscle mass and OXPHEN in the context of disease-related factors that might be associated with increased activity of GSK-3\u03b2 in skeletal muscle including physical inactivity, malnutrition and hypoxia. From a clinical perspective, the therapeutic potential of GSK-3\u03b2 inhibitors or TFEB agonists is highlighted to treat or prevent muscle abnormalities during ageing and in chronic diseases such as chronic obstructive pulmonary disease, chronic heart failure, chronic kidney disease and type II diabetes (diseases that are often characterized by both loss of muscle mass and deterioration of muscle oxidative capacity). Furthermore, we highlight our novel finding that inactivation of GSK-3\u03b2 phosphorylates TFEB, resulting in its nuclear translocation, which results in increased promoter activation and gene expression of PGC-1\u03b1. Ultimately, inactivation of GSK-3\u03b2 potentiates skeletal muscle oxidative substrate metabolism and mitochondrial biogenesis via this process.","auteur":"Wessel Theeuwes","auteur_slug":"wessel-theeuwes","publicatiedatum":"3 april 2020","taal":"NL","url_flipbook":"https:\/\/ebook.proefschriftmaken.nl\/ebook\/wesseltheeuwes?iframe=true","url_download_pdf":"https:\/\/ebook.proefschriftmaken.nl\/download\/66da400c-fe8e-4513-9c87-3d9dfdd00736\/optimized","url_epub":"","ordernummer":"FTP-202604131047","isbn":"978-94-6380-738-8","doi_nummer":"","naam_universiteit":"Universiteit Maastricht","afbeeldingen":11446,"naam_student:":"","binnenwerk":"","universiteit":"Universiteit Maastricht","cover":"","afwerking":"","cover_afwerking":"","design":""},"_links":{"self":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/11444","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio"}],"about":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/types\/us_portfolio"}],"author":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/comments?post=11444"}],"version-history":[{"count":1,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/11444\/revisions"}],"predecessor-version":[{"id":11447,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/11444\/revisions\/11447"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media\/11445"}],"wp:attachment":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media?parent=11444"}],"wp:term":[{"taxonomy":"us_portfolio_category","embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio_category?post=11444"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}