93 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model as pharmacological treatment for the protection of peroxisomal matrix and mitochondrial proteins in malnutrition. RESULTS Liver progenitor organoids generated from mice were differentiated into hepatocyte-like organoids and used for the study of amino-acid restriction, as previously described5. Prolonged amino-acid deprivation leads to severe peroxisomal loss in mature hepatic organoids To investigate the effect of the duration of amino-acid restriction on peroxisomal loss, hepatic organoids were exposed to amino-acid free medium for 48 or 96 h. Clearly, 96 h of amino acid restriction led to a more severe and significant decline in the levels of the peroxisomal marker proteins than 48 h (Figure 1a and 1b). Levels of PMP70, an ATP binding cassette transporter of fatty acids into the peroxisome, and one of the most abundant peroxisomal membrane proteins, were strongly reduced after amino acid starvation. AcylCoA oxidase 1 (ACOX1), the first enzyme of the peroxisomal β-oxidation, and catalase, the enzyme metabolising the hydrogen peroxide produced by ACOX1 and multiple other oxidases, were also significantly reduced after 48 or 96 h of amino-acid restriction compared to control cells (Figure 1a and 1b). To obtain a more comprehensive insight into the different peroxisomal proteins affected by amino acid deprivation, we quantified several peroxisomal proteins by targeted proteomics30. For the proteomic analysis, we designed and used as internal standards a panel of 13C-labelled peptides with the QconCAT technology32. These standards target 57 murine peroxisomal proteins as well as their human orthologues. Up to 28 peroxisomal proteins (depending on the experimental condition and amount of organoids), 4 of which have dual localization, were sufficiently abundant to be detected and quantified in the hepatic organoids. These included metabolite transporters, as well as enzymes of the β-oxidation and α-oxidation (Supplementary Table 1). Overall, a timedependent reduction of these peroxisomal proteins was observed in response to amino-acid deprivation (Figure 1c). Immunofluorescence analysis using antibodies against PMP70 revealed a reduced number of green punctae in the starved organoids, indicating a reduced number of peroxisomes, but only after 96 hours of amino-acid
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