Active autophagy but not lipophagy in macrophages with defective lipolysis.
Bottom Line: We therefore generated mice lacking both ATGL and HSL (A0H0).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.
Affiliation: Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.Show MeSH
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Mentions: It has been recently shown that autophagy is involved in the regulation of intracellular lipid content . 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 . 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.
Affiliation: Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.