Retinol esterification in Sertoli cells by lecithin-retinol acyltransferase

Esterification of retinol occurs during the metabolism of vitamin A in the testis. An acyl-CoA:retinol acyltransferase (ARAT) activity has been described for microsomes isolated from testis homogenates. That activity was also observed here in microsomal preparations obtained from cultured Sertoli cells from 20-day-old (midpubertal) rats. ARAT catalyzed the synthesis of retinyl laurate when free retinol and lauroyl-CoA were provided as substrates. However, in the absence of exogenous acyl-CoA, retinol was esterified by a different activity in a manner similar to the lecithin:retinol acyltransferase (LRAT) activity described recently for liver and intestine. Microsomal preparations obtained from enriched Sertoli cell fractions from the adult rat testis had 75-fold higher levels of LRAT than the preparations from midpubertal animals, but ARAT activity was the same in both these preparations. LRAT utilized an endogenous acyl donor and either unbound retinol or retinol complexed with cellular retinol-binding protein (CRBP) to catalyze the synthesis of retinyl linoleate, retinyl oleate, retinyl palmitate, and retinyl stearate. The addition of exogenous dilaurylphosphatidylcholine (DLPC) resulted in the synthesis of retinyl laurate. The esterification from both exogenous DLPC and endogenous acyl donor was inhibited by 2 mM phenylmethanesulfonyl fluoride (PMSF). ARAT activity was not affected by similar concentrations of PMSF. Furthermore, retinol bound to CRBP, a protein known to be present in Sertoli cells, was not an effective substrate for testicular ARAT. When retinol uptake and metabolism were examined in cultured Sertoli cells from 20-day-old rats, the cells synthesized the same retinyl esters that were produced by microsomal LRAT in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinol esterification activity contributes to retinol transport in stellate cells.

The mechanisms of retinol transport and accumulation in hepatic stellate cells (HSC) remain to be elucidated. Our previous studies suggested that retinol esterification activity, particularly lecithin:retinol acyltransferase (LRAT) activity, in liver retinoid metabolism is important to elucidate the relationship between retinol uptake by HSC and the esterification of retinol. In the present study, using a human HSC-like cell line, LI90, we demonstrated that retinol esterification activity of LI90 cells is similar to that of primary cultures of rat HSC and higher than that of a human hepatoma cell line. Further, since progesterone or diphospho-lauroyl-phosphatidylcholine increased retinol esterification activity of LI90 cells, it is likely that LRAT contributes to retinol esterification in LI90. We examined retinol esterification in LI90 cells and clearance of retinol from culture medium. The percentages of both retinol and esterified retinol in LI90 cells increased in a manner dependent on retinol concentration in medium, whereas that of retinol in medium decreased. The percentages of esterified and unesterified retinol in LI90 cells and of retinol in medium were linearly dependent on the logarithm of the initial concentration of retinol in the medium. These results suggest that retinol esterification activity contributes to retinol uptake by HSC and maintenance of non-toxic retinol levels in plasma.     

Eicosapentaenoic acid inhibits cholesterol esterification in cultured parenchymal cells and isolated microsomes from rat liver

The effects of eicosapentaenoic acid on synthesis and secretion of cholesterol and cholesterol ester by cultured rat hepatocytes were studied. In the presence of eicosapentaenoic acid cellular cholesterol esterification was decreased by 50-75% compared to oleic acid as measured by radioactive precursors and mass. Secretion of cholesterol ester was reduced by 50-60% in the presence of eicosapentaenoic acid as evaluated by radiolabeled fatty acids, mevalonolactone, and mass measurement. Oleic, palmitic, and stearic acid increased, whereas eicosapentaenoic and docosahexaenoic acid decreased synthesis and secretion of cholesterol ester as compared to a fatty acid-free control. Cellular and secreted free cholesterol were unaffected by eicosapentaenoic acid in comparison with oleic acid. The reduced cholesterol esterification was observed within 1 h and lasted for at least 20 h. Eicosapentaenoic acid caused lower cholesterol esterification than oleic acid in the concentration range 0.2-1.0 mM fatty acid and reduced the stimulatory effect of oleic acid on cholesterol ester formation. Cholesterol esterification and release of cholesterol ester were markedly increased by 25-hydroxycholesterol in the presence of eicosapentaenoic acid as well as oleic acid. Experiments with liver microsomes revealed that radioactive eicosapentaenoic acid and eicosapentaenoyl-CoA were poorer substrates (7-30%) for cholesterol esterification than oleic acid and oleoyl-CoA. Reduced formation of cholesterol ester was also observed when eicosapentaenoyl-CoA was given together with labeled oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linolenoyl-CoA, and arachidonoyl-CoA had no inhibitory effect. In conclusion, eicosapentaenoic acid reduced cellular cholesterol esterification by inhibiting the activity of acyl-CoA:cholesterol acyltransferase. The lowered cholesterol esterification caused by eicosapentaenoic acid secondly decreased secretion of very low density lipoprotein cholesterol ester.     

Retinol binding protein in rat testicular cells

Cellular retinol-binding protein (CRBP) was identified in the cytosols of cultured Sertoli cells and peritubular cells from the testes of 20-day-old rats. CRBP was not detected in spermatids or spermatocytes obtained from the testes of 60-day-old rats. Cultured Sertoli cells and peritubular cells contained up to a 5-fold enrichment of CRBP/mg protein compared to whole testis homogenates. FSH- or FSH + testosterone-treated cultures of Sertoli cells showed a 60% increase in the specific activity of CRBP when compared to untreated cultures.     

The esterification of dolichol by rat liver microsomes.

The incubation of 1-[3H)dolichols with cell-free preparations from various rat tissues resulted in the formation of a labeled material which possessed the characteristics of synthetic dolichol palmitate. Rat liver microsomes were found to be a good source of the acyltransferase activity, and the properties of the reaction were investigated using microsomal preparations. The reaction did not require ATP, CoA, or Mg2+ and was stimulated by the addition of phosphatidylcholine. The esterification of dolichol appears to be similar to the esterification of retinol. The fact that the esterification of dolichol is not depressed even in the presence of a several-fold excess of retinol is evidence that the two reactions are catalyzed by different enzymes.     

Purification and characterization of alkaline phosphatase in cultured rat liver cells.

Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.     

Palmitate activation and esterification in microsomal fractions of rat liver.

Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl--AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.     

Growth dependence of phosphoglyceric acid dehydrogenase activity in cultured rat liver cells.

Rat liver epithelial cells in culture (WIRL-3C) have the enzymes that synthesize serine from 3-phophoglyceric acid. Both phosphoglyceric acid dehydrogenase (PGAD) and serine-phosphate (serine-P) forming activities fluctuate with time after subculture and are higher in growing than confluent cells. This activity pattern was not common for other dehydrogenases in WIRL-3C cells, nor was it common for PGAD activity in other cultured cells. At time of subculture, cells are removed from spent medium, treated with trypsin, and fed fresh medium. None of these parameters causes the rise in activity; in contrast, reduction in cell density and the accompanying stimulation of growth do. PGAD activity decreases when growth is slowed either as the cells progress to the end of the culture cycle, when cells are treated with dexamethasone-phosphate (Dx-P) or dibutyryl cyclic AMP(cAMP) and theophylline or when the serum concentration of the medium is reduced to 0.2%. Under these conditions, decreased PGAD activity is paralleled by a decline in growth and DNA accumulation. PGAD activity in WIRL-3C cells is regulated in a manner closely resembling what has been observed previously in rat liver from the whole animal. The possible use of this system in studying regulation of gene expression in mammalian cells is discussed.     

Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells

The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture. Tümen Mansuroglu and József Dudás contributed equally to this work.     

5'-Nucleotidase activities in cultured rat liver epithelial and fibroblast cells

Cell cultures of adult rat liver produced two distinct morphologic cell types: epithelial cells polygonal in shape and growing in nests of closely apposed cells, and fibroblast cells stellate in shape with little cell-cell contact at low density growth, but aligning in parallel arrays at high density. These two morphologic variants displayed dramatic differences in histochemically demonstrable 5'-nucleotidase activities. Fibroblast cells exhibited great activity throughout the cytoplasm with no concentration of activity in the cell membrane. The lesser activity in epithelial cells was concentrated on the cell membrane. The importance of this finding to the interpretation of data derived from experiments with whole liver homogenates is discussed.     

Biochemical changes in cultured foetal rat liver explants.

Liver explants from 20-day-old foetuses cultured for 48h in the absence of serum released 70% of their total soluble protein content into the medium. In the presence of serum this loss still amounted to 60%. The concentration of total particulate protein remained unchanged but there was some translocation of mitochondrial enzymes to the cytosol, and enzymes expected to increase during this stage of development failed to do so. The addition of cortisol plus glucagon (to serum-containing media) did not decrease the loss of total soluble protein from the explants but induced considerable tyrosine aminotransferase activity which was not released into the medium. The observations suggest that under the usual culture conditions a minority of the cells retain their functional integrity. The extent of deterioration, not reflected in histologically visible necrosis or cell damage, can be conveniently monitored by the malate dehydrogenase activity released to the medium.     

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. ³H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol·mg protein⁻¹·min⁻¹ when ³H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of ³H-labeled all-trans retinol and ³H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of ³H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 ± 3 ng CRBP·μg cytosol protein⁻¹. In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of ³H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific ³H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.     

Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.

The multicatalytic proteinase (MCP) prosome or proteasome is a large multifunctional complex which is believed to play a major role in non-lysosomal pathways of intracellular protein degradation and has recently been implicated in antigen processing. In this study, affinity-purified antibodies against rat liver MCP were used to investigate the localization of the proteinase both in rat liver and in growing human L-132 cells in culture, using electron microscopic immunogold techniques. Quantitation of the MCP in different subcellular localizations by morphometric analysis of electron micrographs showed the proportion in the nucleus to be 17% for hepatocytes and 51% for L-132 cells, demonstrating differences in the distribution of MCP in different cell types. In hepatocytes, 14% of the total MCP was found associated with the endoplasmic reticulum. The remainder was localized in the cytoplasmic matrix. Immunofluorescence studies with L-132 cells also showed a reaction in nuclei and cytoplasm. The localization of MCP is consistent with its proposed multiple functions in protein turnover, in the production of peptides for antigen presentation, and in RNA processing.     

The effects of O-acetylsterigmatocystin and related compounds on rat liver and cultured chicken embryonal liver cells.

Fine structural nucleolar changes induced in rat liver and primary tissue culture cells from 10-day-old chicken embryonal liver by O-acetylsterigmatocystin (AcO-stg), related compounds and aflatoxin B1 were compared. (1) Male Wistar rats were given a single i.p. injection of sterigmatocystin (stg), AcO-stg, and aflatoxin B1. 3 days after the injection of 15 mg/kg of stg, sporadic single cell necrosis was observed in rat liver, whereas rats treated with 8 mg/kg AcO-stg or more, and 3 mg/kg of aflatoxin B1 showed massive liver necrosis. Acetylation resulted in a marked increase in solubility in polar organic solvents. This increased solubility could play an important role in determining toxicity. (II) Treatment with the compounds with an unsaturateddelta1,2-furobenzofuranring system, such as AcO-stg, demethyl-diacetyl-stg (deMe-diAc-stg), and aflatoxin B1, resulted in nucleolar segregation and fragmentation of primary culture cells. Both parenchymal and mesenchymal cells in culture were susceptible to AcO-stg and deMe-diAc-stg, while the mesenchymal cells were more resistant to aflatoxin B1 than the hepatocytes. The inhibition of RNA synthesis in both cell types as determined in radioautography was in accordance with the electron-microscopic observations. Acetyldihydrosterigmatocystin (AcO-dihyd-stg), a saturated delta1,2-furobenzofuranring compound, was less toxic to primary tissue culture cells.     

Metabolic activation of procarcinogens to genotoxic products in cultured rat liver cells

A recently established rat liver cell line, RL-12, exhibits an unusually high sensitivity to the genotoxic effects of a number of selected procarcinogens. Significant reductions in cell survival (D37%) and induction of sister chromatid exchanges were obtained with 1 X 10(-9) M benzo[a]pyrene and 2 X 10(-8) M 7,12-dimethylbenz[a]anthracene. This rat liver epithelial cell line may serve as a useful model system to study the metabolic activation of procarcinogens to their ultimate genotoxic form.     

Spectrophotometric determination of intracellular pH with cultured rat liver epithelial cells

A spectrophotometric method using 6-carboxyfluorescein (CF) was developed to determine intracellular pH in anchorage-dependent monolayers of control cells of rat hepatic origin. Until now, such studies have been carried out with ascites cells in suspension, which lack specific controls for comparative studies. The rat cell line is grown on plastic Leighton tube slides which fit directly into 3 cm spectrophotometer cuvettes. One sample, without CF, serves as a control for the light-scattering properties of the cell monolayers. Steady-state determinations show a decline in intracellular pH from 7.3 to 6.8 ten minutes after the addition of glucose and quercetin. Kinetic determinations show that with the addition of glucose to substrate-free cells the rate of acid formation is -0.02 pH units/min; the addition of quercetin results in a further acceleration of the kinetic rate to -0.10 pH units/min. In both types of analyses, the change in intracellular pH is standardized with nigericin and external buffers, based on the decrease in the maximum absorption of CF at 492 nm. The results demonstrate that even with anchorage-dependent monolayers of a control hepatocyte line which produces very little acid, this spectrophotometric method permits determinations sufficiently sensitive for analysis of intracellular pH.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

The effects of insulin on choline kinase activity in cultured rat liver cells

The effect of insulin on phosphatidylcholine biosynthesis in cultured rat liver cells was assessed by measuring changes in the activity of the first enzyme in the choline pathway of phosphatidylcholine biosynthesis, choline kinase (ATP: cholinephosphortransferase, EC 2.7.1.32), in the presence or absence of the hormone. Choline kinase specific activity in liver cells incubated for 18 hours in the presence of 10^?7M insulin increased two-fold from 3.4 ± 0.3 nmoles phosphorylcholine formed/min/mg protein to 7.5 ± 0.6 nmoles/min/mg protein. This effect was dose dependent and reversed by the addition of actinomycin D and cycloheximide. It is concluded that the increase in specific activity is due to synthesis of new enzyme rather than activation of existing enzyme.