Changes in cholesterol metabolism in cultured fibroblasts from patients with Niemann-Pick disease

Cultured skin fibroblasts from 6 patients with Niemann-Pick disease type A were investigated for cholesterol metabolism. An increase in cholesterol synthesis from ¹⁴C-sodium acetate was observed in all cases. A decrease in ¹⁴C-oleic acid incorporation into cholesteryl esters was found in 5 of 6 cases. ¹²⁵I-low density lipoprotein binding was significantly reduced in 3 of 4 investigated cases.     

Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts

One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.     

Type C Niemann-Pick disease. A parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts

Low density lipoprotein (LDL) internalization by mutant type C Niemann-Pick (NPC) fibroblasts results in uptake of excess total cholesterol. Uptake of excess lipoprotein cholesterol appears to be mediated by the specific LDL receptor pathway. Associated with excessive LDL-cholesterol uptake is a lesion in early intracellular cholesteryl ester synthesis. In vitro acylCoA:cholesterol acyltransferase activity is normal in cell-free extracts of mutant cells. The ability of exogenous sterols to enhance intracellular esterification of [3H]mevalonate-derived [3H]cholesterol was severely limited in mutant cell cultures suggesting that in vivo activation and/or expression of activated acylCoA:cholesterol acyltransferase may be compromised by the primary mutation of type C Niemann-Pick disease. After 2 days of LDL uptake, rates of intracellular cholesteryl ester synthesis in mutant cells paralleled the rates of esterification in normal cells suggesting that specific early in vivo expression of the acyltransferase may be affected in this disorder.     

The effects of low-density lipoprotein and cholesterol on acyl-coenzyme A: cholesterol acyltransferase activity in membranes from cultured human fibroblasts.

Membranes prepared from cultured fibroblasts were assayed for acyl-coenzyme A: cholesterol acyltransferase (ACAT) by a method that relied exclusively on the cholesterol already present on the membranes as the sterol substrate. Changes in membrane ACAT activity during incubation of fibroblasts under a variety of conditions were similar to the changes in the rate of incorporation of oleic acid into cholesteryl esters by the intact cells. The addition of low-density lipoprotein (LDL) to fibroblasts pre-incubated with lipoprotein-deficient serum led to a transient increase in membrane ACAT activity, which reached its peak after 7h and was related to the receptor-mediated uptake and degradation of the lipoprotein by the cells. However, after incubation of the membranes with a cholesterol-rich donor lipoprotein, which resulted in an equilibration of cholesterol between membranes and donor, each preparation exhibited the same activity. In contrast with these effects of LDL, incubation of the cells with non-esterified cholesterol produced a prolonged increase in ACAT activity and an increase in the activity observed after equilibration. Furthermore, ACAT activity in cells grown with linoleic acid was higher, both before and after the addition of LDL, than that of cells grown in normal medium or with palmitate. The increase in activity produced by LDL was also greater, reflecting the greater rate of degradation of LDL by the cells, and was associated with an increase in the activity observed after equilibration with donor. The results suggest that although fibroblasts can increase the amount of active enzyme on their membranes to accommodate an exceptionally high or prolonged supply of cholesterol, under normal circumstances the increase in membrane ACAT activity produced by LDL can be explained entirely by an increase in the amount of cholesterol in the substrate pool.     

Niemann-Pick C1-Like 1 and cholesterol uptake

Niemann-Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage

A central feature of Niemann-Pick Type C (NPC) disease is sequestration of cholesterol and glycosphingolipids in lysosomes. A large phenotypic variability, on both a clinical as well as a molecular level, challenges NPC diagnosis. For example, substantial difficulties in identifying or excluding NPC in a patient exist in cases with a "variant" biochemical phenotype, where cholesterol levels in cultured fibroblasts, the primary diagnostic indicator, are only moderately elevated. Here we apply quantitative microscopy as an accurate and objective diagnostic tool to measure cholesterol accumulation at the level of single cells. When employed to characterize cholesterol enrichment in fibroblasts from 20 NPC patients and 11 controls, considerable heterogeneity became evident both within the population of cells cultured from one individual as well as between samples from different probands. An obvious correlation between biochemical phenotype and clinical disease course was not apparent from our dataset. However, plasma levels of HDL-cholesterol (HDL-c) tended to be in the normal range in patients with a "variant" as opposed to a "classic" biochemical phenotype. Attenuated lysosomal cholesterol accumulation in "variant" cells was associated with detectable NPC1 protein and residual capability to upregulate expression of ABCA1 in response to LDL. Taken together, our approach opens perspectives not only to support diagnosis, but also to better characterize mechanisms impacting cholesterol accumulation in NPC patient-derived cells.     

Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous fibroblasts

The esterification of cholesterol derived from human low density lipoprotein (LDL) or fetal bovine serum (FBS) was deficient in cultured fibroblasts from subjects with heterozygous and homozygous type C Niemann-Pick (NPC) disease. Failure to significantly esterify LDL-derived cholesterol resulted in abnormal accumulation of predominantly unesterified cholesterol in homozygous NPC fibroblasts. Compared with normal and homozygous fibroblasts, heterozygous NPC fibroblasts synthesized intermediate levels of cholesteryl ester during the initial 6 h of incubation with LDL. The rate of cholesterol esterification in heterozygous cells was normal when measured over a 24-h period of incubation with LDL. In addition to demonstrating a defect in cholesterol esterification, homozygous NPC fibroblasts accumulated more total cholesterol when incubated with LDL or FBS than normal fibroblasts accumulated. When heterozygous NPC fibroblasts were incubated with LDL or FBS, cellular accumulation of cholesterol reached levels that were high-normal or intermediary between levels observed in normal and homozygous NPC fibroblasts. The partial expression of these metabolic errors in the heterozygous genotype relevantly links these errors to the primary mutation of this disorder.     

Dynamics of lysosomal cholesterol in Niemann-Pick type C and normal human fibroblasts

The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.     

Multiple effects of serum low-density lipoprotein on cultured human fibroblasts.

The effects of serum low-density lipoproteins (LDL) were studied in cultures of human skin fibroblasts grown in medium supplemented with human serum deficient in lipoproteins and in platelet factor. The LDL led to a temporary increase in the rate of cell replication, to increases in the cell content of protein and cholesterol, to an increase in average cell size, and to an increased secretion of glycosaminoglycans. The increases in cholesterol and protein were proportional to the increase in cell size, suggesting that the additional protein and cholesterol were of a structural, rather than a storage, nature. The increase in cell protein during the first few days of exposure to LDL was due to a decrease in the rate of protein degradation. Ultrafiltration of the serum to remove substances of molecular weight less than 30,000 did not reduce the basal rate of cell proliferation but did prevent the stimulation of proliferation by LDL; it did not alter the effect of LDL on cell protein and cholesterol, indicating that the latter responses are independent of the mitogenic action. The response of cells from diabetic donors did not differ from that of normal cells.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-¹⁴C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the $\text{in situ}$ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in $\text{vitro}$ by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.     

Aspirin induces alterations in low-density lipoprotein and decreases its catabolism by cultured human fibroblasts

Aspirin interacts in vitro with human low-density lipoprotein (LDL), which results in a decrease in free amino groups of apolipoprotein B and an increase of electrophoretic mobility of the particle. The aspirin-treated LDL was less efficiently recognized than native LDL by the apo B/E receptor of fibroblasts. These results suggest that aspirin in long-term treatment could influence the LDL-receptor pathway. However, aspirin-treated LDL did not bind to the scavenger receptor of macrophages.     

Receptor-mediated binding and degradation of subfractions of human plasma low-density lipoprotein by cultured fibroblasts

The receptor-mediated metabolism of human plasma low-density lipoprotein (LDL) subfractions was studied. LDL was isolated from healthy donors and further fractionated by density gradient ultracentrifugation into three subfractions: (I) d = 1.031-1.037, (II) d = 1.037-1.041 and (III) d = 1.041-1.047 g/ml, comprising 24 +/- 7%, 46 +/- 8% and 30 +/- 9% of the total LDL protein, respectively. As assessed by electron microscopy and gradient gel electrophoresis, the LDL particle size decreased and the relative protein content increased from fraction I towards fraction III. Fraction II had the highest (Kd 2.6 micrograms/ml) and fraction I the lowest (Kd 5.8 micrograms/ml) binding affinity to LDL receptors of human fibroblasts at 4 degrees C. The rate of receptor-mediated degradation of fraction II was also higher than that of the other two fractions at 37 degrees C. These results suggest that LDL subfractions have different rates of receptor-mediated catabolism depending on particle size or composition, and therefore their metabolic fate and atherogenic properties may also differ.     

cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells

The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of beta-arrestin-2 (betaarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr(-/-) MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.     

Correction of apolipoprotein A-I-mediated lipid efflux and high density lipoprotein particle formation in human Niemann-Pick type C disease fibroblasts

Impaired cell cholesterol trafficking in Niemann-Pick type C (NPC) disease results in the first known instance of impaired regulation of the ATP-binding cassette transporter A1 (ABCA1), a lipid transporter mediating the rate-limiting step in high density lipoprotein (HDL) formation, as a cause of low plasma HDL-cholesterol in humans. We show here that treatment of human NPC1(-/-) fibroblasts with the liver X receptor (LXR) agonist TO-901317 increases ABCA1 expression and activity in human NPC1(-/-) fibroblasts, as indicated by near normalization of efflux of radiolabeled phosphatidylcholine and a marked increase in efflux of cholesterol mass to apoA-I. LXR agonist treatment prior to and during apoA-I incubation resulted in reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes, as well as cholesterol mass, in NPC1(-/-) cells. HDL species in human NPC disease plasma showed the same pattern of diminished large, cholesterol-rich alpha-1 HDL particles as seen in isolated heterozygous ABCA1 deficiency. Incubating NPC1(-/-) fibroblasts with the LXR agonist normalized the pattern of HDL particle formation by these cells. ABCG1, another LXR target gene involved in cholesterol efflux to HDL, also showed diminished expression in NPC1(-/-) fibroblasts and increased expression upon LXR agonist treatment. These results suggest that NPC1 mutations can be largely bypassed and that NPC1 protein function is non-essential for the trafficking and removal of cellular cholesterol if the down-stream defects in ABCA1 and ABCG1 regulation in NPC disease cells are corrected using an LXR agonist.