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Active autophagy but not lipophagy in macrophages with defective lipolysis.

Goeritzer M, Vujic N, Schlager S, Chandak PG, Korbelius M, Gottschalk B, Leopold C, Obrowsky S, Rainer S, Doddapattar P, Aflaki E, Wegscheider M, Sachdev V, Graier WF, Kolb D, Radovic B, Kratky D - Biochim. Biophys. Acta (2015)

Bottom Line: Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation.Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages.We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

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Bafilomycin A1 treatment does not affect lipid turnover in macrophages. Macrophages were cultured in DMEM/1% P/S containing 300 μM OA-BSA and 1 μCi [3H]OA-BSA per ml for 24 h. Thereafter, macrophages were incubated with 10 nM bafilomycin A1 (Baf) +/− Atglistatin and HSL-inhibitor for 14 h. (A, C) FA release into the medium and (B, D) intracellular FA incorporation were determined by liquid scintillation counting and normalized to protein content. Data are presented as mean (A, B) (n = 3–5) and (C, D) (n = 5) + SEM compared to Wt +/− Baf. (E) Macrophages were pulsed with C12-BODIPY for 24 h, washed (control), and chased with HBSS (starved) and Baf for 1 h. Lysosomes were labeled using cathepsin D antibody. (F) Relative cellular colocalization of C12-BODIPY with cathepsin D in pools of cells from 2 mice per genotype was quantified by Pearson’s coefficient analysis. Data are expressed as means (n = 7–10 fields/genotype) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.
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Figure 6: Bafilomycin A1 treatment does not affect lipid turnover in macrophages. Macrophages were cultured in DMEM/1% P/S containing 300 μM OA-BSA and 1 μCi [3H]OA-BSA per ml for 24 h. Thereafter, macrophages were incubated with 10 nM bafilomycin A1 (Baf) +/− Atglistatin and HSL-inhibitor for 14 h. (A, C) FA release into the medium and (B, D) intracellular FA incorporation were determined by liquid scintillation counting and normalized to protein content. Data are presented as mean (A, B) (n = 3–5) and (C, D) (n = 5) + SEM compared to Wt +/− Baf. (E) Macrophages were pulsed with C12-BODIPY for 24 h, washed (control), and chased with HBSS (starved) and Baf for 1 h. Lysosomes were labeled using cathepsin D antibody. (F) Relative cellular colocalization of C12-BODIPY with cathepsin D in pools of cells from 2 mice per genotype was quantified by Pearson’s coefficient analysis. Data are expressed as means (n = 7–10 fields/genotype) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.

Mentions: To investigate the role of autophagy in LD turnover in macrophages, we loaded macrophages with [3H]OA-BSA and analyzed the flux in the absence and presence of bafilomycin A1. In A0 and A0H0 macrophages, there was a trend toward decreased FA release into the medium after 14 h of bafilomycin A1 treatment. However, we failed to observe any differences in untreated compared to bafilomycin A1-treated cells (Fig. 6A). Furthermore, we found increased FA incorporation into TGs in A0 and A0H0 macrophages, which was unaffected by bafilomycin A1 treatment (Fig. 6B). We observed no differences in incorporation of [3H]OA-BSA into other lipid classes between the different genotypes or after bafilomycin A1 incubation. Next, we treated Wt cells with inhibitors against ATGL (Ai) and HSL (Hi), resulting in significantly decreased FA release (Fig. 6C). This finding suggests that the inhibitors efficiently blocked lipolysis. Comparable to the results from A0 and A0H0 macrophages, we observed increased FA incorporation into TGs of inhibitor-treated Wt cells, which was unaffected by bafilomycin A1 treatment (Fig. 6D). In summary, results from these pulse-chase experiments revealed that incorporation of FAs into lipid classes was independent of bafilomycin A1 in macrophages from all genotypes.


Active autophagy but not lipophagy in macrophages with defective lipolysis.

Goeritzer M, Vujic N, Schlager S, Chandak PG, Korbelius M, Gottschalk B, Leopold C, Obrowsky S, Rainer S, Doddapattar P, Aflaki E, Wegscheider M, Sachdev V, Graier WF, Kolb D, Radovic B, Kratky D - Biochim. Biophys. Acta (2015)

Bafilomycin A1 treatment does not affect lipid turnover in macrophages. Macrophages were cultured in DMEM/1% P/S containing 300 μM OA-BSA and 1 μCi [3H]OA-BSA per ml for 24 h. Thereafter, macrophages were incubated with 10 nM bafilomycin A1 (Baf) +/− Atglistatin and HSL-inhibitor for 14 h. (A, C) FA release into the medium and (B, D) intracellular FA incorporation were determined by liquid scintillation counting and normalized to protein content. Data are presented as mean (A, B) (n = 3–5) and (C, D) (n = 5) + SEM compared to Wt +/− Baf. (E) Macrophages were pulsed with C12-BODIPY for 24 h, washed (control), and chased with HBSS (starved) and Baf for 1 h. Lysosomes were labeled using cathepsin D antibody. (F) Relative cellular colocalization of C12-BODIPY with cathepsin D in pools of cells from 2 mice per genotype was quantified by Pearson’s coefficient analysis. Data are expressed as means (n = 7–10 fields/genotype) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.
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Figure 6: Bafilomycin A1 treatment does not affect lipid turnover in macrophages. Macrophages were cultured in DMEM/1% P/S containing 300 μM OA-BSA and 1 μCi [3H]OA-BSA per ml for 24 h. Thereafter, macrophages were incubated with 10 nM bafilomycin A1 (Baf) +/− Atglistatin and HSL-inhibitor for 14 h. (A, C) FA release into the medium and (B, D) intracellular FA incorporation were determined by liquid scintillation counting and normalized to protein content. Data are presented as mean (A, B) (n = 3–5) and (C, D) (n = 5) + SEM compared to Wt +/− Baf. (E) Macrophages were pulsed with C12-BODIPY for 24 h, washed (control), and chased with HBSS (starved) and Baf for 1 h. Lysosomes were labeled using cathepsin D antibody. (F) Relative cellular colocalization of C12-BODIPY with cathepsin D in pools of cells from 2 mice per genotype was quantified by Pearson’s coefficient analysis. Data are expressed as means (n = 7–10 fields/genotype) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.
Mentions: To investigate the role of autophagy in LD turnover in macrophages, we loaded macrophages with [3H]OA-BSA and analyzed the flux in the absence and presence of bafilomycin A1. In A0 and A0H0 macrophages, there was a trend toward decreased FA release into the medium after 14 h of bafilomycin A1 treatment. However, we failed to observe any differences in untreated compared to bafilomycin A1-treated cells (Fig. 6A). Furthermore, we found increased FA incorporation into TGs in A0 and A0H0 macrophages, which was unaffected by bafilomycin A1 treatment (Fig. 6B). We observed no differences in incorporation of [3H]OA-BSA into other lipid classes between the different genotypes or after bafilomycin A1 incubation. Next, we treated Wt cells with inhibitors against ATGL (Ai) and HSL (Hi), resulting in significantly decreased FA release (Fig. 6C). This finding suggests that the inhibitors efficiently blocked lipolysis. Comparable to the results from A0 and A0H0 macrophages, we observed increased FA incorporation into TGs of inhibitor-treated Wt cells, which was unaffected by bafilomycin A1 treatment (Fig. 6D). In summary, results from these pulse-chase experiments revealed that incorporation of FAs into lipid classes was independent of bafilomycin A1 in macrophages from all genotypes.

Bottom Line: Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation.Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages.We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

Show MeSH
Related in: MedlinePlus