102 Chapter 4 Effects of DHA on peroxisomal biogenesis and autophagy Given that DHA supplementation increased the overall pattern of peroxisomal and mitochondrial proteins, we investigated the effect of DHA on both peroxisomal biogenesis and autophagic degradation. DHA nor any of the other compounds tested showed any effect on the peroxins involved in peroxisomal biogenesis (Supplementary figure 2). In a first test on Huh7 cells transduced with the pMRX-IP-GFP-LC3-RFP-LC3ΔG construct, 48 hours of amino-acid restriction induced autophagic flux, similarly to what was observed in the hepatic organoids. Autophagic flux decreased to control levels after DHA treatment in a dose-dependent manner (Figure 4f), but was not affected by the other PPAR‑α agonists, WY-14643 or LA (Supplementary Figure 3). These results are in agreement with what was previously observed in oxidative stressinduced autophagy and TNF-α-induced autophagy41,42. In the GFP-LC3-RFPLC3Δ hepatic organoids, the activation of autophagic flux by 96 h of amino-acid deprivation was replicated, but it was not rescued by DHA (Figure 4g). The effect of DHA on autophagy, at least in the Huh7 cell line, goes in line with the observed prevention of peroxisomal and mitochondrial protein loss. The results suggest, however, that in the organoids the rescue of peroxisomal and mitochondrial proteins by DHA might occur via an autophagy-independent mechanism. DISCUSSION In this study we have demonstrated that prolonged amino acid restriction leads to peroxisomal loss with increased autophagic degradation and alterations in peroxisomal biogenesis in a murine hepatic organoid model. To assess autophagic flux we have implemented the use of the novel GFPLC3-RFP-LC3ΔG probe in the hepatic organoids. Using targeted proteomics, we then demonstrated that DHA supplementation prevented the loss of both peroxisomal and mitochondrial proteins in the context of amino acid restriction. Mechanisms of peroxisomal loss in amino acid restriction and potential therapeutic interventions Amino-acid deficiency has been previously linked with peroxisomal loss43, however, the mechanisms behind this process in malnutrition still remain unclear. While the main cause of peroxisomal loss in amino-acid starvation has been described to be pexophagy43, these studies focused on the effect of
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