210 Chapter 7 hepatic organoids is caused by increased degradation or reduced biosynthesis. While impaired biogenesis could be a potential explanation, little information is available on the topic. Only one in vivo study using rats on an LPD reported no changes in the expression of any peroxins (PEX) involved in peroxisomal biogenesis and maintenance14. During this chapter I showed how amino-acid restricted organoids showed regulation of several PEX genes. This differential regulation indicates, that although it is hard to determine whether biogenesis is up- or downregulated, it is clearly affected by amino acid restriction. In the case of PEX5, linked to pexophagy in nutrient starvation43, we observed a clear upregulation under the amino-acid restricted conditions. Further studies should be done in order to fully understand the effect of amino-acid restriction on peroxisomal biogenesis. In contrast, several groups have focused on the effects of LPD on autophagic degradation in rodents. It is important to emphasize that different experimental conditions lead to different results, and in some cases comparison between models might be misleading. Low protein diets have been shown to induce peroxisomal and mitochondrial loss as well as dysfunctional mitochondria both in children and laboratory animals13,14,44. Most of the in vivo work on malnutrition has reported that long exposure to LPD leads to an impairment in autophagic turnover12,13. However, the results found in this thesis, using in vitro models, opposed those found in in vivo studies. In chapter 4, I reported that amino-acid restriction leads to a clear upregulation of autophagic degradation, as measured with an autophagic probe. An interesting aspect of this model is the use of amino-acid restriction instead of a complete amino-acid starvation. While it has been previously described that complete amino-acid starvations induce autophagy in vitro45,46, there is no information of the effect of long periods of time of amino-acid restriction. If we try to compare our results with the in vivo models, they are more in line with what was observed in the liver of rats fed a LPD for 1 week14. However, in that same study, the authors reported a block in autophagy after 4 weeks of LPD. These results, together with those of the mouse models which used 2 weeks of LDP, might indicate that autophagy is differentially regulated according to the time of exposure to LPD. On that note, in chapter 4, I also reported a different regulation of autophagy in organoids deprived of amino acids for 48 h and 96 h. These results also point towards a differential regulation based on time. However, in the case of the amino-acid restricted hepatic organoid model, amino-acid restrictions of longer than 4 days were hard to recapitulate using the organoids due to technical limitations. On this note, it is important to emphasize the challenges of translating time length from organoid work to animal models.
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