49 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition was not significantly reduced, but it was nevertheless increased upon resupplementation. This is in line with the observed re-growth of crypts. Restoration of growth and new crypt formation after amino acid resupplementation indicates that intestinal stem cells have the capacity to recover from prolonged amino-acid starvation. Re-growth of crypts may be attributed to rapidly cycling Lgr5+ crypt base columnar (CBC) cells, which were previously found resilient to glutamine starvation49. Another possibility is the mobilization of another stem cell population referred to as quiescent ‘reserve’ stem cells (+4 position) that are activated upon injury to restore tissue homeostasis51. Moreover, de-differentiation of progenitor cells52,53 as well as differentiated epithelial cells can contribute to repopulation of the stem cell niche54,55. Yet, the impact of prolonged amino-acid deprivation on intestinal stem cells and on de-differentiation has not been studied. Finally, the barrier dysfunction and reduction of claudin-3 in starved intestinal organoids was in agreement with reported findings in severely malnourished children56–58 and in low-protein fed rodents36. Impact of amino acid starvation on organelle homeostasis in hepatic and intestinal organoids We found that amino-acid starvation impacted peroxisomes earlier than mitochondria in hepatic organoids, whereas both peroxisomes and mitochondria were affected in intestinal organoids after 48 hours. The disparate impact on mitochondria plausibly reflects metabolic differences between the intestine and the liver. In the liver, mitochondria are important for fatty acid oxidation, whereas in the intestine mitochondria are essential for proper stem cell function and thereby epithelial turnover27,59. In addition, nutrient requirements may be higher in the intestine due to the high turnover rate, which could lead to more severe mitochondrial changes when exposed to a similar nutritional insult. In starved hepatic organoids we found that peroxisomal marker proteins were significantly decreased, which corresponded to reduced peroxisomal numbers in the liver of low-protein fed rodents29,30. It has been shown that peroxisomes are selectively degraded upon amino acid starvation60,61. Reduction of peroxisomal proteins, and particularly of Acox-1, is in line with the observed accumulation of pristanic acid in the starved organoids. Mitochondrial marker proteins and mitochondrial oxygen consumption were unaffected in 48 hours starved hepatic organoids, which was very similar in rats that were fed a low-protein diet for 1 week30. However, after 4 weeks mitochondria
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