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G Rogler

Publications and source records attributed to G Rogler.

67 records · Page 4Linked to original sources
PubMed1995

HDL-mediated efflux of intracellular cholesterol is impaired in fibroblasts from Tangier disease patients.

To further elucidate the cellular mechanisms leading to HDL deficiency in Tangier disease, HDL-mediated cholesterol efflux was studied in cultured skin fibroblasts from Tangier patients. Both Tangier and control fibroblasts show specific saturable binding of HDL3 to the cell membrane (Bmax = 70 and 52 ng/mg protein, respectively; Kd = 8.8 and 10.6 micrograms/mL, respectively). There was no appreciable uptake of HDL3 by Tangier and control fibroblasts, indicating that cholesterol efflux from fibroblasts occurs at the cell membrane. When cellular cholesterol was labeled to equilibrium by [14C]cholesterol incubation for 48 hours at 37 degrees C, HDL3-mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnormality in HDL3-mediated cholesterol efflux more precisely, several additional experiments were performed. First, membrane desorption of cholesterol was determined after cell membranes were labeled with [14C]cholesterol for 3 hours at 15 degrees C. With this labeling protocol, there was no difference in HDL3-mediated cholesterol efflux between control and Tangier fibroblasts. Second, efflux of newly synthesized sterols was determined after incorporation of the precursor [14C]mevalonolactone. Under these conditions, specific HDL3-mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were labeled by incubation with reconstituted [3H]cholesteryl-linoleate-LDL. Efflux of LDL-derived cholesterol was only slightly reduced for the first 4 hours of incubation. After 12 hours, there was no difference between control and Tangier cells. The combined data indicate that the reduced efflux of cholesterol from Tangier fibroblasts observed after homogeneous labeling is due to severely reduced efflux of newly synthesized sterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1994

Improved separation of radioactively labelled cellular phospholipids by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the separation and determination of radioactively labelled cellular phospholipids is described. The method is based on separation of phospholipids on a 250 x 4 mm I.D. LiChrospher DIOL 100 (5 microns) column, fitted with a 50 x 4 mm I.D. LiChrospher Si 60 (5 microns) precolumn and a gradient of 5% H3PO4 and acetonitrile. It allows the determination of small amounts of labelled phosphatidylcholine and sphingomyelin due to the sharp elution profile in spite of long retention times.

Cells, Cultured↗
PubMed1994

HDL3-mediated cholesterol efflux from cultured enterocytes: the role of apoproteins A-I and A-II.

High density lipoproteins (HDL) were recently demonstrated in an enterocyte model (CaCo-2 cells) to mediate reverse cholesterol transport by retroendocytosis. The present study was carried out to define the role of the major HDL apoproteins (apo) A-I and apo A-II in this pathway. HDL3 was fractionated by heparin affinity chromatography into the two main fractions containing either apo A-I only (fraction A) or both apo A-I and apo A-II (fraction B). In addition, liposomes were reconstituted from purified apo A-I or apo A-II and dimyristoyl phosphatidylcholine. The cell binding properties and cholesterol efflux potential were studied in the lipoprotein fractions and the liposomes. Both fractions exhibited similar maximal binding capacities of 4427 (A) and 5041 (B) ng/mg cell protein, but their dissociation constants differed (40.5 and 167.7 micrograms/mL, respectively). Fraction A induced cholesterol efflux and stimulated cholesterol synthesis more than did fraction B. Fraction A mobilized both cellular free and esterified cholesterol, whereas fraction B preferentially mobilized cholesteryl esters. Liposomes, containing either apo A-I or apo A-II, showed specific binding, endocytosis and endosomal transport, and were released as intact particles. Apo A-I liposomes also mediated cholesterol efflux. In conclusion, there is evidence that the HDL3 subfractions A and B, as well as reconstituted liposomes containing either apo A-I or apo A-II, were specifically bound and entered a retroendocytosis pathway which was directly linked to cholesterol efflux. Quantitatively, the apo A-I subfraction appeared to play the dominant role in normal enterocytes. The apo A-II content of fraction B was related to the mobilization of cholesteryl esters.

Adenocarcinoma↗
PubMed1993

Polyamine deficiency impairs proliferation and differentiation of cultured enterocytes (CaCo-2).

The polyamine dependence of enterocyte growth and differentiation was studied in the human intestinal cell line CaCo-2 using a specific inhibitor of the key enzyme ornithine decarboxylase (ODC), difluoromethylornithine (DFMO). ODC was highest during the initial phase of rapid growth and was inhibited in a dose dependent fashion by DFMO at 0.06-2 mM. At low levels DFMO only delayed cell replication without affecting final cell count whereas at concentrations of 0.125 mM and above the final cell number was diminished by at least 53% compared to controls. In contrast, DFMO even at 0.03 mM reduced sucrase activity to 44% of controls when added at day 2 but was ineffective when supplemented at day 7 of culture or later. The inhibitor also diminished the number and length of microvilli in a dose dependent fashion, although this effect required higher DFMO levels than the reduction of sucrase activity. The DFMO mediated suppression of cell replication, enzymatic and morphologic differentiation was reversible in the presence of the ODC product putrescine. Putrescine alone did not affect any of the above parameters. In conclusion, the present data suggest that ODC and polyamines are involved both in enterocyte growth and differentiation.

Cell Differentiation↗
PubMed1992

High-density lipoprotein 3 retroendocytosis: a new lipoprotein pathway in the enterocyte (Caco-2).

The present study in Caco-2 cells, derived from a human colon carcinoma and capable of enterocyte differentiation in culture, describes a retroendocytotic pathway for high-density lipoprotein 3 (HDL3). These cells exhibit specific binding of apolipoprotein E-free HDL3 which was competed for by HDL3 but not by low-density lipoproteins. At 37 degrees C, degradation was negligible and intact particles were internalized and resecreted into the medium within 2 hours. Electron microscopy showed binding and internalization of gold-labeled HDL3 in coated pit regions and transport in endosomes distinct from lysosomes to lipid droplets. The fusion of these endosomes with lipid droplets was followed by their dissolution and the subsequent extrusion of HDL particles from the cells. Fluorescence labeling studies of HDL3 supported cytosolic transport in vesicles. Specific binding showed negative feedback regulation by HDL3, was modulated by alterations in cellular cholesterol content, and increased with the cellular state of differentiation. HDL3 mediated efflux of endogenously labeled cholesterol. It is concluded that intact HDL3 is bound specifically by Caco-2 cells, leading to a subsequent intracellular passage and resecretion through a process of retroendocytosis effecting the efflux of cellular cholesterol.

Cholesterol↗
PubMed1992

Regulation of cholesterol metabolism and low-density lipoprotein binding in human intestinal Caco-2 cells.

In the present paper, the regulation of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, acylcoenzyme A cholesterol acyltransferase (ACAT) and low-density lipoprotein (LDL) binding was studied in the human colon cancer carcinoma cell line Caco-2. LDL down-regulated HMG-CoA reductase activity in a dose-dependent fashion to a minimum of 28% of control at 200 micrograms/ml and LDL binding to 52% of control. The activity of ACAT was stimulated by LDL. High-density lipoprotein 3 (HDL3) increased HMG-CoA reductase activity, whereas cholesteryl ester formation was slightly decreased. Inhibition of the endogenous cholesterol biosynthesis by mevinolin increased both LDL binding and activity of HMG-CoA reductase. This effect was reversed by the addition of mevalonolactone but not by LDL. It is concluded that regulation of HMG-CoA reductase and LDL binding is subject to the availability of non-sterol products of mevalonic acid and of exogenous cholesterol. ACAT is regulated mainly by the level of its substrate cholesterol.

Cholesterol↗
PubMed1991

HDL3-retroendocytosis in cultured small intestinal crypt cells: a novel mechanism of cholesterol efflux.

The present study in IEC-6 crypt-derived rat epithelial cells describes a retroendocytotic pathway for HDL3. These intestinal cells exhibited specific binding of apoE free HDL3 with a maximal binding capacity of 2980 ng/mg cell protein and a Kd of 36.4 micrograms/ml. Specific binding was competed for by HDL3 but not by LDL. Apparent internalisation of HDL3 was low, degradation was negligible and intact particles were resecreted into the medium within 2 h. Electron microscopic studies showed binding and internalisation of gold-labeled HDL3 in coated pit regions and transport in endosomes distinct from lysosomes to lipid droplets. De novo cholesterol synthesis from [14C]octanoate was enhanced nearly 2-fold by HDL3 and the surplus of newly formed cholesterol was recovered in the medium. It was concluded that intact HDL3 was bound specifically to intestinal cells and was resecreted through a process of retroendocytosis probably mediating efflux of cellular cholesterol.

Animals↗
PubMed1990

[Regulation of intestinal cholesterol metabolism].

The small bowel epithelium is of major importance in the cholesterol homeostasis of the organism. The morphological and functional heterogeneity of the gut, complicates studies on intestinal cholesterol metabolism. Cholesterol from diet, de novo synthesis and lipoproteins is strictly compartmentalized intracellularly and supports different metabolic needs. Interactions of cholesterol and lipoproteins were studied in cultured intestinal cells (IEC-6, CaCo-2). Newly synthesized cholesterol is mainly utilized for local purposes like membrane synthesis and is essential for cell growth. In contrast to other cell systems it cannot be replaced by LDL cholesterol in case of blocked synthesis. LDL is specifically bound, internalized and degraded. HDL3 also displays specific binding, internalisation and retroendocytosis, but is not degraded. The observed induction of HMG-CoA reductase, suppression of ACAT, as well as cholesterol efflux after contact of HDL3 with lipid droplets argue for intracellular cholesterol transfer to intact HDL3 and finally resecretion into the medium. HDL3 therefore appears as a mediator of reverse cholesterol transport also in the small intestinal epithelial cell.

Carrier Proteins↗
PubMedDate not supplied

Mevalonate is essential for growth of porcine and human vascular smooth muscle cells in vitro.

The effects of mevalonate depletion on growth and cell cycle kinetics of porcine and human vascular smooth muscle cells (SMC) were studied by growth curves and flow cytometric determination of cell cycle distribution. Porcine and human SMC were growth arrested by serum depletion for 48 h. Subsequently, they were stimulated to maximal growth and DNA synthesis by addition of serum. There was a concentration dependent decrease in the proliferation of human and porcine SMC, when cells were incubated in the presence of fluvastatin, a new, fully synthetic HMGCoA-reductase inhibitor. The reduction of cell number was significant with 10(-5) M and 10(-4)M fluvastatin. The highest concentration induced cell loss after prolonged incubation (> 4 days). The G/S-phase transition of human and porcine vascular SMC was reduced to 50% of controls by 10(-4) M fluvastatin as revealed by cell cycle analysis. The effects of fluvastatin on growth kinetics and cell cycle distribution could be completely reversed by the addition of 1 mM mevalonolactone. This indicates that the inhibitory effect of fluvastatin is caused by the inhibition of HMGCoA-reductase and depletion of mevalonate rather than being unspecific. Addition of LDL to supply cholesterol failed to support cell growth and transition of smooth muscle cells from G(zero)/G1-phase to S-phase, even though LDL was taken up by the cells as shown by confocal fluorescence microscopy. Neither did the addition of squalene or cholesterol to the culture medium normalize cell growth. It is concluded that nonsterol products that are synthesized from mevalonate are necessary for growth of smooth muscle cells. HMGCoA-reductase inhibitors like fluvastatin block the synthesis of these nonsterol precursors in human and porcine vascular SMC in vitro and are therefore growth inhibitory.

Animals↗
PubMedDate not supplied

Post-traumatic pseudocyst of the spleen: sclerotherapy with ethanol.

We report a case of successful percutaneous treatment of a chronic post-traumatic splenic pseudocyst using alcohol as the sclerosing agent. A 26-year-old man presented with a symptomatic cystic mass located in the spleen. Aspiration of 300 ml of fluid was only temporarily effective, and therefore a drainage catheter was placed 3 days later. After histopathologic and microbiologic exclusion of a malignant or infectious origin, local sclerotherapy with alcohol was performed because of recurrence after percutaneous drainage. This therapy was repeated six times within 2 weeks. Two weeks later, the remaining volume was determined to be 16 ml. Six months after treatment the cyst was no longer visible. To our knowledge this is the first case of a chronic post-traumatic splenic cyst treated with alcohol. Percutaneous sclerotherapy of a symptomatic post-traumatic splenic pseudocyst may be an alternative to surgical treatment.

Adult↗