103 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model complete amino-acid starvations. However, little is known about the longerterm effects of exposure to low amino-acid levels in contrast to complete and shorter starvations. Moreover, there is little information available on the effect of amino-acid restriction on peroxisomal biogenesis. In this study, hepatic organoids were grown in amino acid restricted medium but not in complete starvation conditions. Organoids were still exposed to low levels of amino acids coming from the Matrigel, in which the organoids are cultured, or potentially from the degradation of growth factors present in the medium as specified in previous literature5. These conditions mimic low protein diets (LPD) like in in vivo conditions better. Several groups have demonstrated that autophagy is altered in rodents in response to low protein diets at a very young age1–3. The results reported here are in agreement with those observed in rats on a 1 week low protein diet. However, when rodents were subjected to a prolonged LPD, autophagy was hampered, both in liver and intestine. In the liver of rats fed a low protein diet for 1 week, LC3II was increased while P62 was downregulated, suggesting autophagy to be increased. In contrast, exposure to LPD for 4 weeks led to an increase in p62, which was interpreted as a block in autophagy1. Similarly, studies using mice on LPD for 2 weeks reported a reduction of autophagy. However, while in both cases the protein levels of LC3-II were downregulated, one of the studies also reported a downregulation of the LC3II/I2 while the other did not3. Given the nature of in vivo work, all of these studies assessed autophagy by western blotting and only a snapshot of the pathway was provided at a given time point. Here we demonstrated that organoids are a dynamic and accessible tool to study autophagic flux in the context of amino acid restriction. To do so, we implemented the use of a dynamic probe (GFP-LC3-RFP-LC3ΔG)36, developed for the study of autophagy in live cells. We reported that 96 h amino acid restriction led to an induction of autophagy, as also observed in rats on LPD for a short time and in other in vitro models under amino acid starvation33. With respect to peroxisomal biogenesis, rodents fed a low protein diet for 4 weeks did not show any regulation in gene expression of peroxins involved in peroxisomal biogenesis1. In the present study, we have demonstrated that amino acid restriction has an impact on the expression of genes involved in peroxisomal biogenesis. Since some genes were upregulated and others downregulated we could not draw any clear conclusions. However, further characterization of this differential regulation might help identify the functional impact. Conspicuously, both catalase and ACOX1 are imported
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