Fig. 7: Itaconate reduces lipid accumulation by hepatocytes and enhances their oxidative phosphorylation.
Targeted lipidomics showing triglycerides (a) and acyl carnitines (b) between untreated and itaconate-treated hepatocytes. For each metabolite, a ratio was computed by dividing the metabolite level by the relative mean for all six samples (three wild-type and three Irg1−/−). The heat map depicts log2-transformed ratios for metabolites. Significance was determined by two-sided multiple t-tests (one per row between genotypes) and is denoted on the side of the heat maps. c, Primary cultures of hepatocytes were treated with the indicated doses of itaconate and/or the indicated dilution of chemically defined lipid mixture. After 24 h, the treated hepatocytes were collected and analyzed for BODIPY staining. The graph depicts hepatocytes treated with itaconate and/or lipid (n = 3 from one experiment shown representative of seven similar experiments; *P = 0.028; **P = 0.005). d,e, Hepatocytes seeded in 96-well plates were treated with 10 mM itaconate and/or 1:100 lipid. The next day, cells were washed and analyzed using a Seahorse Bioanalyzer. d,e, The OCR (d) and extracellular acidification rate (ECAR) (e) is shown for one experiment (four replicate wells). Similar results were obtained in five similar experiments. At the indicated times, drugs were injected as (1) medium only or 40 μM etomoxir; (2) 1 μg ml−1 oligomycin; (3) 1 μM FCCP; (4) 100 nM rotenone + 1 μM antimycin A. f–h, The basal (f), maximal (g) and ATP OCR (h) are graphed (n = 4 for each; *P < 0.05; **P = 0.003). A two-sided ANOVA with multiple comparisons was used for statistical analyses in c,f–h. i–l, For detection of fatty acid carbon utilization into TCA-cycle intermediates hepatocytes were cultured overnight in 1:10 lipid mix ± 10 mM itaconate. Uniformly (U-13C16) labeled palmitate was added for the final 4 h and intracellular levels of labeled TCA intermediates were measured by mass spectrometry. Open bars depict the mean ± s.e.m. for M + 2 isotopologs and dark bars depict the mean ± s.e.m. of M + 4 isotopologs (*P = 0.04; **P = 0.006). No M + 4 was detected for aspartate. Statistical differences for (i–l) were determined by two sample t-test (n = 3 per group).
