50 Chapter 2 had an aberrant structure and compromised fatty-acid β-oxidation in these animals, which together with a decline in peroxisomal number was thought to contribute to the observed hepatic steatosis30. Since it has been suggested that peroxisomal decline precedes mitochondrial decline30, we decided to increase the starvation time in the organoids up to 96 hours. This delayed onset of a mitochondrial phenotype was reproduced in the organoids: after 96 hours of amino-acid starvation mitochondrial protein markers as well as functional state 3 respiration were significantly reduced. These results are consistent with findings that defects in peroxisomal biogenesis may cause mitochondrial decline40,62. The decrease of acyl carnitines (already after 48 hours) preceding the reduction in oxygen consumption could be explained by the system relying on fatty acids to obtain energy after removal of amino acids. In starved intestinal organoids both mitochondrial membrane and matrix markers were lower, reminiscent of reduced mitochondrial numbers in the intestine of mice on a low-protein diet (manuscript in preparation). The decline of peroxisomal marker proteins in intestinal organoids is a novel finding for the intestine. As mitochondria play an important role in maintaining the stem cell niche27,59, reduction in mitochondrial markers proteins could contribute to the observed crypt atrophy in the organoids. Although peroxisomes are highly abundant in the small intestine63, little is known about the functional significance of these organelles in this organ. In Drosophila, peroxisomes are required for homeostasis of intestinal epithelium64. More recently, Du et al. reported that elevated peroxisome numbers are required for stem cell differentiation and epithelial repair in human, mouse and drosophila intestine41. Reduced or dysfunctional peroxisomes could result in epithelial instability64 via redox stress, and compromise epithelial turnover41. Intestinal organoids are a versatile model to further explore the reduction of peroxisomes upon aminoacid starvation and its consequences for intestinal homeostasis. Translation into potential treatments for severely malnourished children We tested if targeting mitochondria and peroxisomes could preserve organ homeostasis under amino- acid deprived conditions. In starved hepatic organoids, addition of PPAR-α agonist fenofibrate partially recovered the protein expression levels of peroxisomal markers but showed no effect on pristanic acid metabolism. Fenofibrate did not recover intracellular triglyceride levels when administered during amino acid starvation. In contrast, fenofibrate treatment in low-protein fed rats significantly improved mitochondrial
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