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.     

Ontogenic changes in lung cholesterol metabolism,lipid content,and histology in mice with Niemann–Pick type C disease

Niemann–Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1^−/− mice (Npc1^nih) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70 days of age). Similar measurements were made in Npc2^−/− mice at 70 days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1^−/− mice and remained so at 70 days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1^−/− mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1^−/− mice starting as early as 28 days. Similar metabolic and histologic changes were evident in the lungs of the Npc2^−/− mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time.     

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter,Niemann–Pick type C1

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1^−/−), and subsequently in Cav-1^−/− mice that also lacked the lysosomal cholesterol transporter Niemann–Pick C1 (Npc1) (Cav-1^−/−:Npc1^−/−). In 50-day-old Cav-1^−/− mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1^+/+ controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1^−/−:Npc1^−/− mice (0.356 ± 0.022) markedly exceeded that in their Cav-1^+/+:Npc1^+/+ controls (0.137 ± 0.009), as well as in their Cav-1^−/−:Npc1^+/+ (0.191 ± 0.013) and Cav-1^+/+:Npc1^−/− (0.213 ± 0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74 ± 0.17, 0.71 ± 0.05, 0.96 ± 0.05 and 3.12 ± 0.43, respectively, with the extra cholesterol in the Cav-1^−/−:Npc1^−/− and Cav-1^+/+:Npc1^−/− mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1^−/−:Npc1^−/− mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.     

Alkylphospholipids deregulate cholesterol metabolism and induce cell-cycle arrest and autophagy in U-87 MG glioblastoma cells

Glioblastoma is the most common malignant primary brain tumour in adults and one of the most lethal of all cancers. Growing evidence suggests that human tumours undergo abnormal lipid metabolism, characterised by an alteration in the mechanisms that regulate cholesterol homeostasis. We have investigated the effect that different antitumoural alkylphospholipids (APLs) exert upon cholesterol metabolism in the U-87 MG glioblastoma cell line. APLs altered cholesterol homeostasis by interfering with its transport from the plasma membrane to the endoplasmic reticulum (ER), thus hindering its esterification. At the same time they stimulated the synthesis of cholesterol from radiolabelled acetate and its internalisation from low-density lipoproteins (LDLs), inducing both 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) genes. Fluorescent microscopy revealed that these effects promoted the accumulation of intracellular cholesterol. Filipin staining demonstrated that this accumulation was not confined to the late endosome/lysosome (LE/LY) compartment since it did not colocalise with LAMP2 lysosomal marker. Furthermore, APLs inhibited cell growth, producing arrest at the G2/M phase. We also used transmission electron microscopy (TEM) to investigate ultrastructural alterations induced by APLs and found an abundant presence of autophagic vesicles and autolysosomes in treated cells, indicating the induction of autophagy. Thus our findings clearly demonstrate that antitumoural APLs interfere with the proliferation of the glioblastoma cell line via a complex mechanism involving cholesterol metabolism, cell-cycle arrest or autophagy. Knowledge of the interrelationship between these processes is fundamental to our understanding of tumoural response and may facilitate the development of novel therapeutics to improve treatment of glioblastoma and other types of cancer.     

Abrogation of neutral cholesterol ester hydrolytic activity causes adrenal enlargement

We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Hepatic uptake and metabolism of phosphatidylcholine associated with high density lipoproteins

Background

Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.

Methods and results

The absence of scavenger receptor-BI resulted in a 30% decrease in cellular incorporation of [³H]PC whereas [³H]cholesteryl ether uptake was almost completely abolished. Although endocytosis is not involved in the uptake of cholesteryl esters from HDL, we demonstrate that HDL internalization accounts for 40% of HDL-PC uptake. Extracellular remodeling of HDL by secretory phospholipase A₂ significantly enhances HDL lipid uptake. HDL-PC taken up by hepatocytes is partially converted to triacylglycerols via PC-phospholipase C-mediated hydrolysis of PC and incorporation of diacylglycerol into triacylglcyerol. The formation of triacylglcerol is independent of scavenger receptor-BI and occurs in extralysosomal compartments.

Conclusions and general significance

These findings indicate that HDL-associated PC is incorporated into primary hepatocytes via a pathway that differs significantly from that of HDL-cholesteryl ester, and shows that HDL-PC is more than a framework molecule, as evidenced by its partial conversion to hepatic triacylglycerol.     

Degradation of chylomicron remnant cholesteryl ester by rat hepatocyte monolayers. Inhibition by chloroquine and colchicine

Rat hepatocytes in monolayer cultures take up and degrade cholesteryl ester of isolated chylomicron remnants. The cholesteryl ester of native chylomicrons was metabolized at a slower rate. The uptake of cholesteryl ester was decreased by the presence of serum. The hydrolysis of cholesteryl ester but not the uptake or binding of chylomicron remnants by the cells was inhibited by chloroquine, which is known to inhibit the lysosomal degradation of protein and of low density lipoproteins by fibroblasts. Colchicine, which inhibits the hydrolysis of chylomicron cholesteryl ester after the uptake by the liver in vivo, had the same effect in hepatocyte monolayers.     

Involvement of lipid droplets in hepatic responses to lipopolysaccharide treatment in mice

Infection and inflammation induce important changes in lipid metabolism, which result in increased free fatty acids and triacylglycerol in plasma and altered high density lipoprotein (HDL) metabolism. Our aim was to elucidate whether hepatic lipid droplets (LDs) are involved in the adaptations of lipid metabolism to endotoxemia. We characterized the lipid content and several enzymatic activities in subcellular fractions and subpopulations of LDs from livers of mice 24 h after lipopolysaccharide (LPS) treatment and analyzed the expression of key genes involved in lipid management. Endotoxemic mice showed lower lipid content in LDs with decreased molar fraction of cholesteryl ester and higher diacylglycerol/triacylglycerol ratio as compared to their controls. They also showed a decrease in cytosolic triacylglycerol hydrolase activity, specifically in dense LDs, and in microsomal and cytosolic diacylglycerol hydrolase activity; concomitantly neutral lipid biosynthetic capacity and triacylglycerol levels in plasma lipoproteins increased. Together with the overexpression of genes involved in lipogenesis and HDL formation our results suggest that altered hepatic management of LD lipids in LPS-treated mice might be related to the channeled mobilization of triacylglycerol for very low density lipoprotein assembly and to the induction of cholesterol export.     

Particle size interconversion of human low density lipoproteins during incubation of plasma with phosphatidylcholine vesicles

Incubation of plasma (37°C, 6hr) in the presence of increasing amounts of phosphatidylcholine (PC) vesicles, above a threshold concentration, results in an increase in particle diameter of LDL relative to that from nonincubated plasma. With further PC addition, the major peak of LDL in the gradient gel electrophoretic pattern is transformed, first, into a bimodal and, subsequently, into a single peak distribution. PC-induced interconversion of LDL requires factors(s) in the d > 1.20 g/ml fraction and, at PC concentrations below approximately 2 mg/ml, is not inhibited by p-chloromercuriphenylsulfonic acid. Plasma incubation with increasing PC levels also leads to characteristic particle size transformations in HDL₃ species, with the transformation products ultimately converging to form a single peak pattern within the HDL_2a size interval. In certain subjects, incubation of plasma, in the absence of added PC, shifts the particle size distribution of LDL towards smaller species; this can be prevented by addition of PC. We propose that incubation-induced shifts of LDL towards larger or smaller species result from changes in phospholipid (PL) content of LDL.     

Two genetic variants in FABP1 and susceptibility to non-alcohol fatty liver disease in a Chinese population

Liver fatty acid-binding protein (FABP1) serves as a key regulator of hepatic lipid metabolism, and polymorphisms within the FABP1 gene have been associated with several metabolic traits. To investigate the association between FABP1 polymorphisms and the risk of non-alcohol fatty liver disease (NAFLD) in a Chinese population, the genotypes and haplotypes of FABP1 (rs2241883 T/C and rs1545224G/A) were determined in 553 patients with NAFLD and 553 healthy controls. The results showed that individuals with at least one copy of the rs2241883 C allele (TC or CC genotype) had an elevated risk for developing NAFLD (odds ratio [OR]=1.32, 95% CI: 1.01-1.71), and individuals with at least one copy of the rs1545224 A allele (GA or AA genotype) also had a significantly increased risk for NAFLD (OR=1.52, 95% CI: 1.14-2.02). Cumulative effect analysis of the two SNPs revealed that individuals with two risk genotypes were at significantly higher risk of NAFLD than those without risk genotype, and a significant trend of increased risk with increasing numbers of risk genotype was observed. Stratification analysis showed that the rs2241883 C allele carriers had higher level of LDL-C and the rs1545224 A allele carriers had higher level of FPG than those without this allele. In addition, haplotype analysis revealed that the one composed of the rs1545224 A and rs2241883 C variants was significantly associated with an increased risk for NAFLD (OR=1.34; 95% CI=1.05-1.40) compared to the GT haplotype. Taken together, the present study suggests that genetic variations within FABP1 influence susceptibility to NAFLD independently or jointly.     

Endoplasmic reticulum-localized hepatic lipase decreases triacylglycerol storage and VLDL secretion

Hepatic triacylglycerol levels are governed through synthesis, degradation and export of this lipid. Here we demonstrate that enforced expression of hepatic lipase in the endoplasmic reticulum in McArdle RH7777 hepatocytes resulted in a significant decrease in the incorporation of fatty acids into cellular triacylglycerol and cholesteryl ester accompanied by attenuation of secretion of apolipoprotein B-containing lipoproteins. Hepatic lipase-mediated depletion of intracellular lipid storage increased the expression of peroxisome proliferator-activated receptor α and its target genes and augmented oxidation of fatty acids. These data show that 1) hepatic lipase is active in the endoplasmic reticulum and 2) intracellular hepatic lipase modulates cellular lipid metabolism and lipoprotein secretion.     

Squalene-based oil-in-water emulsion adjuvants perturb metabolism of neutral lipids and enhance lipid droplet formation

Oil-in-water emulsions are used as vaccine adjuvants, but the mechanism of action remains unknown. In this paper we used phagocytes (monocytes, macrophages, dendritic cells) and non-phagocytic cells (fibroblasts, skeletal muscle cells) to study internalization of emulsions in vitro, and to characterize the influence of emulsion uptake on cellular metabolism of neutral lipids. We found that all tested cell types endocytose the emulsion droplets, and that the uptake leads to an acute accumulation of neutral lipids in the form of cytoplasmic lipid droplets. The accumulated lipids comprise not only the delivered squalene, but also cholesteryl esters, triacylglycerols, fatty acids, and diacylglycerols. Lipid metabolism and innate immunity are closely linked, and accumulation of lipids in non-adipose tissues is known to induce inflammatory conditions. We propose that one aspect of o/w emulsion adjuvanticity could depend on their ability to rapidly change lipid metabolism of the target cells.     

The C57BL/6J Niemann–Pick C1 mouse model with decreased gene dosage has impaired glucose tolerance independent of body weight

The human Niemann–Pick C1 (NPC1) gene has been found to be associated with extreme (early-onset and morbid-adult) obesity and type 2 diabetes independent of body weight. We previously performed growth studies using BALB/cJ Npc1 normal (Npc1^+/+) and Npc1 heterozygous (Npc1^+/−) mice and determined that decreased Npc1 gene dosage interacts with a high-fat diet to promote weight gain and adiposity. The present study was performed using both BALB/cJ and C57BL/6J Npc1^+/+ and Npc1^+/− mice to determine if decreased Npc1 gene dosage predisposes to metabolic features associated with type 2 diabetes. The results indicated that C57BL/6J Npc1^+/− mice, but not BALB/cJ Npc1^+/− mice, have impaired glucose tolerance when fed a low-fat diet and independent of body weight. The results also suggest that an accumulation of liver free fatty acids and hepatic lipotoxicity marked by an elevation in the amount of plasma alanine aminotransferase (ALT) may be responsible for hepatic insulin resistance and impaired glucose tolerance. Finally, the peroxisome-proliferator activated receptor α (PPARα) and sterol regulatory element-binding protein-1 (SREBP-1) pathways known to have a central role in regulating free fatty acid metabolism were downregulated in the livers, but not in the adipose or muscle, of C57BL/6J Npc1^+/− mice compared to C57BL/6J Npc1^+/+ mice. Therefore, decreased Npc1 gene dosage among two different mouse strains interacts with undefined modifying genes to manifest disparate yet often related metabolic diseases.     

Intracellular sterol dynamics

We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated.     

Role of lecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor-acetyl hydrolase-deficient plasma

To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-¹⁴C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.