Lymphoid cell lines as a model system for the study of Wolman's disease: Enzymatic, metabolic and ultrastructural investigations

Nègre, Anne; Salvayre, Robert; Maret, Arlette; Vieu, Claude; Bes, J. C.; Borrone, C; Durand, P.; Douste‐Blazy, Louis

doi:10.1007/bf01799458

Cited by 22 publications

(16 citation statements)

References 20 publications

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“…These conclusions do not agree with the hypothetical role of the lysosomal lipase in cytoplasmic triacylglycerol catabolism of differentiating myoblasts suggested by Sauro et al [18]. Our previous enzymatic data, briefly summarized in Materials and Methods [25], and the present results suggest the existence in fibroblasts of a neutral lipase able to hydrolyse cytoplasmic triacylglycerols: this enzyme could be identical to that described by Hoeg et al [31]. The metabolic data reported here demonstrate that such a lipase is active in Wolman fibroblasts in spite of the observation of Messieh et al [32] who reported an abnormal neutral lipase activity in these cells.…”

Section: Discussioncontrasting

confidence: 84%

“…Experiments were performed in duplicate using a radiochromatographic scanner (Berthold LB 2842 automatic TLC linear analyser). Non-labelled cellular lipids were analysed as previously reported [25]: free and total cholesterol from the lipid extract were determined by gas chromatography (Intersmat, model 120 DFL; methylsilicon capillary column SP2100, 20 m, 0.3 nm internal diameter) according to the procedure of Ambert et al [26] using 5-a-cholestane as internal standard. Triacylglycerols were separated by TLC on silica gel (petroleum ether/diethyl ether/acetic acid, 80/20/1, v/v/v), eluted and determined by gas chromatography (Intersmat 120DFL, SP2100 capillary column; oven temperature programmed from 290°C to 335"C, 6"C/min; flame ionization detector Fibroblasts were cultivated during 6 days in culture medium containing 10% fetal calf serum without (FCS) or with (LP-MI and LP-M2) hyperlipemic serum (pool of sera from hypertriglyceridemic type IV patients, heat-treated for 1 h at 56°C and sterilized by filtration on micropore 0.22 pm).…”

Section: Fig I Time-dependent Kinetics Of Radiolabelled Lipids Aftementioning

confidence: 99%

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

Salvayre

Nègre

Maret

et al. 1987

European Journal of Biochemistry

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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 Iipid 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.Wolman's disease is an inherited lysosomal storage disorder characterized by a massive accumulation of triacylglycerols and cholesteryl esters in the tissues caused by a severe deficiency in lysosomal acid lipase activity [l -61. Theoretically, the stored neutral lipids can be of extracellular origin (lipoproteins) or from endogenous cellular biosynthesis. The exogenous lipoproteins taken up by the cells via a receptor-mediated or a receptor-independent endocytosis [7, 81 reach an endosomal compartment which fuses in part with the lysosomes [9, 101. On the other hand, the endogenous synthesis of triacylglycerols and cholesteryl esters is well known: cholesterol synthesized de novo by the cells, or liberated from the lysosomal compartment can be esterified by the cellular acyl-CoA: cholesterol acyltransferase [l 1, 121. The cytoplasmic triacylglycerol synthesis (through the phosphatidic acid pathway) is considerably increased when the cells receive a large influx of fatty acid which can induce a major cytoplasmic triacylglycerol storage [I3 -171. The relations between this cytoplasmic compartment containing endogenously synthesized triacylglycerols and the lysosomal compartment are poorly understood but some hypotheses have been formulated concerning the role of acid lysosomal lipase in the resorption of the cytoplasmic-induced triacylglycerol lipidosis [18].We report in this paper experimental data supporting the hypothesis that the neutral lipids accumulated in lysosomes of cultured fibroblasts from Wolman's disease (compared to normal) are mainly from exogenous (lipoprotein) origin, whereas endogenously synthesized neutral lipids do not parCorrespondence to R.

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Section: Discussioncontrasting

confidence: 84%

Section: Fig I Time-dependent Kinetics Of Radiolabelled Lipids Aftementioning

confidence: 99%

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

Salvayre

Nègre

Maret

et al. 1987

European Journal of Biochemistry

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“…The use of LCLs has been previously proven to be instrumental in uncovering important pathogenic mechanisms in other diseases, such as hypertension, glycogen and lipid storage diseases, chronic granulomatous disease, hematologic disorders, and solid tumors (27)(28)(29)(30)(31)(32)(33). One illustrative example is essential hypertension.…”

Section: Discussionmentioning

confidence: 99%

Sphingosine kinase 1–mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells

Pi¹,

Tan²,

Hayes³

et al. 2006

Arthritis & Rheumatism

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Objective. It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas-mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls.Methods. Cell death was determined by the MTS assay, and apoptosis was detected by the TUNEL assay and DNA laddering. Proteolytic activation of caspase 3 was determined by immunoblotting, and its enzymatic activity was determined by a fluorometric technique. Messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction (PCR) analysis. The functional role of sphingosine kinase (SPHK) was determined by measuring its enzymatic activity, by quantifying the levels of its product, sphingosine 1-phosphate (S1P), and by investigating the ability of the SPHK inhibitor N,N-dimethylsphingosine and isozyme-specific small interfering RNA (siRNA) oligonucleotides to reverse signaling aberrations.Results. LCLs from patients with RA displayed disease-specific Fas-mediated signal transduction impairment with consequent resistance to cell death. RA LCLs displayed high constitutive SPHK activity and increased levels of S1P. Real-time PCR analysis showed higher SPHK-1 mRNA expression levels in RA patients compared with paired controls. Increased SPHK-1 (but not SPHK-2) mRNA levels were observed in synovial tissue from RA patients. Competitive inhibitors of SPHK reversed the resistance of RA LCLs to Fasinduced apoptosis. Additionally, resistance to Fasmediated signaling was reversed by siRNA oligonucleotides specific for SPHK-1 but not by oligonucleotides specific for SPHK-2.Conclusion. These findings demonstrate diseasespecific resistance to Fas-mediated death signaling in patients with RA and implicate increased SPHK-1 activity as the cause of this aberration.

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“…The fatty acids liberated were quantified radiometrically and fluorometrically as in [18,19]. Proteins were determined by the method of Lowry et al [20].…”

Section: Methodsmentioning

confidence: 99%

Independence of triacylglycerol‐containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy

Salvayre¹,

Nègre²,

Radom³

et al. 1989

FEBS Letters

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The functional relationship between the two subcellular compartments involved in catabolism of triglycerides, i.e. lysosomes and lipid-containing cytoplasmic vacuoles, has been investigated using cultured fibroblasts from patients affected with two different genetic lipid (triacylglycerol) storage disorders: Wolman disease and multisystemic lipid storage myopathy. As shown by metabolic studies in intact cultured cells, lysosomal degradation of exogenous labelled triacylglycerols (incorporated into lipoproteins and internalized via the apo B/E receptor pathway) was blocked in Wolman cells, whereas catabolism of endogenously biosynthesized triacylglycerols was in the normal range. In contrast, in fibroblasts from multisystemic lipid storage myopathy, the degradation of endogenous triacylglycerols was blocked, whereas that of exogenous triacylglycerols (i.e. from lipoproteins) was normal. This comparative study demonstrates that the lysosomal and cytoplasmic compartments are functionally independent. Enzymatic studies allow one to discriminate clearly between 3 lipases and 2 carboxylesterases the role of which is discussed.

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Lymphoid cell lines as a model system for the study of Wolman's disease: Enzymatic, metabolic and ultrastructural investigations

Cited by 22 publications

References 20 publications

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

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

Sphingosine kinase 1–mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells

Independence of triacylglycerol‐containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy

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