92 Chapter 4 factor within the family of nuclear receptors known to induce proliferation of peroxisomes and highly expressed in tissues involved in fatty acid metabolism, such as the liver18. Activation of PPAR-α increases the expression of genes involved in peroxisomal and mitochondrial β-oxidation, thereby upregulating the uptake, activation and oxidation of fatty acids19–22. Therefore, PPAR-α activation might recover peroxisomal number and mitochondrial health by upregulation of β‑oxidation genes, as observed in malnourished rats supplemented with fenofibrate1, and thereby reduce fat accumulation, at least in rodents. Some of the most commonly described PPAR-α ligands are synthetic compounds, such as fibrates. In addition, naturally occurring compounds, including long-chain polyunsaturated fatty acids (LCPUFA), have also been reported to be PPAR- α agonists23. Promising results in improvement of cognition of severely malnourished children highlight the latter compounds as interesting dietary supplements 24. Discovery of the organoid technology in 200925 has transformed the field of biomedicine, providing scientists with physiologically relevant in vitro disease models. Organoids are proliferative 3D structures, derived from primary tissue, adult stem cells or pluripotent stem cells. These structures contain different cell types and mimic the functions of the organ of origin26. Organoids are valuable tools for the study of organ development, disease modelling and drug screening27,28. They have also been shown to be a relevant system to study nutrient metabolism29,30 and metabolic diseases31. We have previously used organoids to model severe malnutrition using long-term exposure to low amino-acid levels. Although no amino acids were added to the medium, all amino acids were found in low concentrations, probably coming from degradation of the Matrigel or the cytokines and growth factors present in the medium5. The latter study recapitulated liver-specific phenotypes of malnutrition, including peroxisomal loss and mitochondrial dysfunction. The aim of the present study was to use the previously established murine hepatic malnutrition in vitro model5 to gain more insight into the mechanisms of peroxisomal loss under different durations of exposure to low-amino acid conditions and assess the role of autophagy therein. Moreover three different PPAR-α agonists, including a synthetic ligand (WY-14643) and two long-chain polyunsaturated fatty acids (linoleic acid and docosahexaenoic acid), were tested to determine their ability to prevent peroxisomal loss. We used a GFP/ RFP autophagic flux reporter system to quantify the effect of amino acid restriction and PPAR-α activation on autophagic flux. We conclude that the natural lipid docosahexaenoic acid (DHA) is a promising compound to explore
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