145 5 Establishing a peroxisomal β-oxidation computational kinetic model to understand the effects of amino-acid restriction Moreover, this also lead to a reduction in peroxisomal functionality in murine hepatic organoids as measured by the metabolism of phytanic acid66. However, the results could not be directly linked to a decreased flux of the β-oxidation of fatty acids. To assess the potential of the above-presented model, we have combined it with targeted proteomics data from a previously published malnutrition in vitro model (chapter 4). Murine hepatic organoids were exposed to amino-acid restricted media for 96 hours, which led to a decrease in the levels of peroxisomal proteins measured by targeted proteomics (Chapter 4). Moreover, the organoids showed an accumulation of triglycerides (TGs), both after 48 and 96 hours, which was hypothesized to be caused by defects in the metabolism of fatty acids due to the impairments in both mitochondria and peroxisomes (Chapter4)66. To understand the effect of amino-acid restriction on the peroxisomal β-oxidation flux, we integrated the proteomics data in the model. To this end the Vmax values of the enzymes were decomposed in the corresponding Kcat value and enzyme concentration [E]: While all the enzymes included in the model were present in the targeted proteomic panel, not all enzymes were present in high enough concentrations to be detected. Here we integrated the enzyme concentrations that were detected (Chapter 4), namely ACOX1, catalase and CROT. The overall flux of the pathway was predicted to decrease substantially and significantly upon amino-acid restriction. While the absolute values are different for the different biological replicates involved in the experiments, the overall trends are the same (Figure 3a). Interestingly, neither the acylCoA nor the acyl-carnitine profile changed in the amino-acid restricted conditions (Supplementary Figure 1) while the levels of enoyl-CoAs and ketoacyl-CoAs were decreased for all the chain lengths (Figure 3b and c, and Supplementary File 1). These results suggest that while the flux of the pathway is downregulated, homeostasis of acyl-CoAs and acyl-carnitines is maintained.
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