44 Chapter 2 ± SEM (*P<0.05, ** P< 0.01. One-way ANOVA with Tukey’s post-hoc test). (F) Representative immunoblot images. (G) Mitochondrial protein levels relative to β-actin. Quantification of data shown in (A) Organoids were grown in complete culture medium throughout (control, grey), amino-acid-free medium for 48 hours (starved, blue), ), starved from 0h to 48h and re-supplemented with complete medium from 48h to 96h (re-supplementation, orange) or in amino-acid-free medium for 48 h supplemented with 100uM fenofibrate (fenofibrate, green). Data represent mean ± SEM from 7-10 biological replicates and 3 biological replicates for Fenofibrate treatment (biological replicates are obtained from independent experiments) (*P<0.05, ** P< 0.01, two-way ANOVA with Tukey’s post-hoc test). (H) Free carnitines and acylcarnitines measured in supernatant of organoids grown in complete culture medium (control), amino-acid-free medium (starved), amino-acid-free medium for 48 h followed by complete culture medium for 48 h (re-supplementation). Data represents mean of 3 biological replicates (from independent experiments) ± SEM. (*P<0.05, ** P< 0.01 Ordinary one-way ANOVA). (I) Triglyceride levels in control, starved and fenofibrate treated organoids (as per colour legend). Data represents 4-5 biological replicates from independent experiments ± SEM (*P<0.05, ordinary one-way ANOVA with Tukey’s post hoc test). With respect to mitochondria, the levels of TOM-20 (a component of the ‘translocase of outer membrane’ (TOM) receptor complex) did not differ between control and amino acid starvation for 48 hours, nor did the protein complexes of the electron transport chain (Figure 4f, g). Total carnitine levels were unaffected after 48 hours, but significantly decreased upon reintroduction of amino acids. Long-chain acyl carnitines (C14-C18) and acetyl-carnitine (C2) were decreased after 48 hours of amino-acid starvation and not restored by reintroduction of amino acids (Figure 4h). Since in vivo studies hinted that peroxisomal loss precedes mitochondrial events30,40, we increased the duration of the amino acid starvation to 96 hours. This longer amino acid deprivation resulted in a clear reduction of mitochondrial protein markers. After 96 hours of amino acid starvation, electron transport chain complexes I to V were significantly downregulated. TOM-20 showed a weaker downward trend (p = 0.06) (Figure 5a,b). At the functional level, the longer starvation reduced ADP-stimulated respiration in the presence of palmitoyl carnitine and malate (state 3), while basal respiration remained unaffected (state 1). Uncoupled respiration (State U) showed a downward trend upon starvation, but this was not significant (p = 0.06) (Figure 5c).
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