Acid lipase cross-reacting material in Wolman disease and cholesterol ester storage disease

Material cross-reacting with antibodies to acid lipase was demonstrated in fibroblasts of three patients with Wolman disease and three with cholesterol ester storage disease. Quantitation of the immunologically cross-reacting material (CRM) by a single radial immunodiffusion method revealed normal levels in both mutant cell types. CRM specific activity toward triolein and cholesteryl oleate was reduced about 200-fold in the Wolman disease fibroblasts and 50- to 100-fold in the cholesterol ester storage disease cells when compared to normal.     

Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease

Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p < 0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann–Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.     

Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases.

Molecular cloning of a full-length cDNA for human lysosomal acid lipase/cholesteryl ester hydrolase (EC 3.1.1.13) reveals that it is structurally related to previously described enteric acid lipases, but lacks significant homology with any characterized neutral lipases. The lysosomal enzyme catalyzes the deacylation of triacylglyceryl and cholesteryl ester core lipids of endocytosed low density lipoproteins; this activity is deficient in patients with Wolman disease and cholesteryl ester storage disease. Its amino acid sequence, as deduced from the 2.6-kilobase cDNA nucleotide sequence, is 58 and 57% identical to those of human gastric lipase and rat lingual lipase, respectively, both of which are involved in the preduodenal breakdown of ingested triglycerides. Notable differences in the primary structure of the lysosomal lipase that may account for discrete catalytic and transport properties include the presence of 3 new cysteine residues, in addition to the 3 that are conserved in this lipase gene family, and of two additional potential N-linked glycosylation sites. Transfection of the cDNA into Cos-1 cells resulted in the expression of acid lipase activity with the substrate range of the native enzyme at a level that was greater than 40 times the endogenous activity.     

Characterization of plasma lipids and lipoproteins in cholesteryl ester storage disease

Cholesteryl ester storage disease, caused by the loss of lysosomal acid ester hydrolase (EC 3.1.1.13), has been previously associated with hyperlipidemia and premature atherosclerosis. We identified a 23-month-old female with cholesteryl ester storage disease and characterized the plasma lipids and lipoproteins in the proband and her family. These studies illustrate several important points about this disease. First, a high index of suspicion is required to diagnose this disease since the major physical manifestation of the disorder, mild hepatomegaly, is subtle. Second, the Type II hyperlipoproteinemia in the proband is paralleled by a reduction in the concentration of high density lipoproteins. Third, analysis of the plasma lipids and lipoproteins in family members revealed both Type II and Type IV hyperlipoproteinemia with an inheritance pattern similar to that of familial combined hyperlipoproteinemia. Fourth, the parents and brother of this patient had 50% normal fibroblast acid ester hydrolase activity. These results raise the possibility that deficiency of the lysosomal acid ester hydrolase may be linked to familial combined hyperlipoproteinemia and that this enzyme deficiency may be more common than previously appreciated.     

Restoration of a regulatory response to low density lipoprotein in acid lipase-deficient human fibroblasts.

Previous studies have shown that cultured fibroblasts derived from patients with genetic defects in lysosomal acid lipase (i. e. the Wolman Syndrome and Cholesteryl Ester Storage Disease) are defective in their ability to hydrolyze the cholesteryl esters contained in plasma low density lipoprotein (LDL). As a result, these mutant cells show a reduced responsiveness to the regulatory actions of LDL, as evidenced by a decreased LDL-mediated suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and by a decreased LDL-mediated activation of cellular cholesteryl ester formation. In the current studies, the Wolman Syndrome and Cholesteryl Ester Storage Disease cells were grown in the same Petri dish with mutant fibroblasts derived from a patient with the homozygous form of Familial Hypercholesterolemia. Whereas pure monolayers of either the Familial Hypercholesterolemia cells (lacking cell surface LDL receptors) or the acid lipase-deficient cells (lacking cholesteryl ester hydrolase activity) responded poorly to LDL, the mixed monolayers developed lipoprotein responsiveness as measured by an enhancement of both LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and LDL-mediated stimulation of cholesteryl ester formation. This effect was shown to result from the release of the lysosomal acid lipase from the Familial Hypercholesterolemia homozygote cells into the culture medium and its subsequent uptake by the acid lipase-deficient cells. The acquisition of this acid lipase activity enhanced the ability of the Wolman Syndrome and Cholesteryl Ester Storage Disease cells to respond to the lipoprotein by suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and activation of cellular cholesteryl ester formation. These data emphasize the importance of the lysosomal acid lipase in the cellular metabolism of LDL cholesteryl esters and, in addition, demonstrate that delivery of this enzyme to genetically deficient cells can enhance the regulatory response to the lipoprotein.     

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.     

Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing.     

Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease.

The hydrolysis of cholesteryl esters contained in plasma low density lipoprotein was reduced in cultured fibroblasts derived from a patient with cholesteryl ester storage disease, an inborn error of metabolism in which lysosomal acid lipase activity is deficient. While these mutant cells showed a normal ability to bind low density lipoprotein at its high affinity cell surface receptor site, to take up the bound lipoprotein through endocytosis, and to hydrolyze the protein component of the lipoprotein in lysosomes, their defective lysosomal hydrolysis of the cholesteryl ester component of the lipoprotein led to the accumulation within the cell of unhydrolyzed cholesteryl esters, the fatty acid distribution of which resembled that of plasma lipoprotein. When the cholesteryl ester storage disease cells were incubated with low density lipoprotein, the reduced rate of liberation of free cholesterol by these mutant cells was associated with a delay in the occurrence of two lipoprotein-mediated regulatory events, suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and activation of endogenous cholesteryl ester formation. In contrast to their defective hydrolysis of exogenously derived lipoprotein-bound cholesteryl esters, the choleseryl ester storage disease cells showed a normal rate of hydrolysis of cholesteryl esters that had been synthesized within the cell. These data lend support to the concept that in cultured human fibroblasts cholesteryl esters entering the cell bound to low density lipoprotein are hydrolyzed within the lysosome and that one of the functions of this intracellular organelle is to supply the cell with free cholesterol.     

New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases

Trinitrophenylaminolauric acid (TNPAL) was linked to glycerol or cholesterol and the resulting yellow compounds were used as substrates for several lipases and cholesteryl esterase in cells from normal individuals and patients with Wolman's or cholesteryl ester storage diseases. Normal cells (lymphoid cell lines or skin fibroblasts) showed two peaks of lipase or cholesteryl esterase activity at about pH 4.0 and 6.0 each. The activity of the most acidic enzyme, which hydrolyzed natural or synthetic triacylglycerols as well as cholesteryl esters, was considerably reduced in cells derived from patients with Wolman's or cholesteryl ester storage diseases. Simple spectrophotometric procedures were developed for using tri-TNPAL glycerol or TNPAL cholesterol to identify homozygotes of these two respective diseases.     

Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Genetic LAL mutations lead to Wolman disease (WD) and cholesteryl ester storage disease (CESD). An LAL-null (lal(-/-)) mouse model resembles human WD/CESD with storage of CEs and TGs in multiple organs. Human LAL (hLAL) was expressed in Nicotiana benthamiana using the GENEWARE expression system (G-hLAL). Purified G-hLAL showed mannose receptor-dependent uptake into macrophage cell lines (J774E). Intraperitoneal injection of G-hLAL produced peak activities in plasma at 60 min and in the liver and spleen at 240 min. The t(1/2) values were: approximately 90 min (plasma), approximately 14 h (liver), and approximately 32 h (spleen), with return to baseline by approximately 150 h in liver and approximately 200 h in spleen. Ten injections of G-hLAL (every 3 days) into lal(-/-) mice produced normalization of hepatic color, decreases in hepatic cholesterol and TG contents, and diminished foamy macrophages in liver, spleen, and intestinal villi. All injected lal(-/-) mice developed anti-hLAL protein antibodies, but suffered no adverse events. These studies demonstrate the feasibility of using plant-expressed, recombinant hLAL for the enzyme therapy of human WD/CESD with general implications for other lysosomal storage diseases.     

Compound heterozygosity for a Wolman mutation is frequent among patients with cholesteryl ester storage disease

Cholesteryl ester storage disease and Wolman disease are rare autosomal recessive lipoprotein-processing disorders caused by mutations in the gene encoding human lysosomal acid lipase. Thus far we have elucidated the genetic defects in 15 unrelated CESD patients. Seven were homozygotes for the prevalent hLAL exon 8 splice junction mutation which results in incomplete exon skipping, while eight probands were compound heterozygotes for E8SJM and a rare mutation on the second chromosome. In this report, we describe the molecular basis of CESD in three compound heterozygous subjects of Czech and Irish origin. RFLP and DNA sequence analysis revealed that they were heteroallelic for the common G(934)-->A substitution in exon 8 of the hLAL gene and a mutation which, if inherited on both alleles, would be expected to result in complete loss of enzyme activity and to cause Wolman disease. In patients A. M. and J. J., two nucleotide deletions in exons 7 and 10 were detected, involving a T at position 722, 723, or 724 and a G in a stretch of five guanosines at positions 1064;-1068 of the hLAL cDNA. Both mutations result in premature termination of protein translation at residues 219 and 336, respectively, and in the production of truncated, inactive enzymes. Subject D. H., in contrast, is a compound heterozygote for the Arg(44)-->Stop mutation previously described in a French CESD proband. Combined with data in the literature, our results demonstrate that compound heterozygosity for a mutation causing Wolman disease is common among cholesteryl ester storage disease patients.     

Characterization of lysosomal acid lipase mutations in the signal peptide and mature polypeptide region causing Wolman disease.

Wolman disease results from an inherited deficiency of lysosomal acid lipase (LAL; EC 3.1.1.13). This enzyme is essential for the hydrolysis of cholesteryl esters and triacylglycerols derived from endocytosed lipoproteins. Because of a complete absence of LAL activity, Wolman patients accumulate progressive amounts of cholesteryl esters and triacylglycerols in affected tissues. To investigate the nature of the genetic defects causing this disease, mutations in the LAL gene from three subjects of Moslem-Arab and Russian descent living in Israel were determined. Two homozygotes for a novel 1-bp deletion introducing a premature in-frame termination codon at amino acid position 106 (S106X) were identified. A third subject was a homozygote for a G-5R signal peptide substitution and a G60V missense mutation. The functional significance of these mutations was tested by in vitro expression of single and double mutants in Spodoptera frugiperda cells. Single mutants G60V and S106X and double mutant G-5R/G60V displayed a virtual absence of lipase activity in cell extracts and culture medium. Signal peptide mutant G-5R retained lipase activity in cell extracts and showed a drastically reduced enzyme activity in culture supernatant, indicating that the mutation may affect secretion of active enzyme from cells. These results support the notion that Wolman disease is a genetically heterogeneous disorder of lipid metabolism.     

Possible role for intracellular cholesteryl ester transfer protein in adipocyte lipid metabolism and storage

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.     

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysosomal acid lipase deficiency impairs regulation of ABCA1 gene and formation of high density lipoproteins in cholesteryl ester storage disease

ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.     

The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.     

Synthesis and hydrolysis of cholesteryl esters by isolated rat-liver lysosomes and cell-free extracts of human lung fibroblasts

The objective of this study was to examine and characterize the cholesteryl ester synthesizing [S] and hydrolyzing [H] properties of the acid cholesteryl ester hydrolase (acid cholesteryl ester hydrolase), both in isolated rat liver lysosomes and in cell-free extracts from cultured fibroblasts. For both liver lysosomes and fibroblasts extracts, the major synthesizing activity was found around pH 4 and did not require exogenous ATP. The rate of hydrolysis was measured at pH 4.5. Several different inhibitors were used in order to characterize the reactions. Ammonium chloride did not markedly affect the activity of acid cholesteryl ester hydrolase at pH 4 [S] or 4.5 [H], whereas chloroquine was a potent inhibitor of acid CEase in both liver lysosomes and fibroblast extracts. The [S] activity of the acid cholesteryl ester hydrolase in either material was not affected by the acylCoA:cholesterol acyltransferase inhibitor Compound 58-035 from Sandoz. Progesterone, on the other hand, which is an often used acylCoA:cholesterol acyltransferase inhibitor, markedly blocked both activities of the acid CEase. Our results indicate that the lysosomal compartment of both studied tissues, in addition to hydrolysis activity, also have a significant esterification activity. It appears that both activities are carried out by the same enzyme.     

Extracellular origin of the lipid lysosomal storage in cultured fibroblasts from Wolman's disease

The experiments reported here allowed us to compare the metabolism of neutral lipids from extracellular origin (lipoproteins) and endogenous origin (triacylglycerol biosynthesis induced by feeding cells with high levels of free fatty acid) in normal and acid-lipase-deficient fibroblasts (Wolman's disease). When the cells were grown in hyperlipemic-rich medium, a major neutral lipid storage appeared in normal as well as in acid-lipase-deficient cells; this storage disappeared rapidly in normal cells during the 'chase', whereas in Wolman cells, the storage of cholesteryl esters and triacylglycerols remained unchanged, or only decreased very slowly. When the cells were fed with high levels of radiolabelled oleic acid, a major accumulation of radiolabelled triacylglycerols was observed. These cytoplasmic triacylglycerols were similarly degraded in normal and Wolman fibroblasts during the 'chase' period. From these results it was concluded that the neutral lipids stored in lysosomes of Wolman fibroblasts are only of extracellular origin (lipoproteins), whereas triacylglycerols biosynthesized by the cells do not participate in this accumulation. Therefore, both cellular compartments involved in triacylglycerol metabolism (lysosomes containing exogenous lipids and cytoplasmic granules of endogenously biosynthesized triacylglycerols) are strictly independent.     

Chemical screen to reduce sterol accumulation in Niemann–Pick C disease cells identifies novel lysosomal acid lipase inhibitors

Niemann–Pick C disease (NPC) is a lysosomal storage disorder causing abnormal accumulation of unesterified free cholesterol in lysosomal storage organelles. High content phenotypic microscopy chemical screens in both human and hamster NPC-deficient cells have identified several compounds that partially revert the NPC phenotype. Cell biological and biochemical studies show that several of these molecules inhibit lysosomal acid lipase, the enzyme that hydrolyzes LDL-derived triacylglycerol and cholesteryl esters. The effects of reduced lysosomal acid lipase activity in lowering cholesterol accumulation in NPC mutant cells were verified by RNAi-mediated knockdown of lysosomal acid lipase in NPC1-deficient human fibroblasts. This work demonstrates the utility of phenotypic cellular screens as a means to identify molecular targets for altering a complex process such as intracellular cholesterol trafficking and metabolism.     

Intracellular processing of exogenously derived non-lipoprotein [3H)cholesterol in normal and mutant human skin fibroblasts deficient in acid sterol ester hydrolase

By studying the incorporation and esterification of non-lipoprotein, free [³H]cholesterol in normal and acid sterol ester hydrolase-deficient human fibroblasts, it was examined whether the esterification reaction of the lysosomal acid sterol ester hydrolase contributed to the formation of cellular [³H|cholesteryl esters. Both the normal and the acid sterol ester hydrolase-deficient cells incorporated exogenous, vesicle-derived free [³H]cholesterol linearly as a function of time. Also, the rate of [³H]cholesteryl ester formation was almost the same in normal and mutant fibroblasts, indicating that the apparent esterification activity of the acid sterol ester hydrolase in normal fibroblasts did not contribute to the formation of [³H]cholesteryl esters in intact cells. To examine whether the incorporated [³H]cholesterol was transported into the endoplasmic reticulum and esterified by the acyl-CoA: cholesterol acyltransferase, the rate of [³H]cholesteryl ester formation was measured in the presence or absence of the acyl-CoA: cholesterol acyltransferase-inhibitor 58-035 (Sandoz Inc.). Results showed that the formation of [³H]cholesteryl esters was reduced markedly when cells were co-incubated with the acyltransferase inhibitor. Maximal inhibition (i.e., 75%) was obtained at an inhibitor concentration of 1 μg/ml. Since the inhibitor 58-035 is very specific for acyl-CoA: cholesterol acyltransferase, this finding clearly shows that exogenous, exchangeable [³H]cholesterol can reach and mix with the intracellular substrate pool of the enzyme.