98 Chapter 4 Screening PPAR-α agonist for the prevention of peroxisomal loss in hepatic organoids Peroxisome proliferator-activated receptor α (PPAR-α) is a nuclear receptor that works as a ligand-regulated transcriptional factor. PPAR-α is activated by a wide range of ligands, of which synthetic fibrates are most widely described37. It can, however, also be activated by natural ligands such as various fatty acids and eicosanoid derivatives21. Here, we assessed the effect of three PPAR-α agonists on peroxisomal proliferation and fatty acid metabolism in amino-acid restricted organoids. We tested one synthetic compound (WY-14643) and two polyunsaturated fatty acids: linoleic acid (18:2ω6 fatty acid) and docosahexaenoic acid (DHA, C26:ω3). Linoleic acid (LA) did not rescue any of the peroxisomal protein markers PMP70, ACOX1, or catalase (Figure 3a,b), whereas PPAR-α agonist WY-14643 had only a minor rescuing effect on ACOX1. ACOX1, is synthesized as 75 kDa polypeptide, which upon import into the peroxisome is proteolytically cleaved into two smaller peptides, 21 kDa and 55 kDa38. Supplementation of the highest dose (200 μM) of WY-14343 prevented the loss of the uncleaved, 75 kDa protein, while the smaller (53 kDa) was almost significant. (Figure 3c,d). In contrast, the natural compound DHA prevented the loss of catalase and the 55 kDa form of ACOX1 in dose dependent manner, with a complete rescue to control levels at the highest dose. DHA did not affect levels of PMP70 nor of the uncleaved form of ACOX1 (75 kDa) (Figure 3e, f). Since LA and WY-14643 did not show any protection against peroxisomal loss, the effect of DHA might be mediated via a PPAR-α-independent mechanism.
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