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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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Reduced acid TG hydrolase activity in A0H0 macrophages. (A) Western blot analysis of LAL protein expression in macrophage lysates. Macrophages from Lal-deficient (L0) mice were used as negative control. Protein expression of β-actin was determined as loading control. Acid (B) TG and (C) CE hydrolase activities were measured in Wt, H0, A0, and A0H0 macrophages. Macrophages from L0 mice were used as control. Data are presented as mean (n = 3–8) + SEM. (D) L0 macrophages were starved for 1 h in HBSS and incubated with 10 μg/ml DQ-BSA for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry after 0, 2, and 6 h, respectively. Data are presented as mean (n = 4–7) + SEM. ***, p ≤ 0.001. (E) Macrophages were incubated in HBSS for 90 min and intracellular TG concentrations were determined. Data are presented as mean + SEM. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
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Figure 5: Reduced acid TG hydrolase activity in A0H0 macrophages. (A) Western blot analysis of LAL protein expression in macrophage lysates. Macrophages from Lal-deficient (L0) mice were used as negative control. Protein expression of β-actin was determined as loading control. Acid (B) TG and (C) CE hydrolase activities were measured in Wt, H0, A0, and A0H0 macrophages. Macrophages from L0 mice were used as control. Data are presented as mean (n = 3–8) + SEM. (D) L0 macrophages were starved for 1 h in HBSS and incubated with 10 μg/ml DQ-BSA for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry after 0, 2, and 6 h, respectively. Data are presented as mean (n = 4–7) + SEM. ***, p ≤ 0.001. (E) Macrophages were incubated in HBSS for 90 min and intracellular TG concentrations were determined. Data are presented as mean + SEM. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

Mentions: Finally, we investigated whether the terminal step in autophagic lipid degradation is induced in lipase-deficient macrophages. To this extent, we first determined the protein expression of LAL, the major TG and CE hydrolase in lysosomes. As shown in Fig. 5A, LAL protein expression was comparable between macrophages from all genotypes. Lal-deficient (L0) macrophages were used as control. Acid TG hydrolase activity, which reveals the activity of the lysosomal TG hydrolase LAL, was slightly decreased in H0 and ~80% reduced in A0 and A0H0 compared to Wt macrophages (Fig. 5B). Acid CE hydrolase activity was comparable in macrophages from all genotypes (Fig. 5C). As a control, L0 macrophages showed drastically reduced acid TG and CE hydrolase activities (Fig. 5B, C). Comparable with A0H0 macrophages, L0 macrophages showed increased dequenched DQ-BSA (Fig. 5D), suggesting an elevated lysosomal proteolysis even in cells lacking the major acid hydrolase. Starvation, a known inducer of autophagy, failed to decrease intracellular TG concentrations in macrophages of all genotypes (Fig. 5E). We starved the cells for 1.5 h since a longer starvation period drives lipase-deficient cells into cell death (unpublished observation). In summary, our data suggest that although autophagy is active in macrophages lacking both ATGL and HSL, degradation of LDs by LAL is less effective.


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)

Reduced acid TG hydrolase activity in A0H0 macrophages. (A) Western blot analysis of LAL protein expression in macrophage lysates. Macrophages from Lal-deficient (L0) mice were used as negative control. Protein expression of β-actin was determined as loading control. Acid (B) TG and (C) CE hydrolase activities were measured in Wt, H0, A0, and A0H0 macrophages. Macrophages from L0 mice were used as control. Data are presented as mean (n = 3–8) + SEM. (D) L0 macrophages were starved for 1 h in HBSS and incubated with 10 μg/ml DQ-BSA for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry after 0, 2, and 6 h, respectively. Data are presented as mean (n = 4–7) + SEM. ***, p ≤ 0.001. (E) Macrophages were incubated in HBSS for 90 min and intracellular TG concentrations were determined. Data are presented as mean + SEM. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
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Figure 5: Reduced acid TG hydrolase activity in A0H0 macrophages. (A) Western blot analysis of LAL protein expression in macrophage lysates. Macrophages from Lal-deficient (L0) mice were used as negative control. Protein expression of β-actin was determined as loading control. Acid (B) TG and (C) CE hydrolase activities were measured in Wt, H0, A0, and A0H0 macrophages. Macrophages from L0 mice were used as control. Data are presented as mean (n = 3–8) + SEM. (D) L0 macrophages were starved for 1 h in HBSS and incubated with 10 μg/ml DQ-BSA for 15 min at 37 °C. Red-fluorescent DQ-BSA was analyzed by flow cytometry after 0, 2, and 6 h, respectively. Data are presented as mean (n = 4–7) + SEM. ***, p ≤ 0.001. (E) Macrophages were incubated in HBSS for 90 min and intracellular TG concentrations were determined. Data are presented as mean + SEM. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Mentions: Finally, we investigated whether the terminal step in autophagic lipid degradation is induced in lipase-deficient macrophages. To this extent, we first determined the protein expression of LAL, the major TG and CE hydrolase in lysosomes. As shown in Fig. 5A, LAL protein expression was comparable between macrophages from all genotypes. Lal-deficient (L0) macrophages were used as control. Acid TG hydrolase activity, which reveals the activity of the lysosomal TG hydrolase LAL, was slightly decreased in H0 and ~80% reduced in A0 and A0H0 compared to Wt macrophages (Fig. 5B). Acid CE hydrolase activity was comparable in macrophages from all genotypes (Fig. 5C). As a control, L0 macrophages showed drastically reduced acid TG and CE hydrolase activities (Fig. 5B, C). Comparable with A0H0 macrophages, L0 macrophages showed increased dequenched DQ-BSA (Fig. 5D), suggesting an elevated lysosomal proteolysis even in cells lacking the major acid hydrolase. Starvation, a known inducer of autophagy, failed to decrease intracellular TG concentrations in macrophages of all genotypes (Fig. 5E). We starved the cells for 1.5 h since a longer starvation period drives lipase-deficient cells into cell death (unpublished observation). In summary, our data suggest that although autophagy is active in macrophages lacking both ATGL and HSL, degradation of LDs by LAL is less effective.

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