90 Chapter 4 ABSTRACT Introduction Acute and chronic exposure to low amino acid conditions have been shown to lead to a reduction in hepatic peroxisomal and mitochondrial content. There is limited understanding of the underlying mechanisms behind this loss, but data suggests degradation through autophagy. Both organelles play a key role in fatty acid metabolism, which may explain why dysfunction in either one of them might lead to hepatic steatosis. Methods Using a previously established murine hepatic organoid model of severe malnutrition, we characterized the effects of prolonged amino-acid restriction on peroxisomal and mitochondrial protein levels and on autophagic flux. To do so, we developed concatemers of 13C-labelled peptide standards for quantification of over 50 different peroxisomal proteins. To assess the autophagic flux, we transduced hepatic organoids with a GFP-LC3-RFPLC3ΔG probe. Finally, the effect of PPAR-α activation on peroxisomal loss was determined with various agonists. Results Prolonged (96 h) amino-acid restriction led to a more severe loss of peroxisomes than a 48 h restriction, and with a substantial induction of autophagic flux. This was accompanied by accumulation of intracellular triglycerides, loss of mitochondrial and peroxisomal proteins, and loss of peroxisomal functionality. While PPAR-α agonists WY-14643 and linoleic acid (LA) had no effect, docosahexaenoic acid (DHA) supplementation partly prevented peroxisomal and mitochondrial loss under amino-acid restricted conditions and partly inhibited autophagy. Discussion The potential of DHA to prevent loss of peroxisomes and mitochondrial functions in low protein diets and severe malnutrition warrants further causal and translational testing in preclinical models and clinical trials, including its use as nutritional supplement.
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