The rate of sphingomyelin synthesis de novo is influenced by the level of cholesterol in cultured human skin fibroblasts

Plasma membrane sphingomyelin (SM) is known to affect the cellular distribution of cholesterol. The aim of this work was to examine how SM homoeostasis in human skin fibroblasts is affected by alterations in the level of cholesterol in the cell. The cellular cholesterol level was decreased by exposing cells to 2-hydroxypropyl-β-cyclodextrin, and increased by exposing cells to cholesterol–methyl-β-cyclodextrin inclusion complexes. A lowering of the cellular unesterified cholesterol content by 20% was shown to increase the incorporation of [14C]palmitic acid into SM by 70%. Subsequently, the cellular SM mass was shown to be increased (24% increase after a 24 h period). Since l-cycloserine completely abolished the increased incorporation of [14C]palmitic acid into SM in cholesterol-depleted cells, we concluded that the de novo synthesis of the sphingosine backbone of SM was activated in cholesterol-depleted cells. This conclusion was further verified by performing a cell-free assay of serine C-palmitoyltransferase (SPT) in cholesterol-depleted cells, which showed that the activity of the enzyme was increased by 30% after cholesterol depletion. Most of the newly synthesized SM in cholesterol-depleted cells was susceptible to degradation by sphingomyelinase, indicating that it was transported efficiently to the cell surface. Loading of fibroblasts with cholesterol had essentially the opposite effects on SM homoeostasis to those of cholesterol depletion, i.e. 20–30% decreased incorporation of [14C]palmitic acid into SM and decreased activity of SPT. The results of this study show that cellular cholesterol levels have marked effects on the homoeostasis of SM.

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A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid: diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/s1388-1981(02)00296-2 ◽

2002 ◽

Vol 1584 (2-3) ◽

pp. 91-98 ◽

Cited By ~ 48

Author(s):

Jürgen G Okun ◽

Stefan Kölker ◽

Andreas Schulze ◽

Dirk Kohlmüller ◽

Katharina Olgemöller ◽

...

Keyword(s):

Fatty Acid ◽

Palmitic Acid ◽

Fatty Acid Oxidation ◽

Human Skin ◽

Skin Fibroblasts ◽

Acid Oxidation ◽

Human Skin Fibroblasts ◽

Mcad Deficiency ◽

Fatty Acid Oxidation Disorders

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Glucocorticoid regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts: Evidence for a receptor-mediated mechanism involving effects on specific de novo protein synthesis

Metabolism ◽

10.1016/s0026-0495(98)90015-4 ◽

1988 ◽

Vol 37 (2) ◽

pp. 179-184 ◽

Cited By ~ 20

Author(s):

Terry J. Smith

Keyword(s):

Protein Synthesis ◽

Human Skin ◽

De Novo ◽

Skin Fibroblasts ◽

Human Skin Fibroblasts ◽

Glycosaminoglycan Synthesis ◽

De Novo Protein Synthesis

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Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts

International Journal of Biochemistry Research & Review ◽

10.9734/ijbcrr/2015/14058 ◽

2015 ◽

Vol 5 (3) ◽

pp. 214-224 ◽

Cited By ~ 4

Author(s):

David Akopian ◽

Ryoko Kawashima ◽

Jheem Medh

Keyword(s):

Human Skin ◽

Skin Fibroblasts ◽

Human Skin Fibroblasts ◽

Cellular Cholesterol

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Cholesterol-mediated regulation of HMG-CoA reductase in microsomes from human skin fibroblasts and rat liver

Biochemistry and Cell Biology ◽

10.1139/o90-097 ◽

1990 ◽

Vol 68 (3) ◽

pp. 674-679 ◽

Cited By ~ 6

Author(s):

R. George ◽

P. J. Davis ◽

L. Luong ◽

M. J. Poznansky

Keyword(s):

Tissue Culture ◽

Enzyme Activity ◽

Human Skin ◽

Rat Liver ◽

Reductase Activity ◽

Cholesterol Content ◽

Skin Fibroblasts ◽

Human Skin Fibroblasts ◽

Hmg Coa Reductase ◽

Coa Reductase

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was determined in microsomes from human skin fibroblasts and rat liver that had been variously manipulated in vivo or in tissue culture to up- and down-regulate the enzyme. The cholesterol content of these microsomal preparations was then altered by depletion to or enrichment from either cholesterol-free or cholesterol-rich lipid vesicles. Microsomes from human skin fibroblasts responded to cholesterol depletion by increasing HMG-CoA reductase activity and by decreasing it in response to cholesterol enrichment. This was independent of the initial enzyme activity or the tissue culture conditions. Alterations in cholesterol content of rat liver microsomes in vitro failed to demonstrate any significant changes in HMG-CoA reductase activity whether the microsomes started with low enzyme activity (cholesterol-fed rats) or with high enzyme activity (cholestyramine-treated rats). The results are discussed in relation to previously published data and in respect to differences in the control of the human skin fibroblast and rat liver enzymes.Key words: cholesterol, HMG-CoA reductase, microsomes, fibroblasts, rat liver.

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Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

Biochemical Journal ◽

10.1042/bj1680091 ◽

1977 ◽

Vol 168 (1) ◽

pp. 91-103 ◽

Cited By ~ 21

Author(s):

Christopher H. J. Sear ◽

Michael E. Grant ◽

David S. Jackson

Keyword(s):

Human Skin ◽

De Novo ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Skin Fibroblasts ◽

Human Skin Fibroblasts ◽

Cultured Fibroblasts ◽

Cscl Density Gradient ◽

Before And After

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.

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