OBJECTIVES: Lysosomal acid lipase (LAL) is the key enzyme, which degrades neutral lipids at an acidic pH in lysosomes. The role of LAL in various cellular processes has mostly been studied in LAL-knockout mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 and Lalistat-2. METHODS: We performed lipid hydrolase activity assays and serine hydrolase-specific activity-based labeling combined with quantitative proteomics to investigate potential off-target effects of Lalistat-1 and -2. RESULTS: Pharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis as well as neutral triglyceride and cholesteryl ester hydrolase activities. Apart from LAL, Lalistat-1 and -2 also inhibit major cytosolic lipid hydrolases responsible for lipid degradation in primary cells at neutral pH through off-target effects. Their binding to the active center of the enzymes leads to a decrease in neutral lipid hydrolase activities in cells overexpressing the respective enzymes. CONCLUSIONS: Our findings are critically important since they demonstrate that commonly used concentrations of these inhibitors are not suitable to investigate the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy. The interpretation of their effects on lipid metabolism should be taken with caution and the applied inhibitor concentrations in cell culture studies should not exceed 1 microM.
Lysosomal acid lipase (LAL) is essential for cholesteryl ester (CE) and triacylglycerol (TAG) hydrolysis in lysosomes. Clinically, an autosomal recessive LIPA mutation causes LAL deficiency (LAL-D), either Wolman Disease or Cholesterol Ester Storage Disease (CESD). LAL-D is associated with ectopic neutral lipid accumulation in the liver, small intestine, spleen, adrenal glands, and blood. Considering the importance of unesterified cholesterol and fatty acids in bone metabolism, we hypothesized that LAL is essential to bone formation, and ultimately, skeletal health. To investigate the role of LAL in skeletal homeostasis, we used LAL-deficient ((-/-)) mice and osteoblast cell cultures. Male LAL(-/-) mice had lower trabecular BV/TV (12%) compared to WT mice (21%), due to decreased trabecular number and increased trabecular separation; this change was not apparent in the females. While both sexes of LAL(-/-) mice displayed decreased cortical bone thickness and polar moment of inertia, only the female LAL(-/-) mice showed increased cortical porosity. Histological analyses revealed that LAL(-/-) mice tended to have less osteoblasts but no change in osteoclast numbers. In studying the cell-autonomous role of LAL, we observed impaired osteoblastogenesis of LAL(-/-) calvarial osteoblasts and in bone marrow stromal cells treated with the LAL inhibitor lalistat. Consistent with LAL's role in other tissues, lalistat resulted in profound lipid puncta accumulation and an altered intracellular lipid profile. Finally, we analyzed a large de-identified national insurance database (i.e. 2016/2017 Optum Clinformatics(a)) which revealed that adults (<=18 years) with CESD (n=3,076) had a higher odds ratio (OR=1.21; 95% CI=1.03-1.41) of all-cause fracture at any location compared to adults without CESD (n=13.7 M) after adjusting for demographic variables and osteoporosis. These data demonstrate that alterations in LAL have significant clinical implications related to fracture risk and that LAL's modulation of lipid metabolism is a critical for osteoblast function.
Lalistat inhibits growth of Mycobacterium tuberculosis in bacterial culture as well as in infected macrophages. Target identification by quantitative proteomics revealed a cluster of 20 hydrolytic proteins including members of the lipase family. Lipases are essential for M. tuberculosis fatty acid production and energy
storage thus representing promising antibiotic targets.
        
Title: Physiological difference in autophagic flux in macrophages from 2 mouse strains regulates cholesterol ester metabolism Robinet P, Ritchey B, Smith JD Ref: Arterioscler Thromb Vasc Biol, 33:903, 2013 : PubMed
OBJECTIVE: DBA/2 apoE(-/-) mice have =10-fold larger lesions than AKR apoE(-/-) mice. The objective of this study was to determine whether macrophages from these 2 strains had altered cholesterol metabolism that might play a role in their divergent atherosclerosis susceptibility. APPROACH AND RESULTS: AKR and DBA/2 macrophages incubated with acetylated low-density lipoprotein resulted in higher cholesterol ester (CE) and lower free cholesterol accumulation in the DBA/2 cells. However, these strains had equivalent acetylated low-density lipoprotein uptake and cholesterol esterification activity. Cholesterol efflux from unloaded cells to apolipoprotein A-I or high-density lipoprotein was similar in the 2 strains. However, on acetylated low-density lipoprotein loading, cholesterol efflux was impaired in the DBA/2 cells, but this impairment was corrected by loading in the presence of an inhibitor of cholesterol esterification. Thus, the cholesterol efflux capabilities are similar in these strains, but there seemed to be a defect in lipid droplet-stored CE mobilization in DBA/2 cells. Lalistat 1, a specific inhibitor of lysosomal acid lipase, completely blocked the hydrolysis of lipid droplet-stored CE, implying that lipid droplet autophagy is responsible for CE turnover in these cells. CE turnover was 2-fold slower in DBA/2 versus AKR cells. Autophagic flux, estimated by a fluorescent light chain 3-II reporter and the increase in p62 levels after chloroquine treatment, was higher in AKR versus DBA/2 macrophages, which had an apparent decrease in autophagosome fusion with lysosomes. When autophagy was activated by amino acid starvation, CE levels decreased in DBA/2 cells. CONCLUSIONS: Physiological regulation of autophagy in macrophages controls CE accumulation and may modify atherosclerosis susceptibility.