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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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Increased autophagosome formation in A0H0 macrophages. (A) Representative electron micrographs of Wt, H0, A0, and A0H0 macrophages, showing degradative autophagic vacuoles (AVd) in cells from all phenotypes and LDs in A0 and A0H0 macrophages. Right panel: Electron micrographs after high-pressure freezing (HPF). Scale bars: 0.5 μm. (B, D) Western blotting of macrophage lysates using (B) anti-LC3B, anti-p62, and (D) anti-cathepsin B specific antibodies. Protein expression of β-actin was determined as loading control. (C) Densito-metric quantification of LC3-II/β-actin (n = 7–11) and p62/β-actin (n = 6–7) of independent experiments + SEM, relative to the expression in Wt macrophages. *, p < 0.05; ***, p ≤ 0.001.
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Figure 3: Increased autophagosome formation in A0H0 macrophages. (A) Representative electron micrographs of Wt, H0, A0, and A0H0 macrophages, showing degradative autophagic vacuoles (AVd) in cells from all phenotypes and LDs in A0 and A0H0 macrophages. Right panel: Electron micrographs after high-pressure freezing (HPF). Scale bars: 0.5 μm. (B, D) Western blotting of macrophage lysates using (B) anti-LC3B, anti-p62, and (D) anti-cathepsin B specific antibodies. Protein expression of β-actin was determined as loading control. (C) Densito-metric quantification of LC3-II/β-actin (n = 7–11) and p62/β-actin (n = 6–7) of independent experiments + SEM, relative to the expression in Wt macrophages. *, p < 0.05; ***, p ≤ 0.001.

Mentions: It has been recently shown that autophagy is involved in the regulation of intracellular lipid content [2]. Utilizing electron microscopy we found a substantial amount of structures resembling degradative autophagic vacuoles (AVd) in macrophages from all genotypes (Fig. 3A). In A0 macrophages, we observed LDs in very close proximity to autophagic vesicle-like structures. Closer inspection revealed that LDs are not enclosed within these membrane structures, which are morphologically not comparable to AVd presented in other images. To investigate whether autophagy might regulate intracellular TG levels in A0H0 macrophages, we determined LC3 degradation. During autophagy, cytosolic LC3 (LC3-I) is modified to its membrane-bound form (LC3-II) located on pre-autophagosomes and autophagosomes, which makes it a commonly used autophagosome marker [25]. As shown in Fig. 3B, macrophages from all genotypes showed a substantial amount of LC3-II, indicating that autophagy is a general recycling mechanism in macrophages. The ratio of LC3-II to β-actin trended to be increased in A0 and A0H0 macrophages compared to controls (Fig. 3C). In addition, we analyzed protein expression of p62, a chaperone that shuttles intracellular protein aggregates into autolysosomes for degradation. Since the entire p62-protein aggregate is degraded after engulfment by the autolysosome, expression levels of p62 are inversely correlated with autophagic flux. Protein expression of p62 trended to be reduced in macrophages from H0 and A0 mice, however, this reduction was statistical significant in A0H0 macrophages (Fig. 3C). Reduced expression of p62 and increased abundance of the mature form of the lysosomal protease cathepsin B (Fig. 3D) in A0H0 macrophages suggest that autophagy is highly active in macrophages deficient in both ATGL and HSL.


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)

Increased autophagosome formation in A0H0 macrophages. (A) Representative electron micrographs of Wt, H0, A0, and A0H0 macrophages, showing degradative autophagic vacuoles (AVd) in cells from all phenotypes and LDs in A0 and A0H0 macrophages. Right panel: Electron micrographs after high-pressure freezing (HPF). Scale bars: 0.5 μm. (B, D) Western blotting of macrophage lysates using (B) anti-LC3B, anti-p62, and (D) anti-cathepsin B specific antibodies. Protein expression of β-actin was determined as loading control. (C) Densito-metric quantification of LC3-II/β-actin (n = 7–11) and p62/β-actin (n = 6–7) of independent experiments + SEM, relative to the expression in Wt macrophages. *, p < 0.05; ***, p ≤ 0.001.
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Related In: Results  -  Collection

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Figure 3: Increased autophagosome formation in A0H0 macrophages. (A) Representative electron micrographs of Wt, H0, A0, and A0H0 macrophages, showing degradative autophagic vacuoles (AVd) in cells from all phenotypes and LDs in A0 and A0H0 macrophages. Right panel: Electron micrographs after high-pressure freezing (HPF). Scale bars: 0.5 μm. (B, D) Western blotting of macrophage lysates using (B) anti-LC3B, anti-p62, and (D) anti-cathepsin B specific antibodies. Protein expression of β-actin was determined as loading control. (C) Densito-metric quantification of LC3-II/β-actin (n = 7–11) and p62/β-actin (n = 6–7) of independent experiments + SEM, relative to the expression in Wt macrophages. *, p < 0.05; ***, p ≤ 0.001.
Mentions: It has been recently shown that autophagy is involved in the regulation of intracellular lipid content [2]. Utilizing electron microscopy we found a substantial amount of structures resembling degradative autophagic vacuoles (AVd) in macrophages from all genotypes (Fig. 3A). In A0 macrophages, we observed LDs in very close proximity to autophagic vesicle-like structures. Closer inspection revealed that LDs are not enclosed within these membrane structures, which are morphologically not comparable to AVd presented in other images. To investigate whether autophagy might regulate intracellular TG levels in A0H0 macrophages, we determined LC3 degradation. During autophagy, cytosolic LC3 (LC3-I) is modified to its membrane-bound form (LC3-II) located on pre-autophagosomes and autophagosomes, which makes it a commonly used autophagosome marker [25]. As shown in Fig. 3B, macrophages from all genotypes showed a substantial amount of LC3-II, indicating that autophagy is a general recycling mechanism in macrophages. The ratio of LC3-II to β-actin trended to be increased in A0 and A0H0 macrophages compared to controls (Fig. 3C). In addition, we analyzed protein expression of p62, a chaperone that shuttles intracellular protein aggregates into autolysosomes for degradation. Since the entire p62-protein aggregate is degraded after engulfment by the autolysosome, expression levels of p62 are inversely correlated with autophagic flux. Protein expression of p62 trended to be reduced in macrophages from H0 and A0 mice, however, this reduction was statistical significant in A0H0 macrophages (Fig. 3C). Reduced expression of p62 and increased abundance of the mature form of the lysosomal protease cathepsin B (Fig. 3D) in A0H0 macrophages suggest that autophagy is highly active in macrophages deficient in both ATGL and HSL.

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