211 7 General Discussion During the work on this thesis, I also illustrated the potential of 3D in vitro models to test different therapeutic approaches to prevent peroxisomal and mitochondrial loss in the context of amino-acid restriction. In chapter 2, I briefly focused on the therapeutic effects of fenofibrate and rapamycin in the liver and the intestine, respectively. Fenofibrate prevented the loss of peroxisomal markers in 48h amino-acid restricted hepatic organoids, but failed to prevent loss of peroxisomal function. In chapter 4 I tested the effects of different PPAR-α agonists on preventing peroxisomal and mitochondrial loss during longer amino-acid restrictions (96h). When comparing the effects of docosahexaenoic acid (DHA) supplementation with other PPAR-α agonists we observed disparate results. DHA supplementation prevented the loss of multiple peroxisomal and mitochondrial proteins, whilst the rest of the compounds showed little to no effect. Interestingly, the results were different from those of fenofibrate in chapter 2. However, it is not possible to compare these results directly as the amino-acid restriction time was different between experiments in the two chapters. Given the differences between DHA and the other agonists, we hypothesized a PPAR-α-independent effect of DHA leading to the prevention of peroxisomal and mitochondrial loss. While in this chapter I only focused on a small panel of compounds, this platform is suitable for larger screenings of other compounds to either stimulate peroxisomal biogenesis or to prevent their degradation. The use of targeted proteomics could be applied to larger screenings of compounds, assessing their effect on prevention of peroxisomal loss in the context of amino-acid restriction. Optimization of the number of organoids needed for the protocol would greatly benefit the upscaling process and minimize associated costs. Potential roles of peroxisomes in MCADD Finally, in chapter 6, I focused on the study of medium chain acyl-CoA dehydrogenase deficiency. This inherited disease affects the metabolism of medium chain acyl-CoA leading to accumulation of medium-chain fatty acids and fatty-acid esters under different circumstances. One interesting point of discussion raised in chapter 6 is the heterogeneity in symptomatology observed amongst MCADD patients21,22,47. While the main aim of this chapter was to develop an in vitro model to study and characterize the disease, we also explored a potential compensatory role of peroxisomes. Here, we hypothesized that different compensatory mechanisms involving peroxisomes could explain the differences in adaptation to conditions such as fasting or illness. In this chapter we observed no regulation of the peroxisomal β-oxidation of fatty acids in MCADD organoids. However, we identified a clear regulation of several genes
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