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. 2020 Feb 18;11:131. doi: 10.3389/fimmu.2020.00131

Figure 4.

Figure 4

Inhibition of mitochondrial respiration by iNOS induces lipid droplet accumulation. (A) Maf-DKO macrophages were incubated for 24 h in medium containing FCS plus or minus etomoxir (200 μM) or oligomycin (10 μM). Oxygen consumption rate (OCR) was then measured in the presence of drug. Three consecutive basal OCR measurements were averaged. Data shown as mean ± SEM of triplicates, and is representative of two experiments. (B,C) LipidTOX MFI of cells incubated in medium with delipidated FCS plus or minus lipid mixture, with or without etomoxir (200 μM) or oligomycin (10 μM). Data shown as mean ± SEM of 3 replicates. Data is representative of two experiments. (D) OCR of cells incubated for 24 h without or with IFNγ plus DETA/NO (100 μM). Three consecutive basal OCR measurements were averaged. Data shown as mean ± SEM of triplicates, and is representative of two experiments. (E) LipidTOX MFI levels in cells incubated in medium containing delipidated FCS plus or minus lipid mixture, with or without DETA/NO (100 μM). Data shown as mean ± SEM of triplicates, and is representative of two experiments. (F) Cells were incubated for 24 h without or with IFNγ plus SEITU (500 μM). OCR was then measured before and after sequential addition of oligomycin, CCCP and rotenone plus antimycin. The blue trace shows OCR of cells in which etomoxir (200 μM) was added after 24 h of incubation with IFNγ plus SEITU, and 30 min before measurements started to be obtained. (G) LipidTOX MFI levels in cells incubated in FCS without or with IFNγ plus indicated concentrations of SEITU. Data shown as mean ± SEM of triplicates, and is representative of two experiments. (H) Histogram and quantification of Mitosox MFI in Maf-DKO cells incubated for 24 h with or without IFNγ or with IFNγ plus SEITU (S) (500 μM). Data shown as mean ± SEM of triplicates, and is representative of two experiments. ns, not significant *p < 0.05; **p < 0.01; ***p < 0.001. (I) Model of metabolic changes leading to TAG accumulation in activated macrophages. Macrophages oxidize fatty acid under basal conditions. Upon activation with IFNγ, nitric oxide produced by INOS inhibits mitochondrial respiration, and as a consequence, β-oxidation. External fatty acid that would otherwise be oxidized in mitochondria is esterified with glycerol 3-phosphate originating from glucose leading to synthesis of TAG and its accumulation in lipid droplets. Numbers in red indicate the inhibition site of the following compounds: 1, etomoxir: inhibits carnitine palmitoyltransferase-1; 2, oligomycin: inhibits ATP synthase; 3, DETA/NO: inhibits respiratory complexes I and IV; 4, SEITU: inhibits iNOS; 5, triacsin: inhibits fatty acyl-CoA synthetase.