47 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition Mitochondrial marker proteins TOM20 and Hsp60 were also downregulated by amino acid starvation (Figure 6a-b). Yet, the complexes of the electron transport chain were not affected by amino-acid starvation (Figure 6a-b). Mitochondrial copy number was also significantly decreased in starvation conditions (Figure 6d). Amino acid re-supplementation normalized all peroxisomal and mitochondrial characteristics, except the level of TOM20, which was even increased after re-supplementation (Figure 6a-d). Interventions to preserve mitochondria and peroxisomes in starved organoids We investigated if amino-acid-deprived organoids could be used as a model to test pharmacological interventions to preserve organ function. As a proof of principle, we tested interventions that have already been applied in animal studies. Fenofibrate, a PPAR-α agonist, was previously shown to preserve peroxisomes and mitochondrial function in the liver of rats on a low-protein diet30. When administered to amino-acid starved mature hepatic organoids, fenofibrate increased levels of PGC1-α, PMP70 and catalase to values similar as the controls (Figure 4a, b). Acox-1, the first enzyme of the peroxisomal β-oxidation was not affected by fenofibrate, nor was its substrate pristanic acid (Figure 5a). No change was observed in the levels of mitochondrial proteins with fenofibrate treatment (Figure 4a, b). Triglycerides were not restored upon fenofibrate treatment. Instead, addition of fenofibrate in starvation medium led to a higher accumulation of intracellular TGs (Figure 4i). We recently showed that rapamycin, an mTORC1 inhibitor, preserved intestinal barrier function and mitochondrial number and morphology in mice on a low protein diet (Manuscript in preparation). Likewise, in amino-acidstarved intestinal organoids, rapamycin increased the levels of mitochondrial marker protein HSP-60, although it remained lower than in control organoids (Figure 6a,b). In addition, protein levels of PGC1-α were increased in starved rapamycin-treated organoids to a similar level as in amino-acid-resupplemented organoids (Figure 6a,b). Although PGC1-α is also known as an activator of peroxisomal biogenesis, rapamycin treatment did not affect the protein levels of peroxisomal proteins (Figure 6a,b). Rapamycin treatment did preserve claudin-3 protein levels in starved organoids, suggesting preserved intestinal barrier function (Figure 3d). However, rapamycin did not appear to decrease the FITC levels in the organoid lumen, nor the number of FITC-dextran positive organoids (Figure 3b, c and Supplementary Figure 6).
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