Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover

Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis

Genomics and proteomics of vertebrate cholesterol ester lipase (LIPA) and cholesterol 25-hydroxylase (CH25H)

Cholesterol ester lipase (LIPA; EC 3.1.1.13) and cholesterol 25-hydroxylase (CH25H; EC 1.14.99.48) play essential role in cholesterol metabolism in the body by hydrolysing cholesteryl esters and triglycerides within lysosomes (LIPA) and catalysing the formation of 25-hydroxycholesterol from cholesterol (CH25H) which acts to repress cholesterol biosynthesis. Bioinformatic methods were used to predict the amino acid sequences, structures and genomic features of several vertebrate LIPA and CH25H genes and proteins, and to examine the phylogeny of vertebrate LIPA. Amino acid sequence alignments and predicted subunit structures enabled the identification of key sequences previously reported for human LIPA and CH25H and transmembrane structures for vertebrate CH25H sequences. Vertebrate LIPA and CH25H genes were located in tandem on all vertebrate genomes examined and showed several predicted transcription factor binding sites and CpG islands located within the 5′ regions of the human genes. Vertebrate LIPA genes contained nine coding exons, while all vertebrate CH25H genes were without introns. Phylogenetic analysis demonstrated the distinct nature of the vertebrate LIPA gene and protein family in comparison with other vertebrate acid lipases and has apparently evolved from an ancestral LIPA gene which predated the appearance of vertebrates

Impaired Bile Acid Metabolism and Gut Dysbiosis in Mice Lacking Lysosomal Acid Lipase

Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of cholesteryl esters and triglycerides at an acidic pH in lysosomes, resulting in the release of unesterified cholesterol and free fatty acids. However, the role of LAL in diet-induced adaptations is largely unexplored. In this study, we demonstrate that feeding a Western-type diet to Lal-deficient (LAL-KO) mice triggers metabolic reprogramming that modulates gut-liver cholesterol homeostasis. Induction of ileal fibroblast growth factor 15 (three-fold), absence of hepatic cholesterol 7α-hydroxylase expression, and activation of the ERK phosphorylation cascade results in altered bile acid composition, substantial changes in the gut microbiome, reduced nutrient absorption by 40%, and two-fold increased fecal lipid excretion in LAL-KO mice. These metabolic adaptations lead to impaired bile acid synthesis, lipoprotein uptake, and cholesterol absorption and ultimately to the resistance of LAL-KO mice to diet-induced obesity. Our results indicate that LAL-derived lipolytic products might be important metabolic effectors in the maintenance of whole-body lipid homeostasis

Macrophage colony-stimulating factor regulates both activities of neutral and acidic cholesteryl ester hydrolases in human monocyte-derived macrophages

Macrophage colony-stimulating factor (M-CSF) regulates cholesterol metabolism in vivo and in vitro. We studied the effects of M-CSF on enzyme activities of acidic cholesteryl ester (CE) hydrolase, neutral CE hydrolase, and acyl-coenzyme A:cholesterol acyltransferase (ACAT), all of which are involved in cellular cholesterol metabolism in macrophages. During the differentiation of monocytes to macrophages, these enzyme activities were induced and further enhanced in response to M-CSF. M-CSF (100 ng/ml) enhanced acidic and neutral CE hydrolase and ACAT activities by 3.2-, 4-, and 2.3-fold, respectively, in the presence of acetyl LDL. The presence of acetyl LDL influenced these enzyme activities. ACAT and acidic CE hydrolase activities were increased and neutral CE hydrolase activity was decreased, indicating that these enzymes are regulated by intracellular cholesterol enrichment. M-CSF increased the ratios of acidic CE hydrolase to ACAT activity and of neutral CE hydrolase to ACAT activity. The results suggest that M-CSF enhances net hydrolysis of CE by stimulating the two CE hydrolases to a greater extent than ACAT, and M-CSF may reduce the rate of atherogenesis

Lysosomal acid lipase: at the crossroads of normal and atherogenic cholesterol metabolism

Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL) plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL) and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression

Lysosomal acid lipase regulates VLDL synthesis and insulin sensitivity in mice

AIMS/HYPOTHESIS: Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). METHODS: We studied metabolic adaptations in Lal (-/-) mice. RESULTS: Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. CONCLUSIONS/INTERPRETATION: Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply

Acid Lipase Deficiency and Lipid Storage in Wolman\u27s Disease and E600-Treated Cells in Culture

Fibroblasts obtained from a child with Wolman’s disease and maintained in culture demonstrated acid lipase deficiency, reached senescence prematurely and exhibited an abnormal #5 chromosome. When cytogenetic analysis was repeated on frozen and stored cells, the chromosomal defect could no longer be demonstrated, but other anomalies were present. The karyotypes of other Wolman’s disease cell cultures and the parents of the proband were normal. The mother’s fibroblasts had reduced acid lipase activity, consistent with a carrier-state, but the father’s fibroblasts had normal acid lipase activity. It was possible to classify a culture as Wolman’s disease, carrier or normal by the ability of medium from the culture to reduce the lipid stored in a Wolman’s disease cell culture. The culture of the proband stored more lipid than other Wolman’s disease cultures. Two enzymes present in fetal calf serum possessing paraoxonase activity could be differentiated by their sensitivity to heating at 56°C. The esterase inhibitor E600 was slightly more toxic to Wolman’s disease than to normal cells, and was also toxic to E. coli. Normal cells exhibited lipid storage when treated with 10(-4)M E600, but a more pronounced time-dependent accumulation of lipid occurred at 10(-3)M E600. p-Nitrophenol, the major metabolite of E600, had little effect on lipid storage. The amount of lipid stored varied directly with the serum concentration and was unaffected by heat inactivation of the serum. Histochemical stains and biochemical analyses were performed on cultured fibroblasts. Both Wolman’s disease and E600-treated cells showed storage of triglycerides and cholesteryl esters, although one Wolman’s disease culture had a normal level of triglyceride. The E600-treated cells also showed a large increase in phospholipid and small increases in free cholesterol and free fatty acid. Wolman’s disease cells treated with E600 showed increases comparable to the normal E600-treated cells, but no increase of free fatty acid was seen. Wolman’s disease heterozygotes appeared to have normal amounts of lipid. Lipid in Wolman’s disease and E600-treated cells fluoresced in ultraviolet light. Cholesteryl esters of untreated normal cells had more stearic (18:0) than oleic (18:1ω9) acid, whereas cholesteryl esters in Wolman’s disease, E600-treated cells, and fetal calf serum had three times more oleic than stearic acid, suggesting that serum lipid was taken up, but not degraded, in Wolman’s disease and E600-treated cells. Three Wolman’s disease cell cultures exhibited genetic deficiency of acid lipase. Normal fibroblasts had acid lipase activity, but possessed little neutral lipase activity. Acid lipase activity of normal cells was inhibited by E600. Wolman’s disease cells resembled normal cells by scanning electron microscopy. Except in a perinuclear zone which also had not stained with oil red O or neutral red, the cytoplasm of E600-treated cells had numerous bumps which appeared to form ridges along the path of actin stress filaments. Normal cells treated with free fatty acid or cholesterol, and Wolman’s disease cells treated with free fatty acid, exhibited lipid storage in large, peripheral lipid droplets. Lipid was generally stored in smaller granules closer to the nucleus in E600-treated and Wolman’s disease cells. E600-treated cells showed increased numbers of lysosomes by neutral red staining and had increased numbers of dense bodies ultrastructurally. The dense bodies of Wolman’s disease and E600-treated cells sometimes contained lipid clefts which probably consisted of cholesteryl ester. The dense bodies accumulated colloidal gold, suggesting their identity with secondary lysosomes. Although some differences were observed, E600-treated cells resembled Wolman’s disease cells histochemically, biochemically, and ultrastructurally

Novel LIPA mutations in Mexican siblings with lysosomal acid lipase deficiency

We would like to thank Radhika Tripuraneni, MD, MPH, for critical reading of the manuscript, Angelica TorizOrtiz, MD, for ultrasound imaging, and the medical staff of the Endoscopy and Pathology Department of CMN “20 de Noviembre”, along with all the personnel involved in the care of the patients. This work was presented in abstract form at 2013’s National Week of Gastroenterology (Semana Nacional de Gastroenterologia de la AMG) in Veracruz, Mexico and at Ⅸ Congress of SLEIMPN in Medellín, Colombia.Peer reviewedPublisher PD

New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease(WD) or the more benign cholesteryl ester storage disease (CESD). To identifythe molecular basis of the different phenotypes we have characterised the LALgene mutations in three new patients with LAL deficiency. A patient with WD washomozygote for a null allele Y303X. The other two patients, with CESD, presentedeither homozygosity for T267I or compound heterozygosity consisting of Q64R andan exon 8 donor splice site substitution (G→A in position–1). The mutants T267I and Q64R and the previously reported L273S, G66V,and H274Y CESD substitutions, overexpressed in stable clones, were found to befully glycosylated and show an enzymatic activity of 3–8% of that ofnormal LAL. On the other hand, the Δ254–277 mutant proteinderived from exon 8 skipping and the Y303X protein were totally inactive. Bytransient transfection of hybrid minigene constructs, the CESD G→A(–1) substitution resulted in partial exon inclusion, thus allowing theproduction of a small amount of normal LAL mRNA and hence of a functionalenzyme. In contrast, a G→Asubstitution observed in WD at position +1 of the same exon 8 donor siteresulted in complete exon skipping and the sole production of an inactiveΔ254–277 protein.In conclusion,LAL genotypes determine the level of residual enzymatic activity, thusexplaining the severity of the phenotype.—Pagani, F., R. Pariyarath, R.Garcia, C. Stuani, A. B. Burlina, G. Ruotolo, M. Rabusin, and F. E. Baralle. Newlysosomal acid lipase gene mutants explain the phenotype of Wolman disease andcholesteryl ester storage disease. J. Lipid Res. 1998. 39:1382–1388