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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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Intact autophagic flux in A0H0 macrophages. Macrophages were cultured in DMEM/10% LPDS for 24 h. (A–D) Macrophages were incubated with 10 nM bafilomycin A1 (Baf) for 0 and 14 h and assayed for (A, C) LC3 (upper blot: short exposure time to quantify LC3-II; lower blot: longer exposure time to visualize LC3-I) and (B, D) p62 protein expression. Protein expression of β-actin was determined as loading control. Representative Western blot analysis of at least 4 independent experiments is shown. Densitometric quantification of LC3-II/β-actin and p62/β-actin of (A) n = 4–7, (B) n = 4–8, (C) n = 6–10, and (D) n = 5–11 + SEM. (E) Cells were starved for 1 h in HBSS and then incubated with DQ-BSA (final concentration 10 μg/ml) for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer after 0, 2, and 6 h, respectively. Data are presented as mean (n = 3–9) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.
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Figure 4: Intact autophagic flux in A0H0 macrophages. Macrophages were cultured in DMEM/10% LPDS for 24 h. (A–D) Macrophages were incubated with 10 nM bafilomycin A1 (Baf) for 0 and 14 h and assayed for (A, C) LC3 (upper blot: short exposure time to quantify LC3-II; lower blot: longer exposure time to visualize LC3-I) and (B, D) p62 protein expression. Protein expression of β-actin was determined as loading control. Representative Western blot analysis of at least 4 independent experiments is shown. Densitometric quantification of LC3-II/β-actin and p62/β-actin of (A) n = 4–7, (B) n = 4–8, (C) n = 6–10, and (D) n = 5–11 + SEM. (E) Cells were starved for 1 h in HBSS and then incubated with DQ-BSA (final concentration 10 μg/ml) for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer after 0, 2, and 6 h, respectively. Data are presented as mean (n = 3–9) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.

Mentions: To analyze the autophagic flux we next incubated the macrophages with bafilomycin A1, which inhibits the fusion between autophagosomes and lysosomes, thereby preventing maturation of autophagic vacuoles and degradation of LC3-II and p62. Bafilomycin A1 treatment resulted in comparable increase in LC3-II (Fig. 4A, C) and p62 (Fig. 4B, D) expression in macrophages from Wt, A0, H0, and A0H0 mice with a trend to decreased p62 expression in H0 (Fig. 4B) and A0H0 (Fig. 4D) macrophages. Chloroquine treatment resulted in similarly increased protein expression of LC3-II and p62 in Wt and A0H0 macrophages (Supplemental Fig. 2A, B).


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)

Intact autophagic flux in A0H0 macrophages. Macrophages were cultured in DMEM/10% LPDS for 24 h. (A–D) Macrophages were incubated with 10 nM bafilomycin A1 (Baf) for 0 and 14 h and assayed for (A, C) LC3 (upper blot: short exposure time to quantify LC3-II; lower blot: longer exposure time to visualize LC3-I) and (B, D) p62 protein expression. Protein expression of β-actin was determined as loading control. Representative Western blot analysis of at least 4 independent experiments is shown. Densitometric quantification of LC3-II/β-actin and p62/β-actin of (A) n = 4–7, (B) n = 4–8, (C) n = 6–10, and (D) n = 5–11 + SEM. (E) Cells were starved for 1 h in HBSS and then incubated with DQ-BSA (final concentration 10 μg/ml) for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer after 0, 2, and 6 h, respectively. Data are presented as mean (n = 3–9) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.
© Copyright Policy - open-access
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Figure 4: Intact autophagic flux in A0H0 macrophages. Macrophages were cultured in DMEM/10% LPDS for 24 h. (A–D) Macrophages were incubated with 10 nM bafilomycin A1 (Baf) for 0 and 14 h and assayed for (A, C) LC3 (upper blot: short exposure time to quantify LC3-II; lower blot: longer exposure time to visualize LC3-I) and (B, D) p62 protein expression. Protein expression of β-actin was determined as loading control. Representative Western blot analysis of at least 4 independent experiments is shown. Densitometric quantification of LC3-II/β-actin and p62/β-actin of (A) n = 4–7, (B) n = 4–8, (C) n = 6–10, and (D) n = 5–11 + SEM. (E) Cells were starved for 1 h in HBSS and then incubated with DQ-BSA (final concentration 10 μg/ml) for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer after 0, 2, and 6 h, respectively. Data are presented as mean (n = 3–9) + SEM. **, p ≤ 0.01; ***, p ≤ 0.001.
Mentions: To analyze the autophagic flux we next incubated the macrophages with bafilomycin A1, which inhibits the fusion between autophagosomes and lysosomes, thereby preventing maturation of autophagic vacuoles and degradation of LC3-II and p62. Bafilomycin A1 treatment resulted in comparable increase in LC3-II (Fig. 4A, C) and p62 (Fig. 4B, D) expression in macrophages from Wt, A0, H0, and A0H0 mice with a trend to decreased p62 expression in H0 (Fig. 4B) and A0H0 (Fig. 4D) macrophages. Chloroquine treatment resulted in similarly increased protein expression of LC3-II and p62 in Wt and A0H0 macrophages (Supplemental Fig. 2A, B).

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