48 Chapter 2 DISCUSSION Advances in three-dimensional cultures, in particular organoids, have opened new avenues for the development of more physiological in vitro models of organ function and diseases1–4. In this paper, we presented two organoid models to study pathophysiological processes and potential treatments for intestinal and hepatic dysfunction in severe malnutrition. Organ-specific manifestations of severe malnutrition were recapitulated in amino-acid-starved organoids. Concurrent mitochondrial and peroxisomal changes largely reflect in vivo findings and were found to be organ-specific. We showed that aminoacid-starved organoids cannot only be used to gain mechanistic insights, but also to test pharmacological interventions. Functional impact of amino acid starvation on hepatic and intestinal organoids Mature hepatic organoids showed no morphological or size changes upon amino-acid starvation. This could be attributed to their low proliferative state, as indicated by lower stemness markers than in hepatic progenitor organoids. Their functionality was, however, substantially impaired. Albumin production dropped upon starvation, which was expected as albumin synthesis requires amino acids as precursors and is stimulated by dietary amino acids42. The same was observed in low-protein fed rats30 and severely malnourished children43. Enlarged fat droplets, increased levels of triglycerides and elevated levels of pristanic acid point to lipid accumulation in starved hepatic organoids. Hepatic steatosis has also been commonly observed in severely malnourished children44 and rodents on a low-protein diet30,45. We did not study glucose production, even though impaired hepatic glucose production reported in severely malnourished children increases the risk of hypoglycaemia and death15,17. Functional glucose production has rarely been studied in organoids since glucose 6-phosphatase (G6PC) levels are typically low in vitro46,47. It was recently shown that organoids derived from primary hepatocytes have a higher G6PC expression33 than organoids derived from cholangiocytes, and a normalized glycogen storage48. This provides a perspective to study functional glucose production in vitro. Intestinal organoids starved for amino acids were smaller than those in complete medium, with fewer crypts and atrophy of cryptlike domains. This could point to suppressed enterocyte proliferation, as has also been shown in intestinal organoids deprived of glutamine, methionine or valine49,50. In starved organoids gene expression of stem cell and proliferation markers
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