Molecular and Cellular Biochemistry 254: 37–46, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Effect of pentoxifylline on arachidonic acid metabolism, neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell Carla C.A. Cardoso,1 Ernani R. Paviani,1 Lavínia A. Cruz,1 Fátima C.R. Guma,1 Radovan Borojevic2 and Regina M. Guaragna1 1
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS; 2Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil Received 19 September 2002; accepted 10 February 2003
Abstract In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A2 (PLA2), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1–2.5 µM), it increased the quantity of stored lipids. Retinol and PTF (5 µM and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA2 activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE2, but not of PGF2a. However, when in presence of retinol, we observed a slightly higher increase in PGE2 and PGF2a production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC. (Mol Cell Biochem 254: 37–46, 2003) Key words: pentoxifylline, retinol, phospholipase A2, arachidonic acid, prostaglandin, neutral lipid, hepatic stellate cell
Introduction Hepatic stellate cells (HSC), also known as lipocyte, Ito cells, or liver fat-storing cells, are responsible for the increased synthesis of extracellular matrix (ECM) in liver fibrosis. They are the main site of vitamin A storage in characteristic lipid
droplets. Their long, contractile cytoplasmic processes encompass the sinusoids and can affect sinusoidal blood flow. Because of their location in the Space of Disse and connection with other sinusoidal and parenchymal cells, they are probably involved in local neurotransmission and paracrine regulation of numerous liver functions. As a source
Address for offprints: R.M. Guaragna, Departamento de Bioquímica, ICBS, UFRGS, Rua Ramiro Barcelos 2600-anexo, Porto Alegre, RS, Brasil (E-mail:
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38 of cytokines, prostaglandins and other bioactive substances, produced in the response to noxious agents, they play a crucial role in the mechanisms of liver response to injury, regeneration and fibrosis [1]. An intriguing aspect of stellate cell function is the capacity to express two distinct phenotypes. In healthy liver, the cells show the ‘quiescent phenotype’, that is to say, they contain vitamin A-rich lipid droplets, underdeveloped organelles and show a low proliferation rate. In chronically diseased liver, the cells acquire the ‘activated phenotype’, they differentiate into myofibroblast-like cells with high proliferative capacity, thereby losing their vitamin A-containing fat-droplets, and developing a prominent rough endoplasmic reticulum, extensive microtubules and bundles of actin filaments with local condensations [2]. The transition of stellate cells into myofibroblast-like cells is regulated by an intricate network of intercellular communication between stellate cells and activated Kupffer cells, damaged hepatocytes, platelets, endothelial and inflammatory cells, involving cytokines and non-peptide mediators such as reactive oxygen species, eicosanoids and acetaldehyde. To understand the molecular basis of the multifaceted cellular changes in HSC activation, increasing efforts have been made to characterize alterations in specific gene regulation or intracellular signaling that may generate some of the activation events. Inhibition of HSC activation and the related subsequent events, such as increased production of ECM components and enhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade [3]. The synthesis of prostanoids is thought to be regulated by the availability of free arachidonic acid (AA) in the activated cells [4]. AA is normally bound by an ester linkage in the sn-2 position of phospholipids. Thus, the activation of an intracellular phospholipase A2 (PLA2) is considered to be crucial for the release of AA and for the subsequent prostanoid synthesis. Prostanoids, which are released from non-parenchymal liver cells in response to proinflammatory stimuli, are involved in the regulation of hepatic functions during inflammation. They exert their effects on their target cells via heptahelical receptors in the plasma membrane [5]. In rat liver, the five major prostanoids, prostaglandin (PG) D2, F2, F2a, PGI2 (prostacyclin) and thromboxane A2, are produced by non-parenchymal sinusoidal cells which comprise Kupffer cells, endothelial cells and stellate cells [6]. Pentoxifylline (PTF), a methyl xanthine phosphodiesterase inhibitor, is one of several agents that have been tested as a potential antifibrogenic treatment. PTF may act as a scavenger of free radicals, which may be an important mechanism in the beneficial effect of the drug on liver fibrosis [7]. It has been proposed that PTF acts as an antifibrogenic agent by reducing the synthesis of extracellular matrix components, and this possibility has been confirmed in animal models of hepatic fibrosis [8].
In previous studies, we have established a permanent cell line, named GRX cells, from primary cultures of liver myofibroblastic cells engaged in granulomatous reactions to schistosome eggs [9]. Both morphological and immunocytochemical studies have shown that these cells originate from typical HSC that lose their fat droplets, migrate, proliferate and form the fibrotic component of granulomas [10, 11]. Beyond the production of fibrotic extracellular matrix, granuloma myofibroblasts are a rich source of pro-inflammatory and myelopoietic growth factors and the accessory molecules, having a relevant function in control of liver tissue inflammatory cell infiltration, similar to other models of liver reaction to injury [12–15]. The GRX cells can be induced to convert into the fat-storing phenotype by retinoids, and by other agents such as combined insulin and indomethacin treatment [16, 17]. In fact, depletion of retinoids occurs in activation of HSC, both in culture and in vivo [2]. Thus, it is plausible that deficiencies of retinoids may permissively upregulate of autocrine fibrogenic cytokines, such as TGF-β, TGF-α and interleukin-10 [14]. In the present study, we examined the conversion of murine HSC expressing the myofibroblastic phenotype into the quiescent lipocytic HSC, by treatment with PTF. Since quiescent HSCs are characterized by intracellular storage of lipids including fat-soluble vitamin A, we monitored the effects of PTF on neutral lipid metabolism. We also examined the action of PTF, reported to be a hepatic cytoprotector and antifibrogenic agent, with or without retinol, which physiologically induces the lipocyte quiescent phenotype, on the PLA2 activity, synthesis and secretion of bioactive lipids, such as arachidonic acid, prostaglandin PGE2 and PGF2α, which participate in the development of inflammatory and tissue repair processes. We have used the GRX cell line (murine HSC) as a model of lipocyte phenotype induction. These results suggest the pivotal role of retinol and PTF on lipid metabolism in molecular regulation of HSC in liver fibrogenesis.
Materials and methods Cell cultures GRX cells were obtained from the Rio de Janeiro Cell Bank (Federal University of Rio de Janeiro, Brazil). They were maintained in the Dulbecco’s minimum essential medium (DMEM, Sigma Chemical Company, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil) and 3 g/L HEPES buffer, pH 7.4, under normal air atmosphere, at 37°C. For arachidonic acid release and characterization of lipids, the cells were maintained in DMEM supplemented with 5% fetal bovine serum (FBS) and 2 g/L HEPES buffer, pH 7.6, under the atmosphere containing 5% CO2 [17].
39 Induction of lipocyte phenotype Cells were plated in 25 cm2 tissue culture flasks (5 × 105 cells/ flask) obtained from Nunc, Roskilde, Denmark. After 24 h the fat-storing phenotype was induced by incubation of cultures in medium supplemented with 5% FBS, pentoxifylline (0.01, 0.1, 0.5, 0.7 and 1 mg/mL) and/or retinol (1, 2.5, 5; 7.5 and 10 µM) for 8 days. The viability of the cells was checked by the Trypan blue exclusion assay.
was removed, the released arachidonic acid was extracted with chloroform: methanol: HCl (100:100:1; v/v/v) [41] and the lipids were analyzed by TLC: chloroform: methanol: acetic acid: water (90:8:1:1; v/v/v/v) [26], which resolves AA from different oxidation metabolites. Radiolabeled lipids, separated by TLC were analyzed by autoradiography on Kodak-X-Omat films. Arachidonic acid spot was cut from TLC and their radioactivity was quantified by liquid scintillation. Under these assay conditions there was no significant conversion of [14C]AA into oxidation products.
PLA2 activity assay Characterization of neutral lipids Cells were washed and scraped from the culture flasks, homogenized and sonicated (Ultrasonic processor, 2 × 30 sec) [4] in 50 mM Tris-HCl buffer (pH 8.5) on ice [18]. This homogenate was centrifuged at 100,000 g for 1 h at 4°C. The microsomal and the supernatant fractions were used for the enzyme activity assays [19, 20]. Protein was determined by the method of Lowry [21] using serum albumin as a standard. Phosphatidylethanolamine vesicles were prepared with 0.275 µCi of the radiolabeled phospholipid (55 mCi/mmol) (Amersham Life Science) and 100 µg phosphatidylethanolamine, dried under nitrogen, and sonicated (60 sec) in 50 mM Tris-HCl buffer pH 8.5 on ice [18, 22]. PLA2 activity was assayed as follows: 100 µg of the cell lysate supernatant were added to 250 µL reaction buffer (50 mM Tris-HCl, pH 8.9; 7.5 mM CaCl2; 5 mM MgCl2; 1 mg/mL fatty acid free-BSA) and to 100 µL of the vesicles substrate, and incubated at 37°C for 30 min [18]. The reaction was stopped by the addition of chloroform: methanol: HCl (100/100/1; v/v) [23]. Arachidonic acid release was extracted in the lower phase and analyzed by thin-layer chromatography (TLC) on silica-gel 60 (TLC Aluminium Sheets, Merck, Darmstadt, Germany) with: hexane:ethyl ether:acetic acid (90:10:1; v/v/v). Lipids were stained by iodine vapor. Radiolabeled arachidonic acid separated by TLC was analyzed by autoradiography on Kodak XOmat films. Lipid spots were cut from TLC plates and their radioactivity was quantified by liquid scintillation. Reference arachidonic acid used in TLC was obtained from Sigma.
Cells were plated in 25 cm2 tissue culture flasks (5.105 cells/ flask). After 24 h, induction of the fat-storing phenotype was initiated by incubation of cultures with DMEM supplemented with 5% FBS, in presence of 0.01, 0.1, 0.5 and 1 mg/mL pentoxifylline and/or 1, 2.5, 5, 7.5 and 10 µM retinol, for 8 days. In the last 24 h, cells were incubated in the presence of 0.6 µCi/mL [14C]acetate (51.0 mCi/mM specific activity; New England Nuclear, Boston, MA, USA). Lipid synthesis was quantified by radiolabeled acetate incorporation. Total lipids were extracted with chloroform: methanol (2:1, v/v). The lower phase was pooled, dried under nitrogen and analyzed by TLC on G-60 silica-gel aluminium plates with hexane:ethyl ether:acetic acid (90:10:1; v/v/v). The neutral lipids analysis has been previously described in detail [27].
Release of prostaglandins Cells were cultured for 3 days with 0.5 mg/mL pentoxifylline and/or 5 µM retinol. The cells were washed 3 times in phosphate-buffered saline (PBS) and they were incubated with DMEM without serum, with or without (1 µM) Ca2+ ionophore A23187. After 2 h at 37ºC, the medium was removed and the supernatants were used for the prostaglandin E2 and prostaglandin F2α assay. The prostaglandin levels were then assayed with an EIA kit (Cayman Chemical Company), following the manufacturers protocols. Protein was determined by a modified Lowry’s method, and the prostaglandin concentrations were expressed as pg per mg protein.
Measurement of arachidonic acid release Cells cultivated in plates (Nunc) were incubated for 24 h with [14C]arachidonic acid (1 µCi/mL) (55.0 mCi/mmol – Amersham Life Science). At the conclusion of the labeling period, the medium was removed and the cells were gently rinsed twice with Ca2+/Mg2+ free phosphate buffered saline (pH 7.4). After rinsing, the cells were incubated with DMEM supplemented with 5% FBS with or without (1 µM) Ca2+ ionophore A23187 [24, 25]. After 2 h at 37ºC, the medium
Cell cycle analysis Cells were removed from culture dishes using trypsin, centrifuged (500 g, 5 min) and resuspended in calcium-free buffer (BSS) saline and centrifuged. The cells were fixed in 70% ethanol and stored [25]. They were analyzed in the FacScan, with Cell Cycle Plus DNA reagent Kit (BectonDickinson, Mountain View, CA, USA).
40 Statistical analysis Results are expressed as means ± S.D. Statistical calculations were done using a one-way ANOVA. Differences among groups were examined using the Bonferroni t-test. Differences between groups were considered significant at p < 0.05.
Results and discussion Hepatic stellate cells play a crucial role in the development of liver fibrosis. Given the highly proliferative capacity of myofibroblastic HSC, several studies have focused on factors that may limit their growth. Mallat et al. [28] have shown that PGE2 and cAMP inhibit proliferation of human myofibroblastic HSC. In the present study we have monitored the action of PTF, an inhibitor of phosphodiesterase, in association or not with retinol, which is a physiologic inducer of HSC differentiation, upon the induction of the lipocyte phenotype and production of cellular mediators such as AA and PGs in the GRX cell line. This cell line is considered to be representative of murine liver connective tissue cells, and it may be used as an experimental model to study the physiology of hepatic lipocytes [29]. GRX cell line can be induced in vitro to display the fat-storing phenotype by treatment with retinol [16] insulin and indomethacin [17, 27]. The proliferation of GRX cells in the presence of these lipocyte inducers has been described: their growth attained the stationary phase after 2 days of treatment concomitantly with the beginning of fat droplets accumulation [16, 17]. In our experimental model, the growth of GRX cells, maintained under standard conditions and in the presence of 0.05, 0.5 and 1 mg/mL PTF is presented in the Table 1. The cell number observed in cultures treated with 0.05 mg/mL PTF was increased similarly to the control cultures, but the proliferation was suppressed at 0.5 mg/mL PTF after 5 days of treatment. Signs of acute toxicity became evident at a PTF concentration of 1 mg/mL for 5 days. These results are similar to those observed by Ventura and Bohnke [30]. Chronic toxicity, proliferation suppression, and degenerative morphological changes were observed at drug concentrations of 1–100 mg/L, on monolayers of highly proliferative cells of epithelial origin. In order to monitor the relation between PTF and/or retinol and the cell cycle, we measured the percentage of cells in the G0-G1, G2-M and S phase of the cell cycle. We concluded that the treatment with PTF with/without retinol after 3 days did not modify the percentage of cells in the G2-M phase when compared to the control cells (approximately 3%). However, after 6 days treatment this value decreased 1.5% as opposed to the control culture that increased to 40%. These results confirmed that PTF with or without retinol decreases the cell proliferation.
Table 1. Proliferation of GRX cells after 5 days of treatment with PTF Cell number (106/mL) Time: 0 Control
0.5 (± 0.05)
Time: 5 days Control PTF 0.05 mg/mL PTF 0.5 mg/mL PTF 1 mg/mL
5.56 (± 0.04) 5.11 (± 0.09) 2.54 (± 0.14) 0.34 (± 0.07)
The cells were mainted for 5 days under standard conditions (control) or in the presence of 0.05; 0.5; 1 mg/mL PTF. Cells were harvested in 1 mL of PBS and counted. Mean and S.D. for 3 independent experiments are shown.
When maintained in standard culture conditions, the GRX cells were stellate (myofibroblasts phenotype), and when reaching confluence, they were not fully contact-inhibited. (Fig. 1A). When induced to convert into the fat-storing phenotype with 5 µM retinol for 8 days, GRX cells began to lose their fibroblastoid morphology, shortened the cell extensions, and progressively acquired a polygonal shape. They accumulated refringent fat droplets, stainable with Oil Red O (Fig. 1B). The number and the size of fat droplets progressively increased until they occupied most of the cell volume. When the cells were treated with 0.5 mg/mL PTF, they maintained the myofibroblast phenotype like control cultures (Fig. 1C). However, when the cells were treated with 0.5 mg/mL PTF associated with 5 µM retinol, the accumulation of fat droplets was increased when compared to the treatment with retinol only (Fig. 1D). Clusters of GRX cells in the confluent monolayer accumulated refringent lipid droplets in the perinuclear area, stainable with the Oil Red O. They progressively increased in size but never fused in a single fat droplet, such as observed in adipocytes [16]. The conversion was not synchronous and, at this stage, fibroblastoid cells could be seen together with the lipocytes. In previous studies we have analyzed the fact that the reorganization of the cytoskeleton precedes the formation of lipid droplets [31]. Early stages of fat droplets in HSCs were also reported to be surrounded by membrane, while mature droplets have no membrane but have a regular intermediate filament cage [17, 31]. The analysis of neutral lipids stored in lipocytes was done by relative concentration of retinol and PTF (Figs 2A and 2B). The stored neutral lipids, analyzed by TLC, were identified as triacylglycerols (TG), monoalkyl-diacylglycerol (AKDAG) [27], diacylglycerol/cholesterol (DAG/Cho) and ester-cholesterol (ECho). After 8 days of treatment, 5 µM retinol alone induced an increase in TG and AK-DAG accumulation (Fig. 2A). It is to be noted that PTF alone did not induce a discernible increase of neutral lipids accumulation when compared to controls (Fig. 2B), but the synergistic effect of the two drugs (PTF and retinol) was very prominent until 0.1 mg/
41
Fig. 1. Oil red O staining of fat droplets formed in GRX cells. After 7 days under standard culture conditions ×200 (A), treated with 5 µM retinol ×200, fat droplet stainging in one cell (arrow) (B), with 0.5 mg/mL PTF ×200 (C) and with 5 µM retinol and 0.5 mg/mL PTF x200, fat droplet stainging in one cell (arrow) (D).
mL PTF with 5 µM retinol (Fig. 2A). When we increase the relative concentration of PTF (0.5, 0.7 and 1 mg/mL) TG and AK-DAG accumulation was lower after the 8-day treatment period (Fig. 2A). It may be inferred that, in low concentrations of PTF, the lipid accumulation in cells increases through the decrease of intracellular mobilization of lipids. In adipo-
A
B
Fig. 2. Thin-layer chromatography of neutral lipids extracted from GRX cells. (A) Effect of increasing relative concentration of PTF (mg/mL) on lipid accumulation of GRX cells treated with 5 µM retinol. The cells were maintained in culture for 8 days in the standard medium (C = control) or medium supplemented with 5 µM retinol (R) with or without 0.05, 0.1, 0.5, 0.7, 1 mg/mL PTF. (B) Effect of increasing relative concentration of retinol (µM) on lipid accumulation of GRX cells treated with 0.5 mg/mL PTF. The cells were maintained in culture for 8 days in standard medium (C = control) or medium supplemented with 0.5 mg/mL PTF with or without 1, 2.5, 5, 7.5, 10 µM retinol. Lipids were identified as diacylglycerols/cholesterol (DAG-Cho) triacylglycerols (TG), monoalkyl-diacylglycerols (AKDAG) and ester-cholesterol (ECho).
cytes, the anti-lipolytic activity acts through dephosphorylation of the hormone-sensitive cAMP-regulated lipase [32]. The same mechanism may act in our model, since PTF in higher concentration (0.5–1 mg/mL) increases the intracellular cAMP content through inhibition of phosphodiesterase [33]. In previous studies, we analyzed the induction of retinol-binding proteins, and the enzyme systems required for accumulation of triglycerides and the associated lipids that constitute the lipid droplets [34, 35]. One of the major mediators was shown to be the retinoic acid (RA), which controls the expression of a vast array of genes of lipid metabolism. It is well recognized that RAR (retinoic acid receptor) and RXR (retinoid X receptor) serve as nuclear receptors for RA and that the induction of mitochondrial, peroxisomal and microsomal genes involved in fatty acid metabolism requires the formation of PPAR heterodimers with RXR [36]. Miyahara et al. [37] demonstrated that HSC activation is associated with the reduction in PPAR-γ expression and PPAR-responsive element binding. These findings indicates that a decrease in PPAR-γ signaling is one of the molecular mechanisms underlying activation of HSC in liver fibrogenesis and the potential therapeutic value of PPAR-γ ligands for liver fibrosis. However, the action of PTF on PPAR receptor has not been yet investigated. The relative activity of PTF and retinol on acetate incorporation in total lipids of GRX cell line is shown in the Figs 3A and 3B. In the presence of 0.5 mg/mL PTF, a high stimulation of acetate incorporation was observed with physiologic
42 concentrations of retinol (1 and 2.5 µM), but a full effect of retinol on lipocyte induction could only be observed in concentrations of 5 µM retinol, that stabilized between 7.5 and 10 µM (Fig. 3A). In the presence of 5 µM retinol, a significant stimulatory effect was observed at 0.1 mg/mL of PTF but a high effect could be observed only in concentrations
of 0.7 mg/mL PTF. Higher concentrations of PTF, supplementing the same quantity of retinol had an opposite effect, decreasing the acetate incorporation into total lipids. This suggests that the toxic effect of PTF reflects on lipid metabolism (Fig. 3B). The de novo synthesis of diacylglycerols/cholesterol, free fatty-acid, triacylglycerols, monoalkyl-diacylglycerol, estercholesterol and phospholipids was monitored by [14C]acetate incorporation. The Table 2 shows only the effect of PTF and/ or retinol on [14C]acetate incorporation into phospholipids, triacylglycerols and monoalkyl-diacylglycerol of GRX cells. PTF alone did not induce significant modifications of acetate incorporation into GRX cells, as compared to controls, except in phospholipids. Retinol induced a significant increase of phospholipids, triacylglycerols and monoalkyl-diacylglycerol synthesis. The relative association of PTF and retinol increased acetate incorporation in all the analyzed lipids. This effect was not discernible in the presence of 5 µM retinol with higher concentration (1 mg/mL) of PTF. The best concentration for diacylglycerols/cholesterol, free fatty-acid, triacylglycerols, ester-cholesterol and phospholipids synthesis was 0.7 mg/L of PTF with 5 µM of retinol, except for monoalkyl-diacylglycerol that increased with low concentration of PTF (0.1 mg/mL). In the presence of 0.5 mg/mL PTF, low concentrations of retinol (1 and 2.5 µM) were sufficient
Table 2. Effect of PTF and/or retinol on [14C]acetate incorporation into lipids of GRX cells Treatment Control PTF (0.5 mg/mL) + RET (1 µM) + RET (2.5 µM) + RET (5 µM) + RET (7.5 µM) + RET (10 µM) Fig. 3. [14C]Acetate incorporation into total lipids of GRX cells. (A) Effect of increasing relative concentration of retinol (µM) on [14C]acetate incorporation into total lipids of GRX cells treated with 0.5 mg/mL PTF. GRX cells maintained in culture for 8 days in standard medium (control) or medium supplemented with 0.5 mg/mL PTF with or without 1, 2.5, 5, 7.5, 10 µM retinol. (B) Effect of increasing relative concentration of PTF (mg/ mL) on [14C]acetate incorporation into total lipids of GRX cells treated with 5 µM retinol. GRX cells maintained in culture for 8 days in standard medium (control) or medium supplemented with 5 µM retinol (Ret) with or without 0.05, 0.1, 0.5, 0.7, 1 mg/mL PTF. The cells were labeled with [14C]acetate (0.6 µCi/mL) during the last 24 h. Lipids were extracted with chloroform: methanol (2:1, v/v) and the total acetate incorporation into cells was quantified by liquid scintillation. Incorporation of acetate is expressed as cpm/µg protein. Mean values ± S.D. for 3 independent experiments are given. ap < 0.05; a’p < 0.01; a”p < 0.001 and NS with respect to control; bp < 0.05; b’p < 0.01; b”p < 0.001 with respect to PTF; cp < 0.05; c’p < 0.01; c”p < 0.001 with respect to retinol.
RET (5 µM) + PTF (0.05 mg/mL) + PTF (0.1 mg/mL) + PTF (0.5 mg/mL) + PTF (0.7 mg/mL) + PTF (1 mg/mL)
Incorporation of [14C] acetate (cpm/µg of protein) Phospholipids TG AK-DAG 5.0 (.1) 8.1 (1.0)e 15.4 (.5)c 31.9 (1.7)a 37.4 (2.9)a 45.8 (5.3)a 49.5 (2.3)a
2.2 (.1) 1.9 (.2)NS 6.3 (.6)b 10.6 (1.0)a 24.2 (.9)a 9.6 (.6)a 6.1 (1.1)b
1.0 (.1) 1.0 (.1)NS 2.6 (.3)d 3.7 (.2)a 4.2 (.7)a 3.9 (.3)a 2.6 (.3)b
14.8 (.6)g 15.0 (.6)g 27.4 (2.0)i 45.4 (3.1)h 102.5 (18.0)h 21.6 (4.7)e
10.8 (.1)g 17.4 (1.0)i 31.3 (2.2)h 24.2 (.9)h 56.0 (11.0) h 10.7 (1.8)f
3.0 (.01)g 4.0 (.1)i 6.8 (.5)h 5.1 (.2)h 6.4 (1.0)i 1.8 (.3)e
The cells were maintained in culture for 8 days in standard medium (control) or medium supplemented with 0.5 mg/mL PTF with/without 1; 2.5; 5; 7.5; 10 µM retinol; or 5 µM retinol with/without 0.05; 0.1; 0.5; 0.7; 1 mg/ mL PTF. Results represent mean ± S.D. of 3 separate experiments. ap < 0.001; b p < 0.01 with respect to control and to PTF; cp < 0.001; p < 0.01 with respect to control and to PTF, respectively; dp < 0.01; p < 0.05 with respect to control and to PTF, respectively; ep < 0.05; fp
