100 Chapter 4 supplementation prevented this loss of peroxisomal and mitochondrial proteins (Fig. 4a and b). Enzymes involved in glycolysis, gluconeogenesis and glycogen metabolism showed a milder downregulation upon amino acid removal, some of which were normalised by DHA (Supplementary Figure 4). Effects of DHA supplementation on peroxisomal function and health Since DHA supplementation recovered peroxisomal and mitochondrial proteins, including enzymes involved in fatty acid oxidation (FAO), we hypothesized that it could also decrease the levels of intracellular triglycerides (TG). In line with our earlier observations5 amino-acid restriction led to a mildly elevated TG level in the hepatic organoids. Contrarily to what was expected, DHA supplementation did not reduce the TG level. However, given that the organoids were stimulated with a fatty acid, we cannot rule out an increase in β-oxidation. The higher concentration of DHA even led to a further accumulation of intracellular triglycerides when compared to the restriction conditions (Figure 4c). This could be caused by incorporation of DHA into phospholipids, inducing other fatty acids to be used to TG synthesis or even by direct incorporation of DHA into TG. To continue assessing peroxisomal functionality, we studied the ability of peroxisomes to metabolize phytanoyl-CoA in control and starved organoids as we previously reported5. Phytanic acid is a branched-chain fatty acid that is primarily oxidized by the peroxisomal α-oxidation, yielding pristanic acid as a product, which subsequently undergoes peroxisomal β-oxidation40,4 (Figure 4d). Mature hepatic organoids were incubated with 25 μM of phytol. Aminoacid restricted organoids had substantially lower levels of phytanic and pristanic acid than control organoids, suggesting that peroxisomal fatty-acid oxidation is affected. Since pristanic acid is an intermediate metabolite in the pathway, however, the effect on the flux of peroxisomal fatty oxidation is unclear. Moreover, DHA supplementation did not affect the levels of phytanic or pristanoic acid at any concentration (Figure 4e).
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